TOXICOLOGICAL CHEMISTRY

LVIV – 2009

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Посібник складено і підготовлено до друку на кафедрі токсикологічної та

аналітичної хімії Львівського національного медичного університету імені

Данила Галицького старшим викладачем Бідниченком Ю.І.

Рецензенти:

- завідувач кафедри токсикологічної та неорганічної хімії Запорізького державного

медичного університету, доктору фармацевтичних наук, професор Буряк В.П. - завідувач кафедри токсикологічної хімії Національного фармацевтичного

університету, доктору хімічних наук, професор Бондар В.С. - завідувачу кафедри медичної хімії Одеського державного медичного університету,

доктор біологічних наук Мардашко О.О.

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INTRODUCTION INTO TOXICOLOGICAL CHEMISTRY

Toxicology is an interdisciplinary scientific branch, which studies toxicological

aspects of biology (toxicokinetics, toxicodinamics, experimental and clinical toxicology) and chemical sciences (chemical structure and analysis of poisons). The discipline is

focused on toxicological aspects of all factors affecting human organism. Special attention

is paid to toxic substances, which are present in the air, water, food, plants, and entire environment, and to industrial toxins. General analytical methods for measuring

concentration of toxic substances in the air, water, food and in biological materials and the procedures in estimating drug toxicity have primary significance and are taught in practical

training of this subject. Toxicological chemistry is a science, which works out new and develops present

methods of detection and determination of poisonous substances in various objects, create

theoretical fundamentals of these methods. A chemical-toxicological analysis is a complex of scientifically grounded methods,

employed in practice for isolation, detection and quantitative determination of toxic substances.

The contemporary toxicological chemistry has the following tasks: 1. Elaboration new and improvement of already employed isolation methods of toxic

substances from various objects.

2. Elaboration the effective purification methods of extracts taken from objects of chemical-toxicological analysis.

3. Introduction in practice of chemical-toxicological analysis new sensitive and specific reactions and methods (chromatography, spectroscopy etc.) for detection of toxic

substances isolated from appropriate objects. 4. Elaboration and introduction in practice of chemical-toxicological analysis new

sensitive methods of quantitative determination of toxic substances.

5. Study of metabolism of toxic substances in organism and elaboration methods of metabolites analysis.

Poison is a substance, natural or synthetic, that causes damage to living tissues and has an injurious or fatal effect on the body, whether it is ingested, inhaled, or absorbed or

injected through the skin.

Although poisons have been the subject of practical lore since ancient times, their systematic study is often considered to have begun during the 16th century, when the

German-Swiss physician and alchemist Paracelsus first stressed the chemical nature of poisons. It was Paracelsus who introduced the concept of dose and studied the actions of

poisons through experiment. It was not until the 19th century, however, that the Spaniard Matthieu Orfila, the attending physician to Louis XVIII, correlated the chemistry of a toxin

with the biological effects it produces in a poisoned individual. Both concepts continue to

be fundamental to an understanding of modern toxicology. Poisoning involves four elements: the poison, the poisoned organism, the injury of

cells, and the symptoms and signs or death. These four elements represent the cause, subject, effect, and consequence of poisoning. To initiate poisoning, the organism is

exposed to toxic chemical. When a toxic level of the chemical compound is accumulated in the cells of the target tissue or organ, the resultant injury to the cells disrupts their normal

structure or function. Symptoms and toxic signs then develop, and, if the toxicity is severe

enough, death may result.

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Definition of Poison

A poison is a substance capable of producing adverse effects on an individual under appropriate conditions. The term "substance" is almost always synonymous with "chemical"

and includes drugs, vitamins, pesticides, pollutants, and proteins. Even radiation is a toxic substance. Though not usually considered to be a "chemical," most radiations are generated

from radioisotopes, which are chemicals. The term "adverse effects" above refers to the

injury, such as structural damage to tissues. "Appropriate conditions" refers to the dosage of the substance that is sufficient to cause these adverse effects. The dose concept is important

because according to it even a substance as innocuous as water is poisonous if too much is ingested. Whether a drug acts as a therapy or as a poison depends on the dose.

Classification of Poisons

Poisons are of such diverse natures that they are classified by origin, physical form,

chemical nature, chemical activity, target site, or use.

1. Classification based on origin Poisons are microbial, plant, animal, or synthetic origin.

Microscopic organisms such as bacteria and fungi produce microbial poisons.

Botulinus toxin, for example, is produced by the bacterium Clostridium botulinum and is capable of inducing weakness and paralysis when present in underprocessed, nonacidic

canned foods or in other foods containing the spores. An example of a plant toxin is the belladonna alkaloid hyoscyamine, which is found in belladonna (Atropa belladonna) and

jimsonweed (Datura stramonium). Animal poisons are usually transferred through the bites and stings of venomous

terrestrial or marine animals, the former group including poisonous snakes, scorpions,

spiders, and ants, and the latter group including sea snakes, stingrays, and jellyfish. Synthetic toxins are responsible for most poisonings. "Synthetic" refers to chemicals

manufactured by chemists, such as drugs and pesticides, as well as chemicals purified from natural sources, such as metals from ores and solvents from petroleum. Synthetic toxins

include pesticides, household cleaners, cosmetics, pharmaceuticals, and hydrocarbons.

2. Classification based on physical form The physical form of chemicals – solid, liquid, gas, vapour, or aerosol – influences

the exposure and absorbability.

Solids are generally not well absorbed into the blood; they must be dissolved in the aqueous liquid lining the intestinal tract if ingested or the respiratory tract if inhaled. Solids

dissolve at different rates in fluids, however. For example, compared with lead sulphate granules, granules of lead are practically non-toxic when ingested, because elemental lead is

essentially insoluble in water, while lead sulphate is slightly soluble and absorbable. Even

different-sized granules of the same chemical can vary in their relative toxicities because of the differences in dissolution rates. For example, arsenic trioxide is more toxic in the form

of smaller granules than is the same mass of larger granules because the smaller granules dissolve faster.

A poison in a liquid form can be absorbed by ingestion or by inhalation or through the skin. Poisons that are gases at room temperature (e.g., carbon monoxide) are absorbed

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mainly by inhalation, as are vapours, which are the gas phase of substances that are liquids

at room temperature and atmospheric pressure (e.g., benzene). Because organic liquids are

more volatile than inorganic liquids, inhalation of organic vapours is more common. Although vapours are generally absorbed in the lungs, some vapours that are highly soluble

in lipids (e.g., furfural) are also absorbed through the skin. Aerosols are solid or liquid particles small enough to remain suspended in air for a

few minutes. Fibres and dust are solid aerosols. Aerosol exposures occur when aerosols are

deposited on the skin or inhaled. Aerosol toxicity is usually higher in the lungs than on the skin. An example of a toxic fibre is asbestos, which can cause a rare form of lung cancer

(mesothelioma). Many liquid poisons can exist as liquid aerosols, although highly volatile liquids,

such as benzene, seldom exist as aerosols. A moderately volatile liquid poison can exist as both an aerosol and as a vapour. Airborne liquid chemicals of low volatility exist only as

aerosols.

3. Classification based on chemical nature Poisons can be classified according to whether the chemical is metallic versus non-

metallic, organic versus inorganic, or acidic versus alkaline.

Metallic poisons are often eliminated from the body slowly and accumulate to a greater extent than non-metallic poisons and thus are more likely to cause toxicity during

chronic exposure.

Organic chemicals are more soluble in lipids and therefore can usually pass through the lipid-rich cell membranes more readily than can inorganic chemicals. As a result,

organic chemicals are generally absorbed more extensively than inorganic chemicals. Classification based on acidity is useful because, while both acids and alkalis are

corrosive to the eyes, skin, and intestinal tract, alkalis generally penetrate the tissue more deeply than acids and tend to cause more severe tissue damage.

4. Classification based on chemical activity Electrophilic (electron-loving) chemicals attack the nucleophilic (nucleus-loving)

sites of the cells' macromolecules, such as deoxyribonucleic acid (DNA), producing mutations, cancers, and malformations.

Poisons also may be grouped according to their ability to mimic the structure of certain important molecules in the cell. They substitute for the cells' molecules in chemical

reactions, disrupting important cellular functions. Methotrexate, for example, disrupts the

synthesis of DNA and ribonucleic acid (RNA).

5. Other classifications of poisons Unlike the classifications described above, there is usually no predictive value in

classification by target sites or by uses. Such classifications are done, however, to systematically categorize the numerous known poisons. Target sites include the nervous

system, the cardiovascular system, the reproductive system, the immune system, and the

lungs, liver, and kidneys. Poisons are classified by such uses as pesticides, household products, pharmaceuticals, organic solvents, drugs of abuse, or industrial chemicals.

6. Forensic-chemical classification of poisons Toxicological chemistry describes all toxic substances in accordance to their

isolation technique from objects of investigation (internal organs of corpses, human body

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fluids – blood, urine etc.). This classification is grounded on chemical, physical and

toxicological properties of toxic substances.

Methods of poisonous substances isolation from biological material

Autopsy One of the stages of chemical-toxicological investigation is an autopsy. Autopsy,

also called Necropsy, Post-mortem, or Post-mortem Examination, dissection and examination of a dead body and its organs and structures to determine the cause of death, to

observe the effects of disease, and to establish the sequences of changes and thus to establish evolution and mechanisms of disease processes.

The early Egyptians did not study a dead human body for an explanation of disease

and death, though some organs were removed for preservation. The Greeks and the Indians cremated their dead without examination; the Romans, Chinese, and Muslims all had taboos

about opening the body; and human dissections were not permitted during the Middle Ages. The first real dissections for the study of disease were carried out around 300 BC by

the Alexandrian physicians Herophilus and Erasistratus, but it was the Greek physician Galen, in the late 2nd century AD, who was the first to correlate the patient's symptoms

Volatile poisons Alkaloids, medicines

Distillation Headspace

analysis

Extraction by polar solvents

(miscible with water)

OBJECT Dialysis

Acids, bases,

salts

Extraction by

nonpolar

organic

solvents

(immiscible

with water) Pesticides

Mineralisation

Metallic

poisons

Mercury

compounds Destruction

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(complaints) and his signs (what can be seen and felt) with what was found upon examining

the "affected part of the deceased." This was the great leap forward that eventually led to the

autopsy and broke an ancient barrier to progress in medicine. It was the rebirth of anatomy during the Renaissance, as exemplified by the work of

Andreas Vesalius (De Humani Corporis Fabrica, 1543) that made it possible to distinguish the abnormal, as such (e.g., an aneurysm), from the normal anatomy. Leonardo da Vinci

dissected 30 corpses and noted "abnormal anatomy"; Michelangelo, too, performed a

number of dissections. Earlier, in the 13th century, Frederick II ordered that the bodies of two executed criminals be delivered every two years to the medical schools, one of which

was at Salerno, for an "Anatomica Publica," which every physician was obliged to attend. The first forensic or legal autopsy, wherein the death was investigated to determine presence

of "fault," is said to have been one requested by a magistrate in Bologna in 1302. Antonio Benivieni, a 15th-century Florentine physician, carried out 15 autopsies explicitly to

determine the "cause of death" and significantly correlated some of his findings with prior

symptoms in the deceased. Theophile Bonet of Geneva (1620-1689) collated from the literature the observations made in 3,000 autopsies. Various observers, thus opening the

door to modern practice, then defined many specific clinical and pathologic entities. The autopsy came of age with Giovanni Morgagni, the father of modern pathology,

who in 1761 described what could be seen in the body with the naked eye. In his voluminous work On the Seats and Causes of Diseases as Investigated by Anatomy, he

compared the symptoms and observations in some 700 patients with the anatomical findings

upon examining their bodies. Thus, in Morgagni's work the study of the patient replaced the study of books and comparison of commentaries.

With Karl von Rokitansky of Vienna (1804-1878), the gross (naked eye) autopsy reached its apogee. Rokitansky utilized the microscope very little and was limited by his

own humoral theory. The French anatomist and physiologist Marie F.X. Bichat (1771-1802) stressed the role of the different generalized systems and tissues in the study of disease. It

was the German pathologist Rudolf Virchow (1821-1902), however, who introduced the

cellular doctrine – that changes in the cells are the basis of the understanding of disease – in pathology and in autopsy. He warned against the dominance of pathologic anatomy – the

study of the structure of diseased tissue – alone as such and stressed that the future of pathology would be physiologic pathology – study of the functioning of the organism in the

investigation of disease. The modern autopsy has been expanded to include the application of all knowledge

and all of the instruments of the specialized modern basic sciences. The examination has

been extended to structures too small to be seen except with the electron microscope, and to molecular biology to include all that can be seen as well as what still remains unseen.

Procedure. The autopsy procedure itself has changed very little during the 20th century. The first

step is a gross examination of the exterior for any abnormality or trauma and a careful

description of the interior of the body and its organs. This is usually followed by further

studies, including microscopic examination of cells and tissues. The main incisions in the body remain the same. For the torso, a Y-shaped incision is

made. Each upper limb of the "Y" extends from either the armpit or the outer shoulder and is carried beneath the breast to the bottom of the sternum, or breastbone, in the midline.

From this point of juncture at the bottom of the sternum the incision is continued down to the lower abdomen where the groins meet in the genital area.

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There are different schools as to procedure beyond this point. In one method, each

organ is removed separately for incision and study. In the so-called en masse methods the

chest organs are all removed in a single group and all of the abdominal organs in another for examination. The great vessels to the neck, head, and arms are ligated – tied off – and the

organs removed as a unit for dissection. The neck organs are explored in situ only or removed from below. Dissection then proceeds usually from the back, except where

findings dictate a variation in the procedure. Usually groups of organs are removed together

so that disturbances in their functional relationships may be determined. After study of the brain in position, it is freed from its attachments and removed in toto. The spinal cord also

can be removed. The dissector proceeds to examine the external and cut surface of each organ, its

vascular structures, including arteries, lymphatics, fascial or fibrous tissue, and nerves. Specimens are taken for culture, chemical analysis, and other studies. Immediately upon

completion of the procedure, all of the organs are returned to the body and all incisions

carefully sewn. After the body's proper restoration, no unseemly evidence of the autopsy need remain.

After the gross examination of the body the findings are balanced one against another and a list of pathological findings is compiled; this list comprises the tentative or

"provisional anatomical diagnoses." Such diagnoses are grouped and arranged in the order of importance and of sequence. On occasion a quick microscopic study is done to confirm a

diagnosis so as to assure its proper listing.

Finally the examiner lists as the cause of death the one lesion without which death would not have occurred. Though obviously all-important in forensic cases, this aspect of

the autopsy analysis is also required in cases not required by law. After all studies – histological, chemical, toxicological, bacteriological, and viral – are completed, any errors

of the provisional anatomical diagnoses are corrected and the final anatomical diagnoses and the final cause of death are listed. A statement of analysis of the autopsy that correlates

the findings with the clinical picture, the "clinical pathological correlation," concludes the

record of the autopsy.

Forensic autopsy. The forensic pathologist goes beyond the mere cause of death; he must establish all

the facts, both lethal and no lethal, with any potential bearing whatsoever on the criminal or civil litigation. The cause of death is not automatically revealed when the body is opened; it

is not an isolated tangible and delimited entity; it is a concept – an opinion – as to

mechanism or happening and as such is subject occasionally to differences in interpretation. The legal autopsy requires meticulous detailed descriptions, measurements, and

documentation. Experience in the investigation of the scene of a death in medico-legal cases is

important, for the evaluation of circumstances of death may be critical in establishing the mode of death – e.g., suicide. The autopsy may not be able, of itself, to determine intent,

whereas the scene and the circumstances may provide unmistakable evidence. Photographic

documentation is important in the medico-legal autopsy. The medico-legal post-mortem examination must always be complete to rule out any other potential contributory cause of

death and therefore must never be limited to a partial study. The identification of the deceased and of all specimens taken from the body is critical; the time of death and the

blood grouping must, if possible, be established. In all autopsies, but especially in forensic cases, findings must be dictated to a stenographer or recording instrument during the actual

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performance of the procedure. The record often becomes legal evidence and therefore must

be complete and accurate.

Purposes of Autopsy

The autopsy deals with the particular illness as evidenced in one individual and is

more than simply a statistical average. Every autopsy is important to expose mistakes, to delimit new diseases and new patterns of disease, and to guide future studies. Morbidity and

mortality statistics acquire accuracy and significance when based on careful autopsies; they also often give the first indication of contagion and epidemics. Nor can the role of the

autopsy in medical education be understated. It is the focal point at which the profession learns to assess and to apply medical knowledge. Thus, the autopsy does more than merely

determine the cause of death. While the medico-legal autopsy in particular has this

important primary objective, most autopsies have a larger purpose.

Objects of Chemical-Toxicological Examination

Chemical-toxicological laboratories carry out investigation of urine and blood,

vomiting mass, stomach washes, dialysates after dialysis, and other objects on toxic

substances presence. Objects of forensic-toxicological examination are controlled substances, poisonous

substances, objects used for poisoning, poisoned persons and another subjects: human or animal corpses, stomach aspirate or wash-out liquid, blood, vomiting masses, excrement,

hair, nail, rests of food, drinks, pesticides, part of plants pesticides processed, water from reservoirs, test of air of industrial enterprises, ground, housekeeping things, clothes etc.

Blood examinations can determine the presence or absence of blood in stains.

Examinations can also determine whether blood is human or nonhuman and can determine the animal species. Blood examinations cannot determine the age or the race of a person.

Conventional serological techniques are not adequately informative to positively identify a person as the source of a stain. Investigations for an "unknown" drug or poison are usually

carried out on specimens of urine (30 ml – for qualitative tests) and blood (10 ml – for quantitative tests) and carried out on a 24-hour as usually.

The internal bodies from corpse take in amounts not less than 2 kg. They should not

be washed by water and to be subject of pollution by chemical substances and mechanical impurity.

All objects of suspicion on unknown toxin poisoning should be taken in separate glasses: stomach with contents, one meter thin and thick intestines with contents from the

most changed sites, not less than one third of liver and gall-bladder with contents, one kidney and all urine, one third of head brain, heart with blood contained in it, spleen and not

less than one quarter of lung.

On suspicion of poisoning by substances introduced through vagina, per rectum, subcutaneous or intramuscular, except for the listed objects of forensic-chemical research, in

addition are directed uterus with vagina, rectum with contents, sites of skin and muscles from places of probable introduction of substance.

On suspicion of a chronic poisoning with arsenic compounds for forensic-chemical research hair, nails and bones in addition are directed.

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After exhumation of corpse except internal organs for forensic-chemical research are

directed: till 1 kg of ground taken from six places around the tomb (direct under a coffin,

above a coffin, at lateral surfaces and ends of a coffin). On research are directed also objects from the coffin, trimmings, and a piece of the coffin bottom (size 400 cm

2) .

The internal bodies, parts of corpse and other objects are directed on research in separate clean and dry glasses. Use of metal or ceramic utensils is inadmissible.

Preservation of objects of chemical-toxicological analysis by formalin or phenol is

undesirable, because these substances are poisons too and can be objects of investigation on presence in biological material. Therefore, it is necessary to know what preserving agent is

used. For the same reasons preservation of biological material by ethanol is undesirable

too. Ethanol can cause poisoning. Preservation of biological material by ethyl alcohol excludes possibility of it detection as probable poisoning caused substance. Besides, the

ethyl alcohol prevents destruction of biological material in research on metal poisons

presence. However, preservation by ethyl alcohol is acceptable if transportation of internal

bodies is made in hot season and can takes over five day, except in cases of poisoning by alcohols and nitrites. Thus internal organs in glass should be layered by alcohol not less than

1 cm. Simultaneously with preserved internal organs to laboratory must be directed for control test 300 ml of used alcohol. Alcohol for the control test takes from the same

container as for the internal organs preservation.

All requests for evidence examinations should be in writing, addressed to the Forensic Chemical Laboratory, and contain the following information:

– The submitting contact person's name, agency, address, and telephone number; – Previous case identification numbers, evidence submissions, and communications

relating to the case; – Description of nature and main facts concerning the case as they pertain to the

Laboratory examinations;

– The name or names of and descriptive data about the individual or individuals involved (subject, suspect, victim, or a combination of those categories) and the agency-assigned

case identification number; and – A list of the evidence being submitted herewith (enclosed) or under separate cover.

Herewith is limited to small items of evidence that are not endangered by transmitting in an envelope. Write on the envelope before placing evidence inside to avoid

damaging or altering the evidence. The written communication should state: Submitted

herewith is the following item of evidence. Separate cover is used to ship numerous or bulky items of evidence or both. Include

a copy of the communication requesting the examinations. The written communication should state: Submitted under separate cover by (list the method of shipment) are the

following items of evidence. – State what types of examinations are requested.

– State where the evidence should be returned and where the Laboratory report should be

sent. – Attach a statement if another expert in the same field examined the evidence, if there is

local controversy, or if other law enforcement agencies have an interest in the case. – State the need and reason or reasons for an expeditious examination. Do not request an

expeditious examination routinely. – Submit a separate communication for multiple cases.

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Packaging and Shipping Evidence

1. Prior to packaging and shipping evidence, call the pertinent unit of the Laboratory for specific instructions.

2. Take precautions to preserve the evidence.

3. When requesting latent print examinations, place nonporous evidence in individual protective coverings such as thick transparent envelopes or suspend in a container so that

there is minimal surface contact. Place porous evidence in individual protective coverings such as paper envelopes. Stabilise the evidence to avoid movement or friction

during shipment. 4. Wrap and seal each item of evidence separately to avoid contamination.

5. Place the evidence in a clean, dry, and previously unused inner container.

6. Seal the inner container with tamper-evident or filament tape. 7. Affix EVIDENCE and appropriate BIOHAZARD or HAZARDOUS MATERIALS

labels to the inner container. 8. If any of the evidence needs to be examined for latent prints, label LATENT on the

inner container. 9. Affix the evidence examination request and all case information between the inner and

outer containers.

10. Place the sealed inner container in a clean, dry, and previously unused outer container with clean packing materials.

11. Completely seal the outer container so that opening of the container would be evident. 12. Label the outer container with appropriate BIOHAZARD or HAZARDOUS

MATERIALS labels. 13. Address the outer container.

14. The Shipment of Etiologic Agents provides packaging and labelling requirements for

etiologic agents (viable micro organisms or toxins that cause or may cause human disease) shipped in interstate traffic.

Package and label etiologic agents in volumes of less than 50 ml in glass tube as shown in the drawings below and sealed with waterproof tape. Place each tube containing a

culture inside a capped container packed with absorbent materials. Package this primary container within a secondary capped container that is labelled with the specimen record.

Surround the secondary container with dry ice and seal it within a capped shipping container

marked with the destination address and the appropriate infectious substance or etiological agent label.

Collected objects in laboratories are tried on pharmaceutical and toxicological examinations.

Pharmaceutical examinations can identify constituents, active ingredients, quantity, and weight.

Toxicology examinations can disclose the presence of drugs or poisons in biological

specimens. The examinations can determine the circumstances surrounding drug- or poison-related homicides, suicides, or accidents.

Because of the large number of potentially toxic substances, it may be necessary to screen for classes of poisons. Examples are as follows:

– Volatile compounds (ethanol, methanol, isopropanol) – Heavy metals (arsenic)

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– Non-volatile organic compounds (drugs of abuse, pharmaceuticals)

– Miscellaneous (strychnine, cyanide)

Features of Chemical-Toxicological Analysis

The chemical-toxicological analysis has some peculiarities (features). In the

chemical-toxicological analysis for detection and quantitative determination of toxic substances, apply many reactions and methods from analytical and pharmaceutical

chemistry. However many these reactions and methods are useless for purposes of chemical-toxicological analysis because of small sensitiveness or no specificity.

A chemical-toxicological analysis is characterised by variety of research objects,

containing negligible quantities of toxic substances. These substances are micro components in great numbers biological material. It is necessary to separate toxic

substances from the proper objects before their discovery and quantitative determination. The choice of methods of toxic substances separation relies on the research object nature. In

case of use of unsuitable method of separation of toxic substance from the explored object, it can be partial or fully lost during the chemical-toxicological analysis. Thus in a number

of cases for separation of the same substance from the different objects it is necessary to

apply different methods. One of features of chemical-toxicological analysis is necessity separation the

research of substances that causing poisoning, from biological material and determine their metabolites.

In the dead body material is contained a negligible quantity of substance causing

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poisoning; for discovery of this substance it is necessary to apply highly sensitive reactions.

However in case of use of highly sensitive reactions it is possible to reveal not only causing

poisoning substance, but also some substances being natural component of tissues and organs, and also medicinal substances, administered in urgent moments in therapeutic

doses. The expert-chemist must therefore know how correctly to interpret the results of applied them reactions of explored substances detection.

For chemical-toxicological examination of investigated objects are applied various analytical methods.

Methods of detection of poisonous substances, isolated from biological material

Chemical

Colour tests

Precipitation reactions

Microcrystalline reactions

Volatile poisons, alkaloids, medicines, metal

poisons, pesticides, acids, bases

Physicochemical

Thin layer chromatography

Gas chromatography

High performance liquid chromatography

Alkaloids, medicines, pesticides

UV-VIS Spectroscopy

Mass-spectroscopy Alkaloids, medicines

Methods of quantitative determination of poisonous substances

isolated from biological material

Chemical

Gravimetric Titrimetric Volatile poisons, metal poisons, pesticides, mineral acids, salts, bases

Physicochemical

Photometry Volatile poisons, alkaloids, medicines, pesticides, metal poisons, salts

Direct and differential spectroscopy Alkaloids, medicines

Extraction-photometry Alkaloids, medicines, metal poisons, pesticides

Gas-liquid chromatography Volatile poisons, pesticides

Biochemical

Enzymatic Volatile poisons, pesticides

Landmarks of Forensic Science

1149 – Idea of having a coroner started by King Richard of England 1284 – Descriptions of bodies having died from different causes written in a book by

Chinese His Yuan Lu 1447 – Missing teeth used to identify the body of the French Duke of Burgundy

1590 – Beginnings of the modern microscope developed by Zacharias Janssen of Holland.

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1628 – Birth of Italian Marcello Malpighi, credited with noticing patterns on the skin of

fingers.

1670 – First simple microscope with powerful lenses created by Anton Van Leeuwenhoek of Holland

1732 – The basis of lie detection equipment made possible with Luigi Galvani discovery that the human nervous system transmits information electronically

1776 – Body of US General Warren is identified by Paul Revere who made his false teeth.

1807 – Forensic Science Institute established at the University of Edinburgh, Scotland. 1814 – First scientific paper on the detection of poisons published by Matthieu Orfila of

Spain. 1823 – Whorls, ellipses and triangles described by Czech physiologist Jan Evangelista

Purkinje. 1836 – Method for the detection of arsenic poison developed by James Marsh of England.

1849 – Bones and teeth remains used as evidence of murder given by a forensic team lead

by anatomy professor Dr Jeffries Wyman. 1850 – First private detective agency in the US set up by Allan Pinkerton.

1859 – Spectroscopy is developed. Gustav Kirchoff and Robert Bunson showed substances give off a spectrum of light, which identifies elements in the substance.

1879 – System of identifying people by special body measurements developed by Frenchman Alphonse Bertillon.

1880 – First criminological use of fingerprints made by Henry Fauld in Tokyo.

1880s – Sherlock Holmes detective stories published by Sir Arthur Conon Doyle, describe solutions based on crime solving methods.

1888 – First hand held camera invented by American George Eastman. 1889 – Matching bullets to the gun that fired it, developed by Alexandre Lacassagne.

1892 – scientific classification of fingerprints developed by Englishman Francis Galton 1895 – X-rays first discovered by German physicist Conrad Rontgen.

1896 – System of matching fingerprints to identify people developed by Edward Henry of

England. 1900 – Scotland Yard adopts the Galton-Henry system of fingerprinting.

1901 – Basic human blood groups are identified by Austrian, Karl Lansdsteiner. 1902 – Harry Jackson became the first British person convicted on fingerprint evidence.

1903 – New York City Police Department began fingerprint files of arrested persons. 1906 – Bite marks found at the scene of the crime first used as evidence in court.

1909 – Discovery those chromosomes carry hereditary information, made by US

physiologist Thomas Hunt Morgan. 1910 – World’s first forensic laboratory set up in France by Edmond Locard.

1921 – First polygraph (lie detector) built by John Larson, United States. 1920s – Helixometer to examine the inside of gun barrels, developed by John Fisher.

1920s – A method to calculate the thickness of muscle, flesh and skin over the scull developed by Russian paleontologist Michael Gerasimov.

1922 – Nobel Prize awarded to Francis Aston for developing the first mass spectrometer.

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TRANSPORT OF CHEMICALS THROUGH A CELL MEMBRANE

In order for a poison to produce toxicity, a sufficient quantity of that chemical must

be absorbed into the body. Because the chemical must pass through a number of cell

membranes before it can enter the blood, the ability of the chemical to cross these lipid-rich membranes determines whether it will be absorbed, and that ability depends on the

chemical's lipid solubility. The cell membrane, the most external layer of all animal cells, is composed of two

layers of lipid molecules (the lipid bilayer). The lipid molecules each have a hydrophilic (water-loving, or polar) end and a hydrophobic (water-hating, or nonpolar) end. Because an

aqueous environment surrounds them, lipid molecules of the cell membrane arrange

themselves so as to expose their hydrophilic ends and protect their hydrophobic ends. Suspended randomly among the lipid molecules are proteins, some of which extend from

the exterior surface of the cell membrane to the interior surface. A chemical tends to dissolve more readily in a solvent of similar polarity. Nonpolar

chemicals are considered lipophilic (lipid-loving), and polar chemicals are hydrophilic (water-loving). Lipid-soluble, nonpolar molecules pass readily through the membrane

because they dissolve in the hydrophobic, nonpolar portion of the lipid bilayer. Although

permeable to water (a polar molecule), the nonpolar lipid bilayer of cell membranes is impermeable to many other polar molecules, such as charged ions or those that contain

many polar side chains. Polar molecules pass through lipid membranes via specific transport systems.

The four types of chemical transport systems through cell membranes are diffusion, facilitated diffusion, active transport, and pinocytosis.

As mentioned above, lipophilic, nonpolar chemicals dissolve in the lipid bilayer.

Simultaneously, some of the molecules are leaving the lipid bilayer. The net result is that chemicals cross the membrane until the concentrations of chemical molecules on both sides

of the membrane are equal and there is no net flow of molecules across the cell membrane (diffusion). Therefore, chemicals diffuse across the membrane only when a concentration

gradient exists across the cell membrane. Diffusion is considered to be passive transport because no external energy is used. Polar molecules, such as water and small water-soluble

molecules (e.g., urea, chloride ions, sodium ions, and potassium ions), can diffuse across

membranes through the water-filled channels created by membrane proteins. Large polar water-soluble chemicals, such as sugars, however, do not diffuse through the membrane.

Certain relatively large water-soluble molecules cross the cell membrane using carriers. Carriers are membrane proteins that complement the structural features of the

molecules transported. They bind to the chemicals in order to move them across the cell membrane. Energy is consumed because the transport proceeds against the concentration

gradient.

Active transport systems move chemicals essential to cellular functions through the membrane into the cell. Such essential chemicals include calcium ions, amino acids,

carbohydrates, and vitamins. Because the structures of poisons usually are not similar to those of chemicals essential to cells, few poisons are absorbed by active transport. Active

transport, however, is important in the elimination of organic acids, bases, and foreign compounds by the kidneys and liver.

Molecules of similar structure compete with one another in binding with the carrier

molecule. Thus, the transport of one chemical can be inhibited by another chemical of

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similar structure, a phenomenon called competitive inhibition. The chemical being

transported also competes with itself for a carrier molecule, so that only a limited amount of

the chemical can be transported by the carrier protein during a specific time. Transport systems that use carrier molecules but which do not require energy to

proceed are called facilitated diffusion. A chemical first binds to the carrier protein in the cell membrane and then diffuses through the membrane. Because no energy is used,

facilitated transport into the cell cannot proceed if the concentration of that chemical is

greater inside the cell membrane than outside. The involvement of carriers means that the process is also subject to competitive inhibition and saturation.

Large molecules, such as proteins and solid particles, are often transported by pinocytosis. The cell membrane engulfs a particle or protein molecule outside the cell, and

brings it into the cell. Although inefficient, pinocytosis operates in the slow absorption of proteins and particles in the intestine and respiratory tract.

Conditions of Exposure

Figure 1 summarizes the conditions of exposure to toxicants. Routes of exposure and absorption of chemicals.

Figure 1: Routes of absorption, distribution, and excretion of toxicants in the human body.

Injection

Although not a common route of exposure for poisons, injection is the only route in

which the entire amount exposed is absorbed regardless of the chemical administered, because the chemical is introduced directly into the body. Chemicals may be injected

17

intravenously (directly into a vein), intramuscularly (into a muscle), subcutaneously (under

the skin), and intraperitoneally (within the membrane lining the organs of the abdomen).

Because the blood is the vehicle of chemical distribution in the body, intravenous injection is the most rapid method of introducing a chemical into the body. The almost

instantaneous distribution, together with the irreversibility, makes intravenous injection a dangerous method of chemical exposure, with a fair chance of causing drug overdose if

improperly administered.

Because there is a relatively large flow of blood to the skeletal muscles, chemicals are absorbed into the blood relatively rapidly after intramuscular injection. The slow

absorption of a chemical into the blood after subcutaneous injection is probably due to the low blood flow in the subcutaneous tissues. Intraperitoneal injection is used only in

biomedical research. Absorption is relatively rapid with intraperitoneal injection because of the rich blood supply to the abdomen.

Ingestion

Ingestion is the most common route of exposure to toxic chemicals. Most chemicals

diffuse across the cell membrane in the nonionised form, so that the degree to which the chemical is ionised is important in determining whether a chemical is absorbed (see above

Transport of chemicals through a cell membrane).

Organic acids and bases dissociate into their ionised forms in response to the pH conditions of the environment. Organic acids are in their nonionised form in an acidic

environment (such as the stomach), and they thus tend to diffuse across a membrane, whereas organic bases are nonionised and thus diffuse across a membrane in a basic

environment (such as in the intestine). Because the pH and surface areas differ in different segments of the gastrointestinal

tract, chemical absorbability of these segments also differ. The major sites of absorption of

ingested poisons are the stomach and the small intestine, with most of the absorption-taking place in the latter. The intestine has a greater blood supply and a much larger surface area.

Folds in the mucous of the small intestine house numerous projections on the luminal surface, which increases the surface area of the 280-centimetre-long small intestine to up to

2,000,000 square centimetres. The pH on the mucosal surface of the small intestine is alkaline. Organic bases tend

to be in the nonionised, lipid-soluble form and thus in general are absorbed there. The pH of

the stomach contents is in the range of 1 to 2 (strongly acidic), and weak organic acids tend to be in the nonionised, lipid-soluble form. It might be expected that the poisons would be

absorbed there, but, because the surface area of the stomach is much smaller than that of the small intestine, often the stomach contents (along with the poisons) are passed to the

intestine before the chemicals are absorbed. The acidic environment of the stomach is the main reason for the poor absorption of organic bases by the stomach.

Topical (Skin)

The skin is composed of three layers of tissues – the epidermis, dermis, and

subcutaneous tissues – and is an effective barrier to many substances. The outer skin layer is the epidermis, containing five layers of cells. The stratum corneum, which is the outermost

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epidermal layer, consists of dead cells and is the major barrier to chemical transfer through

the skin. Although nonpolar chemicals cross the skin by diffusion through the stratum

corneum, no active transport exists in the dead cells of this layer. The second layer, the dermis, is thicker and is composed of loosely packed connective tissue cells in a watery

matrix of collagen and elastin fibres, as well as sweat glands, hair follicles, capillaries, and lymphatic vessels. After crossing the epidermis, chemical molecules are absorbed into the

circulatory system via the capillaries. The capillaries drain into venules in the subcutaneous

tissue. The stratum corneum is not very permeable to water-soluble molecules and ions,

although lipid-soluble molecules do cross it to a certain extent. The permeability is directly proportional to the lipid solubility of the chemical (i.e., highly lipid-soluble chemicals are

readily absorbed) and inversely proportional to the molecular weight of the chemical (i.e., the rate of absorption increases as the molecular weight of the molecule decreases).

The rate of percutaneous absorption also varies with the thickness of the stratum

corneum at different sites of the body. The rate of absorption is higher for skin on the forehead, axilla, back, and abdomen than for thicker regions like the plantar surface of the

foot and the palm. The condition of the skin is also important. Percutaneous absorption is faster when the skin is moist rather than dry.

Solids are not absorbed through the skin because the skin is generally not covered with liquid and because pinocytosis does not operate in dead cells. Liquid chemicals

penetrate the skin largely because of their lipid solubility. Gases and certain vapours can be

absorbed through the skin also, although to a much lesser extent than via inhalation.

Inhalation

The absorption of inhaled gases and vapours differs from that of aerosols and thus

will be discussed separately.

Because the same principles govern the absorption of gases and vapours, the word "gases" is used here to represent both gases and vapours. Absorption of inhaled gases takes

place mainly in the lungs. Before the gases reach the lung, however, they pass through the nose, where highly water-soluble, or highly reactive, gas molecules are retained by mucous.

Unlike intestinal and percutaneous absorption of chemicals, respiratory absorption of gases does not depend on the pH of the alveoli, because gas molecules are not ionised. It

also does not depend on the lipid solubility of the gas molecules, for three reasons. First, the

alveolar gas molecules are situated in close proximity to the capillaries. Second, the alveoli form a huge surface for gas absorption. Third, the time it takes for a unit of blood to go

through the lungs is more than adequate for gas molecules to diffuse from the alveolar space to the blood.

Gas molecules move into the blood by partitioning, which is a gas-transfer process between two phases, such as between the air and the blood or the blood and the tissues. In

partitioning, gas molecules move from a phase of high partial pressure to an adjacent phase

of low partial pressure. When an individual first inhales the gas, the partial pressure of the gas is higher in the air than in the blood, driving gas molecules from the alveolar space to

the blood. As more gas molecules are driven into the blood, the blood's partial pressure is raised. Eventually the partial pressure gradient between the air and blood dissipates and gas

transfer stops; equilibrium is then reached, usually before the blood leaves the lungs.

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The blood carries the gas molecules to the rest of the body, where the gas is

transferred from the blood to the tissue until equilibrium is reached. The blood picks up

more gas molecules in the lungs, and the process continues until the gas in each tissue of the body is in equilibrium with that of the blood entering the tissue. At this time, barring

biotransformation, no further net absorption of gas takes place as long as the exposure concentration remains constant. A person can breathe the gas forever and not absorb more, a

unique characteristic of gas exposure.

The particle size and water solubility of an aerosol chemical are the important characteristics determining absorption of aerosols. For an aerosol to be absorbed, it must be

inhaled and deposited on the respiratory tract. If not deposited, the aerosol particles are exhaled. Aerosols of less than 100 micrometers (0.004 inch) can be inhaled.

The aerosol size also determines the tendency of a particle to be deposited on a certain region of the respiratory tract. The larger aerosols (greater than five micrometers)

tend to be deposited in the upper respiratory tract, while the smaller ones (less than five

micrometers) have a greater chance of being deposited on deeper sites of the lung. The nose acts as a "scrubber" for larger aerosols and thus protects the lung from injury.

Once deposited, aerosol particles must dissolve in the liquid lining the respiratory tract in order to be absorbed. For most aerosols of poor water solubility, the particles are

cleared from the respiratory tract by mechanical or cellular means. In the nasopharyngeal region, mechanical methods of clearance include sneezing and nose blowing for particles

deposited on the anterior one-fifth of the nasal cavity. Particles deposited on the remaining

portion of the nasal cavity and on the pharynx are removed by tiny hairs, called cilia, on the surface of these two regions, which beat almost continuously to move a covering layer of

mucous toward the throat (mucociliary apparatus). Any particles deposited on the mucous are carried along and finally swallowed.

In the tracheobronchial region, mechanical clearance includes coughing and the mucociliary apparatus. The trachea, bronchi, and bronchioles, down to the terminal

bronchioles, are covered with mucous and cilia. The mucociliary apparatus moves upward

toward the larynx, where the respiratory tract joins the oesophagus. The particles are eventually swallowed and may be absorbed by the gastrointestinal tract.

The alveolar region has the slowest rate of particle clearance in the entire respiratory system, unless the particles are water-soluble, in which case they are cleared readily by

dissolution. Water-insoluble particles in the respiratory bronchioles and alveoli are removed by cellular means, principally by macrophages – scavenger cells that engulf cellular debris

in the body by a process called phagocytosis. Once phagocytosed, the mucociliary apparatus

in the terminal bronchioles removes macrophages that contain particles. Pinocytosis by the cells lining the alveoli probably move the free particles to the interstitial space, where they

either enter the lymphatic capillaries and are carried to the bloodstream, or they undergo a long process of dissolution. It can take years for water-insoluble particles to dissolve,

depending on the chemical, which is why water-insoluble particles deposited in the alveolar region tend to remain in the interstitial space for a long time and can cause serious harm.

Frequency of Exposure

The second important condition of exposure is frequency: acute (single exposure),

subchronic (repeated exposures that in total last for no more than 10 percent of the lifetime of an individual), and chronic (repetitive exposures that last in total longer than 10 percent

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of the lifetime). The difference between the frequencies of exposure is the length of time a

chemical is maintained in a target tissue. A single exposure of a poison at a certain dose

may be sufficient to produce a toxic concentration in a target tissue, leading to the development of toxicity. Repetitive exposures at the same dose will then enhance the

severity of the injury because of the presence of toxic levels of the chemical in the target tissue. The continuous presence of a toxic amount of poison may impair the ability of the

damaged cells to carry out repair and thus prevent any chance of recovery. Consequently, a

single dose that produces symptoms and toxic signs can lead to death if repeated over time. Repetitive exposures of some chemicals may also produce a different toxic effect than the

acute exposure. Toxic accumulation is one of the reasons repetitive exposures of a chemical produce

toxicity while a single exposure may not. In a hypothetical case, as depicted in Figure 2, a concentration of more than 100 milligrams per gram in a target tissue is required for

chemical A to cause toxic injury.

Figure 2: Accumulation of toxicants in the target organ during repetitive exposures.

If chemical A is administered at a dose that does not produce toxic levels in the tissue

and the elimination of the chemical is essentially complete within 24 hours, repetitive exposures at the same dose once a day will not result in toxicity. With chemical A there will

be no difference in toxicity between acute and repetitive exposures. Suppose, however, that there is a similar chemical, B, with a slower elimination rate so that chemical B is not

completely eliminated from the target tissue within 24 hours. If the exposure to chemical B

is carried out at the same dose as chemical A, the concentration of B in the target tissue will not return to zero after 24 hours. Consequently, daily exposures of B will cause the toxin to

accumulate, so that the peak target concentration of B increases daily (Figure 2). Eventually, the toxic threshold is reached and injury will develop. Therefore, repetitive exposure can

produce toxicity at a dose that does not result in injury if given only once.

Dose of Exposure

The amount of chemical to which a person is exposed is extremely important. The chemical acts at a certain site, called the active site, triggering a biological response in a

target tissue. Because the biological effect is caused by the presence of the chemical at the active site, the higher the concentration of the chemical at the site, the greater the response.

This is the case with all known poisons, a phenomenon called the dose-response

relationship.

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The dose-response curve is sigmoid, with the linear portion between approximately

16 percent and 84 percent. To compare the potency of chemicals causing similar responses,

the dose that produces a biological response in 50 percent of the subject group is chosen, because it can be calculated with the least chance of error. If the biological response is

mortality, the dose that kills 50 percent of the exposed population is known as the lethal dose 50, or LD50. Toxicity ratings for chemicals are based on their LD50s. The toxicity

rating indicates the amount of chemical required to produce death, but it should be

remembered that all chemicals can kill. Thus, all chemicals are toxic. More important than the toxicity of a chemical is its hazard or risk of usage, a concept that incorporates exposure

to dosage. For example, botulinum toxin is not especially hazardous, even though it is supertoxic, because food is well preserved, keeping the exposure or dose very low. In

contrast, ethanol (alcohol) is hazardous even though it is not very toxic, because some people have a tendency to use it to excess.

DISTRIBUTION OF TOXICANTS IN THE BODY

Role of the Lymphatics

After a chemical crosses the transport barrier at the portal of entry, it remains in the interstitial spaces, the spaces between cells that are filled with water and loose connective

tissue. The absorbed chemical can gain entry into the bloodstream directly via the blood capillaries or indirectly via the lymphatic capillaries.

Lymphatic capillaries are minute vessels located in the interstitial spaces, with one end closed and the other end draining into larger lymphatic vessels. Just like blood capillaries,

the walls of the lymphatic capillaries are composed of a thin layer of cells, the endothelial

cells. Unlike the blood capillaries, however, the junctions between the endothelial cells of the lymphatic capillaries are much looser, and as a result lymphatic capillaries are much

more porous than blood capillaries. Plasma proteins and excess fluid in the interstitial spaces from blood capillaries enter the lymphatic capillaries and eventually flow back to the

heart via the lymphatic system. Insoluble aerosols that cross the alveolar wall by pinocytosis may be absorbed into the circulatory system after first entering the porous lymphatic

capillaries.

Role of the Blood

The chemical is distributed via the blood to the various tissues of the body, where the chemical is transported across blood capillary walls. There are four types of blood capillary

walls: tight, continuous, fenestrated, and discontinuous.

Tight junctions between the endothelial cells, which prevent the diffusion of large molecules and impede that of hydrophilic molecules, characterize tight capillary walls. The

capillaries in the brain are typical of this type of capillary and form part of the blood-brain barrier.

In a continuous capillary wall, channels about five nanometres wide exist between endothelial cells, allowing most small molecules to pass through. Capillaries of this type are

found in the skeletal and smooth muscles, connective tissue, lungs, and fat. Chemicals given

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by intramuscular or subcutaneous injection are readily absorbed into the bloodstream, as are

deposited aerosols that dissolve in the fluid lining the respiratory system and cross the

alveolar wall. In a fenestrated capillary wall, holes as large as 100 nanometres are found in the

endothelial cells. Capillaries in the intestine and glomeruli in the kidney have fenestrated capillary walls, which account for the high permeability of blood capillaries for absorption

by the intestine and for filtration of the blood by the kidney.

The discontinuous capillary wall, the most porous of all capillaries, contains large gaps between the cells through which large molecules and even blood cells pass. This type

of capillary is found in the reticuloendothelial system (including the liver, spleen, and bone marrow), which assists in the removal of aged blood cells.

The porous nature of capillaries in most tissues or organs means that a chemical in the bloodstream can be distributed almost freely to most tissues, except for organs with a

barrier. The molecules diffuse from the blood to the interstitial spaces of the tissue and

finally into the cells by either diffusion or active transport.

Role of Tissue Blood Flow

The rate at which a chemical accumulates in a particular tissue is influenced by the

blood flow to that tissue. The well-perfused organs – i.e., organs that receive a rich blood

supply relative to organ weight – include major organs like the liver, brain, and kidney. A middle group receives an intermediate blood supply and includes the skeletal muscle and

skin. The poorly perfused group includes the fat and bone. As a chemical is distributed to the tissues by the bloodstream, the chemical concentrations in the well-perfused organs

rapidly reach a steady state with the blood concentration while the concentrations of the chemical in the poorly perfused tissue lag behind.

Role of Protein Binding

The plasma contains many proteins, the most abundant being albumin. Some

chemicals are known to bind to albumin. Because albumin is too large to cross the blood capillary wall, chemicals that are bound to this plasma protein are confined in the

bloodstream and are not readily distributed to the tissues. Chemicals with a high affinity to

bind with plasma proteins have lower concentrations in tissues than do chemicals that are not bound to plasma proteins.

Role of Distribution Barriers

There are barriers in certain organs that limit the distribution of some molecules. The

blood-brain barrier consists of tight capillary walls with glial cells wrapped around the capillaries in the brain. Molecules must diffuse through two barriers to get from blood to the

nerve cells of the brain. Despite the barrier, water, most lipid-soluble molecules, oxygen, and carbon dioxide can diffuse through it readily. It is slightly permeable to the ions of

electrolytes, such as sodium, potassium, and chloride, but is poorly permeable to large molecules, such as proteins and most water-soluble chemicals. The blood-brain barriers is

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the reason the ions of some highly water-soluble metals, such as mercury and leads, are

non-toxic to the brain of an adult. Children, however, are more sensitive to the toxicity of

lead because the blood-brain barrier is less well developed in children. The second distribution barrier is the blood-testis barrier, which limits the passage of

large molecules (like proteins and polysaccharides), medium-sized molecules (like galactose), and some water-soluble molecules from blood into the seminiferous tubules of

the testis. Water and very small water-soluble molecules, like urea, however, can pass

through the barrier. The lumen of the seminiferous tubules is where sperm cells of more advanced stages develop. It is thought that the barrier protects the sperm cells.

The placental barrier between mother and foetus is the "leakiest" barrier and is a very poor block to chemicals. The placenta is composed of several layers of cells acting as a

barrier for the diffusion of substances between the maternal and foetal circulatory systems. Lipid-soluble molecules, however, can cross readily, while the transfer of large-molecular-

weight molecules is limited.

Elimination of Toxicants. Excretion

An organism can minimize the potential damage of absorbed toxins by excreting the chemical or by changing the chemical into a different chemical (biotransformation), or by

both methods. The body can excrete exogenous chemicals in the urine, bile, sweat, or milk;

the lungs can excrete gases such as carbon monoxide. Urinary excretion, the most common excretory pathway, takes place in the kidney,

where the functional units are the glomerulus (a filter) and the renal tubule. The artery entering the glomerulus divides into capillaries, with fenestrated walls encased in the

Bowman's capsule. Twenty percent of the blood is filtered through the holes in the capillary walls; molecules smaller than 60.000 molecular weight end up in the filtrate, while red

blood cells, large proteins, and chemicals bound to plasma proteins are not filtered.

Chemical exchange can also take place along the renal tubule. As the filtrate flows down the renal tubule, essential molecules, such as amino acids and glucose, are reabsorbed

by active transport in the first portion of the tubule (the proximal tubule). Chemicals in the filtrate are also reabsorbed by active transport if they structurally resemble these essential

molecules. Unlike glomerular filtration, tubular resorption of a chemical is not influenced by whether or not it is bound to plasma proteins.

As the fluid flows down the renal tubule, water and some chemicals are reabsorbed

from the tubular fluid into the blood by diffusion. The tubular fluid emerges from the kidney and is collected in the urinary bladder. Lipid-soluble chemicals are readily reabsorbed in the

renal tubule, and only water-soluble chemicals are excreted in the urine to a significant extent.

The second major excretory route is the bile, which is formed in the liver and flows into the intestinal tract. The liver does not filter chemicals as does the kidney, but the liver

does secrete chemicals into bile. Chemicals excreted in the bile are eventually eliminated in

the faeces. Biliary excretion of a chemical does not necessarily result in the elimination of the

chemical from the body. Bile is dumped into the small intestine; there is a chance that chemicals in the bile may be reabsorbed by the intestine and in turn reenters the liver via the

portal vein. This cycling of a chemical, known as the enterohepatic cycle, can continue for a long time, keeping the chemical in the body.

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During inhalation exposure, absorption of the gas continues until the partial pressure

of the gas in the tissues is equal to that of the inspired gases in the lungs. As soon as the

concentration of inspired gases decreases or the exposure terminates, respiratory excretion of the gas occurs. Because the partial pressure of the inspired gas is lower in the lungs than

in blood, the blood releases some gas molecules into the alveolar space and these molecules are exhaled. The tissues lose gas molecules to the blood, which carries them to the lungs to

be excreted.

The composition of sweat is similar to that of plasma except that sweat does not contain proteins. After secretion, the fluid moves through the sweat duct, where salt and

water are reabsorbed. The exact mechanism of sweat secretion is not known. It appears that sweat is a filtrate of plasma that contains electrolytes (such as potassium, sodium, and

chloride) and metabolic wastes (like urea and lactic acid). Because sweat resembles a filtrate of plasma, water-soluble chemicals, like some drugs and metal ions, are found in sweat.

Sweat is not a major route of excretion of chemicals, however.

Milk is a potential, albeit minor, route of chemical excretion, but more importantly it is a potential means of chemical exposure for breast-fed infants.

Most chemicals enter milk by diffusion. Therefore, only the nonionised, lipid-soluble forms of organic chemicals are found to a significant extent in milk. Chemicals with a

molecular weight less than 200 and that are present in plasma not bound to proteins are more likely to be found in milk. Because the lipid content of milk is higher than that of

plasma, highly lipid-soluble chemicals can exist in a more concentrated level in milk than in

plasma. Therefore, milk can be a significant route of excretion for highly lipid-soluble chemicals in lactating women.

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Poisonings Classification

Poisoning

Accidental

Criminal Suicidal

True (real)

Intentional

Demonstrative

For murder

For helpless

Disasters:

– on industry

– in life

Over dosage

Self-treatment

Toxic mania

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BIOTRANSFORMATION OF CHEMICALS

Biotransformation, sometimes referred to as metabolism, is the structural

modification of a chemical by enzymes in the body. Chemicals are biotransformed in

several organs, including the liver, kidneys, lungs, skin, intestines, and placenta, with the liver being the most important. Chemicals absorbed in the gastrointestinal tract must pass

through the liver, where they can be biotransformed and thus eliminated before being distributed to other parts of the body. This phenomenon is known as the first-pass effect. As

a result, smaller amounts of certain chemicals are distributed throughout the body after oral administration than after other exposure routes, such as intravenous or intramuscular

injections. Biotransformation of a chemical primarily facilitates its excretion into urine or

bile; however, certain chemicals are biotransformed into more toxic forms and, as a result, biotransformation of chemicals is not always beneficial.

Biotransformation of exogenous chemicals (chemicals that are not naturally found in the body) generally occurs in two phases. In phase I, an exogenous molecule is modified by

the addition of a functional group such as a hydroxyl, a carboxyl, or a sulfhydryl. This modification allows phase II, the conjugation, or joining, of the exogenous molecule with an

endogenous molecule (one naturally found in the body), to take place. The major end

product in most cases is a more water-soluble chemical that is easily excreted. Phase I reactions can be classified as oxidation, reduction, or hydrolysis. Oxidation is

carried out by cytochrome P-450 monooxygenases, mixed-function amine oxidases, and alcohol and aldehyde dehydrogenases. The reactions mediated by cytochrome P-450

monooxygenases can make the chemical less toxic or more toxic. The cytochrome P-450 enzymes can, for example, produce epoxides of some chemicals, which are very reactive

and can attack important cellular molecules, such as DNA. The remaining phase I oxidative

enzymes act on a narrow range of substrates. In addition to the oxidation of a chemical, cytochrome P-450 monooxygenases can

catalyze the reduction. Another group of enzymes that can carry out reduction is the aldehyde/ketone reductases. Each of the three groups of hydrolytic enzymes (epoxide

hydrolases, esterases, and amidases, respectively) creates metabolites with a hydroxyl, carboxyl, or amino functional group.

In phase II reactions an altered exogenous chemical binds with an endogenous

molecule, leading to the formation of a final product (the conjugate), which is usually much more water-soluble and easily excreted than the parent chemical. There are four types of

parent compounds whose excretion can be enhanced by conjugation: glucuronic acid, glutathione, amino acids, or sulfate. The first two types are the most common phase II

reactions. Conjugation of glucuronic acid with a hydroxyl, carboxyl, amino, or sulfhydryl

group leads to the formation of oxygen, nitrogen, or sulfur glucuronides, which are more

easily excreted than glucuronic acid because they are more water-soluble and because they contain a carboxyl group. Conjugation with glutathione also enhances excretion.

Glutathione conjugation yields glutathione conjugates and mercapturic acid derivatives, which are excreted by the liver, kidney, or both.

Two types of conjugations, acetylations and methylation, do not enhance the excretion of the parent chemical. Acetylation and methylation decrease the water solubility

of the parent chemical and mask the functional group of the parent chemical, preventing

these functional groups from participating in conjugations that increase their excretion.

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Acetylation acts on chemicals with an amino group and may render them less toxic.

Chemicals with an amino, hydroxyl, or sulfhydryl group can be methylated. Methylation is

not as important a route of biotransformation for exogenous chemicals as it is for endogenous chemicals.

Therapeutic, Toxic, and Lethal Responses

Figure 3: Dose-response curves.

Because the response to a chemical varies with the dose, any substance can be a poison. Medicine can produce responses that are therapeutic (beneficial) or toxic (adverse),

or even lethal. The sigmoid dose-response relationships for the therapeutic and lethal responses typically look like curves A and C, respectively, of Figure 3. If drug X has

therapeutic, toxic, and lethal dose-response curves of A, B, and C, respectively, X is a very safe drug, since there is no overlap of the curves. For some medicinal agents, there is

overlap of the therapeutic and lethal dose-response curves, so that a dose that causes a

therapeutic response in some individuals can kill others. These agents, consequently, are not as safe.

A quantitative measurement of the relative safety of drugs is the therapeutic index, which is the ratio of the dose that elicits a lethal response in 50 percent of treated

individuals (LD50) divided by the dose that elicits a therapeutic response in 50 percent of the treated individuals (TD50). For instance, the therapeutic index of drug X is 9,000

milligrams per kilogram divided by 30 milligrams per kilogram and is equal to 300. The

larger the therapeutic index, the safer the drug. Diazepam and digoxin are examples of drugs with a large and a small therapeutic index, respectively.

Morphological Versus Functional Toxic Responses

Chemicals can elicit various types of toxic responses, which can be classified by the

nature of the response, the site of toxic action, the time it takes for the response to develop, and the chance of resolution of the response. The nature of the toxic response can be

morphological (structural) or functional or both. In most cases, the chemical produces

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morphological changes in an organ, which in turn affects the function of the organ. In a

small number of cases, the chemical produces functional changes in an organ without

changing the structure of the organ. Inhalation exposures to silica dust at a low concentration for 10 years or more can

lead to chronic silicosis, a condition characterized by the formation in the lungs of silicotic nodules, which are egg-shaped lesions composed of layers of fibroblasts (reparative cells)

and inflammatory cells surrounding a central silica particle. Such lesions can be considered

a morphological toxic response; unless the silica exposure is prolonged, there will be little respiratory impairment because the lungs and certain other organs have a large functional

reserve. If the silica exposure is prolonged, however, the silicotic nodules coalesce (complicated silicosis), and the structure of the lungs is altered so drastically that they do

not distend easily during inspiration. Oxygen exchange in the alveoli is impaired, causing such functional toxic responses as breathlessness, chest tightness, and coughing with

sputum.

Malathion exposure, on the other hand, can lead to functional toxic responses without causing any morphological changes. Malathion does not alter the structure of tissues; rather,

it inhibits an enzyme, acetylcholinesterase, which normally degrades acetylcholine, the neurotransmitter of the parasympathetic nervous system. Inhibition of this enzyme leads to

an exaggeration of the actions of the parasympathetic nervous system, including sweating, secretion of saliva, adjustment of pupil size, and defecation. The end results are increased

perspiration, increased salivation, tearing, blurred vision, abdominal cramping, diarrhea, and

if severe enough, death from respiratory depression.

Local Versus Systemic Toxic Responses

Toxic responses are also classified according to the site at which the response is

produced. The site of toxic response can be local (at the site of first contact or portal of

entry of the chemical) or systemic (produced in a tissue other than at the point of contact or portal of entry).

An example of a local toxic effect is the tissue corrosion produced by strong acids (e.g., sulphate acid) and bases (e.g., sodium hydroxide) in contact with tissues. If the

exposure is external, skin burns result; if ingested, the acid or base causes serious local damage to the oesophagus and stomach.

An example of a systemic toxicant is methanol, which is absorbed and

biotransformed into formic acid. The acid is responsible for metabolic acidosis and optic nerve damage in the retina of the eye, leading to visual impairment, a systemic effect.

Immediate Versus Delayed Toxic Responses

Toxic responses may also be classified according to the time it takes for development

of a toxic response. If it takes up to a few days after exposure, the response is considered immediate. There is no universal standard of minimum time for delayed toxic responses, but

generally a response that takes more than a few days to develop is considered delayed. The time it takes for a systemic toxicant to act depends on many factors, such as the rates of

absorption, biotransformation, distribution, and excretion, as well as the speed of action at the target site.

29

Reversible Versus Irreversible Toxic Responses

Toxic responses differ in their eventual outcomes; the body can recover from some

toxic responses, while others are irreversible. Irritation of the upper respiratory tract by

inhaled formaldehyde gas, for example, is rapidly reversible in that as soon as the inhalation exposure terminates, the irritation subsides. In contrast, the response produced by silica dust

is irreversible because, once the silicotic nodules are formed, they remain in the alveolar

region of the lung.

Chemically Induced Immune Responses

The immune system protects the body against foreign substances, especially

microbes and viruses. To be antigenic, a substance is usually both relatively large and

foreign to the body. Large proteins are often strong antigens. Smaller chemicals can become antigenic by combining with proteins in chemicals called haptens.

Cellular and Humoral Immunities

The development of immunity toward an antigen is called sensitisation. After

exposure to an antigen, a combination of cellular and humoral immunity usually develops. Exposure routes that favour slow absorption into the bloodstream, such as percutaneous

injection, often primarily elicit cellular immunity, while rapid routes of exposure, such as intravenous injection, favour the development of humoral immunity.

Cellular immunity utilizes phagocytes (such as macrophages, neutrophils, and eosinophils), which engulf antigens, and T-lymphocytes, which are thymus-derived,

antigen-specific immune cells containing receptors specific for a special antigen. Cellular

immunity is particularly important in defending the body against tumours and infections. Macrophages phagocytise antigens and secrete proteins (monokines) that regulate cells

involved in immune responses. One monokine is interleukin-2, which stimulates an increase in the number of T-lymphocytes. The T-lymphocytes then develop surface receptors for

specific antigens. Because T-lymphocytes survive for months or years, cellular immunity toward the antigen remains with the individual for a long time. If reexposed to the same

antigen, the sensitised T-lymphocytes recognize the antigen and secrete their own proteins

(lymphokines), which stimulate phagocytes to destroy the antigen. If an antigen is located on foreign or tumour cells, certain T-lymphocytes are transformed into cytotoxic T-

lymphocytes, which destroy the target cells. Humoral immunity utilizes antibodies, also known as immunoglobulins (Ig),

produced by B-lymphocytes. B-lymphocytes are lymphocytes derived from the spleen, tonsils, and other lymphoid tissues. They become plasma cells, which make antibodies.

There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. IgG, IgM, and IgA are

involved in humoral immunity, the function of IgD is not known, and IgE takes part in immediate hypersensitivity (see below).

Humoral immunity involves the inactivation, removal, or destruction of antigens. Antibodies can inactivate viruses by binding to them. With two antigen-binding sites per

protein unit, an antibody can also precipitate the antigen by cross-linking in a network formed with other antibodies. Because each IgM has five protein units, and thus five

30

potential binding sites, IgM is particularly efficient in precipitating the antigen. After the

antigen is precipitated, phagocytes can remove it. In addition, antigen binding by IgG or

IgM activates a serum protein, called a complement, which can then initiate antigen precipitation, amplifying the inflammatory response. If the antigen is on the surface of

certain cells, activated complement can also facilitate the lysis of these cells. IgG or IgM can also link the antigen to phagocytes or to killer cells, resulting in lysis of the cell by an

unknown mechanism.

Allergies

Although the immune system generally protects the body, it can respond in certain

ways that are detrimental to some individuals. Allergy, or hypersensitivity, is a condition of

increased reactivity of the immune system toward an antigen that leads to adverse effects.

Substances that cause allergies are known as allergens. Confusion is sometimes caused by the terms hypersensitivity, hypersusceptibility,

and idiosyncrasy. Hypersensitivity is a reaction to a chemical or substance in certain individuals and has a basis in the immune system. Hypersusceptibility is an increased

predisposition of certain individuals to react to a chemical. Because of biological variability among humans, some individuals respond to a chemical at a dose too low to produce a

similar effect in others. Idiosyncrasy is a genetically determined hypersusceptibility.

Allergic responses differ from the usual toxic responses in three ways. First, the allergic response does not occur during the first exposure to an allergen, but is evident only

after at least one previous exposure. In rare occasions, an allergic response can occur on the first exposure to a chemical if the individual has already developed hypersensitivity toward

a closely related chemical. For example, people allergic to one kind of penicillin are usually allergic to other penicillins as well. Second, allergy is specific to both the allergen and the

individual. Unlike in a toxic response, in which everyone exposed develops the response if a

sufficient dose is administered, only a small fraction of the exposed population is sensitised by an allergen, regardless of the dose. Third, the amount of a chemical required to illicit an

allergic response is usually much less than that required to produce a toxic response. There are four types of hypersensitivities (allergies): immediate, cytotoxic, immune-

complex, and delayed. Each differs from the others in the mechanism of induction and the responses produced. Immediate hypersensitivity is the most common form of allergy.

Delayed hypersensitivity is the second most common, whereas cytotoxic and immune-

complex hypersensitivities are relatively rare. Immediate hypersensitivity, also called anaphylaxis, produces IgE in response to an

allergen that binds to the surface of mast cells or basophils. When reexposed to the allergen, the antigen-binding end of IgE on mast cells and basophils binds the allergen, triggering a

release of anaphylactic mediators from these cells. These mediators, such as histamine and serotonin, cause the contraction of certain smooth muscles (e.g., those of the respiratory

tract, leading to bronchoconstriction in asthmatic attacks), relaxation of blood vessels (e.g.,

in the skin, resulting in redness, or in the whole body, causing a fall in blood pressure as in anaphylactic shock), and increased permeability of capillary walls (e.g., in the skin, leading

to local oedema as seen in urticaria). The unique characteristic of immediate hypersensitivity is its rapid onset, with the response initiated within a few minutes of

allergen exposure.

31

The anaphylactic mediators affect tissues differently. Thus, the allergic response

depends on where the immune reaction takes place. In the skin, immediate hypersensitivity

can result in skin eruptions or urticaria, characterized by wheals with redness. In the respiratory system, it can produce hay fever or asthma. In the gastrointestinal tract, allergic

gastroenteritis, an inflammatory condition of the stomach and intestine, may result. Systemic anaphylaxis may involve the entire body, with shock as a key feature.

A second type of hypersensitivity is cytotoxic hypersensitivity, which has a gradual

onset. After reexposure to an allergen, the allergen molecules attach to the surfaces of blood cells, forming an antigen new to the body. IgG or IgM binds to the new antigen on the blood

cells, lysing blood cells via either complement fixation or antibody-dependent cell cytotoxicity. If the lysed cells are red blood cells, hemolytic anemia results. If platelets (the

blood components intrinsic to blood clotting) are lysed, however, the blood clotting mechanism is impaired.

In a third type of allergy, immune-complex hypersensitivity, the allergen-IgG

complex precipitates in tissues, resulting in inflammation via complement fixation. Immune-complex hypersensitivity in the kidney results in an inflammatory injury of the

glomeruli (glomerulonephritis), and in the lung it leads to a pneumonia-like condition known as hypersensitivity pneumonitis.

Delayed hypersensitivity differs from other types in not involving humoral immunity. Upon reexposure to the allergen, sensitised T-lymphocytes release lymphokines, which

trigger a series of inflammatory reactions. The inflammation leads to the development of

allergic contact dermatitis in the skin and a chronic form of hypersensitivity pneumonitis in the lung. Symptoms of allergic contact dermatitis develop gradually, taking a day or two to

reach maximum levels, which is the best way to distinguish allergic contact dermatitis from atopic dermatitis with similar symptoms. In contrast, the chronic form of hypersensitivity

pneumonitis develops insidiously and not in a fixed time.

Teratogenesis

Teratogenesis is a prenatal toxicity characterized by structural or functional defects in

the developing embryo or foetus. It also includes intrauterine growth retardation, death of

the embryo or foetus, and transplacental carcinogenesis (in which chemical exposure of the mother initiates cancer development in the embryo or foetus, resulting in cancer in the

progeny after birth).

Intrauterine human development has three stages: implantation, postimplantation, and foetal development. The first two stages are the embryonic stages and last through the

first eight weeks after conception. The foetal stage begins in the ninth week and continues to birth.

Depending on the developmental stage, chemical exposure in the mother can result in different degrees of toxicity in the embryo or foetus. In the preimplantation period, a toxic

chemical can kill some of the cells in the blastocyst, resulting in the death of the embryo.

During the postimplantation period, chemical-induced cell death leads to one of two outcomes. If death is confined to those cells undergoing active cell division at the moment,

the corresponding organs are affected, resulting in malformation. If the cell death is generalized without significant replication by the remaining cells to sustain life, the embryo

dies. During the third, foetal, period, chemical injury can retard growth or, if severe enough, kill the foetus.

32

The genesis of a particular organ (organogenesis) occurs at a specific time during

gestation and is not repeated. Because organogenesis is a tightly programmed sequence of

events, each organ system has a critical period during which it is sensitive to chemical injury. Chemical exposure in a critical period is likely to produce malformations of that

organ and not others; however, because there is some overlapping of critical periods of organ development and because chemicals frequently remain in the embryo for a period of

time, malformations of more than one organ usually occur. Since organogenesis occurs

mostly in the embryonic stages, chemical exposure in the first trimester should be minimized, if possible.

Little is known about mechanisms of teratogenesis. It is thought that some teratogens produce malformations directly by killing the cells in the embryo. Teratogens can also

produce malformations indirectly by causing maternal toxicity, resulting in oxygen or nutrient deficiency for the embryo. A few well-known examples are discussed below.

Thalidomide is a drug originally marketed to combat nausea and vomiting in

pregnancy. It was discovered in the 1960s in West Germany to cause rare limb defects, among other congenital anomalies. The discoveries about thalidomide triggered legislation

requiring teratogenicity testing for drugs. Chronic alcohol ingestion during pregnancy is the most common cause of congenital

problems in mental development. Ingestion of more than 30 millilitres (1 ounce) of ethyl alcohol per day during pregnancy can lead to the development of foetal alcohol syndrome,

characterized by intrauterine growth retardation and subsequent learning disabilities, such as

distractibility, language disorders, and low IQ. Heavier consumption of alcohol, more than 60 millilitres per day, by a pregnant woman can result in malformations of the foetal brain

and in spontaneous abortions. Diethylstilbestrol (DES) is a drug used primarily from the 1940s to the '50s to

prevent miscarriage. The drug is an example of a chemical that can produce transplacental carcinogenesis. It was discovered in the early 1970s that exposures to diethylstilbestrol

before the ninth week of gestation could lead to the formation of rare vaginal and cervical

cancers in female progenies.

Carcinogenesis

Carcinogens are chemicals that can produce tumours, abnormal tissue growths

caused by a loss of control in cell replication. Most tumours are solid masses (e.g., lung

cancer), but some do not occur as tissue swellings (e.g., leukaemia). Tumours may be benign or malignant. Benign tumours are to a certain degree

controlled in their growth. As a result, benign tumours maintain some form of cellular organization and grow rather slowly over a period of years. In contrast, cell growth in

malignant tumours is almost totally uncontrolled. Cells in malignant tumours grow very rapidly in a disoriented fashion.

Benign tumours are encapsulated by a fibrous layer and so do not invade surrounding

tissue. Malignant tumours invade surrounding tissue. Thus, while a benign tumour grows at one site, a malignant tumour sends out cancerous cells via the blood and lymphatic system

to distant sites of the body, spreading by a process known as metastasis. The invasion of surrounding tissues by a malignant tumour produces various symptoms.

Carcinogenesis is a complicated process in which many factors are known to play significant roles. Certain external environmental factors are important. For instance,

33

cigarette smoking is known to cause predisposition to the development of lung cancer. A

diet low in fibre content and high in fat is correlated with a high incidence of colorectal

cancer. In addition, internal factors, such as hormonal imbalances and immunosuppression, can also increase the chance of developing tumours. Sensitivity to chemical carcinogens is

known to be species-dependent. A chemical carcinogen may induce tumours in one animal species but not another, and a species that is sensitive to one carcinogen may be resistant to

another. Known human carcinogens include some anticancer drugs, aromatic (containing a

benzene ring in its chemical structure) amino and nitro compounds, metals, radio nuclides, and miscellaneous chemicals. In humans the respiratory tract is the most common target for

chemical carcinogens, followed by the liver and the blood. Although there have been many theories on the mechanism of chemically induced

tumour formation, it is now thought that DNA is the target of most chemical carcinogens. The carcinogens interact with the DNA and interfere with its normal function. Because

DNA controls cellular functions, when DNA is damaged, the cell presumably loses control

and divides in a chaotic fashion. A clone of the parent cell is generated, and these cells maintain the chaotic replication, which ultimately leads to the formation of a tumour. In

general it takes 10 to 20 years for the initial DNA damage in one cell to develop into a recognizable tumour.

Carcinogens that are thought to produce cancer in laboratory animals by altering the DNA are referred to as genotoxic carcinogens. They are either direct-acting or indirect-

acting chemicals.

Direct-acting (reactive) genotoxic chemicals can themselves interact with DNA. Indirect-acting genotoxic carcinogens do not bind to DNA until they have been

biotransformed in the body to reactive chemicals. Among the indirect-acting carcinogens, polycyclic aromatic hydrocarbons, nitrosamines, and nitrosonornicotine are found in

cigarette smoke. Polycyclic aromatic hydrocarbons are also formed in charcoal-broiled meat. Nitrosamines can be formed by the nitrosation of nitrite-cured, protein-rich food, such

as nitrite-cured meat and fish, in the intestine.

Chemicals that produce cancer by a mechanism other than by binding to DNA are known as epigenetic carcinogens. The mechanisms by which epigenetic carcinogens

produce tumours are not known with certainty, but various theories have been proposed. Cytotoxins are thought to kill cells in the target organ. The cell death increases cell

replication by the remaining cells, which somehow results in tumour development, possibly by stimulating the division of cells that have previously had their DNA damaged by a

genotoxic carcinogen.

It has been proposed that hormones and chemicals, which modify the activities of the endocrine system, create a physiological imbalance in organs dependent for their

functioning on a particular hormone. With the imbalance, the organ may lose its normal physiological control and tumour growth may occur. This may be the mechanism by which

estrogens in postmenopausal women lead to development of uterine cancer and the reason antithyroid agents, such as 3-amniotriazole, produce thyroid tumours.

Chemicals that depress the immune system are thought to produce tumours by

impairing cell-mediated immunity, which is important in the normal elimination of tumour cells. The development of tumours involves two main steps: initiation and promotion.

Initiation is the creation by genotoxic carcinogens of a cell with abnormal DNA. After initiation, promoters stimulate the replication of these neoplastic cells andfacilitate the

development of the tumour. Initiators include genotoxic chemicals. Although promoters do not produce tumours directly, they are still considered carcinogens because they can lead to

34

the development of tumours in concert with an initiator. Promoters include large chlorinated

hydrocarbon molecules (e.g., DDT, PCBs, TCDD, butylated hydroxy antioxidants, and

saccharin) and tetradecanoyl phorbol acetate in croton oil.

Mutagenesis

Mutagenesis is the alteration of genes. Substances able to produce mutations are

naturally genotoxic substances. Once a gene is mutated in a cell, the altered gene can be

passed on to daughter cells. The body has ways to repair some of these gene alterations so that the genetic damage does not always propagate.

The effect that a mutation has depends on the cell in which the mutation occurs. In the somatic cells of most organs, mutation either has no effect, causes one cell to die, or

causes a cell to divide at an uncontrolled rate so that a tumour develops. If the mutation

occurs in germ cells (egg and sperms), there may be detectable changes or birth defects, or stillbirth may result.

35

GENERAL PATHWAYS OF XENOBIOTICS BIOTRANSFORMATION

Xenobiotics are the chemical substances that are foreign to the biological system.

They include naturally occurring compounds, drugs, environmental agents, carcinogens, insecticide, etc.

Xenobiotic metabolism is the sum of the physical and chemical changes that affect foreign substances in living organisms from uptake to excretion.

Biotransformation is the series of chemical alterations of a compound (for example,

a drug) that occur within the body, as by enzymatic activity. Metabolism is the sum of all the physical and chemical processes by which living

organised substance is produced and maintained (anabolism) and also the transformation by which energy is made available for the uses of the organism (catabolism).

Xenobiotics are metabolised in human body on two-phase process: 1) biotransformation and 2) conjugation. As a result of these processes are created products,

which eliminates with urine, bile or transpired air.

In run of metabolic reactions (chemical or biochemical) the xenobitics get new functional groups, which increase its polarity and which are the centres for second phase of

process. Such metabolic transformations as oxidation, reducing, and hydrolysis occurs

connection of new polar functional groups. In these case xenobiotics can be deactivated (desintoxication) or activated (toxicity ascending). Non-active compounds (“premedcines”)

can transform into pharmacologically active substances (activation).

However in some cases the metabolites can be more toxic, than foreign compounds, from which they were formed.

It is known, that the hexamethylenetetramine has no antibacterial activity, but its metabolite – the formaldehyde – shows the specified activity and is toxic.

The methanol has considerably smaller toxicity, than formaldehyde being a metabolite of this alcohol.

Morphine, a metabolite of codeine, is more toxic than codeine.

A chloral hydra shows soporific action only after transformation it in more toxic metabolite – trichlorethanole.

A metabolite of phenacetin is paracetamol, which has more expressed pharmacological action on an organism, than phenacetin.

Some xenobiotics are very similar to normal endogenous compounds and can participate in biological metabolism and incorporate into tissues. This process causes

poisonings and named lethal synthesis. Products of lethal synthesis are more toxic, than the

native substances. It can be shown on such example: the non-toxic flour acetic acid in an organism

transforms to very toxic flour citric acid:

F–CH2–COOH → НООС–СНF–COH(COOH)–CH2–СООН

Another example of synthesis is biotransformation of paracetamol. Product of

paracetamol oxidation conjugates with SH-groups of peptides in hepatocytes and causes the liver malfunction and general intonxication.

36

NH

OH

CO CH3

N

O

CO CH3

N CO CH3

S-Protein

N CO CH3

S-Glutathion

ConjugationParacetamol Glucuronid

ExcretionOxidation

S-Protein

Less toxic

More toxic

ConjugationParacetamol Sulphate

Excretion

Glutathion

Less toxic

(SH-groupsof hepatocytes proteins)

Scheme of Paracenamol Biotransformation

Examples of xenobiotics activity changes:

– desintoxication:

Phenobarbital (active medicine) → hydroxylation → p-oxyphenylbarbituric acid (non

active)

– activation:

Prontosil (non active “premedcine”) → reducing splitting → sulphanilamide (active

medcine)

– toxicity ascending:

Parathion (non active insecticide) → desulphuration → paraoxon (active insecticide)

THE FIRST PHASE OF BIOTRANSFORMATION

Oxidation by Microsomal Enzymes 1. Hydroxylation of aliphatic compounds

R CH3 CH2OH[O]

37

2. Hydroxylation of aromatic compounds

CH3

CH2OH

[O] [O]COOH

Toluen Benzylic alcohol Benzoic acid

3. Hydroxylation of alicyclic compounds

4. Epoxydation

Cl

Cl

Cl

Cl Cl

CCl2

Cl

Cl

Cl

Cl Cl

CCl2 O

[O]

5. N-hydroxylation of amines

NH2 NHOH N=O

[O] [O]

Aniline Phenylhydroxylamine Nitrobenzen

6. Sulfoxidation

7. O-desalkylation

Phenacetin Paracetamol

8. N-desalkylation

Morphine Normorphin

OH OH

OH

O

R' S RR S R' [O]

+ CH3CH=O[O]

NHCOCH3

OHO-C2H5

NHCOCH3

HO

HO

O

N-CH3

HO

HO

O

NH + CH 2O

[O]

38

9. Desamination

Phenamine Phenylacetone

10. Desulphuration

Thiobarbital Barbital

Nonmicrosomal Oxidation 1. Oxidising desamination

2. Alcohols oxidation

3. Aldehydes oxidation

Benzaldehyde Benzoic acid

4. Alicyclic compounds aromatisation

Reducing with Microsomal Enzymes

1. Nitrocompounds reducing

2. Azocompounds redusing

Non-microsomal Reducing

1. Aldehydes reducing

R CH2NH2

O2 R CH=NHH2O

RCHO + NH 3

CH3CH2OH + NAD CH3CHO + NHAD H 2.

(CH2)n COOH COOHCOOH

R-NO2 R-NH2

R-N=N-R R-NH-NH-R 2R-NH2

CCl3CH(OH)2 CCl3CH2OH

[O] CH2

NH2

CH CH3CH2 CO CH3 + NH3

N

N

H

H

O

OS

C2H5

C2H5

O

N

N

H

H

O

O

C2H5

C2H5

[O]

C6H5COOH + NHAD H 2.C6H5CHO + NAD + H 2O

39

2. Ketones reducing

3. Disulphides reducing

4. Sulphoxides reducing

5. Aromatic dehydroxylation

1. N-Dehydroxylation

Hydrolysis

1. Esters hydrolysis

2. Amides hydrolysis

3. Hydrazides hydrolysis

4. Carbamates hydrolysis

5. Nitrites hydrolysis

6. Reducing dechloration

R-CO-R R-CHOH-R

R-S-S-R 2 R-SH

(CH3)2SO (CH3)2S

R OH

OH R OH

R

OH

R-NH-OH R-NH2

H3COOC-R-OCOC6H5 HOOC-R-OH + C 6H5COOH + CH3OH

R-OOC- -NH2 HOOC- -NH2 + ROH

N N-CONHNH 2-COOH + H 2N-NH2

NH2COOR NH2COOH + ROH

R-CN R-COOH

R-CN R-COOH

40

THE SECOND PHASE OF BIOTRANSFORMATION

Conjugation is the synthesis reactions. In run of conjugation the xenobiotics or its metabolites interact with endogenous molecules (glucuronic or sulphuric acid, amino acids

etc.). Conjugated molecules become more polar and more hydrophilic and, as a result, these

molecules easy eliminate. But some xenobiotics, such as diethyl ether, phthalic acid, barbital, eliminate without

changes. There are biochemical inert substances. Metabolically inert and nonpolar xenobiotics are heavy eliminated substances and cumulate in fat tissues.

Xenobiotics conjugation occurs the functional groups blocking. As a consequence, these molecules deactivate or decrease activity. Such process is called desintoxication.

Examples of desintoxication due to conjugation:

Cyanide → thiocyanate

1. Glucorunides formation (conjugates with glucuronic acid called glucuronides). Glucuronic acid forms from glucose:

O

COOH

OHOH

OH

HO

O

OHOH

OH

CH2OH

HO

Glucose Glucuronic acid

-COOH -COO-C6H9O6

Benzoic acid Benzoylglucuronid

2. Esters formation (with sulphate acid)

Phenol Phenylsulphate

3. Methylation

4. Acetylation

Aniline Acetanilide

-OH -OSO3H

N N-CH3

+

-NH2-NHCOCH3

41

5. Conjugation with amino acids

Nicotinic acid Nicotinuric acid

6. Conjugation with peptides

CONH

Rn

OC NH

CH

2

COOHCOOH

ATP CoA

Glicine

7. Conjugation with glutathione

SNH

CO

NHO COOH

NH2

COOHSHNH

CO

NHO COOH

NH2

COOH

+

Glutathion reduced

Post-mortem Changes of Medicines and Poisons in Corpses After death tissues of corpses decompose, therefore are formed the substances

interfering to detection and quantitative determination of poisons, caused a poisoning. Many substances formed at rotting of corpses give the same reactions, as well as some poisons.

This circumstance can cause the error conclusions about presence of poisons in bodies.

Besides, during corpses decomposing some toxicants that have caused a poisoning are exposed also to chemical changes.

After death under influence of specific cell enzymes, so-called cathepsines, there is an autolysis (self-digestion) of cells; therefore the albuminous substances are decomposed

to more simple compounds. The cathepsines contain in lysosomes of cells of many bodies. At life of an organism cathepsines and some other hydrolases have insignificant

activity. The disintegration of protein in living organism, caused by cathepsines, is quickly

filled by their synthesis. After death the cathepsines activity considerably grows. At a life a tissue of an organism have pH 6,8 – 7,2, and after death рН of tissues is moved together in

more acidic area, favourable for display of activity of cathepsines. Thus, the autolysis is one of the early corpse phenomena; richest cathepsines

(pancreas, liver, kidney etc.) first of all are exposed to autolysis tissues of corpses. Some factors are breaking an autolysis process (presence in corpses of fluorides, cyanides, arsenic,

carboxyhaemoglobin, cardiac glycosides etc.).

N

-CONHCH2COOHglycine

N

-COOH

42

In some hours after death bacteria that are taking place in intestines, will penetrate

through their walls and on blood vessels are distributed almost on all corpses. As a result of

it under influence of enzymes of microorganisms there comes process of putrefaction of corpse’s tissues.

At putrefaction decomposing of albumin’s and other substances in corpses are formed more simple compounds, which chemical properties can be similar to properties of

some poisons. It complicates chemical-toxicological research.

Intensity of putrefaction processes of corpses and composition of formed at it substances depend on the specific composition of microbial flora, temperatures, moisture,

access of air, and series of other factors. At rotting decomposing of carbohydrates are formed the organic acids and products

of it decarboxylation: aldehydes, ketones, lactones, and carbon (IV) oxide. At rotting decomposing of fats are formed the mixture of alcohols, contains

methanol, ethanol and the highest alcohols. These alcohols than are oxidised to aldehydes

and ketones. At putrefaction protein matters the peptides are formed which are decomposed with

formation of amino acids. Amino acids can be exposed to a desamination with excretion of ammonia. The sulphur-containing amino acids are decomposed with excretion of a

hydrogen sulphide. At putrefaction protein can be formed amines, which frequently named as ptomaine

or “cadaver poison” (putrescine, cadaverine, ethylene diamine etc.).

Ptomaine is the one of a class of animal bases or alkaloids formed in the putrefaction of various kinds of albumin’s matter, and closely related to the vegetable alkaloids; a

cadaver poison. The ptomaines, as a class, have their origin in dead matter, by which they are to be distinguished from the leucomaines. Leucomaine is an animal base or alkaloid

appearing in the tissue during life; hence, a vital alkaloid, as distinguished from a ptomaine. Cadaverine is 1,5-pentanediamine. A foul-smelling diamine formed by bacterial

decarboxylation of lysine. Putrescin is a nontoxic diamine, C4H12N2, formed in the

putrefaction of the flesh of mammals and some other animals. The toxicity of “cadaver poison” too has appeared disputable. After ptomaines

clearing were obtained the substances having smaller toxicity, than ptomaine isolated from corpses. Putrescin and cadaverine, that were synthesed in laboratory, too have appeared less

toxicity, than what are isolated from corpses. Therefore toxicity of “cadaver poison” is explained by action of some impurities contained in a rotted biological material with

ptomaine. The bacterial toxins and some products of synthesis formed in a corpse material

under influence of bacterial enzymes concern to impurity. Described above putrefaction occur in corpses basically without access of air (in

tombs). However, on occasion corpses can be on a surface or in places, into which oxygen of air well will penetrate. In these cases the rotting of corpses occurs under influence of

enzymes of aerobic bacteria and small humidity. Such processes of decomposing of corpses refer to as decay.

Depending on conditions of decomposing there can be a mummification of corpses.

Mummification is a complete desiccation of corpses. This process occurs at dry air, increased temperature and good ventilation. In these conditions the processes of rotting stop

and there is a desiccation of corpses. The majority of organic toxicants in corpses are exposed to oxidation, reduction,

desamination, decarboxilation, desulphuration and other transformations. In a corpse material more proof are the inorganic toxicants. The majority of these substances are

43

reduced at rotting corpses. The ions of metals in inorganic poisons are reduced up to ions

with the lowest charge number. The compounds of arsenic, phosphorus, sulphur and other

metalloids can be reduced with to formation of volatile compounds with hydrogen. The compounds of arsenic and thallium can be kept in corpses about 8 – 9 years,

bond of barium and antimony – about 5 years, the bonds of mercury are kept in corpses some months. After that the inorganic poisons penetrate in ground and not can be always

detected in corpses.

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“VOLATILE” POISONOUS SUBSTANCES

To the group of “volatile” poisonous substances are belong many organic substances,

which have toxicological importance: 1. cyanic acid;

2. aliphatic carbohydrates: petrol components; 3. halogen-carbohydrates: industrial and housekeeping solvents;

4. aldehydes and ketones; 5. alcohols;

6. ethers and esters;

7. aliphatic carbonic acids; 8. carbon disulphide;

9. element-organic compounds; 10. aromatic carbohydrates;

11. nitro- and amino-derivates of aromatic compounds; 12. phenols and phenol-acids;

13. phosphorus and its oxidising and reducing products;

14. heterocyclic compounds: pyridine; 15. condensed aromatic compounds: naphthalene, anthracene, bezpyrene;

16. alkaloids: nicotine, coniine, anabasine, arecoline, ephedrine.

These substances as a rule are isolated from biological material by distillation with water vapour. Such properties of chemical compounds defines they ability to distillation with

steam:

– mixable with water; – ability to azeotropic mixtures formation;

– vapour pressure at boiling temperature; – fugacity.

Distillation

Distillation is a process involving the conversion of a liquid into vapour that is

subsequently condensed back to liquid form. It is exemplified at its simplest when steam from a kettle becomes deposited as drops of distilled water on a cold surface. Distillation is

used to separate liquids from non-volatile solids, as in the separation of alcoholic liquors from fermented materials, or in the separation of two or more liquids having different

boiling points, as in the separation of gasoline, kerosene, and lubricating oil from crude oil.

Other industrial applications include the processing of such chemical products as formaldehyde and phenol and the desalination of seawater. The distillation process appears

to have been utilised by the earliest experimentalists. Aristotle (384-322 BC) mentioned that pure water is made by the evaporation of seawater. Pliny the Elder (AD 23-79) described a

primitive method of condensation in which the oil obtained by heating rosin is collected on wool placed in the upper part of an apparatus known as a still.

Most methods of distillation used by industry and in laboratory research are

variations of simple distillation. This basic operation requires the use of a still or retort in which a liquid is heated a condenser to cool the vapour, and a receiver to collect the

45

distillate. In heating a mixture of substances, the most volatile or the lowest boiling distils

first and the others subsequently or not at all. This simple apparatus is entirely satisfactory

for the purification of a liquid containing non-volatile material and is reasonably adequate for separating liquids of widely divergent boiling points. For laboratory use, the apparatus is

commonly made of glass and connected with corks, rubber bungs, or ground-glass joints. For industrial applications, larger equipment of metal or ceramic is employed.

A method called fractional distillation, or differential distillation, has been developed

for certain applications, such as petroleum refining, because simple distillation is not efficient for separating liquids whose boiling points lie close to one another. In this

operation the vapours from a distillation are repeatedly condensed and revaporised in an insulated vertical column. Especially important in this connection are the still heads,

fractionating columns, and condensers that permit the return of some of the condensed vapour toward the still. The objective is to achieve the closest possible contact between

rising vapour and descending liquid so as to allow only the most volatile material to proceed

in the form of vapour to the receiver while returning the less volatile material as liquid toward the still. The purification of the more volatile component by contact between such

counter-current streams of vapour and liquid is referred to as rectification, or enrichment. Multiple-effect distillation, often called multistage-flash evaporation, is another

elaboration of simple distillation. This operation, used primarily by large commercial desalting plants, does not require heating to convert a liquid into vapour. The liquid is

simply passed from a container under high atmospheric pressure to one under lower

pressure. The reduced pressure causes the liquid to vaporise rapidly; the resulting vapour is then condensed into distillate.

A variation of the reduced-pressure process uses a vacuum pump to produce a very high vacuum. This method, called vacuum distillation, is sometimes employed when dealing

with substances that normally boil at inconveniently high temperatures or that decompose when boiling under atmospheric pressure.

Steam distillation (or distillation with inert gas) is an alternative method of achieving

distillation at temperatures lower than the normal boiling point. It is applicable when the material to be distilled is immiscible (incapable of mixing) and chemically nonreactive with

water. Examples of such materials include fatty acids and soybean oils. The usual procedure is to pass steam into the liquid in the still to supply heat and cause evaporation of the liquid.

If the sample that has to be distilled is only slightly soluble in water and may decompose at its boiling point and experience violent bumping with a vacuum distillation, it

is better to steam distillation. Mixtures of oils and tars do not dissolve well in water; thus,

they can be steams distilled. Heating the compound in the presence of steam makes the compound boil at a lower

temperature. This has to do with partial pressures of water and organic oil.

Ttotal = XAPoA + XBP

oB (Raoult's law)

Water and the organic oils do not mix and can be considered to be unmixed in the

distilling flask. Therefore, they are independent of each other and each has a mole fraction value of 1. Assuming XA = XB = 1, equations for PA and PB express which states that at

constant temperature the partial pressure of a component in a liquid mixture is proportional to its mole fraction in that mixture (i.e., each component exerts a pressure that depends

directly on the number of its molecules present). The above equation simplifies to PTotal =

PA + PB (Dalton’s law). Since the distillation will occur when the pressure is equal to

46

atmospheric pressure, the two components must add up to that value. Since the mole

fraction for each is one, their boiling points must be lower for the pressures to add up to

atmospheric value. This phenomenon allows for a successful distillation to be performed. It is unfortunate that the word law is associated with this relation, because only very

few mixtures behave according to the equations for ideal binary mixtures. In most cases the activity coefficient, Pi, is not equal to unity. When Pi is greater than 1, there are positive

deviations from Raoult's law; when Pi is less than 1, there are negative deviations from

Raoult's law. An example of a binary system that exhibits positive deviations from Raoult's law is

represented in Figure 1, the partial pressures and the total pressure being related to the liquid-phase composition; if Raoult's law were valid, all the lines would be straight, as

indicated by the dashed lines. As a practical result of these relationships, it is often possible by a series of repeated vaporisations and condensations to separate a liquid mixture into its

components, a sequence of steps called fractional distillation.

When the vapour in equilibrium with a liquid mixture has a composition identical to

that of the liquid, the mixture is called an azeotrope. It is not possible to separate an azeotropic mixture by fractional distillation because no change in composition is achieved

by a series of vaporisations and condensations. Azeotropic mixtures are common. At the

azeotropic composition, the total pressure (at constant temperature) is always either a maximum or a minimum with respect to composition, and the boiling temperature (at

constant pressure) is always either a minimum or a maximum temperature. Azeotrope – a mixture of liquids that has a constant boiling point because the vapour

has the same composition as the liquid mixture. The boiling point of an azeotropic mixture may be higher or lower than that of any of its components. The components of the solution

cannot be separated by simple distillation because a mass transfer is absent. For azeotropic

mixtures separation can be used vacuum distillation (changed azeotrope composition) or chemical dehydration methods.

Figure 4. Total pressure and partial pressures for the system benzene-

carbon disulphide at 25 °C (see text).

47

Figure 5. Apparatus for steam distillation

1 – steam generator, 4 – flask for distillate collection,

2 – flask with distilled mixture, 5 – water bath 3 – condenser,

Sometime can be used another than water solvents. For example, for acrylonitrile isolation from food can be used methanol. I any co-distillation variant to mixture adds low-temperature boiled solvent, in presence of which the third component is distilled. This technique named “distillation with carrier”. For examples, ethylene glycol is distilled with benzene or some substances can be isolated from lipids with dichloromethane as carrier. In this case also can be formed azeotropic mixtures – ternary. For example, little amounts of ethylene glycol and toluene were isolated from wax using methanol-cyclohexane azeotropic mixture.

Microdiffusion

Microdiffusion is also a form of sample purification and relies on the liberation of a volatile compound from the test solution held in one compartment of an enclosed system

such as the specially constructed Conway apparatus (Figure 3). The volatile compound is subsequently trapped using an appropriate reagent held in a separate compartment.

The cells are normally allowed to stand for 2-5 hours at room temperature for the

diffusion process to be completed. The analyte concentration is subsequently measured in a portion of the trapping solution either by spectrophotometry or by visual comparison with

standards analysed concurrently in separate cells. The Conway apparatus is normally made from glass, but polycarbonate must be used with fluorides since hydrogen fluoride etches

glass. The cover is often smeared with petroleum jelly or silicone grease to ensure an airtight seal. In order to carry out a quantitative assay at least eight cells are needed: one

blank, three calibration samples, two test samples and two positive controls. It is important

to clean the diffusion apparatus carefully after use, possibly using an acid/dichromate mixture, raising it in distilled water before drying.

Examined objects put into outer well. Reagent solution (absorbed liquid) pours into inner well.

Formaldehyde detection. Into outer well (outside chamber) of the device for microdiffusion put blood or urine, or tissues homogenate and acidify with sulphate acid

solution. Solution of sodium hydrosulphite or sodium sulphite puts into inner well of the

device. The device covers and leaves on few hours at room temperature. After diffusion the

48

liquid from the internal chamber (inner well) of the device puts into test tube and research

on formaldehyde presence with chromotropic acid.

Figure 6. Conway diffusion cells (microdiffusion apparatus)

Acetone detection. Filling the outer well and inner well of device for microdiffusion

like for formaldehyde research. After diffusion liquid from the internal chamber (inner well)

of the device put into test tube and research on acetone presence with salicylic aldehyde:

OH

CO

H+ CH3H3C CO

CH

OH

CH C

O

HC

OH

CH

Methanol detection. Into outer well of the device for microdiffusion put blood or urine, or tissues homogenate and alkalise with saturated solution of potassium carbonate. To

the inner well of the device put solution of sulphate acid. The device covers and leaves on few hours.

Research of liquid from inner well is based on methanol oxidation to formaldehyde

and it detection with chromotropic acid.

Ethanol determination. Into outer well of the device for microdiffusion put blood or urine, or tissues homogenate and add saturated solution of potassium carbonate. Into inner

well put solution of potassium dichromate in sulphate acid. The device covers and leaves on few hours at room temperature. At ethanol presence the liquid in inner well change colour

from yellow to green:

3C2H5OH + 2K2Cr2O7 + 8H2SO4 → 3CH3COOH + 2Cr2(SO4)3 + 2K2SO4 + 11H2O.

49

Sulphides detection. Into outer well of the device for microdiffusion put blood or

urine, or tissues homogenate and acidify with sulphate acid solution. Solution of sodium

hydroxide puts into inner well of the device. The device covers and leaves on few hours room temperature. Then liquid from the inner well put into test tube with solution of

bismuth nitrate in acetic acid – forms black precipitate of bismuth a sulphide:

3Na2S + 2Bi(NO3)3 → Bi2S3↓ + 6NaNO3.

Phenol detection. Filling the outer well and inner well of device for microdiffusion

like as for sulphides research. After diffusion liquid from the inner well of the device put into test tube and research on phenol presence with Folin-Ciocalteau’s reagent. This reagent

is specially prepared mixture of sodium tungstate, sodium molybdate, orthophosphate acid,

chloride acid, lithium sulphate, and elemental bromine. At phenol presence arises dark blue colour.

Cyanides detection. Filling the outer well and inner well of device for

microdiffusion like as for sulphides research. After diffusion liquid from the inner well of the device put into test tube and research on cyanides presence. Into test tube add solutions

of sodium hydro phosphate, chloramine Т, and reagent containing barbituric acid and

pyridine. At cyanides presence red colour arises.

Carbon (II) oxide detection. Into outer well of the device for microdiffusion put blood and acidify with sulphate acid solution. Solution of palladium chloride in chloride

acid solution puts into inner well of the device. The device covers and leaves on one hour at room temperature. At carbon (II) oxide presence in blood in the inner well forms brilliant

film of metal palladium:

CO + PdCl2 + H2O = Pd + 2НС1 + CO2.

Static Headspace Analysis by Gas Chromatography

Static headspace gas chromatography is a technique used for the concentration and

analysis of volatile organic compounds. This technique is relatively simple and can provide sensitivity similar to dynamic purge and trap analysis. The popularity of this technique has

grown and has gained worldwide acceptance for analyses of alcohols in blood, volatile compounds in food, poisons in body fluids, and residual solvents in pharmaceutical

products. Other common applications include industrial analyses of monomers in polymers and plastic, flavour compounds in beverages and food products, and fragrances in perfumes

and cosmetics.

Sample matrices like blood, urine, beverages, plastic, and cosmetics contain high molecular weight, non-volatile material that can remain in the gas chromatography system

and result in poor analytical performance. Many laboratory analysts use extensive sample preparation techniques to extract and concentrate the compounds of interest from this

unwanted non-volatile material. These extraction and concentration techniques can become time consuming and costly. Static headspace analysis avoids this time and cost by directly

sampling the volatile headspace from the container in which the sample is placed.

50

Most consumer products and biological samples are composed of a wide variety of

compounds that differ in molecular weight, polarity, and volatility. For complex samples

like these, headspace sampling is the fastest and cleanest method for analysing volatile organic compounds. A headspace sample is normally prepared in a vial containing the

sample, the dilution solvent, a matrix modifier, and the headspace (Figure 4). Volatile components from complex sample mixtures can be extracted from non-volatile sample

components and isolated in the headspace or vapour portion of a sample vial. An aliquot of

the vapour in the headspace is delivered to a gas chromatography system for separation of all of the volatile components.

G=the gas phase (headspace).

The gas phase is commonly referred to as the headspace and lies above the condensed sample phase.

S=the sample phase.

The sample phase contains the compound(s) of interest and is usually in the form of a liquid or solid in

combination with a dilution solvent or a matrix modifier.

Figure 7. Phases of the headspace vial

Once the sample phase is introduced into the vial and the vial is sealed, volatile

components diffuse into the gas phase until the headspace has reached a state of equilibrium as depicted by the arrows. The sample is then taken from the headspace.

Partition Coefficient

Samples must be prepared to maximize the concentration of the volatile components

in the headspace, and minimize unwanted contamination from other compounds in the sample matrix. To help determine the concentration of an analyte in the headspace, you will

need to calculate the partition coefficient (K), which is defined as the equilibrium distribution of an analyte between the sample phase and the gas phase:

Partition Coefficient (K) = Cs/Cg

Cs=concentration of analyte in sample phase Cg=concentration of analyte in gas phase

Compounds that have low K values will tend to partition more readily into the gas

phase, and have relatively high responses and low limits of detection (Figure 5). An example of this would be hexane in water: at 40°C, hexane has a K value of 0.14 in an air-

water system. Compounds that have high K values will tend to partition less readily into the

gas phase and have relatively low response and high limits of detection. An example of this would be ethanol in water: at 40°C, ethanol has a K value of 1355 in an air-water system.

Partition coefficient values for other common compounds are shown in Table 1.

51

K Values of Common Solvents in Air-Water Systems at 40°C

Solvent K Value Solvent K Value

cyclohexane

n-hexane

tetrachloroethylene 1,1,1-trichloromethane

o-xylene toluene

benzene dichloromethane

0.077

0.14

1.48 1.65

2.44 2.82

2.90 5.65

n-butyl acetate

ethyl acetate

methyl ethyl ketone n-butanol

isopropanol ethanol

dioxane

31.4

62.4

139.5 647

825 1355

1618

The K can be lowered by changing the temperature at which the vial is equilibrated or by changing the composition of the sample matrix. In the case of ethanol, K can be

lowered from 1355 to 328 by raising the temperature of the vial from 40°C to 80°C.

Introducing inorganic salt into the aqueous sample matrix can lower it even further. High salt concentrations in aqueous samples decrease the solubility of polar organic volatiles in

the sample matrix and promote their transfer into the headspace, resulting in lower K values. However, the magnitude of the salting-out effect on K is not the same for all compounds.

Compounds with K values that are already relatively low will experience very little change in partition coefficient after adding a salt to an aqueous sample matrix. Generally, volatile

polar compounds in polar matrices (aqueous samples) will experience the largest shifts in K

and have higher responses after the addition of salt to the sample matrix. Are the lists most of the common salts used for salting-out procedures (to decrease matrix effects):

ammonium chloride ammonium sulphate sodium chloride

sodium citrate sodium sulphate potassium carbonate

Phase Ratio

The phase ratio (ß) is defined as the relative volume of the headspace compared to

volume of the sample in the sample vial:

Phase Ratio (ß) = Vg/Vs

Vs=volume of sample phase

Vg=volume of gas phase Lower values for ß (i.e., larger sample size) will yield higher responses for volatile

compounds (Figure 6). However, decreasing the ß value will not always yield the increase in response needed to improve sensitivity. When increasing the sample size decreases ß,

compounds with high K values partition less into the headspace compared to compounds with low K values, and yield correspondingly smaller changes in Cg. Samples that contain

compounds with high K values need to be optimised to provide the lowest K value before

changes are made in the phase ratio.

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Combining K and ß

Partition coefficients and phase ratios work together to determine the final

concentration of volatile compounds in the headspace of sample vials. The concentration of

volatile compounds in the gas phase can be expressed as Cg = Co / (K+ß) (where Cg is the concentration of volatile analytes in the gas phase and Co is the original concentration of

volatile analytes in the sample). Striving for the lowest values for both K and ß will result in

higher concentrations of volatile analytes in the gas phase and, therefore, better sensitivity (Figure 7).

Figure 5.

Sensitivity is increased when

K is minimised.

Figure 6.

Sensitivity is increased when

ß is minimised.

Figure 7.

Lower K and ß result in

higher Cg and better sensitivity.

Derivatisation/Reaction Headspace

Derivatisation is another technique that can be used to increase sensitivity and

chromatographic performance for specific compounds. Compounds such as acids, alcohols,

and amines are difficult to analyse because of the presence of reactive hydrogen. When attempting to analyse these types of compounds, they can react with the surface of the

injection port or the analytical column and result in tailing peaks and low response. In addition, they may be highly soluble in the sample phase, causing very poor partitioning

into the headspace and low response. Derivatisation can improve their volatility, as well as reduce the potential for surface adsorption once they enter the gas chromatography system.

Common derivatisation techniques used in reaction headspace/gas chromatography

are etherification, acetylation, silylation, and alkylation. Any of these derivatisation techniques can be performed using the sample vial as the reaction vessel (see Table 2 for a

list of commonly used reagents). Although derivatisation may improve chromatographic performance and volatility for some compounds, derivatisation reactions may introduce

other problems into the analytical scheme. Derivatisation reagents as well as the by-products from derivatisation reaction may be volatile and can partition into the headspace

along with derivatised compounds. These extra volatile compounds may pose problems by

eluting with similar retention times as the compounds of interest, causing either partial or complete coelutions.

Derivatisation reactions also are typically run at elevated temperatures. Pressures inside the sample vial may exceed the pressure handling capabilities of the vial or the septa.

53

Specially designed septa are available that allow excess pressure to be vented during

derivatisation reactions.

Common reagents used to derivatise compounds of interest.

Compound of Interest Derivatising Reagent Resulting Derivative

Fatty acids Methanol with boron trifluoride etherification

Glycerol Acetic anhydride with sodium carbonate acetylation

Alcohols Sodium nitrite with trichloroacetic acid esterification

Headspace Sample Size

In addition to working with K, ß, and derivatisation reactions, sensitivity also can be

improved by simply increasing the size of the headspace sample that is withdrawn from the

sample vial and transferred to the gas chromatography. Increasing the sample size also means that the amount of time it takes to transfer the sample to the column will increase in

proportion to the column volumetric flow rate. Sample size can be increased only to the

point that increases in peak width, as a result of longer sample transfer times, will not affect chromatographic separations. Larger sample sizes and longer transfer times can be offset by

using cryogenic cooling and sample refocusing at the head of the column.

Sample Preparation

Samples for headspace/gas chromatography must be prepared in such a manner as to

maximize the concentration of the volatile sample components in the headspace while

minimizing unwanted contamination from other compounds in the sample matrix. Sample matrices such as biological samples, plastics, and cosmetics can contain high molecular

weight, volatile material that can be transferred to the gas chromatography system. Water

from the sample matrix also can cause problems by recondensing in the transfer line. Incomplete or inefficient transfer of high molecular weight compounds or water vapour

from sample matrices can produce adsorptive areas in the transfer line or injection port that can lead to split peaks, tailing peaks, or irreproducible responses or retention times. To

minimize matrix problems and prevent water condensation from aqueous samples, use a higher transfer line temperature (~125°C-150°C).

Sample Vial Heater and Mixer

Once the sample is placed inside a clean, non-contaminating vial and the vial is

sealed, volatile compounds from the sample will partition into the headspace until a state of equilibrium is reached. The rate of volatile compounds partition out of the sample matrix

and into the headspace, as well as the equilibrium concentration of volatile compounds in

the headspace depends on several parameters. Temperature, time, and mixing can be used to improve the transfer of volatile

analytes from the sample into the headspace of the vial. Adjusting the temperature of the sample will change the solubility of the analyte in the sample matrix and can be used to

54

drive the equilibrium in favour of the gas phase. Sufficient time must be built into the

sample cycle in order to achieve a constant state of equilibrium. Some sample matrices

require longer equilibration times due to physical characteristics like high viscosity. Shaking or vibrating the vial during heating can assist in achieving equilibrium more quickly by

exposing more sample surface area for the transfer of volatile analytes to the headspace.

Blood Alcohol Analysis

Quantitation Technique for Blood Alcohol Analysis with Internal Standard

The internal standard technique uses one or more designated compounds at known concentrations spiked into the sample. The response of the compounds of interest is then

compared to the results of the internal standard. There are several advantages to this

technique: – multiple injections of the standard are not necessary for concentration

calculations; – small changes in injection volumes or detector response over time can be

determined. Simulated blood alcohol samples were prepared and analysed using an n-propanol as

the internal standard. n-Propanol is used as internal standard because of the absence it in

normal human blood. On Figure 8 is shown chromatogram of real blood sample with added internal standard, obtained in capillary column and flame-ionisation detector:

Figure 8. Chromatogram of real blood sample with added internal standard

Compound Concentration, w/v

1. Methanol

2. Acetaldehyde

3. Ethanol 4. Iso-propanol

5. Acetone 6. n-Propanol (added internal standard)

0.1 %

0.2 %

0.2 % 0.1 %

0.01 % 0.1 %

55

Quantitation Technique for Blood Alcohol Analysis with Internal Standard and

Derivatisation (Esterification)

This determination can be performed with derivatisation of blood alcohols. To blood

sample in vial add standard solution of n-propanol, trichloroacetic acid, and sodium nitrite. Sodium nitrite (NaNO2) is derivatising agent. Trichloroacetic acid (CCl3–COOH) is

promoter of this reaction and creates strong acidic environment:

R–OH + NaNO2 + CCl3–COOH → R–O–NO↑ + CCl3–COONa + H2O

Gas phase (headspace) pick out by medicine syringe and insert to gas chromatograph.

Advantages of derivatisation technique are:

– only alcohols (normally present and added) form derivatives (esters) with sodium nitrite. Another volatile components of blood (acetaldehyde and acetone) not

form derivatives, – derivatives have large thermal conductivity. We can apply more selective

thermal-conductive detector, – derivatives (formed alkylnitrites) are more volatile and not need sample heating.

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“VOLATILE” POISONS INVESTIGATION

ISOLATION OF “VOLATILE” POISONS FROM BIOLOGICAL MATERIAL

Accordance to poisonous substances chemical properties they can be divided on

acidic, neutral, and alkali compounds. Poisonous substances, that are acids, must be isolated from acidified biological

material. Acidifying using for: 1) destroying complexes of poisonous substances with organism peptides;

2) destroying salts of poisonous substances with organic acids; 3) liberated poisonous substances dissociation in solution suppress;

4) poisonous substances transition to molecular state. Only associated molecules can be

distilled, adsorbed or extracted. For biological material acidifying as a rule uses oxalic or tartaric acids. The

application of inorganic acids is undesirable. Phenols produce in organism from aromatic amino acids – phenylalanine, tyrosine,

and, partly, triptophane. This metabolic-created phenol form conjugates with sulphates and eliminates. But mineral acids used for acidifying decompose these metabolic phenol

conjugates with sulphate acid. As a result, an examined sample contains phenol which are

not occurs poisoning. Mineral acids can decompose cyanides with formic acid formation or liberates very

volatile cyanic acid. As a result, an examined sample lost determined poisonous component. In another side, the mineral acids must be used in special case. For example, only in

strong acidic environment an acetic acid not dissociates and can be distilled. And also chlorinated carbohydrates must be acidified for alkali hydrolysis prevention. The most

preferable is sulphate acid, because this acid: 1) is strong electrolyte, 2) not forms

azeotropic mixtures with water, and 3) not volatile – its vapour not present in distillates. Are not recommend use a phosphate acid for biological material acidifying,

especially if sample is analysed on phosphorus and its biotransformation products presence (phosphine or phosphite acid).

Substances with base properties (aniline, nicotine, and pyridine) can be distilled from alkalised environment. For this purpose uses sodium or potassium hydroxides solutions.

Ampholytes and neutral substances are less sensitive to pH changes. But

environment must be acidified/alkalised for dissociation prevention and for decompose peptides–poisonous substances bonds. Created pH must be equal to pH of these ampholytes

association or to pH of destroyed peptides isoelectric point.

CYANIC ACID

Cyanic acid is a gas or colourless liquid (boiling point 25,6 °C, melting point 13,3

°C, density 0,699), has an almond odour and easily mixes with water and many organic

solvents. At 13,3 °C the cyanic acid hardens, forming fibrous crystalline mass. The cyanic

acid is weak acid. Even carbon dioxide and weak organic acids supersede it from salts. Free cyanic acid does not meet in a nature. It meets as chemical combinations, to

which number the glycosides (amygdalin, prunasin, dhurrin, and linamarin) concern. Bitter almonds and apricot seeds have high levels, whereas peanut, plum, pear, and apple pits have

57

smaller amount of the amygdalin. This glycoside under influence of an emulsin enzyme,

and also under influence of acids is decomposed to a glucose, benzaldehyde, and cyanic

acid. Prunasin contains in a cherry laurel. Prunasin is the primary metabolite of orally administered amygdalin. Dhurrin contains in sorghum. Linamarin contains in cassava and

certain lima beans (CN– content varies between species with black Puerto Rican beans being

the most lethal). Linase enzyme or acid hydrolysis yields cyanic acid from linamarin.

The cyanic acid can be formed at burning celluloid. The traces of this acid contain in

a tobacco smoke. The salts of a cyanic acid (cyanides) are easily hydrolysed in water. At storage of

aqueous solutions of cyanides they are decomposed at access of carbon dioxide:

KCN + Н2O + СО2 = HCN + КНСО3; KCN + 2Н2О = NН3 + НСООК

In aqueous solutions are decomposed not only cyanides, but also cyanic acid:

HCN + 2Н2О = HCOONH4

Application. Action on organism. A cyanic acid and its salts are applied to synthesis of organic compounds, at production of gold, for disaffection, for struggle with the wreckers

of plants etc. The most important and usually used are sodium and potassium cyanides.

Cyanic acid and its salts are very toxic. The cyanic acid surpasses in toxicity many known poisons. Therefore with a cyanic acid and its salts it is necessary to revert very

cautiously. It is necessary to remember, that from addition of strong acids to cyanides a cyanic acid at once precipitates out which can cause serious, and sometimes and lethal

poisonings. The poisonings can give and various compounds of a cyanic acid (chlorcyane, bromcyane etc.). Are described the cases of poisonings with almond seeds. On the one data

the adult was died after 40 – 60 almond seeds eating, and at another lethal case child has eat

10 – 12 almond seeds. At inhale of the large concentration of a cyanic acid the death can occur instantaneously from a stopping of respiration and heart. Taking into account a high

toxicity of a cyanic acid and its salts to work with them in laboratory it is possible only in an exhaust with good ventilation.

The cyanic acid oppresses endocellular ferruginous respiratory enzymes. At an oppression of cytochrome oxydase by a cyanic acid of a cell of an organism do not acquire

Oxygen acting with a blood. As a result of it comes cell an oxygen starvation, in spite of the

fact that the blood oxygenated. Cyanides also can block haemoglobin of a blood, breaking its functions.

The cyanic acid can act in an organism with inhaled air and partially through an uninjured skin, and cyanides – through the alimentary canal.

Metabolism. A metabolite of a cyanic acid is the thiocyanate (rhodanide), which is formed in an organism at a conjugation of cyanides with sulphur under influence of a

phodynase enzyme.

Isolation of Cyanic Acid and Cyanides

Isolation of a cyanic acid and cyanides from a biological material make by steam

distillation. For this purpose collect 3 – 5 ml of the first distillate in a test tube containing 2 ml of 2 % sodium hydroxide solution. As the cyanic acid is quickly decomposed in an

58

organism, the examination of a biological material on it presence is desirable to spend

immediately. For examination take a stomach with contents, liver and kidneys. In view of

fast decomposing of a cyanic acid and cyanides in tissues of an organism these poisons can not be found.

In urine of smoking persons the cyanides amount can be in 3 times more, than in a blood of the non-smokers. In a blood the cyanides can be formed and after death.

Detection of a Cyanic Acid in Distillates

Prussian blue formation

HCN + NaOH = NaCN + H2O

2NaCN + FeSO4 = Fe(CN)2 + Na2SO4

Fe(CN)2 + 4NaCN = Na4[Fe(CN)6]

3Na4[Fe(CN)6] + 4FeCl3 = Fe4[Fe(CN)6]↓ + 12NaCl

Arise blue precipitate.

Iron thiocyanate formation

KCN + (NH4)2S2 = KSCN + (NH4)2S KSCN + FeCl3 = Fe(SCN)3 + 3KCl

Arise red colour of solution.

Benzidine blue formation. Cooper(II) salts forms dicyane (CN)2 in reaction with cyanic acid. Dicyan hydrolyses

with oxygen evolving. This oxygen oxidises benzidine:

2HCN + Cu(CH3COO)2 = Cu(CN)2 + 2CH3COOH

2Cu(CN)2 = (CN)2↑ + 2CuCN

(CN)2 + H2O = O + 2HCN

H2N NH2 + O + 2CH 3COOH

H2N NH2

HN NH

2CH3COOH + H 2O

Arises blue spot on indicator paper.

59

Reaction with picric acid

+ KOH + 2HCN

NO2

NO2

O2N

OH OK

NO2

O2N

N C

N

OH

H

C

NH2

O

In this reaction is formed ammonium salt of iso-hyppuric acid, which have red colour. Also cyanic acid can be detected with microdiffusion method on pyridine and

barbituric acid reaction. Arise red colour of reagent.

FORMALDEHYDE

Formaldehyde (aldehyde of a formic acid) is a well water-soluble gas having an acute specific smell. The aqueous solution containing 36,5 – 37,5 % of formaldehyde refers to as

Formalinum. The formaldehyde is formed at poor combustion of methane, at oxidation of methanol etc. The gaseous formaldehyde at ambient temperature is easy polymerised with

paraformaldehyde (paraforme) formation. One of a formaldehyde polymer is

trioxymethylene (СН2O)3. It has melting point 63 – 64 °C. In aqueous solutions also

paraforme forms concerning to polyoxymethylenes, which are products of polymerisation of

the much greater number of molecules of a formaldehyde. Formaldehyde isolates from a biological material by steam distillation. However this

method overtakes only insignificant part of a formic aldehyde. Considered, that the formaldehyde in aqueous solutions is as a hydrate (methyl glycol), which is difficulty

steamed:

НСНО + НОН → CH2(OH)2.

Application. Action on organism. The formaldehyde is widely used in a chemical industry for plastic masses and phenol-formaldehyde resins producing. Formaldehyde also

is using for hardening of skins, preservation of anatomic probes, hexamethylenetetramine

producing, and disinfecting. The formaldehyde has hardening, antiseptic and deodorising action. The

formaldehyde irritates the top respiratory ways at inhalation even small amounts. The large amounts of inhaled formaldehyde can occur death as a result of an oedema and

laryngospasm. A formaldehyde swallowing causes necrotic defeats of a mouth mucous, of a alimentary channel, hypersalivation, nausea, vomiting, diarrhoea. The formaldehyde

represses the central nervous system, as a result of it there can be a loss of consciousness,

there are cramps. Under influence of formaldehyde the degenerative defeats of a liver, kidneys, heart and brain develop. 60 – 90 ml of formalinum is a lethal dose.

Metabolism. Metabolites of formaldehyde are methanol and formic acid, which, in turn, are exposed to the further metabolism.

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Formaldehyde Detection

Reaction with chromotropic acid. The chromotropic acid (1,8-dioxynaphthalen-3,6-disulphoacid) with a formaldehyde at the sulphate acid presence gives violet colouring.

At interaction of formaldehyde with chromotropic acid the concentrated sulphate acid simultaneously is dehydration agent and oxidiser. In the beginning sulphate acid causes

condensation of a formaldehyde with chromotropic acid, and then oxidises the formed

product of condensation:

At formaldehyde presence in assay there is a violet colour.

Reaction with Shiff’s reagent (Fuchsin acid reduced).

For preparation the Shiff’s reagent a solution the acid Fuchsin (I) reduces with

gaseous SO2. This reagent with aldehydes forms a quinoid stain of pink colour. The Shiff’s reagent with formaldehyde forms dark blue or blue-violet colour compound.

C

HN-SO2H

HN-SO2H

NH2H C

HN-SO2-CH2OH

NH

HN-SO2-CH2OH

The solution is sometimes coloured not at once, and through 10 – 15 min. The

colouring can occur not only under influence of formaldehyde, but also under influence of oxidisers (chlorine, oxides of nitrogen, oxygen in air etc.). Therefore occurrence of

colouring through 30 mines after addition of reagents should not be surveyed as a positive take of reaction on formaldehyde.

This reaction is not specific to detection of formaldehyde. It gives an acetaldehyde,

nitrobenzaldehyde etc. But in highly acid environment (pH 0,7) with Shiff’s reagent reacts only the formaldehyde.

61

Reaction with resorcinol. The aldehydes react with Resorcinol in its tautomeric

form (keto-form) with colour compound formation:

+ HCHO

OH

O

OH

O

CH2

+ H2O

OH

OH OH

O

The occurrence of pink or crimson colour specifies formaldehyde presence. This reaction gives acetaldehyde, acrolein, furfurol etc.

Reaction of silver ions reducing. There is a reaction of the “silver mirror”

formation. From ammonia solution of silver salts the formaldehyde evolves metal silver:

[Ag(NH3)2]NO3 + НСНО + Н2O → Ag↓ + НСООNH4 + NH4NO3

This reaction successfully proceeds at рН 8–9. At high temperature the "silver

mirror" is not formed, and drops out the brown precipitate of colloid silver. Except for formaldehyde this reaction is given also with some other reducing

substances.

Reaction with Fehling’s reagent. The Fehling’s reagent heated with formaldehyde

forms the copper oxide precipitate. The oxide of copper (I) has black colour. The colour of copper (I) oxide depends on the size of particles. The very shallow particles of copper oxide

have blue-green colour, and large particles – red. Therefore at interaction the Fehling’s reagent with reducers are formed previously yellow or red precipitate.

The Fehling’s reagent is a complex compound of cooper with Signet’s salt dissolved

in alkali:

+ HCHO + NaOH → Cu2O↓ + 2C4O6Na2 + CO2↑ + H2O

This reaction is not specific. Except for formaldehyde it is given also with other

aliphatic aldehydes and saccharides.

Detection of formaldehyde with a microdiffusion method. For detection of

formaldehyde in tissues, blood and urine are used the microdiffusion method that based on reaction with chromotropic acid.

COOK

COONa

HCO

HCOH

Cu OCH

HOCH

NaOOC

KOOC

62

METHANOL

Methanol is a colourless liquid (boiling point 64,5 °C, density 0,79), mixed with water and many organic solvents. The methanol burns by a pale blue not smoking flame.

The methanol on a smell and taste does not differ almost from ethanol. The cases of a

poisoning with methanol wrongly accepted instead of are known ethyl. In a nature the methanol in a free condition does not meet. There are widespread it

derivative – essential oils, esters etc. Earlier methanol received by dry distillation of a tree. Some industrial synthetic ways of reception of methanol now are used.

Application. Action on organism. The methanol is widely used in an industry as a solvent of varnishes, paints, as mother substance for reception of methyl chloride, dimethyl

sulphate, formaldehyde and other chemical compounds. It is applied for ethanol

denaturation, enters into composition of antifreeze. The methanol can act in an organism through the alimentary canal, and also with

inhaled air containing vapour of this alcohol. In insignificant amounts the methanol can penetrate into an organism through a skin. The toxicity of methanol depends on poisoning

circumstances and individual susceptibility. Under influence of methanol there is a defeat of eye retina and visual nerve, and sometimes there comes incurable blindness. The methanol

breaks oxidising processes and acid-alkaline equilibrium in cells and tissues. As a result of

it there comes an acidosis. The poisoning with methanol in many cases comes to death. The danger of occurrence of blindness arises already after reception 4 – 15 ml of methanol. The

lethal dose of methanol is 30 – 100 ml. The death comes as a result of respiration break, brain and lungs oedema, collapse or uraemia. The local action of methanol on mucous is

more strongly, and the narcotic action is more weak, than ethanol. The simultaneous entering of methyl and ethyl alcohols in an organism reduces a

toxicity of methanol. It is explained that ethanol reduces oxidation rate of methanol almost

50 %, and consequently, reduces its toxicity. Metabolism. The greatest amount of methanol collects in a liver, and then in kidneys.

The smaller amounts of this alcohol collect in muscles and brain. A metabolite of methanol is the formaldehyde, which is oxidised to a formic acid. The part of this acid is decomposed

to carbon(IV) oxide and water. The nonmetabolised methanol eliminates with exhaled air. It also can eliminate with urine as a glucuronide. However with urine can eliminate small

amounts of the not changed methanol. The methanol oxidises in an organism more slowly,

than ethanol. In an organism (in norm) can contain 0,01 – 0,3 mg-% of methanol and about 0,4

mg-% of formic acid.

Detection of Methanol

Taking into account fugitiveness of methanol it is necessary to cool the distillate by

cold water or ice during steam distillation. The obtained distillate in most cases contains insignificant quantities of methanol. Therefore this distillate is a subject of two or three-

aliquot distillations with a Vigreux distilling column. Only after concentration in distillate detect the methanol presence.

63

Reaction of methyl salicylate formation. At methanol presence is felt a

characteristic smell of a methyl salicylate. This reaction is not specific, because ethanol

forms ethyl salicylate with similar smell.

OH OH

COOH COOCH3

+ H2OCH3OH +

Oxidation of methanol. The majority of methanol detection reactions are based on it oxidation to formaldehyde and detection last with appropriate reactions. Before methanol

oxidation it is necessary to check a formaldehyde presence in examined solution. For methanol oxidation apply potassium permanganate or other oxidisers:

5СН3ОН + 2КMnO4 + 3Н2SО4 = 5НСНО + 2MnSO4 + K2SO4 + 8Н2О.

Detection of methanol after its oxidation. After methanol oxidation to formaldehyde last detect by reactions with chromotropic acid, Shiff’s resgent and with

resorcinol. The specific is the reaction with chromotropic acid. Do not give this reaction ethyl, propyl, butyl, amyl and isoamyl alcohols.

Method of microdiffusion. This method is based on methanol oxidation to

formaldehyde and detection it with chromotropic acid.

Preliminary test on methyl and ethyl alcohols presence in urine and blood. To 1

ml of urine or blood add 1 ml of 10 % potassium dichromate solution 50 % solution of sulphate acid. The green colour appearance testifies about methyl or ethyl alcohols presence

in sample. The colour occurs on an extent 10 – 45 sec.

ETHANOL

Ethanol C2H5OH is a colourless fugitive liquid with a characteristic smell; acre on

taste (density 0,813 — 0,816, boiling point 77 – 77,5 °C). The ethanol burns by a bluish

flame. Ethanol mixes with water and many organic solvents.

Ethanol is produced by fermentation of starch-containing products (grain, potatoes), fruit, sugar etc.

Application of ethanol. The ethanol is widely used in industry as a solvent and starting product for synthesis of many chemical compounds. This alcohol is used in

medicine as a disinfectant. In chemical laboratories it is applied as a solvent, enters into composition of many spirits drinks.

Action on organism and toxicity. The ethanol can act in an organism by several

ways: at reception inside, at intravenous introduction, and also through lungs with inhaled air (as vapour).

The ethanol in an organism acts on a brain cortex. Thus there comes intoxication with characteristic alcoholic "exaltation". This exaltation does not grow out intensifying

excitative process, and arises because of weakening process of inhibition. Thus, under

64

influence of alcohol the prevalence of exaltation processes over inhibition processes is

shown. In the large doses the ethanol causes an oppression of spinal and brain functions.

Thus comes a protractive narcosis with loss of reflexes and oppression of the vital centres. Death is a result of a paralysis of respiratory centre by ethanol.

The acute poisonings with ethyl alcohol borrow the first place (about 60 %) among poisonings with other toxic substances. The alcohol not only causes acute poisonings, but

also promotes sudden deaths from other diseases (first of all, from diseases of

cardiovascular system). The degree of ethanol toxicity depends on a dose, it concentration in drinks, from

presence in drinks other odour and taste additives. The lethal dose for the man is 6 – 8 ml of pure ethanol per 1 kg of body weight (or 200 – 300 ml per all body weight). The long

abusing by ethanol results in a chronic poisoning (alcoholism). Distribution in organism. The ethanol is irregularly distributed in tissues and

biological liquids of organism. It depends on quantity of water in a body or biological

liquid. The quantitative content of ethanol is directly proportional to amount of water and in inverse proportion to fatty tissues in a body. The most amount of ethanol collects in blood.

There is certain dependence between amount of ethanol in a blood and urine. In first 1-2 hours after reception of ethanol (spirits drinks) it concentration in urine is lower, than in

blood. In the elimination period the contents of ethanol in urine exceeds it contents in blood. The large importance in diagnostics of intoxication and poisonings with ethanol has

the results of quantitative definition of this alcohol, which express in promille (‰) that

means a thousand parts. This alcohol can be formed at corpse putrefaction. Amount of ethanol in corpses can

be 2,4 ‰. Metabolism. The part of ethanol (2 — 10 %) eliminates from organism in the not

changed kind with urine, exhaled air, saliva, and faeces. Other amount of this alcohol is metabolised. Some amount of ethanol is oxidised to water and carbon(IV) oxide. Another

part is oxidised to acetaldehyde, and then to acetic acid.

Some medicines (Antabus, Teturam) delay the transformation of acetaldehyde to acetic acid. It results in accumulation of acetaldehyde in organism causes the disgust for

alcohol.

Calculation of Drunk Alcohol Amount and It Concentration in Blood at Given Time

A1 = P⋅r⋅(C + β⋅T1) N1 = A1⋅3,055

CT = CM + β⋅(T1 – 1,5)

CT = C + β⋅T2

A1 – amount of resorbed alcohol (96 %);

N1 – amount of 40 % alcohol in organism, ml; T1 – time from drinking moment to detection moment, hours;

CM – alcohol concentration in blood at maximal resorption (T = 1,5 g), ‰;

CT – alcohol concentration in blood in given time, ‰;

T2 – time from event (accident) to detection moment, hours; P – body weight, kg;

C – alcohol concentration in blood at detection moment, ‰;

r – reduction factor – ratio of alcohol mass in all body to alcohol mass in blood – 0,7;

β – oxidation factor –drop of alcohol concentration per hour – 0,15.

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Calculation of Alcohol Amount in Organism at Corpses Examination

A2 = 1,05⋅[P⋅r⋅C + (a⋅b + c⋅d)/100] N2 = A2⋅3,055

A2 – alcohol amount in organism (96 % in g);

N2 – 40 % alcohol amount in organism, ml; P – body weight, kg;

C – alcohol concentration in blood at detection moment, ‰; r – reduction factor – ratio of alcohol mass in all body to alcohol mass in blood – 0,7;

a – mass of stomach with contains, kg b – alcohol concentration in stomach with contains, ‰;

c – urine amount in urinary bubble, kg;

d – alcohol concentration in urine, ‰.

Dependence of Human Intoxication Degree on the Ethanol Contents in Blood

Ethanol contain in blood, ‰ Intoxication degree

0,3 Absence of intoxication attributes

0,3 – 0,5 Insignificant influence

0,5 – 1,5 Mild intoxication

1,5 – 2,5 Intoxication of average degree

2,5 – 3,0 Strong intoxication

3,0 – 5,0 Very serious intoxication, sometimes death

5,0 – 6,0 Lethal intoxication

Detection of Ethanol

Reaction of iodoform formation. At heating ethanol with alkalised iodine solution

is formed iodoform, having specific smell:

I2 + 2NaOH = NaOI + NaI + H2O;

С2Н5ОН + NaOI = СН3СНО + NaI + Н2O;

CH3CHO + 3NaOI = I3C–CHO + 3NaOH; I3C–CHO + NaOH = СHI3 + HCOONa.

iodoform

This reaction is not specific for ethanol. It gives also acetone, lactic acid etc. Etherification reactions. For ethanol etherification apply sodium acetate and

benzoyl chloride.

1. Reaction of ethyl acetate formation. The ethanol with sodium acetate at the

presence of sulphate acid (as dehydration agent) forms ethyl acetate, having characteristic smell:

2CH3COONa + 2С2Н5ОН + H2SO4 = 3СН3СООС2Н5 + Na2SO4 + 2H2O

66

2. Reaction of ethyl benzoate formation. At interaction of ethanol with benzoyl

chloride forms ethyl benzoate, having characteristic smell:

+ HClC2H5OH +

COO C2H5COCl

Reaction of acetaldehyde formation. Potassium permanganate or potassium

dichromate oxidises ethanol to acetaldehyde:

3C2H5OH + K2Cr2O7 + 4H2SO4 = 3CH3CHO + Cr2(SO4)3 + K2SO4 + 7Н2O

In an examined solution appears an acetaldehyde odour. At this reaction can be formed and acetic acid. The undesirable reaction of acetic acid formation depresses

sensitivity of reaction of ethanol detection.

Oxidation of ethanol and detection it by acetaldehyde. The acetaldehyde formed

after ethanol oxidation can be detected by reaction with sodium nitroprussid and morpholine. At acetaldehyde presence in solution appears dark blue colour. This reaction

gives acrolein, but not formaldehyde. Therefore this reaction can be used for difference of methyl and ethyl alcohols.

Preliminary test on presence of ethanol in urine and blood. This test is the same as for methanol.

Method of a microdiffusion. The ethanol can be found out by a method of a

microdiffusion on the same technique as is described for methanol.

ISOAMYL ALCOHOL

Isoamyl alcohol (СН3)2—СН—СН2—СН2—OH (2-methyl-butanol-4) is an optically

inactive liquid (boiling point 132,1 °С, density 0,814) with unpleasant odor.

The isoamyl alcohol (2-methy-lbutanol-4) is the main component of fusel oils. Into

composition of fusel oils enter also isobutyl alcohol and normal propyl alcohol. Except these alcohols fusel oils contain in insignificant amounts fatty acids, their ethers and

furfurol. The presence 2-methy-lbutanol-4 in fusel oils explains its sharp off odour and high toxicity. The isoamyl alcohol is a product of alcohol fermentation of carbohydrates

contained in beet, potatoes, fruit, grains wheat, rye, barley and other agricultural cultures. Main product of alcohol fermentation is the ethanol containing some amount of fusel

oils. However at alcohol fermentation the fusel oils are formed not from the expense of

carbohydrates, but from the expense of amino acids being hydrolysed of protein products. So, in conditions of alcohol fermentation the isoamyl alcohol (2-methyl-butanol-4) is

formed from leucin. Application. Action on organism. The isoamyl alcohol is applied in industry as a

solvent, and is used for preparation of essences having a pleasant fruit smell. Some of these

67

essences are applied in perfumery. The isoamyl alcohol is used for amyl acetate synthesis at

nitro-cellulose varnishes producing. This alcohol is used for amyle nitrite synthesis.

Isoamyl alcohol is in 10 – 12 times more toxic, than ethyl alcohol. It acts on the central nervous system, has narcotic properties. At reception of isoamyl alcohol there is a

headache, nausea, vomiting. The signs of a poisoning are shown already after reception inside 0,5 g of isoamyl alcohol. The death can come after reception inside 10 – 15 g of this

alcohol. There are cases of lethal poisonings with moonshine and others handicraft

production which contain isoamyl alcohol and other components of fusel oils. Metabolism. The part of a dose of the isoamyl alcohol in an organism transforms to

aldehyde of isovalerianic acetic acid, and then in isovalerianic acid. Some amount of the not changed isoamyl alcohol is eliminated with urine and with exhaled air.

Detection of Isoamyl Alcohol Examination of distillates on isoamyl alcohol presence makes for the decision a

poisoning with moonshine, or other substitutes of ethanol. To detection of isoamyl alcohol apply the Komarovski’s reaction, based on formation

of pigmented compounds with aromatic aldehydes (vanillin, benzaldehyde, p-dimethylaminibenzaldehyde, salicylic aldehyde etc). Except the Komarovski’s reaction for

detection of isoamyl alcohol apply it oxidising reaction to valerianic acid and the formed

product detection. All specified reactions give positive effect only at of water absence in reactants.

Therefore before performance of the listed reactions isoamyl alcohol must be extracted from distillate by ethyl ether. An ethereal extract divide on four parts, each of which places in a

porcelain cup, and then evaporates. In obtained residues detect isoamyl alcohol presence.

Reaction with salicylic aldehyde. The isoamyl alcohol with a salicylic aldehyde at

the presence of concentrated sulphate acid gives colour compound (Komarovski’s reaction). On one data, at this reaction the concentrated sulphate acid of isoamyl alcohol, therefore an

isoamylene is formed (СН3)2—СН—СН== СН2, which interacts with a salicylic aldehyde. According to another data, the concentrated sulphate acid oxidises isoamyl alcohol. The

isovalerianic aldehyde, formed at it, condenses with a salicylic aldehyde. Product of reaction has red colour.

This reaction gives alcohols containing more than three carbon atoms in molecule.

Do not give this reaction methyl and ethyl alcohols.

Reaction with p-dimethylaminibenzaldehyde. The isoamyl alcohol with p-

dimethylaminibenzaldehyde at the presence of concentrated sulphate acid gives colour

compound (Komarovski’s reaction). Product of reaction has dark red colour. At a dilution of a coloured solution by water

the colour passes in violet.

These reactions not give methyl and ethyl alcohols. It gives the higher alcohols.

Reaction of isoamyl acetate formation. The formed isoamyl acetate has smell of

pear:

2CH3COONa + H2SO4 → 2СН3СООН + Na2SO4

(СН3)2—СН—CH2—CH2OH + CH3COOH →

68

→ (СН3)2—СН—СН2—СН2—ООС —СН3 + Н2О

Reaction of isoamyl alcohol oxidation. The isoamyl alcohol under influence of

potassium permanganate at the presence of concentrated sulphate acid oxidises to

isovalerianic aldehyde (СН3)2—СН—СН2—СНО, and then to isovalerianic acid (СН3)2—

СН — СН2—СООН. Both products have specific smell.

ACETONE

Acetone СН3—CO—СН3 (dimethylketon, propanone) is a colourless mobile liquid

(boiling point 56,3 °C) with characteristic odour. It mixes with water, ethanol and diethyl

ether in all parities. The acetone well dissolves salts of many mineral acids and many organic compounds. Acetone is received by dry distillation of tree or coal, and by synthesis.

Application. Action on organism. The acetone is widely used in industry as a solvent for extraction of many substances, for crystalline modification of chemical compounds, for

dry cleaning, for chloroform synthesis etc. The acetone vapours are heavier than air.

Therefore in rooms, in which acetone evaporates, is danger of inhalation poisoning. Acetone shows narcotic action. It has cumulative properties. The acetone is slowly

eliminated from organism. It can pass to organism with inhaled air, and also through the alimentary canal and skin. After entering acetone in a blood the part passes it in a brain,

liver, kidneys, lung and heart. But the acetone contents in these organs are smaller, than in a blood.

Metabolism. The insignificant part of the acetone which has arrived in organism,

transfers to carbon(IV) oxide. Some amount of acetone eliminates from organism in an invariable state with exhaled air and through a skin, and some – with urine.

It is necessary to mean that the certain amount of acetone can be in a blood and in urine of the persons suffering by diabetes and any other diseases. Besides, the acetone is an

isopropanol metabolite.

Detection of Aacetone

Reaction of iodoform formation. At interaction of an acetone with alkaline iodine

solution iodoform formed:

I2 + 2NaOН → NaOI + NaI + Н2O

NaOI + СН3СОСН3 → I3С—CO—СН3 + 3NaOH

I3С—CO—СН3 + NaOH → СНI3 + CH3COONa

At acetone presence form the characteristic yellow crystals of iodoform with specific

smell. This reaction also gives ethanol.

Reaction with sodium nitroprussid. The acetone with sodium nitroprussid in

alkaline environment forms product with intensive-red colour. In acetic acid the colour

passes in red-violet:

СН3СОСН3 + Na2[Fe(CN)5NO] + 2NaOH → Na4[Fe(CN)5ON=СНСООН3] + 2H2O

69

With sodium nitroprussid form pigmented compounds the substances containing

enolising CO-groups:

The ketones, in which molecules are absent methyl or methylene groups connected to

CO– groups, do not give this reaction.

Reaction with furfurol. This reaction is based on acetone ability to be condensed

with furfurol and another aldehydes (vanillin, salicylic aldehyde etc.) with formation of colour compounds:

O OCH

CH CO CH CH

O CH

O

2 CH3COCH3

Formed product has red colour. This reaction is not specific for acetone. It is given by some aldehydes and ketones.

Reaction with o-nitrobenzaldehyde. Acetone with alkaline solution of o-nitrobenzaldehyde formes the indigo having dark blue

colour:

NO2

C OH

H3C C

O

CH3+

NO2

COHH

CH2 CO CH3

NO2

COHH

CH2 CO CH32

N

C

C

H

O

N

C

C

O

H

The small amounts of acetone with o-nitrobenzaldehyde react slowly. Thus at first there is a yellow colour passing in yellow-green, and then in green-blue. The indigo formed

at this reaction is well extracted with chloroform, which gets dark blue colour. At these conditions the acetone solutions in alcohol give blue-red colour.

Microdiffusion method. This method is based on reactions with salicylic aldehyde.

CH C OHCH2 CO

70

PHENOL

Phenol (C6H5–OH) is a thin lengthy needles or colourless crystalline mass with an original smell. On air it gradually change colour to red. Phenol dissolves in water in the

ratio 1:20, and easily dissolves in ethanol, diethyl ether, chloroform, aliphatic oils, and alkalis.

Application. Action on organism. Phenol is applied in medical practice as a

disinfectant. It is widely used in a chemical industry for synthesis many chemical compounds (stains, plastic masses, pharmaceuticals, agents of plants protection).

Phenol penetrates in a blood through mucous and skin, and then distributes in bodies and tissues. Phenol arrived in an organism through the alimentary canal, causes stomach-

aches, vomiting, diarrhoea, sometimes with impurity of a blood. The urine of phenol-poisoned persons has olive or olive-black colour. At per oral entering 10 – 15 g of phenol in

organism occurs death. The largest amount of phenol in corpses of the poisoned persons can

be founded in kidneys, then in a liver. Metabolism. In organism part of phenol is bonded with proteins and is oxidised with

hydroquinone and pyrocatechol formation. Unchanged phenol and its metabolites (hydroquinone and pyrocatechol) are eliminated with urine as conjugates with sulphate and

glucuronic acids. Isolation from biological material. For isolation of conjugated phenol from urine it

acidify by a weak solution of acetic acid, and then phenol distil with steam. Distillate, in

which can pass both phenol and a part of acetic acid, neutralise by sodium carbonate, and then phenol extract with organic solvent.

Phenol Detection

For detection of phenol the part of the distillate put to separatory funnel, add solution

of sodium carbonate to alkaline environment. Contents of a separatory funnel 2 – 3 times shake with 10-ml portions of diethyl ether. The ethereal extracts collect and dry at room

temperature. The dry rest dissolve in 2 – 3 ml of water.

Reaction with bromine water.

OH

+ Br2

OH

Br

Br

Br

+ 3HBr

In examined solution forms the yellow-white precipitate of tribromophenol. This reaction

gives cresols, aniline and another aromatic amines.

NH2 + HOCl + H2ONHCl

71

Reaction of indophenol formation. At oxidation of phenols and amines (including

the ammonia) mixture are formed indophenols having the appropriate colour. The

appearance of dirty - violet colour specifies the phenolum presence in assay:

+ NHCl OH O N OHHOCl

Indophenol form phenols having free para-position, cresols and other compounds containing phenolic group. For this reaction as oxidisers can be used sodium hypochlorite, chlorine or

bromine water, hydrogen peroxide etc.

Liberman’s reaction. This reaction also is based on indophenol formation. As

reagents apply sodium nitrite and sulphate acid. Appearanced dark blue colour pass in red, and then in green, specifies phenol presence in assay. Not gives this reaction nitrophenols

para-constituted phenols.

Reaction with iron(III) chloride. From addition of iron(III) chloride solution to

phenol solution appears violet or blue-violet colour. With iron(III) chloride gives colour products cresols, oxypyridines, oxyquinoline and

other substances containing phenolic groups. Composition and colour of formed products depends on tested substances nature, solvents nature and pH. For example, o-cresol and p-

cresol with iron(III) chloride gives dark blue colour, and m-cresol – red-violet.

Reaction with Millon’s reagent. The Millon’s reagent is a mixture of mono and

divalent mercury nitrates in nitrate acid. Heating accelerates this reaction. Probably, at this reaction form 1,2-quinonmonoxime, which bonds colour inner-complex compound with

mercury ions:

OH

HNO3

N=O

OH O

N OH

N O

OHg

O N

O

O

N OH

2

Hg+2

A small quantity of phenol changes a test solution colour to yellow. The appearance of red colour specifies phenol presence in assay. This reaction is gives aniline. This reaction

frequently is used for detection of p-derivatives of phenol, which cannot be detected with Liberman’s reactions.

72

Reaction with benzaldehyde. Phenol forms the colourless product of condensation

with benzaldehyde in acidic environment. Concentrated sulphate acid at this reaction acts as

dehydrating and oxidising agent. After oxidation the product of reaction change colour in red.

Method of microdiffusion. This method based on reactions with Folin’s reagent.

Phenol can be detected in urine, blood and homogenates of tissues. Appearance blue colour

of the reagent.

CHLOROFORM

Chloroform СНСl3 is a colourless transparent fugitive liquid with a characteristic

smell. Ii mixes with diethyl ether, ethanol and other organic solvents, is weakly dissolves in

water. Under influence of light, air, moisture and temperature chloroform gradually decomposes. Thus can be formed phosgene, formic and hydrochloric acids.

Application. Action on organism. Chloroform is widely used in a chemical industry and in chemical laboratories as a solvent. Earlier it was applied in medicine for narcosis.

The chloroform vapour easily penetrates in organism with inhaled air. Chloroform acts on the central nervous system, causing a narcosis. It collects in tissues, rich fats. A large

amount of chloroform occurs a dystrophy of internal organs, especially a liver. At a

poisoning with chloroform the death comes from a respiration stopping. Metabolism. Chloroform, which has arrived in organism, quickly disappears from a

blood. Through 15 – 20 mines with exhaled air in the not changed kind eliminate 30 – 50 % of chloroform. During the first hour through lungs is eliminated 90 % of chloroform. The

part of chloroform is exposed to a biotransformation. Thus as metabolites are formed carbon(IV) dioxide and hydrogen chloride. The basic objects of the chemical-toxicological

analysis on chloroform presence are exhaled air, fat tissues, and liver.

Chloroform Detection

Reaction of chlorine elimination. At chloroform heating with alcohol alkali solution

there is eliminated chlorine, which can be detected by reaction with silver nitrate:

СНСl3 + 4NaOH → 3NaCl + HCOONa + 2Н2О;

NaCl + AgNO3 → AgCl + NaNO3.

This reaction is not specific. It gives chloral hydrate, perchloromethane, ethylene dichloride,

carbon tetrachloride etc.

Fujiwara’s reaction. Fujiwara’s reaction is based on interaction of halogen-

containing substances with a pyridine at the alkali presence. At interaction of chloroform with a pyridine and alkali is formed a polymethyn stain. At first is formed pyridinium salt:

73

N

CHCl3 +

N

CHCl2

Cl-

Under influence of alkali the pyridinium salt is formed a coloured glutaconic aldehyde:

C-CH=CH-CH2-C

H

O H

O

H

This reaction is not specific. All chlorine-containing aliphatic compounds gives the similar products.

Reaction with resorcinol. At chloroform with resorcinol heating at alkali presence

appears pink or crimson colour.

This reaction except chloroform gives carbon tetrachloride, chloral hydrate etc. Does not give this reaction ethylene dichloride.

Reaction of isonitrile formation. Chloroform with primary amines and alkali forms

isonitrile having unpleasant smell:

СНСl3 + RNH2 + 3КОН → RN=C + 3KCI + 3Н2О

Reaction with Fehling’s reagent. At chloroform interaction with alkali is formed a

formic acid salt:

СНСl3 + 4NaOH → HCOONa + 3NaCl + 2Н2О

The Fehling’s reagent contains complex compound К2Na2[Cu(С4Н3О6)2], which is formed

at interaction of copper (II) ions with Signet salt, oxidises formic acid at heating. As a result of reaction the red precipitate of copper (I) oxide drops:

K2Na2[Сu(С4Н3O6)2] + 2Н2O → 2KNaC4H4O6 + Сu(OH)2

2Сu(OH)2 + HCOONa → Сu2О↓ + СО2 + 2Н2О + NaOH

Except chloroform this reaction gives chloral hydrate, formaldehyde, acetaldehyde. Do not give this reaction 1,2-dichlorethane, carbon tetrachloride etc.

CHLORAL HYDRATE

Cl3C-CH

OH2O

74

Chloral hydrate is a colourless crystals or crystalline powder with characteristic

pungent odour and slightly bitter. It dissolves in water, ethanol, diethyl ether and

chloroform. Chloral hydrate is hygroscopic and escapes on air slowly. Application. Action on organism. Chloral hydrate is applied in medicine as

tranquillising, hypnotic and analgesic agent. In the large doses chloral hydrate can cause a poisoning. On toxic action chloral hydrate is like as chloroform. It is applied at

hyperphrenias and as an anticonvulsant at a tetanus, eclampsia and at other diseases.

Metabolism. Chloral hydrate is quickly absorbed in a blood from the alimentary canal. Metabolites of chloral hydrate are trichloroethanol and trichloroacetic acid. Consider

that the toxic action of chloral hydrate on organism is explained by trichloroethanol formation. The trichloroacetic acid in organism can be formed by two ways: directly from

chloral hydrate and from trichloroethanol. Trichloroethanol is eliminated from organism as a glucuronide. After death some amount of not changed chloral hydrate can be found in a

liver and stomach.

Chloral Hydrate Detection

Chloral hydrate gives all reactions, which are applied for chloroform detection. It is

explained that the used in the chemical-toxicological analysis reactions, are made at the alkali presence. In alkali environment chloral hydrate decomposes with chloroform

formation:

ССl3СНО + NaOH → СНСl3 + HCOONa

For difference of chloral hydrate from chloroform the reaction with Nessler’s reagent can be

used. This reaction gives chloral hydrate containing aldehyde group. Chloroform does not

give this reaction.

Reaction with Nessler’s reagent. At chloral hydrate interaction with Nessler’s

reagent free mercury precipitate forms:

ССl3CHO + К2[HgI4] + 3КОН → Hg↓ + ССl3СООК + 4KI + 2Н2О

At chloral hydrate presence forms a red precipitate, which then becomes dirty green. This reaction does not chloroform, carbon tetrachloride, dichlorethane, and ethylene chloride.

With Nessler’s reagent react aldehydes and some other reducing substances.

CARBON TETRACHLORIDE

Carbon tetrachloride ССl4 is a transparent liquid with an original smell (boiling point

75 – 77 °С). It mixes in any ratio with acetone, benzene, petrol, carbon disulphide and other

organic solvents. In water at 20 °С is dissolved about 0,01 % of carbon tetrachloride. The

carbon tetrachloride is not fire-hazardous, its vapours in some times more heavy air. Application. Action on organism. The carbon tetrachloride is widely applied in an

industry as a solvent of fats, resins, and rubber. The carbon tetrachloride is included into composition of liquids for clothes cleaning and for fire extinguishers filling. At high

temperature as decomposition result of carbon tetrachloride can be formed phosgene and

75

other toxicants causing a poisoning. The carbon tetrachloride is applied in a veterinary

medicine as a helminticide.

The carbon tetrachloride acts in an organism at its vapour inhalation, and also can act through a uninjured skin and alimentary canal. It amount a fat tissues is in some times more,

than in a blood. The contents of carbon tetrachloride in a liver and in an osteal brain are much, than in lung. In blood erythrocytes contains of carbon tetrachloride approximately in

2,5 times more, than in plasma. It has narcotic action, amazes the central nervous system.

The lethal dose of carbon tetrachloride is 30 – 60 ml. Metabolism. The carbon tetrachloride quickly eliminates from organism. After 48

hours in organism cannot be found it in exhaled air. Its metabolites are chloroform and carbon(IV) oxide.

Carbon Tetrachloride Detection In the chemical-toxicological analysis for carbon tetrachloride detection in distillates

apply the same reactions as for others chlorinederivates of hydrocarbons: – reaction of chlorine elimination,

– Fudjiwara’s reaction, – reaction of isonitrile formation,

– reaction with resorcinol.

1,2-DICHLORETHANE

There are two isomers of ethylene dichloride (С2Н4Сl2) 1,1-dichlorethane and 1,2-dichlorethane.

The 1,1-dichlorethane (ethylidene chloride) СН3СНСl2 is a colourless liquid (density

1,189 at 10°С), boiling point – 58°С. 1,2-ethylene dichloride (ethylene chloride) С1—

СН2—СН2—С1 is a liquid (density 1,252 at 20 °С), boiling point – 83,7 °С. In industry 1,2-

dichlorethane is more widely used, than 1,1-dichlorethane.

The 1,2- dichlorethane is weakly dissolved in water, is well dissolved in the majority of organic solvents. It is proof to acids and alkalis action.

Application. Action on organism. The 1,2-dichlorethane is more toxic, than 1,1-dichlorethane. In industry the 1,2-dichlorethane is used as a solvent of fats, wax, resins,

paraffin and other substances. It is applied for extraction of many organic substances from aqueous solutions. The 1,2-dichlorethane vapours penetrate into organism through

respiratory ways. This substance in liquid state can penetrate into organism through a

uninjured skin. There are known cases of poisonings with 1,2- dichlorethane, wrongly accepted inside instead of alcohol drinks. The picture of a poisoning with 1,2-dichlorethane

is similar to a picture of a poisoning with carbon tetrachloride. The 1,2-dichlorethane breaks function the central nervous system, liver, kidneys and cardiac muscle. 15 – 50 ml of 1,2-

dichlorethane in most cases causes death.

1, 2-Dichlorethane Detection In the chemical-toxicological analysis for 1,2-dichlorethane detection apply the same

reactions as for others chlorine-derivates of hydrocarbons. Except for common reactions on

76

chlorine-derivates of hydrocarbons (reaction of chlorine elimination and Fujiwara’s test) are

applied some specific reactions.

Reaction of ethylene glycol formation. At heating of 1,2-dichlorethane with a

solution of sodium carbonate in a soldered ampoule is formed the ethylene glycol:

Cl–CH2–CH2–Cl + Na2CO3 + H2O → HO–CH2–CH2–OH + 2NaCl + CO2

At interaction of ethylene glycol with potassium periodate КIO4 is formed the

formaldehyde:

Cl–CH2–CH2–Cl + КIO4 → 2НСНО + КIO3 + Н2О

The formaldehyde, which is formed at the specified reaction, can be detected with

chromotropic acid. Do not give this reaction chloroform, chloral hydrate, carbon tetrachloride, 1,1-dichlorethane.

Reaction of copper acetylide formation. At heating of 1,2-dichlorethane in a

soldered ampoule with a spirituous solution of sodium hydroxide of sodium is formed

acetylene. It interacts with copper(I) salts and forms the copper acetylide, having pink or cherri-red colouring:

Cl–CH2–CH2–Cl + NaOH → CH≡CH + 2NaCl + 2H2O

CH≡CH + 2CuNO3 + 2NH4OH → CuC≡CCu↓ + 2NH4NO3 + 2H2O

Do not give this reaction chloroform, chloral hydrate, carbon tetrachloride, 1,1-

dichlorethane.

Reaction with quinoline. At heating 1,2-dichlorethane with quinoline is formed

cyanine stain:

N C N+

ClCH2CH2 H CH2CH2Cl

Cl-

At sluggish heating appears a brown or brown-red colour. At fast heating the liquid gets blue-red colour. Except for 1,2-dichlorethane with quinoline give colour products ethyl

chloride, ethyl bromide, and ethyl iodide. Do not give this product chloroform, chloral

hydrate, carbon tetrachloride, and 1,1-dichlorethane. For difference of 1,2-dichlorethane from chloroform, chloral hydrate and carbon

tetrachloride can be applied the reactions with resorcinol, with Fehling’s reagent, and isonitrile formation reaction. These reactions do not give 1,2-dichlorethane.

77

Reaction Allowing to Distinguish Chlorine-Derivates From Each Other

Detected substances

Reaction Chloroform Chloral

hydrate

Carbon

tetrachloride

1,2-Dichlor-

ethane

Chlorine elimination + + + +

Fujiwara’s test + + + +

Resorcinol + + + –

Fehling’s reagent + + + –

Nessler’s reagent – + – –

Ethylen glycol formation – – – +

Copper acetylide formation – – – +

Quinoline – – – +

ACETIC ACID

The anhydrous (glacial) acetic acid СН3СООН is a colourless hygroscopic liquid or

colourless crystals with a sharp smell. It mixes with water, ethanol and ethyl ether in all ratios. Small amounts of acetic acid can contain in a human body.

Application. Action on organism. The acetic acid is applied for synthesis of stains, cellulose acetate, acetone etc. As vinegar and acetic essence it is applied in food industry

and in cooking. 10 - 20 g of an acetic essence or 200 – 300 ml of vinegar is a lethal dose.

The acetic acid acts on a blood and kidneys. At contact with a skin the ice acetic acid causes burns and vesicultion. After reception of a concentrated acetic acid or acetic essence is

amazed the alimentary canal inside, there is a hematemesis, diarrhoea, the hemocatheretic anaemia, hemoglobinuria, anuria and uraemia develops. At inhalation of acetic acid vapour

there is a boring of a respiratory ways mucous, can develop a bronchopneumonia, catarrhal bronchitis, inflammation of a pharynx etc.

Metabolism. A metabolite of acetic acid is acetaldehyde, which partially transforms

into ethanol and partially decomposes to carbon(IV) oxide and water. The acetic acid distils from biological objects after its acidifying by 10 % solution of

a sulphate or phosphoric acid. In view of its fugitiveness distillate collect in a flask containing 0,1 N solution of sodium hydroxide.

Acetic Acid Detection

Reaction with iron(III) chloride. Appearance red colour caused by basic iron

acetate formation:

8СН3CООNa + 3FeCl3 + 2Н2О →

→ [Fe3(OH)2(CH3COO)6]Cl + 2СН3СООН + 8NaCl

At heating of colour solution there is a hydrolysis, as a result of which the brown precipitate

forms.

Reaction with sodium nitrite and lanthanum iodide. At interaction of acetate-ions

with lanthanum nitrate La(NO3) at iodine and ammonia solution presence drops dark blue

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precipitate. It is caused an adsorption of iodine on basic lanthanum acetate. Similar colour

propionates give. This reaction run prevents sulphates, phosphates and cations forming

precipitates with ammonia.

Reaction of indigo formation. At heating acetic acid or acetates with calcium is

formed acetone:

2СН3СООН + СаО → (СН3СОО)2Са + Н2О

(СН3СОО)2Са → СН3СОСН3 + СаО + СО2

The formed acetone at the presence of alkalis interacts with o-nitrobenzaldehyde. A finished product of reaction is the indigo.

Reaction of ethyl acetate formation. At heating acetates with ethanol at the

presence of sulphate acid is formed ethyl acetate with specific smell:

СН3СООН + СН3СН2ОН → СН3СООС2Н5 + Н2О.

79

SUBSTANCES ISOLATED BY MINERALISATION OF

BIOLOGICAL MATERIAL

In chemical-toxicological analysis the method of a mineralisation is applied for

research of biological material (bodies of corpses, biological liquids, plants, foodstuff etc.) on presence of so-called “metal poisons”. These poisons are salts, oxides and other

compounds of barium, bismuth, cadmium, manganese, copper, mercury, lead, silver, thallium, chrome, zinc and some other metals. In toxicology compounds of such metalloids

as arsenic and antimony also concern to group of “metal poisons”.

In most cases these poisonous substances penetrate in organism through the alimentary canal, absorb in blood and cause poisonings.

Any from the listed above chemical elements in small quantities contain in tissues of organism as their normal component. There are named microelements.

Some chemical elements, which compounds are toxic, in small quantities play the important role in physiological processes in organism. For example, the cobalt is a

component of vitamin В12 (cyanocobalamin). This trace substance is coenzyme of some

enzymes (carboxypeptidase, carboxyanhydrase). The copper is included into composition of many enzymes (polyphenoloxidase, cytochrome oxydase, phenolase etc.). It is a component

of protein ciruloplasmine, participates in synthesis of haemoglobin. The manganese is necessary for activation of some enzymes (arginase, prolidase). Zinc also is included into

composition of enzyme lactatdehydrogenase. The quantitative contents of some microelements in tissues of organism are given in table.

Microelements Content in Some Body Tissues

Content, mg-% Microelement

Liver Kidney Spleen Lung Heart Muscle Brain

Cadmium 0.64–

6.68

1.32–

6.48

– – – – –

Cobalt 0.025 – – – – – –

Manganese 0.17–

0.20

0.06 0.022–

0.032

0.022 0.021–

0.032

0.05 0.028–

0.03

Copper 0.71 0.116–0.36

0.12–0.24

0.11 0.19 0.125 0.22–0.46

Arsenic 0.011 – 0.008 0.009 0.01 – –

Tin 0.06 0.02 0.022 0.045 0.022 0.011 –

Mercury 0.002 0.002 – – – 0.0002 0.0002

Lead 0.13 0.027 0.03 0.028 0.038 0.01 0.013

Silver 0.005 – – – – – –

Chromium 0.001–0.013

0.027–0.028

0.0005–0.01

0.0007 0.01 0.0002 0.002

Zinc 5.4–14.5 5.5 1.1 0.65 1.4 3.0–5.15 0.8–1.5

Notwithstanding what the some metals in small quantities contain in organism as its normal component, at raising the contents them in a blood and tissues they cause

poisonings.

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Interaction of “Metal Poisons” with Biological Material

The toxicity of “metal poisons” is explained by bond them with the appropriate function groups of peptides and other vital compounds in organism. As a result of metals

cations conjugation with proteins and other substances break normal functions of the appropriate cells and tissues in organism and there comes a poisoning.

Bond of metals ions with amino acids. The amino acids are structural elements of proteins. All amino acids (except for a proline), included in composition of protein, contain

free carboxylic group and free aminogroup at α-carbon atom. The proline is α-imino-acid. The terminal amino- and carboxy- groups of amino acids can interact with ions of

metals. Free lateral function groups in amino acids can also bond with metal ions: alcohol

groups in molecules of a serine and treonine, phenylic group in thyrosine, sulphhydryl group in cysteine, disulphide group in cystine, second carboxylic groups in aspartic and

glutamic acids, second nitrogen-containing groups in an arginine and histidine. The methionine contains atoms of sulphur in a carbon chain.

Depending on presence of the certain groups of atoms in molecules of amino acids, nature and chemical properties of metals at interaction between them the connection of

various hardness can be formed.

The amino acids in aqueous solutions and in a crystalline condition are as bipolar ions:

R C COOH

H

NH2

R COO-

NH3+

C

H

The amino acids are amphoteric compounds. The dissociation them on ions depends

on pH. In acid environment amino acid dissociate as bases, in alkaline – as acids. The

character of its interaction depends on cation nature. At interaction of the negatively charged atoms of oxygen in carboxylic groups with cations of alkali metals there are ionic

bonds (the salts), and with cations of heavy metals – covalent bonds are formed. The cations of complex-formed metals with amino acids form inner-complex

compounds (chelates). The inner-complex compounds also can be formed by cations of metals contact with lateral reactive function groups of amino acids (–SH, –NH2, –СООН).

For example: of phenyl alanine and Cysteinum;

+ Me+2

CH2 C

NH2

COOH

H

+ H+CH2 C

N

COO

H

Me+2

/2

Phenyl alanine Chelate compound of phenyl alanine with metal ion

Bond of metals with proteins. In formation bonds with metals take part terminal

amino- and carboxylic group of proteins molecules. However numbers of end groups in molecules of protein are insignificant. Each molecule of protein presenting a lengthy

polypeptide chain contains only two considerably removed from each other terminal (–NH2 and –СООН) groups and large number of lateral function groups. Therefore bonds

81

formation between ions of metals and protein occurs basically at the expense of lateral

function groups (–SH, –NH2, –OH, –СООН). Believe that the metals interact with proteins

mainly through histidine oddments containing imidazole ring, and cysteine having lateral sulphhydryl group.

Bond of metal with another compounds. Metals can bind in organism and with

other compounds playing the important role in all living cells. The pteridines (including

folic acid), purines, riboflavine, nucleic acids and many other compounds form such bonds. In most cases ions of metals with the listed above compounds form strong covalent bonds.

Methods of Biological Material Mineralisarion

The bonds of metals with the majority of the specified substances are strong

(covalent). Therefore for investigation of biological material on “metal poisons” presence it is necessary to destroy organic matters, to which the metals are bind, and to transfer them in

ionic state. Methods used for this purpose are divided on two groups: methods of dry ashing and methods of a wet ashing, or wet mineralisation.

The choice of organic matter mineralisation method depends on properties of analysed elements, amount of biological material assay etc.

Dry Mineralisation Methods

Dry mineralisation methods are based on ignition of organic matters to high temperature.

The dry ashing technique is based on ignition of investigated biological material to high temperature at air access. A dry ashing make in porcelain, quartz or platinum crucibles.

For analysis take small samples (1 – 10 g) researched objects (foodstuff) and heat up them

in a crucible up to 300 – 400 °С. This mineralisation method has disadvantages:

– main of which are volatile of some metals or their compounds during heating, – interaction of some metals with a material of crucibles,

– it is difficult to supervise temperature of a researched material immediately in a crucible.

Compounds of mercury and thallium can escape partially or completely during dry

ashing of biological material even at low temperature. If temperature is higher than 400 °С

chlorides of cadmium, lead, silver, zinc, manganese, and arsenic are fugitive. At high temperature zinc, silver, lead and some other metals can interact with walls quartz and

porcelain crucibles, and the cobalt is capable to be alloyed with walls of platinum crucibles

or to interact with a material of porcelain crucibles. The technique of organic matters alloy with nitrates in chemical-toxicological

analysis is applied more often, than method of a dry ashing. The biological material or other organic matters heat with melted nitrates of alkali metals. With temperature raising the

oxidising properties of nitrates amplify. For explosion prevention for melting apply mixtures of nitrates with carbonates of alkali metals. After burning of all mixture a crucible

cool and its contents dissolves in boiling water. Obtained solution applies for “metal

poisons” detection. This mineralisation method is unsuitable for examination of biological objects on mercury presence, because it escapes at heating.

82

Oxidisers Applied for Organic Matters Wet Mineralisation

For wet ashing of organic matters apply acids-oxidisers (nitrate, sulphate and perchlorate), potassium chlorate and perhydrole. These used oxidisers destroy bonds

between metals and protein, peptides, amino acids and some other compounds.

Nitrate acid. Destruction of biological material by heating with concentrated nitrate

acid requires the large expense of time. Concentrated nitrate acid weakly oxidises fats. At biological material heating with concentrated nitrate acid loses some amounts of cobalt,

zinc, and manganese.

Sulphate acid. For destruction of organic matters the concentrated sulphate acid

apply as dehydrating and oxidising agent. The concentrated sulphate acid applies for

destruction of various organic compounds at high temperature. In oxidation process

concentrated sulphate acid can be reduced to sulphur(IV) oxide, free sulphur or hydrogen sulphide.

The concentrated sulphate acid as an oxidiser of organic matters has some disadvantages:

– oxidation process is protracted, – can be formed the carbonised oddments, not decomposed by this acid,

– during destruction with heating can escape mercury compounds.

Mixture of sulphate and nitrate acids. In 1821 Matthieu Orfila for destruction of

organic matters has offered a mixture of concentrated sulphate and nitrate acids. This method has modification – can is used mixture of concentrated sulphate acid and

ammonium nitrate. This technique many years was widely applied in practice of forensic-chemical laboratories USSR.

After destruction of organic matters by mixture of sulphate and nitrate acids or

mixture of concentrated sulphate acid and ammonium nitrate in mineralisate contains the plenty of nitrogen oxides which prevent detection and quantitative definition some of “metal

poisons”. Ii must be used denitration – disposal of nitrate, nitrate acids and nitrogen oxides. For this purpose were applied hydrogen peroxide, urea, sulphaminic acid, formaldehyde etc.

However this method is unsuitable for the chemical-toxicological analysis of biological material on mercury presence, which appreciable amounts escape during a

mineralisation.

Mixture of potassium chlorate and hydrochloric acid. This method is based on

destruction of organic matters by potassium chlorate (KClO3) and hydrochloric acid. At interaction of potassium chlorate with a hydrochloric acid the chlorine having oxidising

properties forms. The chlorine blasts a biological material. Destruction of organic matters occurs slowly and mineralisation of biological

material does not reach up to end. Recently this method has lost the importance and in

chemical-toxicological analysis is not applied.

Perchlorate acid. In the chemical-toxicological analysis the perchlorate acid

(НСlO4) and its mixture with other acids is applied to destruction of organic matters. The

method of destruction of organic matters with perchlorate acid НСlО4 is characterised by high speed of mineralisation, and also ability of this acid to blast substances, which proof or

83

slowly destructing by other oxidisers. Solutions of a perchlorate acid at heating are

decomposed to chlorine, oxygen and water:

4НСlО4 → 2Сl2 + 7О2 + 2Н2О.

Concentrated hydrogen peroxide. For destruction of organic matters in chemical-toxicological analysis sometimes apply concentrated hydrogen peroxide and sulphate acid.

It is explained by interaction of hydrogen peroxide with sulphate acid and formation persulphate acid H2SO5, having large oxidising ability.

Sometimes for mineralisation of organic matters apply ternary mixture (concentrated hydrogen peroxide, concentrated sulphate and nitrate acid). In these cases researched object

in the beginning process by mixture of concentrated sulphate and nitrate acids. After partial

oxidation of organic matters by this mixture add concentrated hydrogen peroxide to completely destruction organic matters.

At use of this method of organic matters destruction the losses of appreciable amounts of arsenic, mercury and other metals are possible. These losses are enlarged at the

contents in a researched biological material of chlorides exceed.

Destruction of Biological Material by Nitrate and Sulphate Acids

In the beginning of mineralisation the concentrated sulphate acid acts as dehydrating agent. Its dehydrating power amplifies with temperature rising. The concentrated sulphate

acid breaks frame of cells and tissues of a biological material. At rising temperature (is

higher 110 °С) and concentration (up to 60 - 70 %) a sulphate acid shows oxidising

properties and is decomposed with excretion of sulphur (IV) oxide.

The nitrate acid is a weak oxidiser. But with formation of nitrogen oxides and nitrite acid and also with raising of temperature the nitrate acid shows itself as a strong oxidiser.

During heating a biological material with mixture of nitrate and sulphate acids occurs not only destruction of organic matters by these acids, but also reactions of sulphating and

nitration of organic compounds. The phenyl groups of amino acids are exposed to a

nitration and sulphating basically. The nitration and sulphating of organic matters is undesirable because mixture of these acids rather difficulty decomposes nitro- and sulpha-

compounds. During destruction of a biological material with the mixture of nitrate and sulphate

acids forms some amount of nitrozylsulphate acid HOSO2ONO, which prevents detection of some metals cations of in mineralisates.

In the first stage of a mineralisation there is a destruction of a biological material

with nitrate and sulphate acids, which proceed 30 – 40 min. In the second stage of a mineralisation there is oxidation of organic substances in a liquid phase (destructate). This

stage of destruction is longer, than the first stage of destruction. However detection and quantitative determination of cations of some metals prevent

nitrate and nitrite acids, and also nitrogen oxides, which contain in mineralisates. In this connection obtained mineralisates are the subjects of denitration.

Denitration is the process of mineralisate remission from nitrate, nitrite,

nitrozylsulphate acids and nitrogen oxides. Denitration can be proceeded by any ways: – hydrolysis method. This method is based on mineralisate dilution with water and

subsequent boiling of received liquids. At diluted mineralisates heating escape nitrate,

84

nitrite acid and nitrogen oxides, and hydrolyses nitrozylsulphate acid. This method requires

15 – 17 hours of operating time;

– urea using. With urea denitration process comes to end for 3 – 5 min (at 135 – 145 °С); – sodium suphite using. With sodium sulphite denitration finishes at 10 – 15 min (if

temperature is higher than 100 °С);

– with formaldehyde. At interaction of formaldehyde with nitrate acid nitrogen eliminates:

4HNO3 + 5НСНО → 2N2 + 5СО2 + 7Н2О

As a result of interaction of nitrite acid with formaldehyde escape nitrogen and nitrogen (II)

oxide:

4HNO2 + 2НСНО → N2 + 2NO + 2СО2 + 4Н2О

The nitrozylsulphate acid at heating with water is decomposed.

Denitration is considered finished then, reaction of mineralisate with solution of diphenylamine will be negative. This test is based on diphenylamine oxidation by nitrate

acid and its decomposition products. Oxidation products have dark blue colour:

85

“METAL POISONS” DETERMINATION

BARIUM

Application and toxicity. Barium hydroxide (barium water) is applied in glass

manufacturing and in production of ceramic. Barium chloride is used in tanning industry and in agriculture as herbicide. Barium carbonate is applied as rodenticide, and also for

ceramic and glass productions. Barium nitrate and chlorate are applied in pyrotechnic. Barium acetate is used for textile manufacturing. Barium sulphate is used in medicine for

roentgenoscopy of stomach. There are marked cases of poisonings by barium carbonate contained as an impurity in barium sulphate. At presence of this impurity in, under influence

of a hydrochloric acid of gastric juice barium carbonate dissolves with barium chloride

formation and causes a poisoning. The soluble compounds of barium irritate mucous of alimentary canal. At poisonings

with barium compounds can come liver degeneration. Death from barium is a result of cardiovascular failure. The barium compounds eliminate from organism mainly through

intestines. The traces of these compounds are eliminated through kidneys and are partially postponed in bones.

Mineralisates examination on barium presence

In the chemical-toxicological analysis for detection of barium compounds are used

BaSO4 precipitates. A precipitate of barium sulphate forms after destruction of biological material by mixture of sulphate and nitrate acids. Except barium sulphate the precipitate can

contain lead sulphate. Research of barium sulphate precipitate make after it separation from

lead sulphate. For separation this precipitate is processed by a hot solution of ammonium acetate acidified by acetic acid.

Crystalline modification of a barium sulphate precipitate. A part of a researched

precipitate put on subject glass and slightly dries. Then to a deposit add 2 – 3 drops of concentrated sulphate acid and heat up to occurrence of SO3 white vapour. Through 10 – 20

min after cooling on subject glass there are colourless crystals shaped of rectangular with

extended angles or the form of lenses, assembled as crosses.

Reaction of barium sulphate reduction. Part of barium sulphate precipitate,

moisturised with chloride acid, is reduced on a flame of the gas or alcohol burner. As a

result of it the barium sulphide BaS is formed and the flame of the burner is coloured in green. After that to reduced precipitate in chloride acid adds crystal of potassium iodate

КIO3. Thus the colourless prismatic crystals of barium iodate are formed:

BaS + 2НС1 + 2 КIO3 → Ва(IO3)2 + H2S + 2KC1.

Reaction with potassium chromate:

2ВаCl2 + К2Сr2O7 + Н2O → 2ВаСrO4 + 2КCl + 2НCl

86

In connection with solubility of barium chromate precipitate in inorganic acids to solution

add sodium acetate:

CH3COONa + НCl → CH3COOH + NaCl

The acetic acid, formed at this reaction, does not dissolve barium chromate precipitate. The strontium-ions do not prevent this reaction, as the strontium chromate precipitate is

dissolved in mineral and acetic acids.

Reaction with sodium rhodizonate. Sodium rhodizonate with barium ions forms a

red-brown precipitate:

O

O

O

O

ONa

ONa

+ Ba+2

O

O

O

O

O

O

Ba

Barium rhodizonate precipitate under influence of chloride acid passes in insoluble acid salt

having bright red colour.

LEAD

Application and toxicity. Lead oxide, carbonate and chromate are applied for preparation of paints. Lead oxide enters into composition of a medical plaster. Lead stearate,

oleate and other organic salts are used as stabilisers of plastic and additives to paints, and

also enter into composition of some lipsticks and liquids for a hair. In the industrial plants using metal lead, and also in shafts, in which receive leaden

ores, there can be poisonings with lead vapour and by an inhaled dust. However basic source of poisonings with lead compounds is the entering them in the alimentary canal.

The lead ions in organism are bridged to sulphhydryl and other function groups of enzymes and some other vital peptides. The lead compounds brake synthesis of a

porphyrine, cause infringement of central and peripheral nervous systems functions. About

90 % of lead ions bound with erythrocytes. The lead compounds eliminate from organism mainly with faeces. The smaller

amounts of these compounds eliminate with bile, and traces – with urine. The lead compounds are partially postponed in an osteal tissue as trisubstituted sodium phosphate.

Mineralisates Examination on Lead Presence

After destruction of a biological material by mixture of sulphate and nitrate acids

lead drops out in mineralisate as a white precipitate of lead sulphate. These precipitate dissolve in acidified solution of ammonium acetate:

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2PbSO4 + 2CH3COONH4 → [Pb(CH3COO)2 ⋅ PbSO4] + (NH4)2SO4

Extraction of lead ions from mineralisate. To acetic solution of lead acetate add a solution of dithizon in chloroform and shake. Thus is formed monosubstituted lead

dithizonate Pb(HDz)2. Chloroform solution has orange-red colour:

Reaction with potassium iodide. Forms yellow precipitate РbI2:

Pb(CH3COO)2 + 2KI → PbI2↓ + 2CH3COOK

It dissolves at heating and again occurs as yellow plates at solution cooling. At reagent excess РbI2 dissolves with K2[PbI4] formation:

PbI2 + 2KI → K2[PbI4]

Reaction with potassium chromate. Forms orange-yellow precipitate of lead chromate:

Pb(CH3COO)2 + K2CrO4 → PbCrO4↓ + 2CH3COOK

Reaction with hydrogen sulphide. Forms black precipitate of lead sulphide:

Pb(CH3COO)2 + H2S → PbS↓ + 2CH3COOH

Reaction with caesium chloride and potassium iodide. Reaction executes on

subject glass. Forms the yellow-green needles assembled in spheroids:

Pb(CH3COO)2 + CsCl + 3KI → Cs[PbI3] + КСl + 2CH3COOK

Reaction with copper acetate and potassium nitrite. Reaction executes on subject

glass. Forms black or brown cubic crystals:

Pb(CH3COO)2 + Сu(СН3СОО)2 + 6KNO2 → К2Сu[Pb(NO2)6] + 4СН3СООК

BISMUTH

Application and toxicity. The poisoning with bismuth can come after reception of its

compounds inside and at inhalation of a dust containing this metal. The bismuth compounds are applied to reception of alloys having of low melting temperature, luminous

compositions, crystal glass etc. Some bismuth compounds are applied in medicine (basic bismuth nitrate, bismuth salicylate etc.) for ointments and cosmetic agents preparation. The

bismuth enters into composition of some drugs for syphilis treatment.

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The bismuth ions, penetrated in blood, long time reside in organism (in liver,

kidneys, lien, lungs and brain).

The bismuth eliminates from organism through kidneys, intestine, and sweat glands. As a result of bismuth accumulation in kidneys their defeat is possible. At excretion of

bismuth from an organism with sweat glands can be an itch of skin and occurrence of dermatomes.

Mineralisates Examination on Bismuth Presence

Extraction of bismuth ions from mineralisate. To mineralisate add sodium

dirthyldithiocarbamate solution. Thus the bismuth ions form inner-complex compound:

For masking another ions add Complexone III (Trilone B) solution. The formed complex of bismuth dirthyldithiocarbamate extracts by chloroform, and then decomposes by nitrate

acid.

Reaction with a thiourea. At interaction of bismuth ions with thiourea are formed of

various thiourea complexes having citric-yellow colour:

Reaction with oxyn (8-oxyquinoline) is based on transition of bismuth ions in

acidic complex [BiI4]–, which interact with oxyn in acidic environment and forms orange-

red precipitate. This complex is ionic associate (iodobithmutate of oxyn):

Bi(NO3)3 + 4КI → K[BiI4] + 4КNO3;

Reaction with brucine and potassium bromide. Reaction executes on subject

glass. Forms the yellow-green crystals assembled in spheroids.

Reaction with caesium chloride and potassium iodide. Reaction executes on

subject glass. Forms the orange-red crystals Cs[BiI4], shaped of hexagons or six-radial

sprockets.

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CADMIUM

Application and toxicity. The cadmium is applied in industry to reception of low-melting alloys, for replacement bismuth in a typographical font or for replacement of tin at

enamel lining of utensils. The cadmium sulphide is one of components of luminous paints, uses for porcelain manufacturing. Cadmium sulphate also is applied for paints

manufacturing.

Metal cadmium and it oxide, using in engineering for reception of alloys, at high temperature can escape and get in an organism with inhaled air.

The adsorption of cadmium compounds occurs through the alimentary canal, and vapour – through respiratory way. The soluble cadmium compounds denature protein

contained in walls of the alimentary canal. In blood cadmium bonds with sulphhydryl groups of enzymes, breaking their functions. The cadmium compounds collect mainly in a

liver and kidneys. They can cause fatty liver degeneration. The bonds of cadmium eliminate

from organism basically through kidneys with urine and walls of intestines. Sometime is marked the intestinal bleeding in cases of cadmium compounds poisonings.

Mineralisates Examination on Cadmium Presence

Extraction of cadmium ions from mineralisate. At research mineralisate on

presence of cadmium ions of them translate in a complex with sodium dirthyldithiocarbamate. This complex extracts by chloroform and then decomposes by

chloride acid:

Reaction with sodium sulphide. Forms yellow precipitate CdS. At negative result

of this reaction the further research of aqueous phase on cadmium presence do not make.

Reaction with a brucine and potassium bromide. Reaction executes on subject

glass. Forms the colourless prismatic crystals assembled in spheroids.

Reaction with pyridine and potassium bromide. Reaction executes on subject

glass. Forms the colourless prismatic crystals assembled in spheroids.

MANGANESE

Application and toxicity. The manganese compounds are applied in engineering and

medicine. There are cases of application of potassium permanganate for criminal abortions. The manganese compounds are the strong protoplasmic poisons. It acts on the central

nervous system, amaze kidneys, lungs, blood circulation. At use of strong solutions of potassium permanganate for throat gargle can come mucous oedema of mouth and pharynx.

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The reception inside of strong solutions of manganese compounds can cause stomach

perforation. At hit of strong solutions of manganese compounds in a uterus, vagina, and the

urinary bubble can appear threat of peritonitis. The manganese compounds collect in liver. It eliminates from an organism through

the alimentary canal and with urine.

Mineralisates Examination on Manganese Presence

Reaction with potassium periodate КIO4.

2MnSO4 + 5KIO4 + 3H2O → 2HMnO4 + 5KIO3 + 2H2SO4

Reaction with ammonium persulphate. Depending on reaction conditions

ammonium persulphate can oxidises manganese ions with formation of various compounds. At boiling in acidic environment without catalysts ammonium persulphate oxidises

manganese ions to manganate acid Н2МnO4:

MnSO4 + 2(NH4)2S2O8 + 4H2O → (NH4)2MnO4 + 2(NH4)2SO4 + 3H2SO4

In an alkaline condition without catalyst ammonium persulphate oxidises manganese ions to

МnО2:

MnSO4 + (NH4)2S2O8 + 4NH4OH → MnO2 + 3(NH4)2SO4 + 2H2O

At the catalyst presence (salt of silver or mixture cobalt, nickel and mercury salts)

ammonium persulphate oxidises manganese ions to permanganate-ions МnО4–:

2MnSO4 + 5(NH4)2S2O8 + 8H2O → 2NH4MnO + 4(NH4)2SO4 + 8H2SO4

COPPER

Application and toxicity. The copper compounds are widely used in industry for

paint preparation and for ceramic manufacturing. The copper compounds in combination with arsenic compounds are used in agriculture as fungicides, for example – Parisian

(Swainfurt) green Cu(CH3COO)2⋅3Cu(AsO2)2. In medicine is applied copper citrate. The metal copper vapours (in metallurgy) can get in organism with inhaled air. The

utensils from metal copper used for cooking of fruit, containing an organic acid, also can

cause poisoning. The adsorption of copper compounds from a stomach in blood occurs slowly.

Arrived in stomach coppers salt cause vomiting and diarrhoea. After adsorption in blood copper compounds act on capillaries, cause a haemolysis, defeat of liver and kidneys. At

introduction of copper salts strong solutions as drops in eyes the conjunctivitis can develop and damage of cornea.

The copper ions are eliminated from organism mainly through intestines and kidneys.

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Mineralisates Examination on Copper Presence

Extraction of copper ions from mineralisate. Detection of copper ions is based on it extraction by chloroform as diethyldithiocarbamate complex. This complex then

decompose by mercury(II) chloride.

Reaction with ammonium tetrarhodanomercurate. With (NH4)2[Hg (SCN)4]

forms yellow-green crystalline precipitate Cu[Hg(SCN)4]. Reagent in presence of zinc ions

forms precipitate Cu[Hg(SCN4)] ⋅ Zn[Hg(SCN)4], having pink or violet colour.

Reaction with potassium hexacyanoferrate(II). Forms red-brown precipitate

Cu2[Fe(CN)6].

Reaction with pyridine and ammonium thiocyanate. With this reagent forms

complex compound [(PyH)2]⋅[Cu(SCN)4], which dissolves in chloroform. Chloroform solution has emerald-green colour.

ARSENIC

Application and toxicity. Arsenic(III) oxide is used as insecticide in agriculture and

for glass manufacturing. Important in toxicology view are organic compounds of arsenic that apply in medicine – Novarsenolum, Osarsolum etc. Very toxic are arsenic containing

element-organic battle poisoning substances – luisite and adamsite. Compounds of arsenic

(V) in organism transform to arsenic (III). Soluble arsenic compounds are well resorpted to organism. Arsenic containing dust

arrives to organism through respiratory ways. Arsenic interacts with sulphhydryl groups of enzymes and brake metabolic processes. Can occur capillary paralysis. Some arsenic

compounds have necrotising action. This property of arsenic (III) oxide is used in dentistry. Arsenic cumulates in organism. At acute poisoning arsenic compounds collect in

internal organs, and at chronic poisoning – in bones and ceratosic tissues (nails, hair).

Arsenic eliminates from organism with urine and through some glands. Arsenic can be detected in corpses on many years after death.

Mineralisates Examination on Arsenic Presence

Sanger-Black’s reaction based on arsenic compounds reducing to arsenic hydride

(arsine) and it detection on reaction with mercury(II) chloride. Reaction makes in special device (apparatus).

Zn + H2SO4 → ZnSO4 + 2H↑

Hydrogen formed at interaction of sulphate acid and zinc, reduces arsenic compounds to AsH3:

Na3AsO3 + 6Н + 3HCl → AsH3↑ + 3NaCl + 3H2O

Na3AsO4 + 8Н + 3HCl → AsH3↑ + 3NaCl + 4Н2О

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The formed hydrogen arsenide (arsine) reacts with mercury(II) chloride, which the filter

paper is impregnated with. At reaction on paper are formed yellow or brown spots:

AsH3 + 3HgCl2 → Аs(HgCl) 3 + 3НCl

After processing paper by weak solution of potassium iodide all paper (except for the spot containing the specified arsenic compound) gets red colour caused by transferring of

mercury chloride in iodide of this metal:

HgCl2 + 2KI → HgI2 + 2KCl

At the further processing of paper by strong solution of potassium iodide the paper is

decolourised (is formed K2[HgI4]), and the spot containing arsenic compounds As(HgCl)3, remains yellow or brown.

Hydrogen sulphide, which can be formed at interaction of hydrogen with sulphate acid, Sanger-Black reaction prevents:

H2SO4 + 8Н → H2S + 4Н2О

Hydrogen sulphide reacts with mercury chloride on a filter paper and forms black spot of mercury, which masks colour spot of arsenic compound. To linkage hydrogen

sulphide apply cotton impregnated with a solution of lead acetate:

H2S + Pb(CH3COO)2 → PbS + 2СН3СООН

Reaction with a solution of silver diethyldithiocarbamate in pyridine. The

arsenic compounds in mineralisate reduce to hydrogen arsenide (arsine), which collect in

test tube with fresh solution of silver diethyldithiocarbamate in pyridine. The solution of silver diethyldithiocarbamate gets steady red violet colour. Mechanism of this reaction is

not found. This reaction carries out in the special device.

Sanger-Black’s apparatus: 1 – flask with sample, 2 – impregnated with lead acetate cotton,

3 – impregnated with mercury chloride paper.

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The March’s reaction is based on reduction of arsenic compounds by hydrogen and

on subsequent thermal decomposition of the hydrogen arsenide (arsine), formed at it:

2Na3AsO3 + 6Н + 3H2SO4 → 2AsH3↑ + 3Na2SO4 + 3H2O

2Na3AsO4 + 8Н + 3H2SO4 → 2AsH3↑ + 3Na2SO4 + 4Н2О

2AsH3 → 2As + 3H2

The arsenic formed at thermal decomposing of hydrogen arsenide, is postponed on walls of reduction tube of the March’s device as scurf (“an arsenic mirror”).

The March’s reaction is the most proof from all reactions recommended for arsenic detection in various objects. It not only allows to find small amounts of arsenic, but also to

distinguish it from antimony.

SILVER

Application and toxicity. The most toxic silver compound is nitrate, which uses in medicine as disinfectant, knitting and cauterising agent. The poisoning with silver can come

at inhalation of dust formed at processing of silver ores. The silver oxide, chloride, bromide

and iodide are not dissolved in water and are not toxic. The silver compounds, which have arrived in stomach, are adsorbed in a blood in

insignificant quantities. These compounds interact with a hydrochloric acid of stomach and transform to insoluble in water chloride. The silver nitrate acts on skin and mucous causing

“chemical” burns. At entering in organism through respiratory ways a dust of silver or it salts, there is danger of capillary defeat. The long reception of silver compounds inside can

Device for arsenic detection with solution of silver diethyldithiocarbamate: 1 – flask with sample, 2 – dropping funnel, 3 – tube,

4 – test-tube with silver diethyldithiocarbamate.

The March’s apparatus 1 – flask with sample and zinc flakes, 2 – dropping funnel with sulphate acid, 3 – calcium chloride tube,

4– reduction tube.

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cause argiria (deposition of silver in tissues), at which the skin gets grey-green or brownish

colour.

The silver compounds are eliminated from organism mainly through intestines.

Mineralisates Examination on Silver Presence

Reaction with dithizon. The silver ions with chloroform solution of dithizon in

acidic environment form yellow monosubstituted dithizonate AgHDz:

Reaction with sodium chloride. The silver ions with sodium chloride form white precipitate, which dissolvs in ammonium hydroxide:

AgNO3 + NaCl = AgCl↓ + NaNO3

AgCl + NH4OH = [Ag(NH3)2]Cl

Reaction with nitrate acid. To solution, containing silver ammonia complex, add

nitrate acid to рН=1. Forms white precipitate:

[Ag(NH3)2]Cl + 2HNO3 = AgCl↓ + 2NH4OH

Reaction with potassium iodide. To solution, containing silver ammonia complex,

add saturated solution of potassium iodide. Forms yellow precipitate:

[Ag(NH3)2]Cl + KI = AgI↓ + 2NH4Cl + KCl

Reaction with thiourea and potassium picrate. Reaction execute on subject glass.

The yellow prismatic crystals or aggregates form.

ANTIMONY

Application and toxicity. The compounds of three-valent antimony are more toxic,

than compounds of five-valent antimony. The antimony compounds are applied for

preparation some grades of glass, paints, rubber products, in matches production, for rubber vulcanisation. The metal antimony is included into composition of some alloys used for

preparation of bearings, typographical font etc.

In medicine potassium antimonyl tartrate (КООС–СНОН–СНОН–COOSbO) as expectorating and vomitive is applied. Wider applications in medicine have the organic

compounds of antimony used as chemiotherapeutic drugs. A toxicity of antimony organic compounds is smaller, than toxicity of inorganic. Action of antimony compounds on

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organism in many respects is similarly to action of arsenic. The arrived in blood antimony

compounds act as “capillary poison”. At poisoning with organic compounds of antimony

the functions of cardiac muscle and liver are broken. The antimony eliminates from organism mainly through kidneys. Therefore can

develop nephritis.

Mineralisates Examination on Antimony Presence

Reaction with malachite green or diamond green. Malachite green is the basic

stain that forms an ionic associate with acidic complex of antimony [SbCl6]. This complex

has dark blue or light-blue colour and is extracted by xylene or toluene. It is possible to apply diamond green instead malachite green.

Malachite green Diamond green

In mineralisate the antimony is in a three-valent condition. Under influence of added

sodium nitrite Sb(III) transform to Sb(V):

HSbO2 + 2NaNO2 + 2НСl → HSbO3 + 2NO + 2NaCl + H2O

At hydrochloric acid presence formes acidic complex [SbCl6]

–:

HSbO3 + 6HCl → H[SbCl6] + 3H2O

Formed ionic associate with malachite green has structure:

(CH3)2N N(CH3)2

C [SbCl6]

+

_

Reaction with sodium thiosulphate. In acidic environment forms orange precipitate

Sb2S3:

2HSbO2 + 3Na2S2O3 → + Sb2S3↓ + 3Na2SO4 + H2O

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THALLIUM

Application and toxicity. The thallium oxide is applied to reception of synthetic precious stones and special grades of glass. The thallium halogenides are used for

luminophores preparation. Metal thallium enters into composition of some alloys and amalgams. Thallium sulphate is rodenticide. Thallium acetate is applied for depilation.

At poisoning with thallium compounds the central nervous system is amazed (there

can be a disintegration of a myelin environment), there comes parasympathetic nervous system paralysis, there is kidneys defeat. At poisoning with thallium compounds the hair

drops out (baldness). On toxicity thallium in many respects reminds action of arsenic and lead.

The thallium compounds after entering in blood quickly distribute in organism. They slowly eliminate from organism mainly through kidneys and intestine (cumulating).

Mineralisates Examination on Thallium Presence

Reaction with dithizon. The thallium dithizonate is well extracted with chloroform,

which phase gets red colour.

Reaction with malachite green is based on interaction acidic complex [ТlСl4]

– with

malachite or diamond green. This dark blue or light-blue ionic associate is extracted by toluene or xylene:

CHROME

Application and toxicity. The chrome compounds are applied in tanning and textile

industries, are used for chrome plating of metal products, for production of matches, paints, and cinema films. In chemical industry chrome compounds are applied as oxidisers. In view

of a toxicity of chrome compounds they are not applied in medicine. From chrome compounds used in various branches of economy, the most toxic are

the chromates and bichromates. The chromates and bichromates render irritating and

cauterising action on a skin and mucous, causing ulceration. Under chromates and bichromates influence can come haemolysis and the methaemoglobin is formed. After

entering chrome compounds in organism through the alimentary canal can come tumescence, and then burns of mucous of mouth, oesophagus and stomach. At entering in

organism a dust containing chrome compounds the pneumonia develops. At acute poisonings the chrome compounds collect in liver, kidneys and endocrine

glandular. The chrome compounds eliminate from an organism basically through kidneys.

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In this connection at poisoning with the chrome compounds the kidneys and mucous of

urinary ways are amazed.

Mineralisates Examination on Chrome Presence

Reaction of perchromate acid formation. The perchromate acid formation can be

presented by the following equation:

K2Cr2O7 + 4H2O2 + H2SO4 = 2H2CrO6 + K2SO4 + 3H2O

The formed perchromate acid quickly decomposes in aqueous solutions. Therefore from aqueous solutions it extract by organic solvents.

ZINC

Application and toxicity. Zinc and its compounds are widely used in economy and in

medicine. Metal zinc is included into composition of alloys (bronze, brass etc.). Zinc is applied for covering iron products for protection from corrosion and for manufacturing zinc

utensils. In industry zinc oxide is applied to preparation of paints (snow white). Zinc

sulphide is applied for manufacturing luminous paints. Zinc phosphide is very toxic. It applies as rodenticide.

In medicine are applied many zinc compounds: zinc oxide as astringent in ointments and pastes, zinc sulphate – in ophthalmology, zinc stearate enters into composition of

powder and some ointments, zinc undecylenate is fungicide agent. Zinc and its compounds can arrive in organism through the alimentary canal, and

also through respiratory way as a dust formed at zinc ores processing. Zinc can arrive in an

organism with inhaled air as vapour of alloys processing. Zinc dust and vapour in organism bond with protein causing attacks of fever (so-called brass fever). Inhalation of zinc dust or

vapour can appear the nausea, vomiting and muscle pains. Zinc compounds, which have arrived in stomach, can cause vomiting, diarrhoea, cramps.

At poisoning the zinc compounds collect in liver and pancreas.

Mineralisates Examination on Zinc Presence

Presence of zinc ions in mineralisate determine with dithizon. If result of this preliminary test is negative, the further mineralisate investigation on zinc ions presencel not

carry out. At a positive take of reaction with dithizon will carry out(spend) the further research mineralisate on ions of Zinc. With this purpose from mineralisate ions of Zinc

evolve as диэтилдитиокарбама-that. Received the dirthyldithiocarbamate of Zinc

decompose by an acid And in an aqueous phase determine the presence of ions of Zincum through the

appropriate reactions.

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Reaction with dithizon. At interaction of zinc ions with dithizon is formed

monosubstituted dithizonate Zn(HDz)2. Zinc dithizonate extractes by chloroform or another

organic solvents. The solution zinc dithizonate in chloroform has purple-red colour.

Extcraction of zinc ions from mineralisate. To mineralisate adds solution of

sodium diethyldithiocarbamate for inner-complex compound formation. Zinc

dirthyldithiocarbamate extracts by chloroform and then decompose by acid.

Reaction with potassium hexacyanoferrate(II). Forms white precipitate:

3ZnSO4 + 2K4[Fe(CN)6] →K2Zn3[Fe(CN)6] + 3K2SO4

Reaction with sodium sulphide. Forms white precipitate ZnS.

Reaction with ammonium tetrarhodanomercuroate. Reaction executes on subject

glass. Forms white precipitate that is colourless single clinoid crystals associated in

dendrites:

ZnSO4 + (NH4)2[Hg(SCN)4] → Zn[Hg(SCN)4]↓ + (NH4)2SO4

MERCURY

Application and toxicity. Mercury and its compounds are applied in engineering,

chemical industry, medicine. Metal mercury is applied in medicine for preparation of ointments. In engineering it is used at manufacturing lamps, thermometers and various

devices. Metal mercury in stomach arriving is non toxic. Toxic are the all its compounds. The mercury vapour and dust can act in organism with inhaled air. Thus the central

nervous system, at the first – brain cortex, is amazed. Mercury in organism bonds with sulphhydryl groups of enzymes and other vital protein. As a result the normal physiological

functions of cells and tissues are broken. The mercury compounds amaze all organs

(stomach, liver, kidneys, and glands), through which mercury is eliminated from organism. Mercury cumulates mainly in liver and kidneys.

Mercury slowly eliminates from organism. In two weeks after acute poisoning the certain quantities of mercury can be detected in tissues. Mercury eliminates from organism

with urine and faeces, and also sweat and salivary glands.

Destructates Examination on Mercury Presence

Reaction with dithizon. This reaction is based on interaction of mercury(II) ions with dithizon. Forms monosubstituted dithizonate, which has orange-yellow colour in acidic

environment and purple-red – in alkali.

Reaction with copper(I) iodide suspension. Forms red or orange-red precipitate

Cu2[HgI4]:

Hg(NO3)2 + 4CuI → Cu2[HgI4] + 2CuNO3

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QUANTITATIVE DETERMINATION OF “METAL POISONS” IN

MINERALISATES

In chemical-toxicological analysis for quantitative determination of “metal poisons”

are applied: – gravimetric method for barium as BaSO4 precipitate;

– titrimetric methods: complexonometry – for bismuth, copper, cadmium, and zinc,

iodometry – for lead, thiocyanometry – for silver, and argentometry – for arsenic; – photocolorimetric method for all metals: with dithizone – mercury, lead, silver,

thallium, with malachite green – antimony and thallium, with diphenylcarbazide – chrome, with diethydithiocarbamate – copper and arsenic, with thiourea –

bismuth; – visual colorimetric method (methods of standard series) for mercury – on colour

intensity of Cu2[HgI4] suspension, and for arsenic – on colour display of filter

paper strips, impregnated by mercury chloride.

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TOXIC SUBSTANCES

ISOLATED FROM BIOLOGICAL MATERIAL BY POLAR SOLVENTS

Now this group of toxic substances is the most numerous. This group contains alkaloids, their synthetic analogues, medicines and venoms. These substances are isolated

from a biological material by various polar organic solvents, which mix with water. The first methods of isolation of toxic substances from biological material were

proposed in the beginning of XIX century. For this purpose was applied acidified ethanol

and acidified water only for alkaloids extraction. In that time the synthetic pharmaceuticals were not applied in medicine and do not cause poisonings. The special methods were not

developed for extraction of these synthetic drugs from biological material, and the methods earlier offered for research of alkaloids were applied.

All earlier offered methods of alkaloid extraction from biological material have series of disadvantages. According to these methods, the isolation of alkaloids and synthetic

nitrogen-containing toxic substances of the basic character was made without the account of

influence of isolating liquids pH (acidified ethanol or acidified water) on extraction degree of researched substances from biological material. The influence pH on impurity extraction

from previously purified extracts was not taken into account. The choice of organic solvents for researched substances extraction from biological material was made empirically.

The certain losses of alkaloids during extraction from biological material always take

place. Besides, the results of extraction of alkaloids by these methods are not reproducing.

Investigated matrices are very complicated. They have some peculiarities:

1. Bonds between poison and peptides are strong. As a rule these bonds are covalent or result of specific interaction enzyme-receptor.

2. Contain of investigated substances in analysed samples is very small. 3. Biological material contains many compounds similar to analyte.

The isolation of toxic compounds from material of biological origin is very complicated and multistage process. There are three stages of poisonous substance isolation

from objects of chemical-toxicological investigation. On each stage there are many factors, which influence on isolation efficiency:

I stage. Infusing of investigated objects with isolating liquids:

– character and method of object preparation,

– nature of isolating liquid and applied electrolyte (acid or base), – pH of solution,

– recurrence and duration of infusing, – mode of peptides precipitation,

– mode of impurities separation.

II stage. Extraction with organic solvents:

– nature of organic solvent, – pKa of substance,

– pH of solution, – distribution coefficient,

For acid substances pH = pKa – 2,

For base substances pH = pKa + 2.

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– nature and concentration of electrolyte,

– ionic strength of solution.

III stage. Concentration and purification (clarification) of isolated substance:

– chosen purification technique, – re-extraction,

– chromatography,

– solid phase extraction, – sublimation.

The proteins are amphoteric compounds. Accordance to pH value they can dissociate

as an acid and as the bases. At the certain pH value the number of positive and negative charges in protein becomes identical. In this case common charge of protein is zero. The pH

value, at which protein matters and other amphoteric compounds have no charge and do not

move in electrical field, refer as an isoelectric point. The isoelectric point of proteins depends on their nature.

At pH value more than isoelectric point the protein has negative charge, at pH less than isoelectric points protein are lower have positive charge. After dissociation alkaloid

ions get positive charge. The protein having negative charge with cations of alkaloids form bonds or complexes. Thus, the alkaloids and their synthetic analogues bonds with protein at

pH above their isoelectric point.

For breaking of peptide-toxin bonds must be applied very active agents. But we can

not mineralise our sample, because the investigated substances are the organic compounds, which easy destroy. We cannot heat our sample, because our toxins are thermally unstable

substances. A degree of isolation of the specified substances, amount and nature of impurities

passing from biological material in extracts, depend on nature and composition of isolating

liquids.

The methods, used in the chemical-toxicological analysis, for extraction from biological material alkaloids and other nitrogen-containing bases are based on isolation of

these substances by polar solvents, which mix with water. Now as isolating agents are used such solvents as methanol, acetone, acetonitrile, dioxane.

There are some demands to isolating liquids: – these liquids should well penetrate into cells and tissues of biological material;

– these liquids should well to break bonds between poisons(venom) and proteins in tissues; – these liquids should well to dissolve good salts of toxic substances, which are formed in

biological material under influence of natural acids or acids/bases that are included in composition of isolating liquids;

– these liquids should well to dissolve as it is possible smaller quantities of impurities

passing from biological material to extracts.

The ability of isolating liquids to penetrate in corpse tissues depends on contents of cells and tissues of researched biological material. The infiltrations into biological material

of organic solvents, which are not mixing with water, prevent water that is included in cells

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and tissues. Therefore, organic solvents, which are not mixing with water, are not suitable

isolation toxic substances from biological material.

On isolation degree of alkaloids and other basic nitrogen compounds from biological material renders influence a nature of used acid. The salts of toxic substances dissolve in

isolating liquid better, then native poisons. We must take into account, that in acidic or alkalic solutions some alkaloids or

medicines can hydrolyse. For example, in strong acidic environment hydrolyse alkaloids –

derivates of tropane, in alkali environment hydrolyse phenothiazine derivates. As a result, we loss toxic compounds and pollute our extracts with analytes decomposition products.

At isolation toxic substances in extracts from a biological material together with

toxic substances pass impurities of protein, products of their hydrolysis (peptides, amino acids), lipids and other compounds. Impurities composition and quantitative depend on

composition and putrefaction degree of biological material, on nature of isolating liquid, pH

value and on some other factors. The lipids, in a kind of their insolubility in water, practically do not pass in acidic

aqueous extracts from biological material. But lipids good dissolve in organic solvents, which are mixed with water.

Solubility of proteins and their precipitation substantially depend on inductivity of solvents. The inductivity of ethanol (D = 24) is lower, than at water (D = 80). At solvent’s

inductivity dropping the attractive force between molecules permeates grow.

On proteins solubility influences also the ionic strength of solutions, which dependent on concentration of electrolytes in these solutions.

At isolation toxic substances by alcohol, acetonitrile or acetone, can occurs denaturation of proteins on surface of biological material slices. As a result of it appear

difficulties for infiltration of isolating liquids into biological material. This complications influence on examined substances isolation efficiency (completeness).

The extracts obtained) by infusion of biological material with polar solvents always contain the certain amount of impurity of proteins, amino acids, lipids and other compounds

interfering to detection and quantitative determination of toxic substances. The extracts from biological material are subjects to clearing of impurity.

At present time are used many methods of extracts from biological material clearing.

Filtration and centrifugation. Filtering apply for clearing extracts of mechanical pollution (shallow particles of biological material). However through pores of filters can

pass more shallow particles of solid matters, which size is less than a diameter of filters pores. Besides, the material of filters can adsorb the certain quantities of toxic substances,

which are isolated from biological material. Taking into account the mentioned above disadvantages of filtering, as method of extracts clearing from impurities apply

centrifugation.

Precipitation of impurities. The appropriate reagents can precipitate impurities in

extracts, which pass through pores of filters and do not settle at a centrifugation. Some reagents apply not only for extracts clearing, but also for precipitation albumin's substances

from blood. Such reagents are trichloroacetic acid, wolframic acid, metaphosphoric acid, and some heteropoly acids – phosphorotungstic, phosphoromolybdic. Phosphorotungstic

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and phosphoromolybdic acids precipitate protein and peptides, and the trichloroacetic acid

precipitates protein, but do not precipitate peptides.

Some proteins precipitate at heating. At temperature raising to 40 °C the solubility of the majority of protein grows (from this rule there are exceptions). At the further

temperature rising occurs denaturation of proteins, their solubility is depressed, and

therefore they drop in sediment. Such way of extracts clearing is applied on Stas-Otto’s method (ethanol acidified by oxalic acid).

As it was marked above, from addition of ethyl or methyl alcohols, acetone and other organic solvents, which are mixing with water, the solubility of proteins is depressed. As a

result of it they can drop in sediment. One of effective methods of remission of proteins and other impurities from extracts

from biological material is the salting-out. The efficiency of salting-out depends from

concentration and nature of electrolytes, added to solutions or extracts from biological material.

At low concentration the neutral electrolytes raise solubility of many proteins. This effect does not depend on a nature of used salt, but depends on their concentration and

charge of ions, which are included in composition salt. Salts that containing divalent ions raise solubility of protein better, than salts consisting of monovalent ions. The rising of

solubility of proteins under influence of low electrolytes concentration is explained by

change their and dissociation degree of ionised (carboxilic, phenolic, sulphhydryl) groups of protein. With rising of added to extracts salt content the solubility of protein is depressed,

and at the further addition of these salts of proteins can drop in sediment. The precipitation of proteins from extracts can be made by change of pH value. At

pH, appropriate to an isoelectric point of protein, it does not carry a common charge. Therefore at specified pH between the neighbour molecules of protein there is no

electrostatic pushing away. As a result of it there is sticking molecules of protein and

abasement it in sediment. Some impurities from extracts cannot be halted by the listed above methods. These

methods of extracts clearing, based on precipitation of impurity, have also some disadvantages. The sediment of impurity can adsorb on the surface researched toxic

substances. The adsorption of toxic substances by sediment of impurity can be one of the reasons of losses of researched substances during the chemical-toxicological analysis.

Extraction of impurities infuses. In the chemical-toxicological analysis the method of extraction is applied to clearing extracts from a biological material more frequently, than

other methods. Through a method of extraction it is possible to purify extracts from lipids, amino acids, products they decarboxilation and desamination, and also from another

impurities. The extraction of impurities from extracts is successful only then, when the organic dissolvent, not mixing with water, is correctly chosen and the appropriate pH value

is fixed. The extraction of impurities should be made at such pH, at which the researched

substances are not extracted.

Solid-phase extraction (SPE) is an extraction method that uses a solid phase and a liquid phase to isolate one, or one type, of analyte from a solution. It is usually used to clean

up a sample before using a chromatographic or other analytical method to quantify the amount of analyte(s) in the sample. The general procedure is to load a solution onto the SPE

phase, wash away undesired components, and then washes (elutes) off the desired analytes

with another solvent into a collection tube. The goal of SPE is to quantitatively remove an

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analyte from a sample matrix with complete recovery in a solvent so that the recovered

analyte is suitable for subsequent analysis. Owing to the limitations of most commercially

available SPE sorbents, this ideal is rarely achieved.

Application of physical-chemical methods for extracts clearing. Recently in the chemical-toxicological analysis for clearing extracts from biological material from impurity

are developed some physical-chemical methods. There are methods of ion-exchange

chromatography, gel-chromatography, and molecular adsorptive chromatography on columns, affinity chromatography and thin layer chromatography. Also are applied for these

purposes the various electro-migration methods (electrophoresis). Physical-chemical methods allow not only concentrating, busing also simultaneously to detect toxic substances

in extracts from biological material.

In the modern chemical-toxicological analysis the extraction is the basic method of

extraction of alkaloids and other toxic substances from extracts, biological liquids (urine, blood), contains of stomach and other objects.

Alkaloids and other toxic substances isolated from a biological material, long time divide on two groups: substances extracted by organic solvents, not mixing with water, from

acidic extracts, and substances extracted by the same solvents from alkalic extracts. However such sectioning of toxic substances on two groups is conditional.

For each alkaloid there is an interval of pH value, at which it is extracted by organic

solvents, not mixing with water, in maximal amounts. This interval of pH value is named as area of extraction maximum (Table).

The area of extraction maximum of alkaloids depends on nature of alkaloids and

nature of organic solvents used for extraction of these substances from aqueous solutions. For example, the alkaloids colchicine, caffeine, narcotine, and theobromine have the area of

extraction maximum in acidic environment or on border of acidic and alkaline environment.

However, the certain quantities of these alkaloids are extracted and from an alkaline environment. Alkaloids, the area of extraction maximum of which is in an alkaline

condition, are partially extracted and from acidic environment. The mentioned above laws of extraction concern not only alkaloids, but also their

synthetic analogues being nitrogen-containing organic compounds with basic properties.

For maintenance of completeness of extraction of alkaloids and other nitrogen-

containing organic compounds with basic properties from solutions it is necessary to extract them at pH, which take place in the field of extraction maximum, using organic solvents

ensuring extraction of maximal amounts of these substances. The degree of extraction of toxic substances, which are not having the basic or acid

properties, basically depends on nature organic solvents and does not depend from pH value or depends a little.

It is necessary to make account of extraction degree, constant of distribution and number of repeated extractions necessary for complete extraction of researched substances

from solutions.

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Dependence of Alkaloids Extraction Degree on pH and Nature of Organic Solvents

Alkaloid Organic solvent

pH of

extraction

beginning

Interval pH of

maximum

extraction

Maximal amount

of extracted

alkaloid, %

Chloroform 1,9 9,7 — 11,7 94 — 95

Ethylene dichloride 1,9 9,7 — 11,7 61 — 71

Diethyl ether 2,4 9,2 — 11,7 21 — 23 Anabasine

Benzene 3,5 9,7 — 11,7 60 — 70

Chloroform 3,0 8,0 — 10,0 97 — 99

Ethylene dichloride 4,0 9,0 — 10,0 91 — 94

Diethyl ether 5,0 10,0 — 11,0 37 — 38 Galantamine

Benzene 5,0 9,0 — 10,0 84 — 88

Chloroform 3,0 7,0 — 8,5 80 — 83

Diethyl ether 4,0 8,0 — 8,5 57 — 62 Cocaine

Benzene 4,0 7,0 — 8,5 68 — 70

Chloroform 1,5 4,0 — 8,0 90 — 96

Ethylene dichloride 1,5 4,0 — 7,0 91 — 93 Colchicine

Benzene 1,5 4,0 — 8,0 20 — 25

Chloroform 1,8 4,0 — 5,5 96 — 98

Ethylene dichloride 1,8 4,0 — 5,5 82 — 86 Caffeine

Diethyl ether 1,8 4,0 — 5,5 3 — 4

Chloroform 1,0 4,0 — 7,0 91 — 93

Ethylene dichloride 1,0 5,0 — 7,0 76 — 78 Narcotine

Diethyl ether 2,4 5,0 — 7,0 83 — 85

Chloroform 4,0 9,0 — 12,0 91 — 94

Ethylene dichloride 5,0 9,0 — 12,0 90 — 93 Platyphylline

Diethyl ether 6,0 9,0 — 12,0 66 — 70

Chloroform 3,0 7,0 — 9,0 43 — 47

Ethylene dichloride 3,0 7,0 — 9,0 48 — 50 Securenine

Diethyl ether 4,0 9,0 — 10,0 45 — 46

Chloroform 5,0 8,8 — 10,5 88 — 90

Diethyl ether 6,7 9,8 — 10,5 40 — 43 Scopolamine

Benzene 4,9 9,0 — 10,0 76 — 78

Chloroform 2,1 4,0 — 7,0 32 — 37

Ethylene dichloride 2,1 6,1 — 8,0 12 — 22 Theobromine

Diethyl ether 2,1 4,0 — 7,0 4 — 6

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QUALITATIVE AND QUANTITATIVE DETERMINATION OF TOXIC

SUBSTANCES ISOLATED FROM BIOLOGICAL MATERIAL

For qualitative determination (detection) of alkaloids and their synthetic analogues isolated from biological material are applied many methods:

1. Chemical methods: – chromogenic reactions (colour tests),

– precipitation reactions, – microcrystalloscopic reactions.

2. Physical-chemical methods:

– thin layer chromatography (TLC screening),

– electro-migration methods (electrophoresis), – absorption spectroscopy,

– mass-spectrometry.

3. Pharmacological method

1. Chemical Methods

Reactions of precipitation. Precipitates formation of alkaloids and their synthetic analogues is one of methods of detection of these substances in the chemical-toxicological

analysis. With this purpose are applied reagents of alkaloids group precipitation. Such reagents are some complexes, heteropoly acids, tannin, picric acid, pycrolonic acid etc. To

complexes being reagents of group precipitation of alkaloids are Buschard’s reagent

(solution of iodine in potassium iodide), Dragendorf’s reagent (potassium tetraiodobithmutate), Mayer’s reagent (potassium tetraiodomercurate), hydrogen

hexachloroplatinate etc. From heteropoly acids as reagents of alkaloids and amines precipitation are applied: a phosphomolybdic acid (Sonnenstain’s reagent), phosphotungstic

acid (Schaibler’s reagent), silicotungsten acid (Bertran’s reagent) etc. All these reagents form with alkaloids, their synthetic analogues, and other organic

compounds with basic properties amorphous precipitates.

Heteropoly acids and organic acids form not dissolving complex compounds or ionic associates. Formation of ionic associate of alkaloid coniine with Dragendorf’s reagent

(K2[BiI4]) can be presented by the following equation:

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The reagents of alkaloids group precipitation also give precipitates with proteins and

products of their hydrolysis. But these reactions are not specific. These reactions can be

applied as preliminary tests on presence in biological material of alkaloids and other nitrogen-containing organic compounds with basic property. At positive results of these

tests it is necessary to carry out the further research investigated substances with appropriate reactions and methods. Therefore in the chemical-toxicological analysis the large

importance have the negative results of reactions with reagents of alkaloids group

precipitation. At negative results of these reactions it is possible to exclude from the plan of the chemical-toxicological analysis research of alkaloids and other nitrogen bases.

Some alkaloids and nitrous substances with these reagents form crystalline precipitates. Characteristic form of these crystals is identification sign of examined

substances. Colour tests. For alkaloids detection can be applied the colouring reactions.

Chemical mechanism of these reactions is not investigated. The reagents used for detection

of alkaloids with colour reactions based on various classes of chemical compounds. To this purpose are applied concentrated sulphate and nitrate acid, and also mixtures of

concentrated sulphate acid with other compounds. For detection of alkaloids and other nitrogen bases apply Erdmann’s reagent (mixture of concentrated sulphate and nitrate

acids), Mandelin’s reagent (concentrated sulphate acid containing vanadate acid), Brand’s reagent (concentrated sulphate acid containing formaldehyde), Freude’s reagent

(concentrated sulphate acid containing molybdenic acid) etc.

Microcrystaloscopic reactions. These reactions are based on precipitation of researched substances with the appropriate reagents and on definition of the formed crystal

forms. The forms of formed crystals depends on many factors: concentration of researched substance, concentration of reagent, ratio of volumes of solutions of researched substance

and reagent, temperature, pH value, presence of impurity, polymorphism of formed crystals etc. Therefore microcrystaloscopic reaction should carry out the persons having necessary

knowledge in crystallochemistry and crystallography.

2. Physical and Physical-chemical Methods

For detection of poisonous substances traditionally is applied thin layer chromatography (TLC). Now this technique is applied for screening of extracts from objects

of biological origin.

Screening – the act or process of one that screens – the examination usually methodically in order to make a separation into different groups or select or eliminate by a

screening process. The aim of the TLC screening is to obtain as much information as possible in a short time and with minimum of sample.

Drugs are extracted from the sample into an organic solvent under acidic and alkaline conditions. The extracts are analysed by thin-layer chromatography on one plate using a

single solvent system (as a rule – ethyl acetate : methanol : concentrated ammonium

hydroxide (85 : 10 : 5). The basic extract is acidified during the evaporation stage to minimise loss of volatile bases such as amphetamines. It is important that the pH change

between extractions is accomplished satisfactory. Extracts of stomach contents may contain fatty material, which makes chromatographic analysis difficult, and purification by re-

extraction into aqueous acid or alkali may be required.

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Although simple examination of the developed chromatogram under ultraviolet light

(254 nm and 366 nm) may reveal the presence of fluorescent compounds such as quinine,

the use of a number of spray (visualisation) reagents the scope of the analysis and increases the confidence of any identification.

The recommended TLC visualisation reagents are as follows: 1. Mercury (I) nitrate – for acidic extracts – gives white spots with a grey centre on a

darker background with barbiturates and related compounds.

2. Acidified iodoplatinate – for basic extracts – gives mainly purple, blue or brown spots with a range of basic and neutral drugs (caffeine, dichlorphenazone).

3. Mandelin’s reagent – for basic extracts – gives colours ranging from blue and green to orange and red with a variety of basic compounds. Some, especially tricyclic

antidepressants such as amitriptyline and nortriptyline, give fluorescent spots if viewed under UV-light (366 nm) after spraying with this reagent.

4. Sulphate acid (50 %) – for basic extracts – alone gives red, purple or blue spots with

many phenothiazines and their metabolites. This is especially valuable since some phenothiaxines (chlorpromazine, for example) are given therapeutically in relatively

high doses and have many metabolites, which can give a very confusing picture if unrecognised.

Of course, many additional mobile phase and spray reagent combinations could be used as well as, or in place of, those suggested here. For example, methanol : concentrated

ammonium hydroxide (99 : 1,5) is widely used in the analysis of basic drugs, and is

especially useful in the detection of morphine and related opioids. Of the spray reagents, Marquis' reagent gives a variety of colours which different basic drugs, and again especially

valuable for the detection of morphine and other opioids which give blue/violet colour. In all cases, the colour obtained from a particular compound may vary depending on

– concentration, – the presence of co-eluting compounds,

– the duration and intensity of spraying, and

– the type of silica gel used in the manufacture of the plate. Performance of TLC screening and it result interpretation have some difficulties:

1. Some compounds may show a gradation or even a change on colour from the edge of the spot toward the centre (usually a concentration effect), while the intensity or even the

nature of the colour obtained may vary with time. 2. A further problem is that the interpretation and recording of colour reactions are

very subjective. It is thus important to analyse authentic compounds, ideally on the same

plate as the sample extracts. 3. Even so, compounds present in sample extracts sometimes show slightly different

chromatograms from the pure compounds owing to the presence of co-extracted material. Interfering neutral compounds, especially fatty acids from stomach contents, can be

removed by back-extraction of acidic or basic compounds into dilute base or acid, respectively (neutral compounds stay in the organic extract), followed by re-extraction into

organic solvent.

4. In difficult cases it may be useful to calculate the Rf value for unknown compounds and to compare the findings with reference values.

Capillary electrophoresis (CE) is a family of related techniques that employ

narrow-bore (10-200 µm internal diameter) capillaries to perform high efficiency

separations of both large and small molecules. Various CE techniques perform separations

based on several mechanisms, as molecular size (sieving), isoelectric focusing and

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hydrophobicity. High voltages are used to separate molecules based on differences in charge

and size.

Separation results from the combination of electrophoretic migration (the movement of charged molecules toward an electrode of opposite polarity) and electroosmotic flow (the

bulk of electrolyte flow caused by a charged inner capillary wall and an applied potential). The electroosmic flow is dependent upon field strength, electrolyte pH, buffer composition

and ionic strength, viscosity and capillary surface characteristic; all of which can be used

singly or in combination to enhance separations. Detection is achieved by monitoring UV absorbency directly on-line through a

window in the capillary. Other detection options include laser-induced fluorescence, diode array and mass spectrometry.

Spectroscopic sensors are a handful of methods based on interaction between the sample and electromagnetic radiation across the spectrum of wavelengths, including

ultraviolet and visual absorption, fluorescence emission, near-infrared and infrared

absorption, Raman scattering, nuclear magnetic resonance, microwave absorption and (ultra)-sound transmission. The spectroscopic methods based on different regions of the

electromagnetic spectrum and different physical principles have naturally different sensing capabilities, but share the ability to provide rapid multivariate information on the sample

being monitored, which in turn makes it possible to determine several quality parameters simultaneously.

Mass spectrometry is an analytical technique that is used to

– identify unknown compounds – quantify known materials and

– elucidate the structural and physical properties of ions. It is a technique associated with very high levels of specificity and sensitivity.

Analyses can often be accomplished with minute quantities - sometimes requiring less than picogram (10

-12 grams) amounts of material.

A mass spectrometer is an instrument that can separate charged atoms or molecules

according to their mass-to-charge ratio. Relative molecular masses of organic compounds and biopolymers can be measured in this way and the instrument is also capable of

generating structural information. The sample is introduced into the mass spectrometer, which is generally kept under

high vacuum (<10-5

mBar). Compounds are converted into gas phase molecules either before or during the charging or ionisation process, which takes place in the ion source.

Many types of ionisation mode are available: the type of compound to be analysed

and the specific information required determine which ionisation mode is the most suitable. Once ionised, the molecule ion may fragment, producing ions of lower mass than the

original precursor molecule. These fragment ions are dependent on the structure of the original molecule.

Having obtained a mass spectrum of a compound, the mass spectrometer is then set such that only one of the ions produced in the ion source is focused and detected. Usually an

intense ion representative of the molecule is chosen, selected and the intensity recorded.

This is known as selected ion recording (SIR) and is a very sensitive technique. For some compounds it is possible to detect at the femtogram (10

-15 gram) level.

By choosing a suitable internal standard, often a stable isotope labelled version of the sample of interest, and setting the mass spectrometer to switch very rapidly between the

sample ion and the equivalent ion for the internal standard, a very selective set-up for monitoring and quantifying a known sample is achieved. This method is generally used with

110

chromatographic and electrophoresis techniques such as gas chromatography, high

performance liquid chromatography or capillary electrophoresis for optimum sensitivity.

3. Pharmacological Method

Some toxic substances at action on organism of animals cause characteristic

physiological reactions. For example, atropine injected in cat’s eye causes a mydriasis. After putting solution of nicotine on frog’s backrest it accepts a characteristic pose.

Strychnine in frog causes tetanic cramps, and frog accepts characteristic pose. The experts – pharmacologists having special knowledge of this area and owning

engineering of experiment can carry out the pharmacological tests of toxic substances isolated from biological material.

The quantitative determination of toxic substances isolated from biological material is a final stage of the chemical-toxicological analysis. The quantitative

determination of toxic substances is made after their identification. Identification the toxic substance allows establishing the cause of poisoning and confirming it nature. Quantity of

determined toxic substance allows to definite medical error (estimate of therapeutic dose) or criminal cause.

Methods of quantitative determination of poisonous substances isolated from biological material are:

1. Physical-chemical:

– photometry, – direct and differential spectroscopy,

– extraction-photometry,

– gas-liquid chromatography.

2. Biochemical:

– enzymatic, – immunochromatographic,

– radioimmune.

Now are applied not only various chromatographic techniques as gas-liquid chromatography, high performance liquid chromatography, but such modern combined

methods as gas / liquid chromatography with mass spectrometry or capillary electrophoresis with mass spectrometry. These combined analytic techniques allow

separating, detecting, and quantitatively determining the investigated toxic substances in

sample simultaneously. For contemporary toxicological analysis develops computation methods, for

example, chemometrics. Chemometrics is a novel technology for exploring and modelling complex and unknown relations in multivariative data that have high level of correlation.

The core toolbox of chemometric models consists of adaptive decompositional models (e.g. component analysis), supplemented with visual analysis to allow for identification of hidden

relations in the data. Although genetic algorithms and artificial neutral networks will not

allow for interpretation of latent relationships, these black-box modeller also popular within chemometrics for optimisation and quantification. Chemometrics is able to handle large data

sets and deals efficiently with real-world multivariate data and, instead of fearing, it takes advantage of the co-linearity of spectral data.

Immunochromatography. Over the past 10 years, many in vitro diagnostic test kits have been commercialised that utilise the principles of immunochromatography. Many of

111

the tests are equipped with a zone at the end of the test strip that will change colour when

the sample front reaches it, thereby telling the user that the test is complete and that it’s time

to interpret the results (end of assay). The majority of the analytes are measured on the basis of presence/absence (yes/no),

and most of them are detected using immunometric assays. For immunometrictype assays, a ligand specific for the analyte (normally, but not necessarily an antibody [Ab]) is

immobilised to the membrane. The detector reagent, typically an antibody coupled to latex

or colloidal metal, is deposited (but remains unbound) into the conjugate pad. When sample (urine, plasma, whole blood, etc.) is added to the sample pad, it rapidly

wets through to the conjugate pad and the detector reagent is solubilised. The detector reagent begins to move with the sample flow front up the membrane strip. The antibody that

is coupled to the detector reagent will bind analyte that is present in the sample. As the sample passes over the zone to which the capture reagent has been immobilised, the analyte

detector reagent complex is trapped. Colour develops in proportion to the amount of analyte

present in the sample. There are also commercially available assays for drugs of abuse and for steroid-based

ovulation prediction that is based on competitive immunoassay protocols. In this type of assay, the detector reagent is typically the analyte (or an analogue of the analyte) bound to

latex or a colloidal metal. As the sample (containing analyte) and detector reagent pass over the zone to which the capture reagent (typically an antibody) has been immobilised, some of

the analyte and some of the detector reagent are bound and trapped. The more analyte

present in the sample, the more effectively it will be able to compete with, and/or displace, the binding of detector reagent. The hallmark of most competitive immunoassays is that an

increase in the amount of analyte in the sample results in a decrease of signal in the readout zone.

Detector reagents are antibody or antigen coupled to latex, colloidal metal, enzyme, etc. The antibody or antibodies that will be used in the development of the

immunochromatographic assay must have sufficient sensitivity (a high enough association

constant, Ka) and specificity to accomplish the performance requirements of the finished product. The degree of purification (e.g., none, IgG fraction, affinity-purified) will depend

on how the antibody is being used (i.e., for detector reagent conjugation or for immobilisation onto the membrane) and the amount of specific immunoglobulin required

for the application. Radioimmunoassay (RIA) is a technique for quantifying minutely small amounts of

biological substances such as enzymes, hormones, steroids, and vitamins in blood, urine,

saliva, or other body fluids. RIA requires three materials: a radioactively labelled preparation of the substance to be measured, antibody to this material, and a biological fluid

containing an unknown amount of the material. For example, to measure the amount of morphine in a blood sample a radioactively labelled solution of morphine is mixed with

antimorphine antibody. The amount of morphine that binds to the antibody is determined by counting the amount of radioactivity combined with the antibody. To measure the amount of

morphine in the blood sample, a small amount of the blood is mixed with the labelled

morphine and the antimorphine antibody. The amount of morphine in the blood sample is shown by the decrease in the amount of radioactive morphine bound to the antibody. The

RIA is now an important research tool, as well as being commonly used in hospitals to help diagnose diabetes, thyroid disorders, hypertension, reproductive problems, and drugs of

abuse.

112

SUBSTANCES EXTRACTED BY POLAR ORGANIC SOLVENTS

FROM ACIDIC AQUEOUS EXTRACTS

This group of toxic substances contains derivatives of barbituric acid (barbiturates),

xanthine derivatives (purine alkaloids) and some synthetic medicines.

BARBITURATES AND METHODS OF THEIR RESEARCH

In modern medicine is applied the large number of barbiturates (derivative of barbituric acid). Barbiturates represent one of groups of substances having the large

toxicological importance. Barbituric acid (malonyl urea) is not applied in medicine, but their

derivatives are widely used. The barbituric acid with alkalis forms salts. The acid properties of a barbituric acid

are caused by presence of hydrogen atoms in —NH-groups which is placed near carbonyl group —CO—.

Barbiturates are 5,5-disubstituted derivatives of barbituric acid (amobarbital, barbital, phenobarbital etc) and 1,5,5-trisubstituted barbituric acid (hexenal, hexobarbital, benzonal

etc.). The structure of barbituric acid and some barbiturates are shown below.

NH

NH

O

O

O

O

O

C 2 H 5

O

N

N

CO

Barbituric acid Benzonal

HN

N

ONa

O

O

C2H5

CH2 CH2 CH(CH3)2

HN

N

O

O

C2H5

C2H5

O

H

Amobarbital Barbital

Compound Chemical name

Amobarbital 5-ethyl-5-isopentylbarbituric acid

Barbital 5,5-diethylbarbituric acid

Benzonal 1-benzoyl-5-ethyl-5-phenylbarbituric acid

Hexenal 1,5-dimethyl-5-(cyclohexen-1-yl)-barbiturate of sodium

Pentobarbital 5-ethyl-5-(1-methylbutyl)-barbituric acid

Phenobarbital 5-ethyl-5-phenylbarbituric acid

Secbutabarbital 5-p-butyl-5-ethylbarbituric acid

Secobarbital 5-allyl-5-(1-methylbutyl)-barbituric acid

Thiopental 5-ethyl-5-(1-methylbutyl)-2-thiobarbituric acid

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Isolation from biological material. The methods were applied to barbiturates isolation

from biological material long time which were developed for alkaloids isolation. Last

decades for barbiturates isolation from objects of biological origin are offered special methods.

1. Isolation of barbiturates by acidified with oxalic or sulphate acid water. An optimal condition is pH 2. Extraction of barbiturates from infuses is provided with

chloroform at the same pH value. For extracts purification are used two techniques: 1) re-

extraction by 0,1 N sodium hydroxide solution or 2) gel-chromatography on sephadex gel. Obtained solutions use for detection and quantitative definition of barbiturates.

2. Isolation of barbiturates by alkalised water. For precipitation of impurity passing from biological material in extracts is applied sodium tungstate. Alkaline aqueous infuses

acidify by sulphate acid solution to рН 2 and shake with diethyl ether. Obtained ethereal extract uses for detection and quantitative definition of barbiturates.

Application. Action on organism. Barbiturates are potent hypnotics and sedative, but

in many countries only phenobarbital and (intravenous) thiopental find wide application nowadays. Barbiturates may also be used for euthanasia in veterinary medicine, and barbital

sodium is used as laboratory chemical, especially in buffer solutions. In acute poisonongs it may important to ascertain whether barbital or phenobarbital

(so-colled long-acting barbiturates), or a short- or medium-acting compounds has been taken. This is because alkaline diuresis can enhance excretion of barbital and phenobarbital,

but not other barbiturates.

Metabolism. The most part of barbiturates accepted dose eliminates from organism with urine in the not changed kind. About 45 % of barbiturates are exposed to various

transformations. Another metabolites of barbiturates are: 5-ethyl-5-(3-hydroxy-3-

methylbutyl)-barbituric acid – established for barbamyl, 5-ethyl-5-β-oxyethylbarbituric

acid, its glucuronide, and 5-ethylbarbituric acid – established for barbital.

Phenobarbital is metabolised by several ways. The basic metabolites of phenobarbital are 5-ethyl-5-p-hydroxyphenylbarbituric acid and p-oxyphenylbarbital. These metabolites

partially eliminate as glucuronides. Thiobarbituratres are exposed to N-desmethylation.

All these metabolites eliminate with urine.

DETECTION OF BARBITURATES

Colour Tests

There is no reliable simple test for these compounds and thin-layer chromatography

or a solvent extract of urine, stomach contents or scene residues best performs a qualitative

analysis. This should also permit identification of the type of barbiturate present if not the actual compound ingested.

Barbiturates presence can be detected with colour tests and precipitating reactions.

Preliminary test. For detection of barbiturates in urine apply reaction with cobalt acetate and lithium hydroxide.

Zwikker in 1931 has established, that after addition cobalt chloride and sodium or

barium hydroxide to barbiturates is formed the coloured compound. An optimal condition

114

for this reaction performance is spirituous environment. The run of this reaction prevents

water, which decomposes coloured compound. Therefore for this reaction performance all

used reagents are dissolved in absolute ethanol or methanol. A shade and intensity of colour depend on used alcohol that is explained various solvating effect. This reaction gives some

hydantoins, sulphanyl amide drugs, purines, pyrimidines etc. The occurrence of light-blue colour specifies on barbiturates presence in urine.

Reaction with isopropyl amine and cobalt salts. At interaction barbiturates with isopropyl amine and salts of cobalt the inner-complex compounds are formed:

At presence barbiturates there is a violet colour.

Reaction of murexide formation. At some barbiturates and thiobarbiturates presence this reaction gives products with pink colour. This reaction gives barbamyl,

barbital, phenobarbital, and thiopentale.

Phenobarbital also can be detected on reaction of p-nitrophenylethylbarbituric acid

formation. This acid has melting point 279 °С. This reaction does not give barbiturates, not containing phenyl group. The nitro-group in p-nitrophenyl-ethylbarbituric acid can be

reduced to amino-group, and then p-aminophenylethyl-barbituric the acid can be detected

by reaction of diazo-compounds formation. This reaction is specific for phenobarbital, but this reaction gives also benzonal.

Precipitation Reactions

Reaction with pyridine and copper salts. At interaction barbiturates with a pyridine

and copper salts are formed slightly soluble complexes. Under influence of a pyridine occurs enolisation and partial ionisation of

barbiturates. The pyridine with ions of copper forms the positively charged complex ion [Cu(Py)]

2+. At interaction of pyridine complex with ions of copper and ionised molecules of

barbiturates the inner-complex compound is formed:

115

The inorganic acids destroy this compound. The precipitate formed by barbiturates with

copper salts and pyridine can be amorphous or crystalline.

Another reagents for barbiturates precipitation are:

– chloro-zinc-iodine solution, – mixture of iron chloride and potassium iodide solutions,

– solution of potassium diiodocuprate in iodine solution. With these reagents barbiturates forms crystalline precipitates with various colours

and forms.

Detection of Barbiturates on Absorption Spectrums in UV-area

To the dry residue after evaporation of extracts from biological material or the

medicinal forms dissolve in ammonium hydroxide solution (рН~10) and quickly scan this solution on UV-spectrophotometer in the region 200 – 450 nm. Add two drops of

concentrated sulphate acid to the cell, mix using the plastic or glass paddle, and check pH (рН~2). Repeat the scan in the region 200 – 450 nm. Absorption maximum of 1,5,5- and

5,5-substitued barbiturates disappears. In these conditions absorption maximum of

thiobarbiturates are displaced to 290 and 239 nm. Addition of aqueous sodium hydroxide to the ammoniac extracts (pH~14) produces further characteristic spectral changes which can

be useful in qualitative work (figure).

Ultraviolet spectra of aqueous sodium barbital at different pH values

116

Quantitative Determination of Barbiturates

The described method will permit measurement of total barbiturate in a solvent extract of the specimen, and relies on the characteristic spectral shift shown by barbiturates

on going from pH 11 to pH 2. However, ideally a double-beam spectrophotometer is required. Accurate measurement of individual barbiturates normally requires gas-liquid or

high-performance liquid chromatography.

To perform a quantitative determination, measure the difference between absorbance at pH 10 and at pH 2, construct a calibration graph by analysis of the standard barbiturate

solutions, and calculate the barbiturate concentration in the sample. Alternatively, use the following formula:

C = [(DpH 10) – (DpH 2)] ⋅ f ⋅ 25

DpH 10 and DpH 2 – optical density at pH 10 and pH 2 relatively; C – concentration of barbiturate (mg/l);

f – dilution factor (if any).

XANTHINE DERIVATIVES

Xanthines are also called purines. These substances contain the condensed ring system of an imidazole and pyrimidine. In medicine are used such xanthine (2,6-

dioxypurine) derivatives as caffeine and theophylline, which are alkaloids:

N

N

N

N

H

H

H

O

O

N

N

N

N

O

O

H3C

CH3

CH3

N

N

N

N

H

O

O

H3C

CH3 Xanthine Caffeine Theophylline

For detection of xanthine derivatives apply reaction of ammonium salt of purpuric acid formation and reactions of group precipitation of alkaloids.

Reaction of formation of purpuric acid ammonium salt. Oxidisers (chlorine

water, bromine water, hydrogen peroxide, potassium chlorate KClO3 etc.) in acidic environment from xanthine derivative form mixture of mesoxalyl urea and dialuric acid.

After ammonia addition this components form methyl derivative of purpuric acid

(ammonium salt of tetramethyl purpuric acid - murexide), which have violet colour. For caffeine example:

117

N

N

N

CH3

CH3

CH3

O

O

N

NO

O

O

O

CH3

CH3

NH3

CH3

CH3

O

N

OO

O

OO

CH3

CH3

NH4

H2O2

CAFFEINE

Caffeine is 1,3,7-trimethylxanthen. Caffeine contains in coffee, tea, cola and other

beverages. Except for a caffeine specified plants contain also others derivative of a xanthine (theobromine, theophylline). Now caffeine not only extracts from plants, but also receives

by a synthetic way. In an alkaline condition the caffeine is decomposed with formation of physiologically

inactive caffeidine:

Caffeine Caffeidine

Application. Action on organism. The caffeine renders exciting action on the central

nervous system, weakens action of soporific and narcotic agents, raises an excitability of

spinal cord, raises respiratory and vasomotoric centers. Under caffeine influence cardiac activity amplifies. Caffeine is also used to treat neonatal apnoea. In medicine are applied the

caffeine base and its soluble double salts (Coffeinum - Natrium Benzoatum, Coffeinum - Natrium Salicylas).

Metabolism. The caffeine is quickly resorpted from the alimentary canal. About 85 % of an oral dose is excreted unchanged in urine. The caffeine is quickly decomposed in

organism (approximately 15 % of the accepted dose is decomposed during 1 hour) by N-

demethylation and oxidation. All caffeine metabolites (1-methylxanthine, 7- methylxanthine, 1,7-dimethylxanthine, a 1-methyl-uric acid, 1,3-methyl-uric acid) are

excreted with urine. Caffeine is important metabolite of theophylline in neonates, and in adults with impaired drug handling.

Detection of Caffeine

1. Caffeine gives positive murexide reaction.

2. Caffeine gives precipitates with Dragendorf's, Sonnechtain's, and Schaibler's reagents. 3. At warming (on a boiling water bath) caffeine solution with Nessler's reagent during 1 –

2 minutes forms a red-brown precipitate.

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THEOPHYLLINE

Theophylline (1,3-dimethylxanthen) is alkaloid of purine derivatives, which contains

in tea leafs. Now theophylline receives by synthesis.

Application. Action on an organism. Theophylline is applied in medicine as powder and tablets. Theophylline is a bronchodilatator widely used in the treatmant of asthma, often

as a mixture with ethylenediamine (aminophylline). Theophylline has the expressed diuretic

action. Theophylline is more toxic than caffeine. After reception of the large doses of theophylline the activity of the central nervous system and cardiovascular system is broken.

Metabolism. In organismt theophylline is metabolised to 3-methylxanthen, 1,3-dimethyluric acid (about 50 % of a dose), and 1-methyluric acid (about 20 % of a dose). All

these metabolites are excreted with urine.

Detection of Theophylline

1. Theophylline gives murexide reaction. 2. Theophylline gives reaction with diazoted sulphanylic acid.

3. Ethylenediamine gives a green colour in the o-cresole/ammonia urine test used to detect paracetamol, but in this case only indicates prior ingestion of aminophylline.

SALICYLIC ACID AND DERIVATIVES

COOH

OH

COOH

OCOCH3

COOH

OHH2N

Salicylic acid Acetylsalicylic acid 4-Aminosalicylic acid

OH

COOCH3

OH

CONH2

Methyl salicylate Salicylamide

Application. Action on organism. Metabolism. Salicylic acid (2-hydroxybenzoic acid). Salicylic acid is used topically to treat

various dermatological disorders. 10 – 20 % solutions of salicylic acid have keratolytic

action. Salicylic acid also suppresses a secretion of sweat glands. Salicylic acid and its derivatives are an active component of linden flores. Salicylic

acid contains in insignificant amounts in a cherry, raspberry, fragaria and other berries. It is the principal plasma metabolite of acetylsalicylic acid and can also arise from

the metabolism of methyl salicylate and salicylamide. Salicylic acid is excreted in the urine, mostly as a conjugate with glycine (salicyluric acid).

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Acetylsalicylic acid (aspirin). Acetylsalicylic acid is a frequently used salicylic acid

derivative. It is used as an analgesic and is also a metabolite of aloxipirin and benorilate.

The estimated minimum lethal dose in an adult is 15 g. Acetylsalicylic acid is rapidly metabolised by plasma esterases in vivo to salicylic

acid, which is then excreted in the urine, mostly as a conjugate with glycine (salicyluric acid).

4-Aminosalicylic acid (PAS). 4-Aminosalicylic acid is used in the treatment of

tuberculosis. Methyl salicylate. Methyl salicylate (oil of wintergreen) is a strong-smelling liquid at

room temperature and is widely used in topical medicinal products. On ingestion it is more toxic than acetylsalicylic acid because it is more rapidly absorbed. Deaths have occurred in

children after ingestion of as little as 4 ml; 30 ml is usually fatal in adult. Methyl salicylate is partially metabolised to salicylic acid in vivo.

Salicylamide. Salicylamide is used as analgesic. On hydrolysis, it forms salicylic

acid.

Detection of Salicylates

Salicylates give a distinctive purple colour with iron (III) ions and this reaction forms the basis of the test described.

Acetylsalicylic acid and methyl salicylate do not themselves react with iron (III) ions,

so that stomach contents and scene residues must be hydrolysed before analysis is performed. Salicylamide is only detectable after hydrolysis, even in urine samples.

Reaction with iron (III) chloride. With iron (III) chloride salicylic acid forms

complex compound, colour of which depends on pH. At pH 1,8–2,5 is formed monosalicylic blue-violet complex, at pH 4– 8 is formed disalicylic red-brown complex, and

trisalicylic yellow complex is formed at pH 8-11.

O

COO-

-

Fe

3-

3

Reaction of methyl salicylate formation. At salicylic acid heating with methanol at

the sulphate acid presence the methyl ether of salicylic acid (methyl salicylate) is formed.

The methyl salicylate has characteristic smell.

Preliminary assays on salicylic acid presence in urine and blood. For detection of salicylic acid in urine and the bloods are offered preliminary test based on reactions with

Trindler’s reagent and on reaction with solution of iron (III) nitrate in nitrate acid. The strong violet or purple colour indicate the presence of salicalates in researched objects.

Azide preservatives react strongly in the test, and weak false positives can be given by urine

specimens containing high concentrations of ketones (ketone bodies). The test is sensitive and will detect therapeutic dosage with salicylic acid,

acetylsalicylic acid, 4-aminosalicylic acid, methyl salicylate and salicylamide.

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PYRAZOLON-5 DERIVATIVES

This group of analgesic and antipyretic remedies consists from three derivatives of pyrasolon-5:

Phenazone (antipyrine) —1-phenyl-2,3-dimethylpyrosolon-5. Aminophenazone (amidopyrinum) is 1-phenyl-2,3-dimethyl-4-dimethylamino-

pyrazolon-5.

Noramidopyrinmethansulphonate (analgin) is 1-phenyl-2,3-dimethyl-4-methylamino-pyrasolon-5-N-mathamsulphonate of sodium.

NN O

H3C

H3C

C6H5

H

NN O

H3C

H3C

C6H5

N(CH3)2

NN O

H3C

H3C

C6H5

N

CH3

CH2SO3Na

H2O.

Phenazone Aminophenazone Noramidopyrinmethansulphonate

PHENAZONE

Application. Action on an organism. Phenazone is applied at neuralgias, rheumatic disease, chorea, and catarrhal diseases. This drug is analgesic, antipyretic and anti-

inflammatory.

Metabolism. Phenazone is quickly penetrates in blood from alimentary canal. The maximal level it in plasma is achieved through 1–2 hours. 5 % of phenazone dosage

eliminates from organism unchanged. About 30–40 % of phenazone conjugates with glucuronic acid and eliminates as glucuronide.

Detection of Phenazone

Reaction of nitrozoantipyrine formation. With nitrite acid is formed

nitrozoantipyrine, having green colour:

Reaction of azo dye formation. If to nitrozoantipyrine solution add α-

naphthylamine, than forms pyrasolic azo dye with red colour. Limit of detection is 2 µg of phenazone. This reaction is specific for phenazone detection. Aminophenazone does not

give this reaction.

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Reaction with iron (III) chloride. With iron (III) chloride phenazone forms

ferropyrine, which has red or orange-red colour.

AMINOPHENAZONE

Aminophenazone (amidopyrinum) is 1-phenyl-2,3-dimethyl-4-

dimethylaminopyrazolon-5. Application. Action on an organism. Aminophenazone is applied at headaches,

neuralgia, myositis, acute rheumatic disease, arthritises. Aminophenazone is an anlgesic and antipyretic, which is now little used since agranulocytosis, and renal tubular nesrosis may

occur after therapeutic dosage. Ingestion of about 10 g can cause severe acute poisoning in an adult. At long-term treatment arise oppression of a hemopoiesis and dermal eruptions.

Metabolism. Aminophenazone metabolised with desmethylation and acetylation.

Aminophenazone metabolites are 4-aminoantipyrine, methyl aminoantipyrine, rubeazonic and methyl rubeazonic acids. These acids have red colour and urine of the persons ingested

large doses of aminophenazone can have red-brown colour.

Detection of Aminophenazone

Reaction with iron (III) chloride. Aminophenazone with iron (III) chloride forms complex compound having violet colour.

Reaction with silver nitrate. Aminophenazone with silver nitrate forms complex compound having violet colour. At small amounts of aminophenazone silver ion can be

reduced to metal silver and falls black precipitate. Reaction with nitrite acid. With sodium nitrite and sulphate acid solutions arise

purple colour.

p-AMINOPHENOL DERIVATIVES

C2H5O NH CO CH3

HO NH CO CH3

Phenacetin Paracetamol

As antipyretic and analgesic media in medicine are used such p-aminophenol

derivatives: Phenacetin (acetophenetidin) – 1-ethoxy-4-acetaminobenzen

Paracetamol (acetaminophen) – N-acetyl-p-aminophenol.

PHENACETIN

Application. Action on an organism. It is applied at headache and neuralgia. Phenacetinum was previously used as an antipyretic and analgesic, but long-term use was

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associated with nephrotoxicity. Sometimes at treatment of phenacetin large dosages cases of

allergic reactions, methemoglobinemia, anemia are observed.

Metabolism. Phenacetin is metabolised with desalkylation and hydroxylation. It is largely metabolised to paracetamol. Paracetamol too is metabolised with p-aminophenol

formation. The part of phenacetin metabolites excreted with urine, and another part excreted as glucuronide or sulphate conjugates with urine.

Detection of Phenacetin

Ingestion of phenacetin can be detected in urine using the o-cresol/ammonia test on a

hydrolysed urine specimen.

Detection of phenacetin after hydrolysis. The majority of phenacetin detection

reactions is reduced to detection of p-aminophenol. p-Aminophenol presence can be detected in reactions of indophenolic and azo dyes formation.

Reaction of indophenolic dye formation. With anhydrite of chromate acid added to

hydrolysate arise cherry-red colour.

Reaction formation of ammonium salt of indophenolic dye. With phenol, lime

chloride and ammonia added to hydrolysate arise dark blue colour.

Reaction of azo dye formation. To hydrolysate add sodium nitrite, chloride acid,

and then β-naphthol. Forms the azo dye with red colour.

Reaction of ethyl acetate formation. At phenacetin heating with sulphate acid the acetic acid is formed. After addition ethanol to this solution is formed ethyl acetate having

characteristic smell. This reaction is not sensitive.

Reaction of 3-nitrophenacetin formation. Phenacetin heated with nitrate acid forms 3-nitrophenacetin, having yellow or orange colour.

In this reaction it is possible to distinguish phenacetin from antipyrin and acetanilide.

PARACETAMOL

Paracetamol (acetaminophen) is widely used analgesic and sometimes occurs in combination with other drugs such as dextropropoxyphene. It is a metabolite of phenacetin

and of benorilate, and is itself largely metabolised by conjugation with glucuronic acid and

sulphate prior to urinary excretion. Antidote for paracetamol intoxication is N-acetylcystein. This amino acid contains

SH-groups which competes with the same groups of S-proteins. Hydrolysis of the glucuronate and sulphate conjugates with concentrated

hydrochloric acid gives p-aminophenol, which can be conjugated with o-cresol to form a strongly royal blue coloured dye. This test is very sensitive and will detect therapeutic

dosage with paracetamol 24–48 hours later.

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Only aromatic amines, such as aniline, which also give rise to p-aminophenol in

urine after hydrolysis are known to interfere. Ethylenediamine (from aminophylline, for

example) gives a green colour in this test. Protein precipitation with trichloroacetic acid and subsequent treatment with nitrate

acid and spectrophotometric measurement of the nitrated derivative give a selective assay for paracetamol in plasma.

ALKALOIDS – INDOLE DERIVATIVES

Strychnine Bricune

Strychnine (strychnidine-10-on) and the related compound bricune (10,11-

dimethoxystrychnine) are highly toxic alkaloids derived from the seeds of Strychnos nux

vomica and other Strychnos species. These alkaloids are indole derivatives. Organic solvents both from acidic, and from alkaline aqueous solutions extract

strychnine and brucine. However lot of these alkaloids are extracted from alkaline aqueous solutions.

Application. Action on organism. In medical practice basically Strychnine nitrate and Tinctura of Nux vomica are applied. Strychnine raises the central nervous system, raises

reflectors excitability. In therapeutic doses strychnine stimulates sense organs, raises

vasomotor and respiratory centres, tonuses a skeletal muscles. Therefore strychnine is applied as a tonic agent. Large doses of strychnine cause strong tetanic cramps.

Brucine is not applied in medicine. However it enters into composition of Tinctura of Nux vomica. In case of the Tinctura poisoning both alkaloids are presented in corpses.

Brucine has the same pharmacological action as strychnine, but is less toxic. There are specific attributes of poisoning with these alkaloids: at first come

frequently replicating cramps, and after that comes death (at the phenomena of asphyxia).

Metabolism. Strychnine and brucine quickly absorb from alimentary canal, easily penetrate into blood through mucous and uninjured skin. About 80 % of indole alkaloids are

metabolised in liver. Other amounts of these alkaloids eliminate unchanged slowly with urine. Strychnine and brucine can cumulate in organism.

All metabolites of strychnine are not identified. Metabolites of brucine are methoxy-2-oxy-3-strychnine and its isomer oxy-2-methoxy-3-strychnine.

Detection of Strychnine and Brucine

Reactions with reagents of alkaloids precipitation. Both alkaloids give the same precipitates with Dragendorf's, Buschard's, Maier's, Schaibler's, and Sonnenstain's reagents,

and with concentrated nitrate acid.

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Reaction with potassium dichromate and sulphate acid. With these reagents both

alkaloids form dark blue compounds, which change later in purple, red, and then the colour

disappears. A limit of detection: 1 µg of strychnine in assay. This reaction prevents

morphine, quinine, nitrate acid.

Detection of Strychnine

Reaction with ammonium vanadate and sulphate acid (Mandelin's reagent). Mandelin's reagent with strychnine gives purple colour, which change to red and than to

yellow over 10 minutes. Large excess of acid prevents this reaction.

Detection of Brucine

Reaction with nitrate acide and tin (II) chloride. Concentrated nitrate acid gives with brucine blood-red colour passing in yellow. If to yellow solution add some drops of tin

(II) chloride solution, arise violet colour. A limit of detection: 14 µg of a brucine in assay.

Difference Strychnine from Brucine

In addition to the simple test with Mandelin's reagent, strychnine can be detected and

differentiated from brucine by thin-layer chromatography.

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SUBSTANCES EXTRACTED BY ORGANIC SOLVENTS

FROM ALKALISED AQUEOUS EXTRACTS

The largest group of toxic compounds is substances, which are extracted by organic

solvents from alkalised aqueous extracts. In this group are included alkaloids, which are derivatives of quinoline (quinine), isoquinoline (morphine, codeine, papaverine), pyridine

(anabasine, nicotine, arecoline), piperidine (coniine), tropane (atropine, scopolamine, cocaine), indole (strychnine, brucine, reserpine) etc.

The synthetic substances maid from morphine (apomorphine, dionine, heroin), synthetic drugs being derivatives of phenothiazine (chlorpromazine, diprazine),

benzodiazepine (chlordiazepoxide, diazepam, nitrazepam), p-aminobenzoic acid (novocaine

and dicaine) are also included in this group.

ALKALOIDS

Historically alkaloids were detected by colour tests with so called “alkaloid reagents”. Some examples are presented on the table.

Colour Tests on Alkaloids

Reagenst

Alkaloid Concentrated

H2SO4

Concentrated

H2SO4 with

HNO3

Concentrated

H2SO4 with

molybdenic

acid

Concentrated

H2SO4 with

vanadic acid

Concentrated

H2SO4 with

formaldehyde

Veratrine

yellow-orange

→ violet-red

(after staying)

yellow →

orange → violet-red

yellow →

violet-red

yellow →

violet-red –

Brucine – bloody-red red → yellow – –

Strychnine – yellowish – blue-violet –

Morphine – red violet red → blue-

violet violet

Heroine

(diacethyl morphine)

–

pale-yellow

→ green → blue

purple – red

Dionine

(ethyl morphine)

– orange green → blue – green → blue

→ blue-violet

Codeine

(methyl morphine)

red-violet

(after heating) brown-red →

yellow

light green →

light blue – violet

Papaverine blue-violet

(after heating) dark red

green → blue

(after heating) –

pink → violet-

red

Narcotine

green-yellow

→ yellow-red

→cherry-red (in few days)

red → violet-

red

blue-green →

cherry-red →

red (after heating)

–

violet →

green →

yellow

126

QUINOLINE DERIVATIVES – QUININE

N

N

CH3O

CH

OH

CH CH2

Quinine is the major alkaloid found in the bark of various species of Cinchona. In this tree except for quinine contains also chinidine, cinchonine and other alkaloids. Into

composition of quinine molecule enter quinolinic and quinuclidinic cycles connected by

group of atoms —CH(OH)—. The quinine is isomer of quinidine. In medical practice are applied quinine hydrochloride and sulphate.

Quinine Biotransformation

N

N

CH3O

CH

OH

CH CH2

N

N

CH3O

CH

OH

COOH

N

CH3O

CO-COOHN

N

CH3O

CH

OHOH

CH CH2

N

N

CH3O

CH

OH

OH

CH CH2

hydroxylation

oxidising break

oxidising side chain

2-oxyquinin

2'-oxyquinin quinetine

gemoquinic acid

hydroxylation

Application. Action on organism. Quinine is one of the most effective and widely

used in the treatment of malaria. It is also used to treat night cramps and is a constituent of tonic water. The fatal dose of quinine in adult may be as little as 8 g. Its isomer quinidine is

127

used as an antiarrhythmic. The quinine is applied in obstetric practice to exaltation and

intensifying of patrimonial activity. Quinine overdose can initiate abortion in pregnant

women. Depending on the accepted dose quinine can cause central nervous system oppression, headache, giddiness, and infringement of vision. The quinine raises a

musculation of a uterus and strengthens its reduction. It causes reduction of a lien. Metabolism. In an organism both compounds are metabolised by oxidation of

quinolinic and quinuclidinic cycles. Thus are formed 2-oxyquinine and dioxyquinine.

Metabolites and unchanged quinine eliminate in urine.

Detection of Quinine

Preliminary test on presence of quinine in urine. Quinine from alkalised urine

extracts to chloroform and than – re-extracts to solution of sulphate acid. Arises blue

fluorescence. The fluorescence becomes more expressed, if acidic extract to irradiate with UV-light. The fluorescence of quinine depends from pH. In acidic solution quinine has

light-blue fluorescence. In an alkaline condition (рH~9) the quinine has violet fluorescence. The products of quinine oxidation have yellow-green fluorescence.

Reactions with reagents of group precipitation of alkaloids. The quinine gives

specific sign precipitates with Buschard's, Dragendorf's, Maier's, Sonnenstain’s reagents.

Taleioquine reaction. On addition to quinine bromine water and then ammonia is

formed emerald-green colour taleioquine, which can be extracted by chloroform:

N

N

CH3O

CH

OH

CH=CH2

N

N

CH

OH

O

O

CHBr-CH2Br

+ NH4OH

- NH4Br

+ Br2

N

CHOH-CH2OH

N

CH

OH

NH

NHN

CHOH-CH2OH

N

CH

OH

N

OHN

CHOH-CH2OH

N

CH

OH

N

OH

Reaction reproducibility depends on concentration of researched substance. The

reaction prevents pyrazolone-5 derivatives, caffeine etc.

Eritroquine reaction. With solution of potassium hexacyanoferrate (III) and

bromine water in weak acidic environment quinine forms compound having pink or red-

violet colour.

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ISO-QUINOLINE DERIVATIVES

OPIUM

The opium represents a complex mixture of alkaloids soporific poppy (Papaver

somnifer). Dried latex (juice) of not ripened boxes (heads) of soporific poppy is applied in

medicine under the name opium. Opium contains more than 20 alkaloids. Opium can

contain from 2–3 to 15–20 % of alkaloids, main of which are morphine, codeine, narcotine, paramorphine and papaverine. Meconic acid and meconine accompany alkaloids in opium.

In medicine are used pure opium alkaloids: morphine, codeine, and papaverine:

O

OH

OH

CH3

O

CH3O

OH

CH3

N

CH2

CH3O

CH3O

O

O

CH3

CH3

Morphine Codeine Papaverine

MORPHINE

Morphine is the principal alkaloid of opium and is a potent narcotic analgesic. In

opium contains 3 – 20 % of this alkaloid. Application. Action on organism. In medicine is used morphine hydrochloride.

Morphine is strong analgesic. It depresses an excitability of pain centres and has antishock action at traumas. Morphine causes euphoria. At repeated application morphine morbid

predilection (morphinism) quickly develops. Morphine slowly absorbs in blood through alimentary system.

Metabolism. Morphine is a metabolite of codeine. Approximately 5 % of dose of

morphine are metabolised to normorphine, but conjugation with glucuronic acid is the major pathway. The principal product is morphine-3-glucuronide, but morphine-6-glucuronide is

also formed. Free morphine in urine accounts for about 10 % of a dose, while morphine-3-glucuronide accounts for 75 %. The estimated minimum fatal dose of morphine or

diamorphine in an adult unaccustomed to taking these compounds is 100-200 mg.

Detection of Morphine

Reactions with reagents of group precipitation of alkaloids. Morphine gives

deposits with reagents of group precipitation of alkaloids (Buschard's, Dragendorf's, Maier's, Sonnenstain’s reagents).

Colour tests. Morphine forms colour compounds with concentrated nitrate acid and

with Mandelin's, Marqui’s, Freude's, and Erdmann's reagents.

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Pellagrie's reaction. At heating with concentrated hydrochloric and sulphate acids

morphine and codeine transform to apomorphine. The obtained compound reacts with

iodine alcohol solution and gives green colour. If reaction product to extract in diethyl ether, it colour changes in purple-red. This reaction gives also ethyl morphine (dionine) and

heroine.

Reaction with iron (III) chloride. Morphine forms dark blue compound with iron

(III) chloride. Reaction with iodate acid (HIO3). With a solution of iodate acid or solution of

potassium iodate (KIO3) morphine forms precipitate. This precipitate dissolves in chloroform giving violet colour.

Reaction with potassium hexacyanoferrate (III) and iron (III) chloride. This

reaction is based on reaction of potassium hexacyanoferrate (III) reducing by morphine.

Formed (reduced) potassium hexacyanoferrate (II) interacts with iron (III) chloride giving Prussian blue.

Photocolorimetric determination of morphine. Quantitative determination of

morphine is based on reaction with silicomolybdenic acid, as a result of which is dark blue colour compound. This method allows determining from 0,2 mg up to 4 mg of morphine in

sample.

CODEINE

Codeine is monomethylic ether of morphine. In opium contains 0,2 – 2 % of codeine. Application. Action on organism. Codeine is a narcotic analgesic. Codeine less than

morphine oppress respiration and used as cough medicine.

Metabolism. Codeine is metabolised by O-demethylation and N-demethylation to give morphine and norcodeine, respectively, and by conjugation to form glucuronides and

sulphates of both parent drug and metabolites. The estimated fatal dose of codeine in an adult is 800 mg. However, codeine is much less toxic than morphine, and death directly

attributable to codeine is rare.

Detection of Codeine

Preliminary tests on presence of Codeine in urine. 1. On filter paper execute reaction with Brand’s reagent. Codeine gives red colour

passing in blue-violet. 2. On filter paper execute reaction with ammonium vanadate solution in sulphate

acid. Codeine gives green colour passing in dark blue.

Reactions with reagents of group precipitation of alkaloids. Codeine forms

specific precipitates with Dragendorf’s, Buschard’s, Maier's reagents.

Colour rtests. Codeine with Mandelin's, Marqui’s and Freude's reagents gives

coloured products.

130

Pellagrie's reaction. Codeine gives Pellagrie’s reaction.

Quantitative determination of codeine. For codeine determination is used

extraction-photocolorimetric assay. The method of photocolorimetric determination of codeine is based on reaction with tropeolin 00, at which is formed purple ionic associate

extracted by chloroform. This method enables to determine from 0,2 mg up to 2,0 mg of

codeine in sample.

PAPAVERINE

Opium contains 0,1 – 1,5 % of papaverine. Now papaverine is received synthetically.

Application. Action on organism. Papaverine is spasmolytic medicine.

Metabolism. Papaverine metabolises mainly by demethylation. Thus the phenolic bonds are formed glucuronides, which eliminate with urine.

Detection of Papaverine

Reactions with reagents of group precipitation. The papaverine forms precipitates

with reagents of group precipitation of alkaloids.

Colour tests. The papaverine gives reactions with Mandelin's, Marqui’s, Freude's,

and Erdmann's reagents.

Reaction with cadmium chloride. With cadmium chloride papaverine forms

crystals having form of thin plates shaped cube.

PYRIDINE DERIVATIVES – NICOTINE

N

N

CH3

Nicotine is an alkaloid derived from the leaves of Nicotiana tabacum. At room

temperature nicotine is liquid and can be distilled with water steam. Application. Action on organism. As little as 40 mg of nicotine can prove fatal in an

adult. Nicotine is commonly encountered in tobacco although usually in concentrations insufficient to cause acute poisoning, except when ingested by young children. Nicotine

occurs in higher concentrations in some herbal medicines, and is also used as fumigant in horticulture.

Metabolism. Nicotine can be absorbed rapidly through the skin, and is metabolised

principally by N-demethylation to give cotinine. In organism nicotine is decomposed mainly in liver. The mentioned above metabolites of nicotine eliminate with urine.

131

Detection of Nicotine

Reaction with Dragendorf's reagent. With Dragendorf's reagent nicotine forms crystalline precipitate. These crystals signed as letter T, or letter X, or as flying birds. Limit

of detection is 1 µg in sample.

Reaction with Raineke's salt. With Raineke’s salt nicotine forms prismatic crystals.

Limit of detection is 1,2 µg in sample. Reaction with iodine solution in diethyl ether. With reagent nicotine forms resin

kind precipitate containing needle ruby-red crystals with a dark blue shade

Reaction with formaldehyde. With formaldehyde and concentrated nitrate acid

nicotine gives red or pink colour.

Reaction with p-dimethylaminobenzaldehyde. With p-dimethylaminobenz-

aldehyde nicotine forms compound with pink colour, which passes in violet. The colour is

kept about day.

Other reactions on nicotine. The nicotine can be detected on reactions with

Buschard’s reagent, solution of vanillin and hydrogen peroxide.

PIPERIDINE DERIVATIVES – CONIINE

NH

CH3

Coniine (1-propylpiperidine) is alkaloid contained in Conium. Coniin is colourless liquid with strong smell of mice urine. Coniine is decomposed on air, as a result it has

brown colour. It can be distilled with water steam without decomposing. Application. Action on organism. In connection with a high toxicity coniine not

apply in medicine. The toxic properties of coniine were known in antiquity. According to

the literary data, in Ancient Greece coniine was applied to poisoning the philosopher Socrates (469 — 399 ad AD).

Now there are casual poisonings with plants containing coniine. It leaves confuse with leaves of parsley.

Coniine quickly absorbs in blood from the alimentary canal. Coniine causes paralysis of terminations motor nerves. Coniine at first excites, and then paralyses the central nervous

system. Death comes from respiration paralysis.

Detection of Coniine

Reactions with reagents of group precipitation of alkaloids. Coniine gives precipitates with Buschard’s, Dragendorf’s, and Maier’s reagents.

132

Reaction of copper dithiocarbamate formation. Coniine and other secondary

amines with carbon disulfide and ammoniac solution of copper sulphate form insoluble in

water dithiocarbamates:

+ CS2 + Cu2+ + OH_

N

CH3

NH

CH3

_

CS

SCu / 2 + H2O

Coniine gives brown or yellow colour in benzene solution. Limit of detection is 1 µg

in sample.

This reaction gives other secondary amines, ephedrine as example.

Reaction with Dragendorf’s reagent. Coniine with Dragendorf’s reagent forms

orange-red crystals shaped of rhombuses or parallelograms. Limit of detection is 3,5 µg in

sample.

Sublimation of coniine hydrochloride. After sublimation from chloroformic

solution coniine condenses in colourless needles. Limit of detection is 0,33 µg in sample.

TROPANE DERIVATIVES

NCH3

O

CH2OH

O

NCH3

O

CH2OH

O

O

Atropine Scopolamine

NCH3

O

CH2OH

O

COO-CH3

Cocaine

ATROPINE

Atropine is an alkaloid contained in plants such as Atropa belladonna and Datura

stramonium. Atropine is ester of alcohol tropine and tropanic acid. A stereoisomer of

atropine is Hyoscyamine is rotating stereoisomer of atropine. L-rotatory hyoscyamine is less physiologically active than atropine.

133

Application. Action on organism. In medical practice is used atropine sulphate. It has

potent anticholinergic activity and is used to reduce bronchial and salivary secretion before

anaesthesia, to treat gastrointestinal spasm and to produce mydriasis in ophthalmic procedures. Atropine is also used as an antidote to poisoning with inhibitors of

cholinesterase, such as some organophosphorous pesticides, carbamat pesticides and some chemical warfare agents. Atropine is very potent and does of 10 mg or more can cause

severe poisoning.

Atropine is quickly absorbed through mucous, skin, intestine (but not through a stomach).

Metabolism. Atropine is decomposed in organism to tropine and tropanic acid. However this decomposing is not the basic pathway of atropine metabolism. Testifies that

only about 2 % of tropanic acid eliminate with urine. In urine it is revealed 3, and in liver 4 metabolites of atropine, which are not identified. About 50 % of atropine eliminate with

urine in the not changed kind.

Detection of Atropine

Reactions with reagents of group precipitation of alkaloids. Atropine gives

specific precipitates with Buschard’s, Dragendorf's, and Maier's reagents.

Vitalie-Moren's reaction. This reaction is based on decomposition of atropine with

nitrate acid. Are formed tropine and 1-phenyl-hydracrylic acid. 1-Phenyl-hydracrylic acid formes trinitroderivative having yellow colour:

At action of alkali on trinitroderivative of 1-phenyl-hydracrylic acid arises violet

colour:

Limit of detection is 1 µg in sample. Except for atropine this reaction gives

hyoscyamine, scopolamine, veratrine, and strychnine.

Reaction with p-dimethylaminobenzaldehyde and sulphate acid. With solution of

p-dimethylaminobenzaldehyde in concentrated sulphate acid atropine gives red colour, which passes in cherry-red, and then in violet. This reaction gives hyoscyamine and

scopolamine. At presence of morphine and codeine arise red colour, which does not pass in violet. Cocaine does not give colour with p-dimethylaminobenzaldehyde.

134

Reaction with Raineke's salt. With Raineke's salt {NH4[Cr(NH3)2(SCN)4]} atropine

forms amorphous precipitate quickly passing in purple colour crystalline. Limit of detection

is 0,1 1 µg in sample.

Reaction with nitroxanthic acid. Atropine with solution of nitroxanthic acid gives

light yellow crystalline precipitate as plates or dendrites from them. Limit of detection is 5

µg in sample.

SCOPOLAMINE

Scopolamine (hyoscine) is an alkaloid contained in plants such as Datura

stramonium and Scopolia carniolica. Scopolamine is an ester of alcohol scopine and tropanic acid. In medicine is used scopolamine hydrobromide.

Application. Action on organism. Scopolamine similarly to atropine causes mydriasis, paralysis of an accommodation, relaxation of sleek muscles, decrease of a

secretion digestive and sweat glands. Scopolamine is used as antiemetic and calmative at

sea and airs illnesses. Metabolism. Scopolamine is metabolised by hydrolysis. The major amount of

scopolamine is decomposed in liver and is eliminated from organism with urine.

Detection of Scopolamine

Scopolamine gives the same reactions, which are applied for atropine detection (Vitalie-Moren's reaction, reaction with p-dimethylaminobenzaldehyde and with Raineke's

salt).

Reaction with hydrogen tetrabromoaurate. Scopolamine with hydrogen

tetrabromoaurrate forms light brown, yellow or orange-red crystals (gear dendrites). A limit

of detection is 1 µg in sample.

COCAINE

Cocaine is an alkaloid obtained from coca, the dried leaves of Erythroxylon coca and

other species of Erythroxylon, or by synthesis from ecgonine. Chemically cocaine is methyl ether of benzoylecgonine.

Application. Action on organism. Cocaine is applied in medicine as a hydrochloride. Cocaine hydrochloride is an effective local anaesthetic when used at concentrations of 10-

200 g/l, but normally only applied topically because of the risk of the systemic toxicity if given by other routes.

Injection or inhalation into the nasal passages (sniffing, snorting) frequently abuses

cocaine. Cocaine free-base (crack) is very rapidly absorbed when inhaled into the nasal passages or smoked. Absorbed cocaine acts on the central nervous system. It causes

euphoria, exaltation, and then oppression of the central nervous system. At often reception of cocaine the morbid predilection (cocainism) develops. Cocaine is administered with

epinephrine for decrease of absorption rate and elongation of the anaesthesia period.

135

Ingested cocaine has less effect owing to hydrolysis in the gastrointestinal tract. The

estimated minimum fatal dose in an adult is 1-2 g, but addicts may tolerate up to 5 g/day.

Metabolism. Cocaine basically is metabolised in liver. Metabolites formed at it eliminate with urine. The principal metabolites are benzoylecgonine, ecgonine and ecgonine

methyl ester. Only 1-9 % of an intravenous dose is excreted in urine as cocaine, while 35-55 % is excreted as benzylecgonine.

Detection of Cocaine Reaction with reagents of group precipitation of alkaloids. Cocaine gives

precipitates with Maier's, Buschard’s, and Dragendorf's reagents and with nitroxanthic acid.

Reaction with potassium permanganate. With solution of potassium permanganate

cocaine forms red-violet crystals shaped of rectangular plates and dendrites. Limit of

detection is 4 µg in sample.

With potassium permanganate the crystalline precipitates give scopolamine, aconitine, cotarnine, berberine and hydrastine. However form of these crystals are different.

Reaction with hydrogen hexachloroplatinate. Cocaine forms light yellow crystals

with hydrogen hexachloroplatinate. Limit of detection is 33 µg in sample.

Reaction of ethyl benzoate formation. Cocaine hydrolyses by concentrated

sulphate acid to benzoic acid. Benzoic acid forms with ethanol ethyl benzoate having

characteristic smell. This reaction has little sensitivity and is used only for detection of cocaine presence in powders.

EPHEDRINE

Ephedrine is acyclic alkaloid, in which amino-group is in lateral chain. Application. Action on organism. Ephedrine is a sympathomimetic agent. It raises

arterial pressure, narrows vessels, dilates a pupil and bronchus, reduces intestine peristalsis, and raises the central nervous system. In medicine the ephedrine is used at bronchial asthma

and in ophthalmology practice. The estimated minimum lethal dose of ephedrine in an adult

is 4 g, but fatalities are rare. Metabolism. Ephedrine is metabolised by N-demethylation to norephedrine

(phenylpropanolamine), and by oxidative deamination and conjugation. Ephedrine is itself a metabolite of methylaphedrine. Ephedrine metabolites eliminate from organism with urine.

136

Detection of Ephedrine

Reaction with copper salts and carbon disulfide. At interaction of ephedrine with carbon disulfide and alkaline solution of copper sulphate forms derivative of dithiocarbamic

acids, soluble in benzene:

At presence of ephedrine the benzene solution gets brown or yellow colour. Limit of

detection is 2 µg in sample. This reaction gives also coniine.

Reaction with 2,4-dinitrochlorbenzene. Ephedrine and other compounds with OH-

group in α-position and amino-group in a β-position at heating undergo a hydramine

decomposition. Thus is formed phenyl ethyl ketone and amine:

The formed methyl amine with 2,4-dinitrochlorbenzene gives compound with yellow colour, which is extracted by chloroform:

Limit of detection is 5 µg in sample.

Reaction with Dragendorf's reagent. Ephedrine with Dragendorf's reagent forms

needle-like crystals. Limit of detection is 1,6 µg in sample.

137

SUBSTANCES EXTRACTED BY ORGANIC SOLVENTS

FROM ALKALISED AQUEOUS EXTRACTS

(Synthetic Medicines)

p-AMINOBENZOIC ACID DERIVATIVES

H2N COO (CH2)2 N(C2H5)2 HCl

Procaine – ether of p-diethylaminoethanole and p-aminobenzoic acid

HN COO (CH2)2 N(CH3)2 HClC4H9

Tetracaine – ether of p-dimethylaminoethanole and p-butylaminobenzoic acid

PROCAINE Application. Action on organism. Procaine will widely be utilised in medicine as

local anaesthetic. It is less active, than cocaine. After absorption in a blood procaine depresses an excitability of peripheral cholinoreactive systems, reduces spastic strictures of

sleek muscles, and depresses an excitability of heart muscle. In toxic doses procaine causes exaltation, and then paralysis of the central nervous system.

Metabolism. Procaine is a non-resistant drug. In an organism it breaks up to p-

aminobenzoic acid and diethyl aminoethanole. The both procaine metabolites eliminate in urine. The part of p-aminobenzoic acid eliminates as glucuronide.

Scheme of Procaine Biotransformation

NH2

CO-NH CH2

N(C2H

5)

2

N R

H

OHRN

H

O

CH3

C

Procainamid

N-Hydroxy-ProcainamidN-Acetyl-Procainamid

138

Detection of Procaine

Reaction of azo dye formation. With sodium nitrite (in acidic environment)

procaine forms diazonium salt. This salt with β-naphthol in alkali environment transform to

red-orange azo-compound.

Reaction with Dragendorff's reagent. Dragendorff's reagent with procaine forms

precipitate of red-brown rectangular plates.

TETRACAINE

Application. Action on organism. Tetracaine is applied to larynx anaesthesia at intubation, at various invasive diagnostic procedures. Also it used in ophthalmology.

Tetracaine is stronger local anaesthetic agent as procaine and cocaine. However, Tetracaine is in 2 times more toxic than cocaine and in 10 times than procaine.

Metabolism. Tetracaine metabolite is the p-aminobenzoic acid.

Detection of Tetracaine Vitalie-Moren's reaction. With nitrate acid at heating dicaine forms compound

having yellow colour. Addition of alcohol alkali solution change colour to red.

Reaction with sodium nitrite. From concentrated solution of sodium nitrite dicaine

crystallise as thin prisms.

PHENOTHIAZINES These compounds are derivatives of phenothiazine, which itself is used as am

anthelminthic in veterinary medicine. Some commonly encountered phenothiazines are listed in table:

Compound Chemical name

Chlorpromazine 3-(2-chlorophenothiazin-10-yl)-N,N-dimethylpropylamine

Chlorprothixene (Z)-3-(2-chlorothioxanthen-9-ylidene)-N,N-dimethylpropylamine

Dimetotiazine 10-(2-dimethylaminopropyl)-N,N-dimethylphenothiazine-2-sulfonamide

Prochlorperazine 2-chloro-10-[3-(4-methylpiperazin-1-yl)-propyl]-phenothiazine

Promazine N,N-dimethyl-3-(phenothizin-10-yl)-propylamine

Thioridazine 10-[2-(1-methyl-2-piperidyl)-ethyl]-2-methylthiophenothiazine

Phenothiazines are widely used as antihistamines, tranquillisers, and in various

psychiatric disorders. There are often extensively metabolised. Chlorpromazine, for example, has over 50

metabolites in human. The main metabolites of phenothiazines are sulphoxides.

139

N

S

H

Phenothiazine

HCl

N

S

H2C (CH2)2N(CH3)2

Chlorpromazine

N

S

CH

2

CH

N

CH3

CH3

CH3

HCl.

Promethazine

HCl.

N CH3

CH3

N

S

CH

2

CH

CH3

CH

2

O CH3

Levopromazine

Preliminary tests on phenothiazines presence in urine. The test with FPN reagent (mixture of iron(III) chloride solution, perchloric and nitrate acids) is based on the reaction

of many of phenothiazines with iron ion under acidic conditions. The occurrence of pink

colour specifies presence of phenothiazine derivatives in urine.

CHLORPROMAZINE

Reaction with concentrated sulphate acid. Chlorpromazine with concentrated

sulphate acid gives purple-red colour.

Reaction with concentrated nitrate acid. Chlorpromazine with concentrated nitrate

acid gives purple-blue colour.

Reaction with concentrated hydrochloric acid. Chlorpromazine with concentrated

hydrochloric acid gives pink, passing in red-violet colour.

Reaction with Marquis reagent. Chlorpromazine with Marquis reagent gives purple

colour.

Reaction with Mandelin's reagent. Chlorpromazine with this reagent gives green

colour passing in purple.

PROMETHAZINE

Reaction with concentrated sulphate acid. Promethazine with concentrated sulphate acid gives purple-red colour.

Reaction with concentrated nitrate acid. Promethazine with concentrated nitrate

acid gives purple-red colour passing in yellow.

140

Reaction with concentrated hydrochloric acid. Promethazine with concentrated

hydrochloric acid gives pink-violet colour passing in purple-violet.

Reaction Vitalie-Moren's. Promethazine gives reaction Vitalie-Moren's.

Reaction with Marquis reagent. Promethazine with Marquis reagent gives purple

colour.

Reaction with Mandelin's reagent. Mandelin's reagent with promethazine gives

green colour passing in purple.

LEVOPROMAZINE

Reaction with Marquis and Freude's reagents. Levopromazine with both Marquis and Freude's reagents gives blue-red colour.

Reaction with Mandelin's reagent. Levopromazine with Mandelin's reagent gives

red-violet colour.

BENZODIAZEPINES

Most of these compounds have the general structure shown below. There are over 60 members of this group. Some common benzodiazepines are listed in table.

Compound Chemical name

Chordiazepoxide 7-chloro-2-methylamino-5-phenyl-3H-1,4-benzodiazepine 4-oxide

Clobazam 7-chloro-1-methyl-5-penyl-1H-1,5-benzodiazepin-2,4(3H,5H)-dione

Clonazepam 5-(2-chlorophenyl)-1,3-dihydro-7-nitro-2H-1,4-benzodiazepin-2-one

Diazepam 7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4- benzodiazepin-2-one

Flurazepam 7-chloro-1-(2-diethylaminoethyl)-5-(2-fluorophenyl)-1,3-dihydro-

2H-1,4-benzodiazepin-2-one

Lorazepam 7-chloro-5-(2-chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one

Nitrazepam 1,3-dihydro-7-nitro-5-phenyl-2H-1,4-benzodiazepin-2-one

Oxazepam 7-chloro-1,3-dihydro-3-hydroxy-5-phenyl-2H-1,4-benzodiazepin-2-one

Temazepam 7-chloro-1,3-dihydro-3-hydroxy-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one

Application. Action on organism. Benzodiazepines are used as tranquillisers, and

clobazam, clonazepam, and diazepam are also used as anticonvulsants. Temazepam especially has been abused, often together with other drugs. All benzodiazepines potent the

action of analgesic, sedative, and hypnotic remedies.

141

N

N

HN-CH3

Cl

OC6H5

Chlordiazepoxide

N

NCl

OCH

3

C6H5

Diazepam

NH

N

O

O2N

C6H5

Nitrazepam

NH

N

OH

O

Cl

C6H5

Oxazepam Metabolism. Most benzodiazepines are extensively metabolised and many members

of this group are in fact metabolites of other compounds. Thus, diazepam gives nordazepam, oxazepam (3-hydroxynordazepam), and temazepam (3-hydroxydiazepam), which are

excreted in urine as glucuronide or sulphate conjugates.

Detection of Benzodiazepines

Preliminary test on benzodiazepines presence in blood and urine. There is no reliable colour test for these compounds. However, on hydrolysis by concentrated

hydrochloride acid most benzodiazepines and their conjugates give rise to

amonibenzophenones. Formed 2-aminobenzophenones with N-1-naphthylethylenediamine form cherry-red azo-dyes. As diazepoxide, for example:

Cl O

NH2N

NCl

CH3

C2H5

O

NaNO2+H+

+

Cl O

N N+

+

Cl O

N N+

+

Na2CO3+

N (CH2)2 NH2

Cl O

N N

N (CH2)2 NH2

Instead of N-1-naphthylethylenediamine can be used α- or β-naphthole. Formed aza-dye has orange colour. This reaction can be applied for photocolorimetric quantification of

benzodiazepines.

142

Reaction with ninhydrin. With spirituous solution of ninhydrin benzodiazepines

form colour compounds: diazepamum – red or orange-red, nitrazepamum – yellow-brown,

and chlordiazepoxide – brown.

Reaction with Marquis reagent. Different benzodiazepines give products with

various colours from yellow to red.

Reaction with Freude's reagent. Different benzodiazepines give products with

various colours from orange to purple.

Also formed on hydrolysis amonibenzophenones can be extracted and analysed by thin-layer chromatography. Two different spray reagents are used to increase the

discriminating power of the method, p-dimethylaminocinnamaldehyde and nitrite acid and N-(1-naphthyl)ethylenediamine (the Bratton-Marshall reaction).

BUTYROPHENONE DERIVATIVES

HALOPERIDOL

(CH2)3C

O

F N

OH

Cl

Application. Action on organism. Haloperidol (4-[4-(4-chlorophenyl)-4-

hyrrohypiperidino]-4'-fluorobutyrophenone) is a neuroleptic used orally or parenterally to treat schizophrenia and a variety of other disorders.

Metabolism. Haloperidol is slowly excreted in urine following oral dosage, about

40 % being eliminated within 5 days, about 1 % as unchanged.

Detection of Haloperidol

Reaction with m-dinitrobenzen. With m-dinitrobenzen haloperidol forms soluble

compound having violet colour.

(CH2)3C

O

F N

OH

Cl

NO2

NO2

+

143

C

O

F

NO2

NO2

CH (CH2)2 N

OH

Cl

KOH+

C

O

F C (CH2)2 N

OH

ClNO2

O

OKN

+

_

Reaction with 2,4-dititrophenylhydrazine. Haloperidol with 2,4-

dititrophenylhydrazine forms yellow crystalline precipitate.

(CH2)3C

O

F N

OH

Cl

NO2

NO2

+

NH NH2

CF (CH2)3 N

OH

Cl

NO2

NO2

N NH

ISONICOTINIC ACID DERIVATIVES

ISONIAZIDE

Isoniazide – hydrazide of isonicotinic acid

N CO NH NH2

144

Application. Action on organism. Isoniazide is used in the treatment of tuberculosis.

Metabolism. The principal metabolic reaction is acetylation, but other reactions

include hydrolysis, conjugation with glycine and N-methylation. Up to 70 % of a dose is excreted in urine, largely as metabolites. Serious toxicity may occur in adults with doses of

3 g.

Scheme of Iproniazide biotransformation

N

CO(NH2)CH(CH

3)

2

N

COOH

+ (CH3)2CHNHNH2

isopropyl hydrazine

isonicotinic acid

N

CONHNH2

acethylation

acetyl isoniazide

aceton

+ CH3COCH3

dealkylation

conjugationwith glycine

isonicotinuric acid

hydrolysis

Detection of Isoniazide Reaction with phosphorusmolybdenic acid. Isoniszide reduces and forms

molybdenic blue.

The colorimetric procedure based on reaction with metaphosphoric acid and

sodium nitroprusside can be used to measure the plasma isoniazid concentration if overdosage with this drug is suspected.

145

Scheme of Nitrazepam Biotransformation

NH

N

O

C6H

5

O2N

NH

N

O

C6H

5

O2N

O

COOH

O

OH

OH

OH

NH

N

O

C6H

5

O2N

OH

NH

N

O

C6H

5

NH2

NH2

C6H

5

O2N O

NH2

C6H

5

NH2

O

NH

N

O

C6H

5

NH2

OH

NH

N

O

C6H

5

NH2

O

COOH

O

OH

OH

OHNH

N

O

C6H

5

NH

CH3

O

OxidationConjugation withglucuronic acid

Hydrolysis

Hydrolysis

Reducing

Oxidation

Conjugation withglucuronic acid

Conjugation (Acetylation)

146

SUBSTANCES DETERMINED DIRECTLY

in BIOLOGICAL MATERIAL

CARBON MONOXIDE

Carbon monoxide (CO) is an important constituent of coal gas, but is not present in natural gas. Nowadays, common sources of carbon monoxide are automobile exhaust

fumes, improperly maintained or ventilated gas or fuel oil heating systems, and smoke from

all types of fires. Carbon monoxide is also produced in vivo from the metabolism of dichloromethane.

Carbon monoxide is highly poisonous and combines with haemoglobin and other haem proteins such as cytochrome oxidase, thereby limiting the oxygen supply to tissue and

inhibiting cellular respiration. The affinity of carbon monoxide for haemoglobin is about 200 times that of oxygen. Thus, severe acute or acute-on-chronic poisoning can occur, when

relatively small quantities of carbon monoxide are present in the inspired air.

Features of acute carbon monoxide poisoning include headache, nausea, vomiting, haematemesis, hyperventilation, cardiac arrhytmias, pulmonary oedema, coma and acute

renal failure. Cyanosis is commonly absent, so that skin and mucosae remain pink even in the presence of severe tissue hypoxia. Death often ensues from respiratory failure. Late

neuropsychiatric sequel are an increasingly recognised complication. Treatment consists of removed from the contaminated atmosphere and administration

of 100 % oxygen via a well-fitting facemask. Hyperbaric oxygen may be indicated in

certain cases, and is especially effective in preventing the development of late sequel, but facilities where this can be given are rare. A simple guide to the interpretation of blood

HbCO results is given in table.

Interpretation of Blood Carboxyhaemoglobin (HbCO) Results

HbCO saturation, % Associated with:

3–8 Cigarette smokers

< 15 Heavy smokers (30-50 cigarettes per day)

20 Danger to heart disease patients

20–50 Progressive loss of mental and physical co-ordination

resembling ethanol intoxication

> 50 Coma, convulsions, cardiorespiratory arrest, death

Chemical Methods of Carbon Monoxide Detection in Blood

The qualitative tests described below are relatively insensitive and are useful only in

the diagnostics of acute carbon monoxide poisoning. If a positive result is obtained then

either the blood carboxyhaemoglobin (HbCO) or the breath carbon monoxide concentration should be measured without delay.

Express-test. To 0,1 ml of whole blood (treated with heparin, edetic acid or fluoride/oxalate) add 2 ml of 0,01 M ammonium hydroxide solution and mix for 5 seconds.

A pink tint in comparison with the colour obtained from a normal blood specimen suggests the presence of carboxyhaemoglobin. Cyanide may give a similar tint, but acute cyanide

147

poisoning is generally much less common that carbon monoxide poisonong. Sensitivity is

20 % of HbCO. The detection carboxyhaemoglobin in blood is the proof of a poisoning with

carbon monoxide.

1. Reaction with sodium hydroxide solution (Goppe-Zeiler’s test). To certain volume of blood add equal or double volume of 30 % sodium hydroxide solution. The

HbCO blood remains bright red, and blood without HbCO change colour in brown.

2. Reaction with ammonium sulphide (Salkovsli-Katajama’s test). To 10 ml of

distilled water add 5 drops of blood and 5 drops of fresh-made ammonium sulphide solution. Mixture cautiously shakes add 30 % acetic acid solution to light acidic

environment and slightly shake. The HbCO blood has crimson-red colour, and blood without HbCO becomes grey-green.

3. Reaction with quinine and ammonium sulphide (Khoroshkevich-Marks test). To 2 ml of blood add 4 ml of 8 % quinine hydrochloride solution and mixture boil short

time. After cooling add 2-3 drops of fresh-made ammonium sulphide solution and strongly shake. The HbCO blood has light-red colour, and blood without HbCO gets dirty red-brown

colour.

4. Reaction with hexacyanoferrate(III) of potassium (Bürker’s test). To 5 ml of

blood adds water to 500 ml and shake. To 5-10 ml of obtained solution add 5 drops of 1 % potassium hexacyanoferrate(III) solution K3[Fe(CN)6]. The HbCO blood remains bright red,

and blood without HbCO change colour in yellow.

5. Reaction with hexacyanoferrate(III) of potassium and potassium dichromate (Sidorov’s test). 1 ml of blood dilutes with water to 10 ml. To 2 ml of obtained solution add

3-5 drops of 20 % potassium hexacyanoferrate(III) solution and the same volume of 0,01 %

potassium dichromate solution. Mixture slightly shakes. The HbCO blood becomes carmine-red, and blood without HbCO gets brown-green colour.

6. Reaction with hexacyanoferrate(III) of potassium and acetic acid (Vetzel’s test). A blood dilute with ten-aliquot volume of distilled water. To 10 ml of obtained solution add 5 ml of 20 % potassium hexacyanoferrate(III) solution and 1 ml of glacial

acetic acid. In HbCO blood forms cherry-red precipitate, and in blood without HbCO forms

grey-brown precipitate.

7. Reaction with tannin (Kunkel-Vetzel’s test). A blood dilute with five-aliquot volume of distilled water. To 5 ml of this solution add 15 ml of 3 % tannin solution and well

shake. In HbCO blood forms light carmine-red precipitate, and in blood without HbCO forms grey-brown precipitate.

8. Reaction with formaldehyde (Libmann’s test). To 5 ml of whole blood add 5 ml of 40 % formaldehyde solution and strongly shake. The HbCO blood remains bright red,

and blood without HbCO in some minutes becomes brown-black.

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9. Reaction with lead acetate (Rubner’s tests). To 5 ml of whole blood add 20 ml

of 5 % basic lead acetate solution and strongly shake during 1 min. The HbCO blood keeps

red colour, and blood without HbCO becomes brown. 10. Reaction with copper sulphate (Zalesski’s test). To 1 ml of blod add water to

100 ml and well shake. To 5 ml of obtained solution dd 5 drops of 10 % - copper sulphate solution. Mixture well shakes. The HbCO blood becomes purple-red, and blood without

HbCO gets green colour.

Spectroscopic Method of Carbon Monoxide Determination in Blood

Blood of poisoned with carbon monoxide persons contain haemoglobin and its bonds: haemoglobin bonded neither with oxygen no with carbon monoxide –

desoxyhaemoglobin (Hb), oxyhemoglobin (OHb) – a haemoglobin bonded with oxygen,

and carboxyhaemoglobin (HbCO) – a haemoglobin bonded with carbon monoxide. Besides in a blood some of a methaemoglobin (HbMt) can contain. At poisonings the

methaemoglobin does not bond with carbon monoxide. Muscle tissues of poisoned persons contain desoxymyoglobin (HbM), oxymioglobin

(HbOM) and carboxymyoglobin (HbCOM). All listed above haemoglobin bonds (desoxyhaemoglobin, oxyhaemoglobin and

carboxyhaemoglobin) have specific absorption spectra in wavelengths from 450 nm to 620

nm. The absorption spectra of oxyhaemoglobin and carboxyhaemoglobin have a little difference from each other. But absorption spectra of desoxyhaemoglobin and

carboxyhaemoglobin considerably differ from each other. Therefore difference of these spectra are used for quantitative determination of carboxyhaemoglobin (HbCO) in blood

(see figure). The quantitative method for determination blood HbCO described below realises on

the fact that both oxygenated haemoglobin and methaemoglobin (oxidised haemoglobin)

can be reduced by sodium dithionite (Na2S2O4⋅2H2O) while HbCO is largely unaffected. For quantitative assay to 0,2 ml of whole blood add 25 ml of ammonium hydroxide

solution (1 ml/l) and takes three approximately equal portions: x, y and z. Keep portion x in a stopped tube while the following procedures are performed:

a) saturate portion y with carbon monoxide (to give 100 % HbCO) by bubbling the

gas through the solution for 5-10 minutes. Take care to minimise frothing; b) saturate portion z with oxygen by bubbling pure oxygen or compressed air through

the solution for at least 10 minutes to remove all bound carbon monoxide (to give 0 % HbCO). Again take care to minimise frothing.

After that add a small amount (about 20 mg) of sodium dithionite to each test solution (x, y and z) and also to 10 ml of ammonium hydroxide solution and mix well.

Measure the absorbances of solutions x, y and z against the dithionite-treated

ammonium hydroxide solution at 540 nm and 579 nm.

The percentage carboxyhaemoglobin saturation (% HbCO) can be calculated from the equation:

% HbCO = z)solution /A(Ay)solution /A(A

z)solution /A(A)solution x/A(A

579540579540

579540579540

−−

× 100

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Approximate normal values are:

(A540/A579 solution y) = 1,5, corresponding to 100 % HbCO,

(A540/A579 solution z) = 1,1, corresponding to 0 % HbCO.

Spectra obtained using a blood sample from a patient poisoned with carbon monoxide

(A), 100 % HbCO (B), and 0 % HbCO (reduced haemoglobin) (C)

Note that the haemoglobin content of blood varies from person to person, and thus

the volume of diluent used to be altered. This method is unreliable in the presence of other

pigments such as methaemoglobin (indicated by a relatively high absorbance in the region 580-600 nm). Lipaemic blood specimens may give turbid suspensions, which also give

unreliable results. The measurements are performed at the point of maximum difference of absorbance

(540 nm, λmax HbCO) and the point of equal absorbance (579 nm, isobestic point).

Sensitivity is approximately 10 % HbCO.

For saturation of blood with carbon monoxide is used special device. Flask 1 contains concentrated sulphate acid (dehydration agent), and dropping funnel 2 – formic

acid. On reaction with these reagents is obtained carbon monoxide:

HCOOH → CO↑ + H2O

Obtained gas bubbles through the Drexel’s flask 3, 4, 5 and 6, which contain: flask 3 – 10 %

sodium hydroxide solution, flasks 4 and 6 – distilled water, flask 5 – solution of researched blood in phosphate buffer.

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The device for blood saturation with carbon monoxide.

TOXIC SUBSTANCES ISOLATED FROM BIOLOGICAL MATERIAL

BY INFUSION WITH WATER

The groups of substances, which are isolated from various objects by infusion with water, include inorganic acids, alkali and salts of inorganic acids. For clearing aqueous

extracts from researched objects apply filtering or centrifugation, and then method of dialysis.

INORGANIC ACIDS

For the proof of inorganic acid presence in dialysates determine pH and presence of appropriate acids anions in investigated liquids.

The detection of sulphate-ions, chloride-ions and ions of other acids in dialysates is not yet the proof of poisonings with the named acids. Anions of the specified acids can be in

organism as tissue components.

For the confirmation of poisonings with inorganic acids it is necessary to distil them from dialysates. Thus the free acids are distilled off only. The salts of these acids, which are

in extracts from researched objects, are not overtaken.

SULPHATE ACID

Distillation of sulphate acid. To dialysates add copper sawdust and heat. Thus is formed anhydrite of sulphite acid SO2, which distil and collect in receiver with iodine

solution. At interaction of anhydrite of sulphite acid with water and iodine is formed sulphate acid:

2H2SO4 + Cu → H2SO3 + CuSO4 + H2O;

H2SO3 → SO2 + H2O;

SO2 + I2 + H2O → H2SO4 + 2HI.

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Reaction with barium chloride. With barium chloride forms white precipitate,

which not dissolves in nitrate and hydrochloride acids, and also in alkalis.

Reaction with lead acetate. With lead acetate forms white precipitate, which not

dissolves in nitrate acid, but dissolves in alkalis and in hot ammonium acetate solution:

PbSO4 + 4NaOH → Na2PbO2 + Na2SO4 + 2H2O;

2PbSO4 + 2СН3СООNН4 → [Pb(СН3СОО)2⋅PbSO4] + (NH4)2SO4.

Reaction with sodium rhodizonate. Barium rhodizonate has red colour. Added

sulphate acid decomposes this coloured compound and the red colour disappears:

NITRATE ACID

Nitrates such as sodium nitrate (NaNO3) are most commonly found in inorganic

fertilisers, but also used as antiseptics, food preservatives and explosives. Death in an adult may follow the ingestion of about 15 g of the sodium or potassium salt. Organic nitrates,

such as glyceryl trinitrate, are used as vasodilators. Nitrates are metabolised to nitrites in the gastrointestinal tract. Nitrates are strong oxidising agents and the test given below will also

detect compounds with similar properties, such as bromates, chlorates, hypochlorites,

iodates and nitrites. Acute poisoning with nitrates can cause nausea, vomiting, diarrhoea, abdominal pain,

confusion, coma and convulsions. In addition, nitrates may give rise to headache, flushing, dizziness, hypotension and collapse. Treatment is symptomatic and supportive.

Methaemoglobinaemia is often produced and this may be indicated by dark chocolate-coloured blood. Blood methaemoglobin can be measured but is unsuitable and the use of

stored samples is unreliable.

Distillation nitrate acid from dialysates. The addition of copper sawdust to

dialysates promotes distillation of nitrate acid:

2HNO3 + 3Cu → 2NO + 3CuО + Н2О;

3CuО + 6HNO3 → 3Cu(NO3)2 + 3H2O;

2NO + O2 → 2NO2;

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2NO2 + H2O → HNO2 + НNО3.

Reaction with diphenylamine. This reaction is based on diphenylamine oxidation by nitrate acid. Arises dark blue colour.

Reaction with brucine. Nitrate acid with brucine gives red colour. The same colour

with brucine gives nitrites, perchlorates and some other oxidisers.

Reaction of wool staining. Concentrated nitrate acid stains white woollen fibres in

yellow colour. From addition of ammonia the yellow colour passes in orange.

CHLORIDE ACID

Reaction with silver nitrate. Forms white precipitate of silver chloride, soluble in ammonia.

HCl + AgNO3 = AgCl↓ + HNO3,

AgCl + 2NH4NO3 = [Ag(NH3)2]Cl + 2HNO3.

Reaction with potassium chlorate. To 1 ml of distillate add crystals of potassium

chlorate (КClO3) and heat. At presence of hydrochloric acid in distillate the free chlorine evolves. Chlorine react with iodine and causes blue colour arising of iodine-starch paper:

6НС1 + КClO3 → 3Сl2 + КСl + 3H2O,

Cl2 + 2KI → I2 + 2КСl.

NITRITES

Nitrites such as sodium nitrite (NaNO2) were formerly used as vasodilators, and are used to prevent rusting, as food preservatives and in explosives. Nitrites may also arise from

the metabolism of nitrates. The fatal dose of sodium nitrite is about 10 g, although ingestion

of as little 2 g has caused death in an adult.

Reaction with sulphaminic acid and р-naphthol. This reaction based on reaction of aza-dyes formation:

NaNO2 + HCl → HNO2 + NaCl;

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Reaction with Greess’ reagent. This reagent consists from sulphaminic acid and α-

naphthylamine. With Greess’ reagent nitrites form azo-dye:

Detection of nitrites with iodine-starchy paper. At nitrites presence in distillate iodine-

starch paper change colour to blue.

ALKALIS and AMMONIA

From alkalis toxicological importance have hydroxides of sodium, potassium and

ammonia. The proof of poisonings with alkalis is the strong alkaline reaction of aqueous extracts from biological material and presence of appropriate metals cations in them.

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PESTICIDES

Some 10,000 species of the more than 1 million species of insects are crop eating,

and of these, approximately 700 species world-wide cause most of the insect damage to

man’s crops, in the field and in storage. Humanoids have been on earth for more than 3 million years, while insects have

existed for at least 250 million years. We can only guess, but the first materials likely used by our primitive ancestors to reduce insect annoyance were mud and dust spread over their

skin to repel biting and tickling insects, a practice resembling the habits of elephants, swine, and water buffalo. Under these circumstances, mud and dust would be classed as repellents,

a category of insecticides.

Historians have traced the use of pesticides to the time of Homer around 1000 B.C., but the earliest records of insecticides pertain to the burning of "brimstone" (sulphur) as a

fumigant. Pliny the Elder (A.D. 23-79) recorded most of the earlier insecticide uses in his Natural History. Included among these were the use of gall from a green lizard to protect

apples from worms and rot. Later, we find a variety of materials used with questionable results: extracts of pepper and tobacco, soapy water, whitewash, vinegar, turpentine, fish oil,

brine, lye and many others.

At the beginning of World War II (1940), our insecticide selection was limited to several arsenicals, petroleum oils, nicotine, pyrethrum, rotenone, sulphur, hydrogen cyanide

gas, and cryolite. And it was World War II that opened the Chemical Era with the introduction of a totally new concept of insect control chemicals – synthetic organic

insecticides, the first of which was DDT. Since the publication of Rachel Carson's Silent Spring in the 1960s, there has been

concern regarding the effects of chemical pesticides on humans and on the environment. In

the environment, the biological concentration of chemical pesticides (the amount retained in an organism through direct contact or consumption of affected plants or animals) tends to

increase the higher the animal is in the food chain. DDT, for example, severely reduced the rate of reproduction in many fish and birds.

Chemical pesticides now undergo exhaustive and expensive trials prior to government registration and release. The carcinogenicity of some pesticide components,

however, is a vigorously debated topic. Government testing often uses massive amounts of

such substances on laboratory animals, creating what some critics feel is an exaggerated assessment of their danger. Humans are heavily exposed to pesticides usually as a result of

acute exposure, such as accidental inhalation, on the job. Potential dangers from pesticide use must be weighed against improved crop quality

and yield and greatly improved human health around the world, as well as the availability of disease-preventing fresh fruits and vegetables that the use of pesticides has made possible.

Nevertheless, many consumers are concerned about the effects of pesticide residues in

foods, especially for infants, whose systems may not be able to convert toxic chemicals into harmless substances as readily as adult systems can. In addition, concerns have been raised

for farm workers in developing countries that lack the protective safeguards required in the different countries; their health is threatened by the continued use of pesticides that are

known health hazards. Efforts are being made to reduce chemical pesticide use in favour of Integrated Pest Management (IPM), biological controls, and plant breeding for inherent pest

resistance.

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Pesticide is biological, physical, or chemical agent used to kill plants or animals that

are harmful to people; in practice, the term pesticide is often applied only to chemical

agents. As a rule, pesticides is weakly dissolved in water, paraffinic and cycloparaffinic

hydrocarbons, it is good – in alcohols, ketones and ethers. Accordance to this specific property all pesticides are contained own group of toxic substances, isolating from

biological material by nonpolar organic solvents.

PESTICIDES CLASSIFICATION

On purposes Insecticides – for destruction of harmful insects.

Fungicides – for struggle with mould (microscopic fungi) illnesses of plants. Zoocides (Rodenticides) – for struggle with rodent.

Acaricides – for struggle with pincers. Algicides – for destruction of algae and other representatives of aqueous vegetation.

Antiseptics – for preservation of non-metallic materials from destruction by micro

organisms. Arboricides – for destruction undesirable wood and shrubbery vegetation.

Nematocides – for struggle with round worms (nematodes). Limacides – for struggle with mollusc.

Bactericides – for struggle with bacteria and bacterial illnesses. Herbicides – for struggle with weeds. The herbicides of contact action amaze plants at

immediate contact with leaves and stalks of plants. These pesticides do not move on

vascular system of plants, therefore they affect only those sites of plants, which were processed by them. The herbicides of systemic action after plants processing will penetrate

into their vascular system, are distributed on all plant and cause destruction. Herbicides work on root system of plants or on sprouting seeds.

Other substances used for stimulation plants grow (grow regulators), leaves dump (defoliants), for plants desiccation before harvesting (desiccants), and also used for scaring

away of insects (repellents) or for their attraction (attractants) also are pesticides.

On Way of Penetration in Organism

1. The contact insecticides act at contact with any part of an insect body. 2. The intestinal insecticides render harmful action on insects after penetration it into

intestines bodies.

3. The systemic they are translocated by a plant from the area of application to other plant parts, where they affect only pests that feed on the crop.

4. The fumigants get in an insect’s organism through respiratory ways and cause poisonings. Fumigants, which may initially have the form of a solid, liquid, or gas, kill

pests while in a gaseous state.

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On Origin

Pesticides can be derived from plants (e.g., pyrethrin, neem) or minerals, or they can be chemically manufactured (e.g., DDT, 2,4-D). Natural predators and other biological

methods are also used. Among the biological agents, parasites and predators feed on pests, pathogens sicken them, and pheromones interfere with insect mating. There are also

genetically engineered pesticides, such as the toxin-producing Bacillus thuringiensis strain

used against moth larvae.

On Chemical Structure: Inorganic

Organic – derivatives of:

– phosphorus acids, – carbaminic acids,

– carbohydrates (chlorine-containing), – carbonic acids,

– chlorophenoxycarbonic acids, – phenol,

– chloroacetanilide,

– urea and thiourea, – symetric trazine,

– cyclopropancarbonic acid, – coumarine.

On Toxicity (Internal Treatment to Laboratory Animals): Extremely toxic – LD50 is less than 15 mg/kg

High toxic – LD50 is 15 – 150 mg/kg Middle toxic – LD50 is 150 – 1500 mg/kg

Low toxic – LD50 is more than 1500 mg/kg

Besides, nonselective pesticides can affect both the targeted pest and other

organisms; selective pesticides affect only the target pest. Persistent pesticides are those that remain in the environment for a long time.

ORGANOCHLORINES

The organochlorines are pesticides (insecticides) that contain carbon (thus organo-),

hydrogen, and chlorine. They are also known by other names: chlorinated hydrocarbons,

chlorinated organics, chlorinated insecticides, and chlorinated synthetics. The

organochlorines are mostly of historic interest, since only a few survive in today’s arsenal.

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DIPHENYL ALIPHATICS

The oldest group of the organochlorines is the diphenyl aliphatics, which included DDT, DDD, dicofol, ethylan, chlorobenzilate, and methoxychlor. DDT is probably the best

known and most notorious chemical of the 20th century. It is also fascinating, and remains to be acknowledged as the most useful insecticide developed. More than 4 billion pounds of

DDT were used throughout the world, beginning in 1940, and ending essentially in 1973,

when the U.S. Environmental Protection Agency cancelled all uses. The remaining First World countries rapidly followed suit. In 1948, Dr. Paul Muller, a Swiss entomologist, was

awarded the Nobel Prize in Medicine for his lifesaving discovery of DDT (1939) as an insecticide useful in the control of malaria, yellow fever and many other insect-vectored

diseases.

Mode of action. The mode of action for DDT has never been clearly established, but

in some complex manner it destroys the delicate balance of sodium and potassium ions within the axons of the neuron in a way that prevents normal transmission of nerve impuses,

both in insects and mammals. It apparently acts on the sodium channel to cause "leakage" of

sodium ions. Eventually the neurons fire impulses spontaneously, causing the muscles to twitch-- "DDT jitters"-- followed by convulsions and death. DDT has a negative

temperature correlation--the lower the surrounding temperature the more toxic it becomes to insects.

HEXCHLOROCYCLOHEXANE (HCH)

Also known as benzenehexachloride (BHC), the insecticidal properties of HCH were

discovered in 1940 by French and British entomologists. In its technical grade, there are five isomers, alpha, beta, gamma, delta and epsilon. Surprisingly, only the gamma isomer has

insecticidal properties. Consequently, the gamma isomer was isolated in manufacture and sold as the odourless insecticide lindane. In contrast, technical grade HCH has a strong

musty odour and flavour, which can be imparted to treated crops and animal products.

Because of its very low cost, HCH is still used in many developing countries.

The hexachlorocyclohexane is 1,2,3,4,5,6-hexachlorocyclohexane is halogen

derivative of alicyclic hydrocarbons. Hexachlorocyclohexane is grey crystalline substance

with a mould smell. Smell of this substance is caused by impurity of pentachlorocyclohexane and benzene tetrachloride. At increased temperature HCH is

sublimed, thus the part of this substance is decomposed with phenyl trichloride and hydrogen chloride formation.

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Action on organism. The effects of HCH superficially resemble those of DDT, but

occur much more rapidly, and result in a much higher rate of respiration in insects. The

gamma isomer is a neurotoxicant whose effects are normally seen within hours as increased activity, tremors, and convulsions leading to prostration. It too, exhibits a negative

temperature correlation, but not as pronounced as that of DDT. HCH can penetrate through the undamaged skin. Has the expressed cumulative

property. Causes a dermahemia, oedema, and occurrence pustules. HCH is long lingers over

in bodies and tissues of an organism (especially in fatty tissues), eliminate out through kidneys, passes in milk of the feeding women.

Isolation of Hexachlorocyclohexane from Biological Material

From a corpse material and food infusing with hexane, petrol, or benzene can isolate

hexachlorocyclohexane. Also this pesticide can be isolates by infusing with aqueous solution of oxalic acid

and the next distillation with steam.

Detection of Hexachlorocyclohexane

Reaction with ethane dicarboxylic (succinic) acid and iron(III) sulphate. To microtest tube bring in a little amount of succinic or phthalic acid and small amount of

researched substance. An aperture (opening) of a test tube cover by a filter paper moistened

with iron(III) sulphate solution and heated up to 200 °C. On the paper appears blue spot.

Limit of detection: 30 µg of HCH in sample. Reaction is not specific. It gives another

chlorinated hydrocarbons.

Reaction of chlorine elimination of and detection it with silver nitrate. All

chlorinated hydrocarbons hydrolyses with sodium hydroxide alcohol solution and liberate

chlorine as chroride-ion. These chloride ion forms white precipitate a silver nitrate solution.

Cl

Cl

Cl

Cl

Cl

Cl

+ 6KCl+ 6KOH + 6H2O

KCl + AgNO3 → AgCl↓ + KNO3

Reaction of HCH dechloration and the formed benzene nitration. At HCH

heating with alcohol alkali solution there is eliminating chlorine (dechloration) from a molecule of this substance and is formed benzene. At action of sodium nitrate and

concentrated sulphate acid (nitration) is formed m-dinitrobenzene. From addition of

potassium hydroxide appears violet colour.

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Cl

Cl

Cl

Cl

Cl

Cl

+ 6KCl+ 6KOH + 6H2O

+ 2NaNO3 + H2SO4 + Na2SO4 + 2H2O

NO2

NO2

NO2

NO2

N+

O-KO

NO2

KOH

CYCLODIENES

The cyclodienes appeared after World War II: chlordane, 1945; aldrin and dieldrin,

1948; heptachlor, 1949; endrin, 1951; mirex, 1954; endosulfan, 1956; and chlordecone (Kepone(r)), 1958. There were other cyclodienes of minor importance developed in the U.S.

and Germany. Most of the cyclodienes are persistent insecticides and are stable in soil and relatively stable to the ultraviolet of sunlight. As a result, they were used in greatest quantity

as soil insecticides (especially chlordane, heptachlor, aldrin, and dieldrin) for the control of termites and soil-borne insects whose larval stages feed on the roots of plants.

Dieldrin Heptachlor (vesicol)

To appreciate the effectiveness of these materials as termiticides, consider that wood

and wooden structures treated with chlordane, aldrin, and dieldrin in the year of their

development are still protected from damage – more than 55 years! The cyclodienes were the most effective, long-lasting and economical termiticides ever developed. Because of

their persistence in the environment, resistance that developed in several soil insects, and in some instances biomagnification in wildlife food chains, most agricultural uses of

cyclodienes were cancelled by the EPA between 1975 and 1980, and their use as termiticides cancelled in 1984-88.

Action on organism. Unlike DDT and HCH, the cyclodienes have a positive

temperature correlation – their toxicity increases with increases in the surrounding temperature. Their modes of action are also not clearly understood. However, it is known

160

that this group acts on the inhibitory mechanism called the GABA (gamma-aminobutyric

acid) receptor. This receptor operates by increasing chloride ion permeability of neurones.

Cyclodienes prevent chloride ions from entering the neurones, and thereby antagonise the "calming" effects of GABA. Cyclodienes appear to affect all animals in generally the same

way, first with the nervous activity followed by tremors, convulsions and prostration. Heptachlor is 4,5,6,7,8,8-heptachloro-endomethylene bicyclo[4.3.0]nonadien-1,5. It

is crystalline substance with a weak camphor smell.

The heptachlor is high toxic. It has skin-resorptive and cumulative properties. It penetrates in organism through the alimentary canal and in a blood it oxidises to

epoxyheptachlor (dieldrin), which is more toxic, than heptachlor. Heptachlor and dieldrin cumulate in tissues. In ground these substances are kept within several years.

Isolation of Heptachlor and Dieldrin from Biological Material

Crushed biological material infuses few times with n-hexane. Obtained n-hexane

extracts clarify by acidified saturated sodium sulphate solution. Purified thus n-hexane extract dry and investigate on named pesticides presence.

From urine and blood heptachlor and dieldrin are extracted by diethyl ether. Obtained extracts dry and use for detection of heptachlor and dieldrin.

Detection of Heptachlor and Dieldrin

Reaction with diethyl amine. To a test tube put solution of researched substance in

ethylene dichloride. Then on a wall of test tube add 5–7 drops of reagent consisting of one volume of diethyl amine and two volumes of 0,1 N potassium hydroxide solution in

methanol. Mixture shakes. Liquid gets green colour, which quickly disappears.

Reaction with diethanol amine. To a test tube put 1–2 ml of researched substance

solution in ethylene dichloride and adds few drops of reagent (mixture of 1 part of diethanol amine and two parts of potassium hydroxide solution in methanol). Arises violet colour.

This reaction is specific for heptachlor and dieldrin.

Reaction with aniline and pyridine. To a test tube put 2–3 ml of researched

substance solution in benzene, add 5 drops of aniline and 2 drops of 0,1 N potassium hydroxide solution in methanol. A test tube place on 15 s in boiling water bath, then put in it

1 ml of pyridine and again heat 10 s in boiling water bath. Through 1–3 min solution obtaines dark-green colour.

CHLOROPHENOXY HERBICIDES 2,4-D (2,4-Dichlorophenoxyacetic acid) and related compounds are used to control

broad-leaved weeds in lawns and in cereal crops and, at higher application rates, for total vegetation control. They are frequently encountered as mixtures, both with other members

of the group and with other pesticides.

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Cl

Cl

O CH2COOH

Action on organism. Absorption of chlorophenoxy herbicides may lead to vomiting,

diarrhoea, araflexia, muscle weakness, pulmonary oedema and coma, with death in severe case. Alkalisation may increase the renal excretion of 2,4-D and other chlorophenoxy

compounds, and also protect against systemic toxicity.

Infusing with toluene performs isolation of chlorophenoxy herbicides from

biological material. Toluene extracts dry and residue use for colour tests.

Detection of 2,4-D

Reaction with sodium nitrite. To dry residue in porcelain cap add freshly prepared

sodium nitrite solution in concentrated sulphate acid. Appears brown colour. Reaction with chromotropic acid. To dry residue in porcelain cap add

chromotropic acid solution in concentrated sulphate acid. Appears purple colour. Described colour tests are common for all group of chlorophenoxy compounds.

Accordance to substituents structure, formed products have different colours. These tests are not specific and can only be used to indicate the presence of chlorophenoxy compounds.

POLYCHLOROTERPENES

Only two polychloroterpenes were developed – toxaphene in 1947, and strobane in

1951. Toxaphene had by far the greatest use of any single insecticide in agriculture, while strobane was relatively insignificant. Toxaphene was used on cotton, first in combination

with DDT, for alone it had minimal insecticidal qualities. Then, in 1965, after several major

cotton insects became resistant to DDT, toxaphene was formulated with methyl parathion, an organophosphate insecticide mentioned later.

Toxaphene is a mixture of more than 177 10-carbon polychlorinated derivatives

(camphene). These materials persist in the soil, though not as long as the cyclodienes, and

disappear from the surfaces of plants in 3-4 weeks. This disappearance is attributed more to

volatility than to photolysis or plant metabolism. Toxaphene is rather easily metabolised by mammals and birds, and is not stored in body fat nearly to the extent of DDT, HCH and the

cyclodienes. Despite its low toxicity to insects, mammals and birds, fish are highly susceptible to toxaphene poisoning, in the same order of magnitude as to the cyclodienes.

EPA cancelled toxaphene’s registrations in 1983.

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Action on organism. Toxaphene and strobane act on the neurons, causing an

imbalance in sodium and potassium ions, similar to that of the cyclodiene insecticides.

ORGANOMETALIC PESTICIDES

ETHYLMERCURY CHLORIDE

Ethylmercury chloride C2H5ClHg is organomercury fungicide. It is white crystalline

substance with specific smell. Ethylmercury chloride weakly dissolves in water, and better – in hot ethanol, acetone and solutions of alkalis. Ethylmercury chloride is volatile compound.

Ethylmercury chloride has skin-resorption action. It cumulates in organism.

Ethylmercury chloride vapours in 2 times more toxic than mercury vapours. At poisoning by ethylmercury chloride the contents of erythrocytes in a blood decreases, the fatty

dystrophy of liver is occurred. On skin there are ulcers and inflammatory infiltrate.

Isolation of Ethylmercury Chloride from Biological Meterial

Ethylmercury chloride isolates from corpses, food, and biological fluids by infusing with concentrated chloride acid. Obtained acidic extracts (infuses) collects and shakes with

chloroform. Chloroform extract research on ethylmercury chloride presence.

Detection of Ethylmercury chloride

Test with copper wire. This test is based on ability of metal copper to liberate mercury from ethylmercury chloride. Clear copper wire or copper plate dips in solution

containing ethylmercury chloride. Ethylmercury chloride decomposes and mercury postponed on metal copper as grey scurf. Copper wire with mercury grey scurf heats with

several crystals of iodine. Thus the red mercury (II) iodide forms. This mercury (II) iodide dissolves in solution of iodine in potassium iodide and forms potassium tetraiodomercurate

K2[HgI4]:

C2H5ClHg + Cu + H2O → Hg↓ + CuCl + C2H5OH

Hg + I2 → HgI2↓

HgI2 + 2KI → K2[HgI4]

Thin layer chromatography. This method is based on reaction of ethylmercury chlorideа with dithizone. At this reaction the ethylmercury chloride dithizonate forms. Formed

compound is chromatographied in system of solvents: n-heptane and chloroform (2:5). On

chromatographic plates appear yellow spots. Sensitivity is 0,1 µg of ethylmercury chloride in sample.

163

PHOSPHOR ORGANIC PESTICIDES

Phosphor organic compounds are one of the most important pesticides used in agriculture. Now for the specified purpose are applied as insecticides, herbicides, acaricides,

nematocides, defoliants, fungicides etc.

The wide application of organophosphates in agriculture is caused by many advantages:

– these compounds have high insecticidal and acaricidal activity, – the majority of these compounds is quickly decomposed in organisms of the human and

animals, – therefore they do not cumulate in bodies and tissues of mammal and almost do not cause

chronic poisonings,

– the majority of organophosphates in plants, ground and in other objects of environment is decomposed within several weeks.

Disadvantage of phosphor organic pesticides is their high toxicity. Some organic compounds of phosphorus can penetrate into organism through an uninjured skin, not

causing on it of any visible changes. Arrived thus in organism phosphor organic compounds the acute poisonings cause.

There are four basic structures of organophosphorus pesticides:

R O P

S

O

S

R

X

Phosphorodithioates

R O P

S

O

O

R

X

Phosphorothioates

R O P

O

O

S

R

X

Phosphorothiolates

R O P

O

O

O

R

X

Phosphates

where: R = alkyl; X = a wide variety of structures.

CHOLINESTERASE ACTIVITY

The high toxicity of phosphor organic compounds is explained by depressing action

of these substances on ferment systems of the human and animals. Especially strongly they

oppress acetyl cholinesterase, which plays the important role in regulation of physiological processes of organism. Phosphor organic compounds phosphorylate active centres of acetyl

164

cholinesterase; therefore it loses ability to adjust processes of acetylcholine decomposing

that results in infringement of many organism functions.

Scheme of Cholinesterase Interaction with Phosphor Organic Compounds

PRO

RO

O

OR'P

RO

RO OR'

ChEO

R'OH PRO

RO

O

ChE

++HChE

Systemic toxicity from carbamate and organophosphorus pesticides is due to largely

to the inhibition of acetylcholinesterase at nerve synapses. Cholinesterase, derived initially from the liver, is also present in plasma, but inhibition of plasma cholinesterase is not

thought to be physiologically important. It should be emphasised that cholinesterase and acetylcholiestarase are different enzymes: plasma cholinesterase can be almost completely

inhibited while erythrocyte acetylcholinestarase still processes 50 % activity. This relative inhibition varies between compounds and with the route of absorption and depending on

whether exposure has been acute or chronic. In addition, the rate at which cholinesterase

inhibition is reserved depends on whether the inhibition was caused by carbamate or organophosphorus pesticides.

In practice, plasma cholinesterase is a useful indicator of exposure to organophosphorus compounds or carbamates, and a normal plasma cholinesterase activity

effectively excludes acute poisoning by these compounds. The difficulty lies in deciding whether a low activity is indeed due to poisoning or to some other physiological,

pharmacological or genetic cause. The diagnosis can sometimes be assisted by detection of

a poison or metabolite in a body fluid, but the simple methods available are relatively insensitive.

Dependence of Blood Cholinesterase Activity on Carbophos Concentration

Organism state Cholinesterase activity, µmol Carbophos concentration, µg/ml

Doorstep 57,08 ± 3,8 0,01 – 0,17

Critical 21,48 ± 1,55 0,2 – 1,5

Mortal 3,89 ± 1,3 1,55 – 10,0

Cholinesterase Assay

Ability of phosphor organic compounds to reduce activity of an acetyl cholinesterase

it is possible to apply for the analytical purposes. Acetyl cholinesterase assay is general for detection of the majority of phosphor organic pesticides, which depress activity of an acetyl

cholinesterase. Acetylcholine under influence of acetyl cholinesterase is decomposed to acetic acid,

which changes pH of substrate environment. Indicators can fix these pH changes and change own colours. For example, widely used for acetylcholine test indicator

Bromothymole Blue changes colour from blue in neutral environment to yellow in acidic

solution. If to mixture of acetylcholine and Bromothymole Blue add an acetylcholinesterase, the phosphor organic compound being an inhibitor of acetylcholinesterase. Acetylcholine is

165

not decomposed by acetylcholinesterase and the colour of the indicator does not change. For

performance of cholinesterase assays to mixture of reactants, instead of

acetylcholinesterase, can be added blood plasma or horse serum containing this enzyme. In this case blood plasma or serum is a source of acetylcholinesterase.

Detection of Phosphorus Pesticides

For establishing of phosphor organic compounds presence in researched objects must

be carried out cholinesterase test and detected presence of phosphorus. For detection of phosphorus presence the organic compounds must be mineralised. Mineralisation of

phosphor organic compounds provided by: calcium oxide, or mixture of concentrated sulphate and nitrate acids, or mixture of sodium carbonate and sodium peroxide. In obtained

mineralisate detects phosphate-ions presence with ammonium molybdate solution:

H3PO4 + 12(NH4)2MoO4 + 21HNO3 = 21NH4NO3 + (NH4)3PO4·12MoO2·2H2O↓ + 10H2O.

At phosphates presence appears blue colour of mineralisate.

Detection of Phosphorylation Activity

Phosphorylation (alkylation) properties of phosphor organic compounds (POC)

demonstrate reaction with 4-(p-nitrobenzyl)-pyridine. Part of extract from biological material evaporate and the residue dissolve in acetone. To obtained solution add solution of

4-(p-nitrobenzyl)-pyridine in acetone. After heating forms Formed product of condensation

has red-violet colour. Reaction runs for scheme:

H3CO

H3COP

S

S CH

CH2

COOC2H5

COOC2H5

N NO2CH2+

H+

H3CO

H3COP

S

+N NO2CH2

+OH-

X-

H3CO

H3COP

S

N NO2CH

This reaction is typical for ethers of phosphoric, thio- and dithio- phosphoric acid.

Sensitivity is 0,5–2 µg of POC in sample.

166

TRICHLORFON (CHLOROPHOS)

Chlorophos is O,O- dimethyl (2,2,2-trichloro-1-hydroxyethyl)phosphonate.

O P

O

O

CH CCl3H3C

H3C OH

Chlorophos is a white crystalline powder. It dissolves in water, benzene, chloroform and other organic solvents, and worse – in paraffin hydrocarbons.

Chlorophos is slowly decomposed in acidic environment and more quickly – in alkaline. It rather quickly decomposes in diluted solutions on light. At chlorophos

decomposition in acidic solutions the methanol and O-methyl-(2,2,2-trichloro-1-oxyethyl) phosphoric acid are formed. In alkaline condition at chlorofos decomposition the more toxic

O,O-dimethyl-O-(2,2-dichlorovinyl) phosphate (DDVP) is formed:

O P

O

O

CH CCl3H3C

H3C OH

KOHO P

O

O

CHH3C

H3C

O CCl2 + KCl + H2O

Chlorophos has irritating action on skin, depresses activity of a cholinesterase in a

blood. At chronic poisonings by chlorophos the infringement of liver function and disease

of cardiovascular system is observed.

Scheme of Chlorophos Biotransformation

P

O CCl3

OHMeO

OMe P

O

OH

MeO

OMeP

O

OHMeO

OH

Cl

Cl

OMe

OH

Cl

Cl

H

H

O

Cl

Cl

P

O

OH

OH

OMeP

O

OH

OH

OH

+

atoms regrouping

dichloro acetaldehyde

+ H2O

+ H2O

+ H2O

Isolation of Chlorophos from Biological Material

167

Chlorophos is good dissolved in water and in organic solvents. Due to this ability

chlorophos can be isolated from objects of forensic-chemical examination by infusing with

acidified water, or by infusing with any organic solvent (acetone, hexane or chloroform).

Detection of Chlorophos

Reaction with pyridine and alkali (Fujiwara’s test). Heated with pyridine and

alkali chlorophos forms compound with red or pink colour. Sensitivity is 10 µg of

chlorophos in sample:

P

O CCl3

OHMeO

OMe

N N

HCCl2

H

O

H

OO

H

N

HCCl2

Cl+NaOH

+H2O

Reaction with resorcinol. At pH 9–11 chlorophos reacts with resorcinol and forms

compound having yellow-green fluorescence. A limit of detection is 40 µg of chlorophos in

sample:

P

O CCl3

OHMeO

OMe

H

CHCl2

O

P

O

ONa

MeO

OMe + NaCl + H2O+ 2NaOH +

H

CHCl2

O

OH

CHCl2

OH OH

OH

OH

OH

2+ + H2O

Reaction of isonitrile formation. Chlorophos forms isonitrile (specific unpleasant

smell) with spirituous solution of aniline in alkali environment. Reaction is not specific. Other chlorine containing substances give this reaction.

P

O CCl3

OHMeO

OMe

H

CHCl2

O

P

O

ONa

MeO

OMe + NaCl + H2O+ 2NaOH +

N=C H

H

O

H

CHCl2

O

NH2

+ 2NaOH+ + 2NaCl + 2H2O +

Reaction with o-tolidine. o-Tolidine (dissolved in acetone) with chlorophos in alkali

environment forms condensation product having yellow or orange colour. This reaction gives metaphos, thiophos etc.

168

Reaction with 2,4-dinitrophenyl hydrazine. Product of chlorophos condensation

with 2,4-dinitrophenylhydrazine has blue or pink colour.

Reaction with acetone. Mixture of acetone and alcohol solution of sodium

hydroxide at chlorophos presence change colour to pink changing in orange.

MALATHION (CARBOPHOS)

Carbophos is diethyl-(dimethoxythiophosphorylthio)-succinate or S-1,2-bis-(ethoxycarbonyl)-ethyl O,O-dimethyl phosphorodithioate.

COOC2H5

COOC2H5

SO P

S

O

CHH3C

H3C CH2

Carbophos is a colourless liquid with characteristic unpleasant smell. It weakly

dissolves in water and good – in the majority of organic solvents, except hydrocarbons.

Scheme of Carbophos Biotransformation

1 – desalkylation; 2 – hydrolysis; 3,6 – alkaline hydrolysis; 4 – oxidation;

5 – acid hydrolysis; 7 – breaking-up by carboxylase enzyme.

From biological material carbophos is isolated by infusing with chloroform.

Detection of Carbophos

To detection of Carbophosum apply colour reactions and method of a chromatography in a shallow layer of sorbent.

Reaction with diazoted sulphanylic acid. With diazoted sulphanylic acid

charbophos forms cherry-red coloured compound.

169

P

S

MeO

S CH-COOC2H

5

CH-COOC2H

5

OMeN

+SO

3HClN P

S

MeO

S CH-COOC2H

5

CH-COOC2H

5

OMe

SO3HN=N

+ NaCl + H2O_

+ NaOH +

Reaction with Marquis reagent. Dry residue of extract from biological material

with marquis reagent gives orange colour, which later passes in dark brown.

PARATHION-METHYL (METAPHOS)

Metaphos is O,O-dimethyl O-4-nitrophenyl phosphorothioate

OO P

S

O

H3C

H3C

NO2

Metaphos is white crystalline substance. It weakly dissolves in water and in

hydrocarbons, but well dissolves in other organic solvents.

Metaphos expresses strong toxicity, local action does not have. At peroral treatment the metaphos quickly penetrates in blood. At metaphos poisoning the haemoglobin

concentration in blood decreases, but methaemoglobin amount decreases. For metaphos isolation from biological material apply methods, which are

described for carbophos isolation from biological material.

Detection of Metaphos

Reaction with o-dianizidine and sodium perborate. To test tube put acetone solution of researched substance, add freshly made acetone solution of o-dianizidine and

solution of sodium perborate (NaBO3·4H2O). The solution gets yellow or red colour. This reaction is give also others phosphorus containing organic compounds

(chlorophos, phosphamide, thiophos etc.).

Detection of Thio- and Dithio- Phosphate Acid Derivatives

Reaction with palladium chloride. Substances, containing thio-аtom (keto-sulphur)

react with palladium chloride and form complex compound. The reaction products have

colour from yellow to brown. Reaction sensitivity is 2-10 µg of POC in sample.

Reaction with copper sulphate. Derivatives of dithiophosphate acid after alkaline hydrolysis form complex compound with copper sulphate:

170

H3CO

H3COP

S

SH2 + CuSO4

S

S

PH3CO

H3COCu

S

S

PH3CO

H3CO

+ H2SO4

At carbophos presence product of reaction has yellow colour.

Chromato-enzyme Method of Phosphor Organic Compounds Detection

Chromatogram spray by enzyme reagent (extract from fresh-frozen calf's liver). After spraying the plate is placed into thermostat on hour at 38°С. In place of phosphor organic

compounds location the cholinesterase is oppressed. After incubation in thermostat the plate spray by solution of indoxyl acetate with mixture of potassium hexacianoferrate (ІІ) and

potassium hexacyanoferrate (ІІІ). Indoxyl acetate is hydrolysed by active cholinesterase to

indoxyle, and further runs reaction of indigo formation. A plate background is coloured into dark blue, and spots containing phosphor organic compounds are colourless.

NH

O

O

C CH3enzyme [O]

NH

OH

Indoxyl acetate indoxyl

N

C

C

H

O

N

C

C

H

O

Indigo (blue)

Thin layer Chromatography

Developing of sulphur containing substances (thio- and dithio- phosphate acid

derivetaives) on chromatograms also can be provided by: – solutions of Bromothymole Blue and silver nitrate. After spraying spots of sulphur

containing substances are coloured into dark blue colour; – solution of palladium chloride spraying. Spots are coloured in yellow.

171

CARBAMATE PESTICIDES

The carbamate insecticides are derivatives of carbamic acid (as the OPC are derivatives of phosphoric acid). And like the OPC, their mode of action is that of inhibiting

the vital enzyme cholinesterase. These compounds have general formula:

C N

R2

R3

O

OR1

Substitution of sulphur for oxygen also occurs, but such compounds generally have low

insecticidal activity. The first successful carbamate insecticide, carbaryl (Sevin), was introduced in 1956.

More of it has been used world-wide than all the remaining carbamates combined. Two

distinct qualities have made it the most popular carbamate: its very low mammalian oral and dermal toxicity and an exceptionally broad spectrum of insect control.

CARBARYL

Carbaryl is 1-naphthyl methylcarbamate.

O CONHCH3

Carbaryl is white crystalline substance. It slightly dissolves in water, well soluble in organic solvents. Carbaryl hydrolyses in neutral, acidic and alkaline solutions. One of

products of its hydrolysis is 1-naphthol. At protracted influence carbaryl occurs liver disfunctions. Carbaryl quickly

penetrates from stomach in blood. Carbaryl metabolite is 1-naphthol.

Action on organism. Carbamates inhibit cholinesterase as OPC do, and they behave in almost identical manner in biological systems, but with two main differences. Some

carbamates are potent inhibitors of aliesterase (miscellaneous aliphatic esterase whose exact functions are not known), and their selectivity is sometimes more pronounced against the

cholinesterase of different species. Second, cholinesterase inhibition by carbmates is reversible. When cholinesterase is inhibited by a carbamate, it is said to be carbamylated, as

when an OPC results in the enzyme being phosphorylated. In insects, the effects of OPC

and carbamates are primarily those of poisoning of the central nervous system, since the insect neuromuscular junction is not cholinergic, as in mammals. The only cholinergic

synapses known in insects are in the central nervous system. (The chemical neuromuscular junction transmitter in insects is thought to be glutamic acid, but that has not been proved).

172

Isolation of Carbaryl from Biological Material

As isolating fluids can be used any nonpolar organic solvent. Widely are used

benzene and hexane.

Detection of Carbaryl

Reaction with picric acid. It is microcrystal test. On subject glass form dark yellow

crystals assembled in fascicles.

Reaction with 4-aminoantipyrine. At first carbaryl participate in condensation

reaction with 4-aminoantipyrine. To obtained product add solution of potassium

hexacyanoferrate(II). Appears orange-red colour, which becomes more intensive in organic

phase (chloroform). This reaction gives also 1-naphthol.

Reaction with mixture of copper chloride and sodium bromide. After alkali

hydrolysis of carbaryl to the test tube add fresh-made mixture of copper chloride and sodium bromide. Appears red-violet or blue-violet colour.

This reaction gives also 1-naphthol.

173

TABLE OF CONTENTS

Introduction to Toxicological Chemistry 3

Transport of chemicals through a cell membrane 15

Distribution of toxicants in the body 21

Biotransformation of chemicals 26

General pathways of xenobiotics biotransformation 35

“Volatile” Poisons Substances 44

“Volatile” Poisons Investigation 56

Isolation of “volatile” poisons from biological material 56

Cyanic acid 56

Formaldehyde 59

Methanol 62

Ethanol 63

Isoamyl alcohol 66

Acetone 68

Phenol 70

Chloroform 72

Chloral hydrate 73

Carbon tetrachloride 74

1,2-dichlorethane 75

Acetic acid 77

Substances Isolated by Mineralisation of Biological Material 79

“Metal Poisons” Determination 85

Barium 85

Lead 86

Bismuth 87

Cadmium 89

Manganese 89

Copper 90

Arsenic 91

Silver 93

174

Antimony 94

Thallium 96

Chrome 96

Zinc 97

Mercury 98

Quantitative determination of “metal poisons” in mineralisates 99

Toxic Substances Isolated From Biological Material by Polar Solvents 100

Qualitative and quantitative determination of toxic substances isolated

from biological material

106

Substances extracted by organic solvents from acidic aqueous extracts 112

Barbiturates and methods of their research 112

Xanthine derivatives 116

Salicylic acid and derivatives 118

Pyrazolon-5 derivatives 120

Alkaloids – indole derivatives 123

Substances extracted by organic solvents from alkalised aqueous extracts 125

Alkaloids 125

Quinoline derivatives – quinine 126

Iso-quinoline derivatives 128

Pyridine derivatives – nicotine 130

Piperidine derivatives – coniine 131

Tropane derivatives 132

Ephedrine 135

Substances extracted by organic solvents from alkalised aqueous extracts

(Synthetic Medicines)

137

p-Aminobenzoic acid derivatives 137

Phenothiazines 138

Benzodiazepines 140

Butyrophenone derivatives 142

Isonicotinic acid derivatives 143

Substances Determined Directly in Biological Material 146

Carbon monoxide 146

Toxic Substances Isolated from Biological Material by Infusion With Water 150

175

Inorganic acids 150

Nitrites 152

Alkalis and ammonia 153

Pesticides 154

Pesticides classification 155

Organochlorines 156

Diphenyl aliphatics 157

Hexchlorocyclohexane (hch) 157

Cyclodienes 159

Chlorophenoxy herbicides 160

Polychloroterpenes 161

Organometalic pesticides 162

Ethylmercury chloride 162

Phosphor organic pesticides 163

Cholinesterase activity 163

Trichlorfon (chlorophos) 166

Malathion (carbophos) 168

Parathion-methyl (metaphos) 169

Carbamate pesticides 171

Carbaryl 171