workbook for practical lessons on drug and analytical...

Workbook for practical lessons

on Drug and Analytical Toxicology

___ YEAR ___ GROUP STUDENT

__________________________________________

MINISTRY OF HEALTH OF UKRAINE

NATIONAL UNIVERSITY OF PHARMACY

DEPARTMENT OF DRUG AND ANALYTICAL OXICOLOGY

Workbook for practical lessons

on Drug and Analytical Toxicology

___ YEAR ___ GROUP STUDENT

__________________________________________

Kharkiv

2018

UDC 54.01:615 (075)

Сompiled by: S.A. Karpushyna Doctor of Chemistry, Professor, Professor of the Drug and Analytical Toxicology Department,

National University of Pharmacy,

S.V. Baiurka Doctor of Chemistry, Professor, Head of the Drug and Analytical Toxicology Department, National

University of Pharmacy,

Reviewers: M.Ye. Blazheyevskiy, Doctor of Chemistry, Professor, Professor of the Physical and Colloid Chemistry

Department, National University of Pharmacy;

S.V. Kolisnyk, Doctor of Pharmacy, Professor, Professor of the Analytical Chemistry Department, National

University of Pharmacy;

L.A. Toryanik,PhD (Pedagogics) , Assistant Professor of the Foreign Languages Department, National

University of Pharmacy.

Approved by the Central Methodological Committee of NPhaU

(dated October, 16, 2018, №2)

Karpushyna, S.A. Workbook for practical lesons on drug and analytical toxicology / S.A. Karpushyna, S.V. Baiurka. – Kh.: NPhaU

Publishing House, 2019. – 87 p.

The Workbook for practical lessons on drug and analytical toxicology is intended to use at practical lessons and self-study work for the

foreign (English-course-language) pharmacy students in specialty 8.12020101 "Pharmacy". This Practice Book is recommended for

pharmaceutical faculties and pharmaceutical universities of the III-IV accreditation levels.

3

CHEMISTRY LABORATORY SAFETY RULES

GENERAL SAFETY RULES

Before you begin any work in the laboratory, you should read

and understand the chemistry laboratory safety rules. If you

do not understand a rule, insist on a satisfactory explanation

from your instructor or a faculty member. Afterwards, sign

the safety rule verification form that will be kept on file with

the toxicological chemistry department.

You must be covered continuously from shoulders to below

the knees and must wear special clothes (coats and caps with

cotton, shoes that cover your feet). For fire safety, flammable

materials, loose clothes, ties should not be worn, and long

hair should be tied back.

EYE PROTECTION AND HANDLING CHEMICALS

Many chemicals are toxic and/or corrosive. Chemical

reagents require careful handling.

Do not work with a chemical above or near your face. Avoid

eyes and skin contact with any chemical.

Do not taste or ingest any chemical in the laboratory. Do not

keep food or drink items at your lab bench.

Never pipet by mouth. Never pipet directly from a reagent

bottle.

Do not inhale reagent fumes. Fume hoods must be used

whenever toxic or corrosive vapours are released during the

work you are doing. Odour tests are to be made only when

specifically directed to do so. Use a waving motion of your

hand to bring the vapour near your nose (this is wafting).

Do not handle broken glass with bare hands.

When heating a substance in a test tube, be careful not to

point the mouth of the test tube at another person or yourself.

BIOHAZARDS

Do not work with a biological sample (biological material,

body fluids) by the unprotected hands. Wear safety

disposable rubber gloves. Aseptic technique (hand washing

with antibacterial soap before and after the lab, disinfection

of tables before and after the lab, and using the proper

procedures for handling the biological samples) should be

followed at all times when working with biological samples.

FLAMMABLE, EXPLOSIVE, AND REACTIVE CHEMICALS

Keep flammable and combustible materials away from open

flames. Some examples of flammable materials include

alcohol, diethyl ether, chloroform, acetone.

Do not heat a test tube containing flammable and explosive

substances over an open flame or directly on a hot plate (a

gas ring). Use water bath for this purpose.

Do not add water to a concentrated reagent, especially

concentrated sulphuric acid.

ALWAYS add the acid slowly to water.

SAFETY RULES TO USE ELECTRICAL DEVICES

Never use damaged electrical devices. Electrical outlets

should be inspected for signs of damage or burning before

use.

Do not leave working electrical appliances unattended.

Do not use or store highly flammable solvents near electrical

equipment.

Avoid contacting electrical devices with wet hands or wet

materials.

4

FIRE SAFETY

Know the location of fire extinguishers, fire blankets, and

safety showers in the case of fire.

THE FIRST AID IN THE CASE OF ACCIDENT

If a chemical splashes into your eye, get help

immediately. Shout out, "I have a chemical in my eye!"

If someone nearby gets a chemical in his/her eye, you should:

(1) shout for help from the instructor, (2) provide help if the

instructor is not there immediately.

Wash the eye thoroughly with a stream of water from the

eye wash fountain, or any other water source. Hold the

eyelids open.

Any chemical that comes in contact with your skin should be

washed off with water right away.

Concentrated sulphuric acid should be removed from the skin

with dry fabric. Then wash the damaged skin with water and

then with sodium bicarbonate solution. Other strong acids

should be washed off with water and then sodium

bicarbonate solution.

Concentrated caustic alkali solutions should be washed off

from the skin with water and then neutralized with acetic or

citric acid diluted solutions.

Phenol, bromine and organic solvents unmixing in water

should be removed from the skin with such organic solvents

as alcohol or diethyl ether.

In the case of a thermal burn you should treat the damaged

skin with glycerol or put the cotton wool saturated with

alcohol. In the case of a severe thermal burn you should put

the cotton wool saturated with 3 % solution of tannin

immediately.

In the case of cutting yourself by glass you should remove

the fragments of broken glass and disinfect the damaged skin

with iodine solution.

In the case of a severe electrical shock (1) call for help; (2)

make sure the victim is no longer in contact with the

electrical current source – turn off all power if this can be

done quickly (Do not touch the person until power is not

shut off ! If you can't shut off the source with dry feet and

hands, use a board, wooden stick, rope, etc., to get the person

away from the source.); (3) check for breathing and heart

beat and apply cardiopulmonary resuscitation (CPR), if

necessary, check for burns – cover burned areas with dry,

sterile dressings, call for medical help. Unless it is absolutely

necessary, don't move the person. He or she could have a

traumatic injury, especially to the head or neck.

To prevent shock lay the person down and, if possible, the

position the head slightly lower than the trunk with the legs

elevated.

HAZARDOUS WASTE

Only neutral aqueous solutions go down the sink drain. All

chemical waste is to be sorted into the appropriate waste

container.

Clean your work bench with a damp sponge. Neutralize all

acid spills with sodium bicarbonate and wash with a wet

sponge. Turn off electrical appliances, shut gas jets

completely. Wash your hands.

Leave the area safe for the next person.

Do not take any chemical things out of the laboratory for

any reason. It is illegal!

http://www.ilpi.com/safety/extinguishers.html

5

CONTENT MODULE 1 DRUG TOXICOLOGY

Practical lesson 1

_____

Date

Topic: TOXICOLOGY OF OPIOID ANALGESICS, NON-NARCOTIC ANALGESICS AND NON-STEROIDAL

ANTI-INFLAMMATORY DRUGS

Practical work

Using the reference literature, scientific and professional publications give a toxicological characteristic to the following groups of drugs:

opioid analgesics (OA) of natural and synthetic origin (morphine, codeine, fentanyl, buprenorphine, tramadol)

non-opioid analgesics (NOA) (paracetamol, a derivative of p-amino phenol; metamizol sodium, a derivative of pyrazolone-5)

non-steroidal anti-inflammatory drugs (NSAIDs) (acetyl salicylic acid (ASA), a derivative of salicylic acid)

To summarize the material is in the form of table (see Table 1).

Table 1 – Toxicological characteristic to opioid analgesics, non-opioid analgesics and non-steroidal anti-inflammatory drugs

Drugs

Toxicokinetic (or

toxicometric)

parameters)

Mechanism of the

toxic action

The main symptoms

of an intoxication

Treatment of a

poisoning

Prevention of an

intoxication

(factors that

increase toxicity)

1 2 3 4 5 6

6

1 2 3 4 5 6

7

1 2 3 4 5 6

8

Independent educational and research work

1. Toxicological characteristics of NSAIDs, the phenylpropionic and phenylacatic acid derivatives (ibuprofen, diclofenac sodium).

2. Toxicological characteristic of NSAIDs, the pyrazolone derivatives (phenylbutazone) and indolacatic acid (indomethacin).

9

Practical lesson 2

_____

Date

Topic: TOXICOLOGY OF NEUROLEPTICS, TRANQUILIZERS AND HYPNOTIC DRUGS. TOXICOLOGICAL

CHARACTERISTICS OF MEDICINES STIMULATING CNS AND LOCAL ANESTHETICS

Practical work

Using the reference literature give a toxicological characteristic to the drugs that suppress the CNS and local anesthetics:

neuroleptics (antipsychotic drugs) (derivatives of phenothiazine: chlorpromazine, diprazine, levomepromazine)

tranquilizers (derivatives of 1,4-benzodiasepine: chlorodiazepoxide, diazepam, nitrazepam)

hypnotic drugs (barbiturates)

To summarize the material is in the form of table (see Table 1).

Table 2 – Toxicological characteristic to the drugs that suppress the central nervous system and local anesthetics

Drugs

Toxicokinetic (or

toxicometric)

parameters)

Mechanism of the

toxic action

The main symptoms

of an intoxication

Treatment of a

poisoning

Prevention of an

intoxication

(factors that

increase toxicity)

1 2 3 4 5 6

10

1 2 3 4 5 6

11

1 2 3 4 5 6

12


1. Toxicology of sedative medicines (bromides). Methods of treating acute poisoning with these drugs.

2. Toxicology of analeptics (strychnine). Targets of toxic action on CNS, CVS and other organs and systems. Help in case of analeptics

poisoning. Toxicodynamics. Prevention of poisoning and first aid during intoxication by this medicine.

3. Toxicology of psychostimulants (amphetamines, caffeine). Targets of toxic action on CNS, CVS and other organs and systems. Help

in case of psychostimulant poisoning. Psychostimulants and their toxicodynamics. Prevention of poisoning and first aid during

intoxication by these medicines.

4. Toxicology of psychotomimetics (cocaine). Targets of toxic action on CNS, CVS and other organs and systems. Help in case of

cocaine poisoning. Toxicodynamics. Prevention of poisoning and first aid during intoxication by this substance.

5. Toxicology of local anesthetics, the derivatives of p-aminobenzoyl acid (benzocaine, procaine hydrochloride, lidocaine, cocaine

hydrochloride). Toxicodynamics and toxicokinetics of local anesthetics. Prevention of poisoning and first aid during intoxication by

these medicines.

13

CONTENT MODULE 2 ANALYTICAL TOXICOLOGY. CHEMICOTOXICOLOGICAL ANALYSIS OF DRUGS

Practical lesson 3

_____

Date

Topic: ANALYTICAL TESTING SCHEME DESIGN. SPECIMEN TESTING, EXTERNAL EXAMINATION AND

PRELIMINARY TESTS

External examination includes:

to determine consistency and morphological composition of the sample;

to determine the presence of preservative;

to determine odour and colour of the sample;

to mark the presence of external inclusions.

Fill the results of the research in Table 3

Table 3 – External examination of the sample

Sample, No. Odour Colour Morphological composition

of the sample Presence of a preservative External inclusions

Preliminary tests

14

1. Determination of the sample pH Method: Place a litlle amount of the sample in a test tube, add the same amount of distilled water, mix up the contents of the tube.

Apply some drops of the aqueous solution obtained on a scrap of the universal indicator paper by a glass stick. Apply a drop of distilled water

on another scrap of the universal indicator paper (blank experiment).

Estimation of the results obtained: the acidic medium suggests the presence of alkalis in the sample, the alkaline medium suggests the

presence of acids. A strongly acidic medium (pH 1–3) can be caused by the presence of mineral acids or a great amount of organic acids

administrated; a strongly alkaline medium (pH 9–10 and more) can be caused by the presence of alkalis, ammonia, carbonates, silicates and

other salts of weak acids and strong bases (cyanides, nitrites, etc.).

2. Difference of alkalis and carbonates

Method: Place 1–2 ml of the aqueous solution examined in a test tube; add 1–2 drops of phenolphtalein ethanol solution (1:1000); a

pink colour appears; add barium chloride solution and shake up, the colour can disappear or can remain.

Estimation of the results obtained: if the colour of the solution remains the alkaline medium is conditioned by the presence of alkalis, if

the colour disappears and the white precipitate appears, the alkaline medium is due to the presence of carbonates or silicates.

3. Test for ammonia and hydrogen sulphide

Method: Place the sample examined in a test tube, attach three paper scraps (a wet red litmus indicator paper; a wet reactive paper

treated by copper sulphate solution and a wet reactive paper treated by lead acetate solution) to the cork and close the tube with the help of

this cork.

Estimation of the results obtained: if all three paper scraps change their colour (the litmus indicator paper and the paper treated by

copper sulphate solution turn blue, reactive paper treated by lead acetate solution turns black), the conclusion about the presence of ammonia

and hydrogen sulphide as a result of the rotting process in the biological material examined can be drawn; if the colour of the litmus indicator

paper and the paper moistened by copper sulphate solution change, the presence of ammonia administrated can be supposed.

Fill the results of the research in Table 4.

15

Table 4 – Results of the preliminary tests

No. of

the

sample

pH

Observation after

addition of

phenolphtalein

ethanol solution

to the sample

Observation after

addition of barium

chloride solution to

the sample

Observation of

reaction of the wet

red litmus indicator

paper and wet

reactive one treated

by copper sulphate

solution with the

sample’s vapour

Observation of

reaction of the

wet reactive

paper treated by

lead acetate

solution with

the sample’s

vapour

Chemical reaction equation(s) and

estimation of the results obtained

The conclusion:

16

Topic: CHEMICOTOXICOLOGICAL ANALYSIS OF INORGANIC ACIDS, ALKALIS, NITRATES AND NITRITES

Preliminary test: determination of the sample’s pH (for mineral acids and alkalies).

Isolation: extraction by water.

Purification of water extracts from proteins by dialysis. Electrodialysis is used to accelerate the process of separation of the analyzed

substances from impurities.

Figure 1 – Device for electrodialysis

Sulphuric acid is distilled from the aqueous extract. Distilation is carried out in the presrnce of copper filings. The distillate is

collected in a receiver which contains a solution of iodine.

Cu + 2H2SO4CuSO4 + SO2 + 2H2O

SO2 + I2 + 2H2O H2SO4 + 2HI

Nitric acid is distilled from the aqueous extract. Distilation is carried out in the presrnce of copper filings. The distillate is collected in

a receiver which contains water.

3Cu + 8HNO33Cu(NO3)2 + 2NO + 4H2O

Cu + 4HNO3Cu(NO3)2 + 2NO2 + 2H2O

2NO + O2 2NO2

1 – Purified aqueous extract

2 – Dialysis (semipermeable) membrane

3 – Funnel

4 – Distilled water

5 – Impurities

1

2

3

4

← 4

5 ←

17

2NO2+ H2O HNO3 + HNO2

Hydrochloric acid is distilled from the aqueous extract. Hydrochloric acid can also be distilled from the dialysate in case where it

contains sulphuric acid (the reaction of sulphuric acid with natural chloride resulted in releasing hydrogen chloride). Therefore, the

detection of hydrochloric acid is carried out after confirming the absences of sulphuric acid in the dialysate.

Table 5 – Detection of inorganic acids, alkalis, nitrates and nitrites

No. Method Observation Chemical reaction equation(s)

Specificity and

sensitivity of

the reaction

Detection of sulphuric acid

1

Reaction with barium chloride

Sensitivity:

Specificity:

2

Reaction with lead acetate

Sensitivity:

Specificity:

3

Reaction with sodium rhodisonate and barium salt

Sensitivity:

Specificity:

Detection of nitric acid

1

Reaction with diphenylamine

Sensitivity:

Specificity:

18

2

Reaction with brucine in concentrated sulphuric acid Sensitivity:

Specificity:

3

Colouring of wool in a yellow colour Sensitivity:

Specificity:

Detection of hydrochloric acid

1

Reaction with silver nitrate

Sensitivity:

Specificity:

2

Reaction with potassium chlorate Sensitivity:

Specificity:

Detection of potassium hydroxide

1

Reaction with sodium hydrotartrate

Sensitivity:

Specificity:

2

Reaction with sodium cobalt nitrite (Na3[Co(NO2)6]) Sensitivity:

Specificity:

Detection of sodium hydroxide

1

Reaction with potassium hydroxystibnate (K[Sb(OH)6])

Sensitivity:

Specificity:

19

2

Reaction with zinc-uranil acetate

(Zn(UO2)3(CH3COO)8)

Sensitivity:

Specificity:

Detection of ammonium hydroxide

1

Reactions with litmus paper and reactive paper

moistened by copper sulphate

Sensitivity:

Specificity:

2 Reaction with Nessler’s reagent

Detection of sodium nitrite

1

Reaction with sulphanilic acid and β-naphtol

Sensitivity:

Specificity:

2

Reaction with Griss reagent (sulphanilic acid and α-

naphthyl amine)

Sensitivity:

Specificity:

3

Reaction with iodine-starch paper Sensitivity:

Specificity:

Detection of sodium nitrite

1

Reaction with diphenylamine Sensitivity:

Specificity:

2

The reaction with brucine Sensitivity:

Specificity:

20

3

The reaction of ferrum (II) with sulfate and concentrated

sulfuric acid

Sensitivity:

Specificity:


1. Sulphuric acid. Mechanism of toxic action. Chemical-toxicological analysis of sulphuric acid.

2. Nitric acid. Mechanism of toxic action. Chemical-toxicological analysis of nitric acid.

3. Hydrochloric acid. mechanism of toxic action. Chemical-toxicological analysis of hydrochlori acid.

4. Toxicity and treatment of nitrate and nitrite poisonings. Antidote therapy.

5. Chemical-toxicological analysis of nitrates and nitrites.

6. Alkalis. Mechanism of toxic action. Chemical-toxicological analysis of caustic alkalis (sodium hydroxide, potassium hydroxide) and

ammonia.

7. Physical basis of the dialysis method.

21

Topic: CHEMICOTOXICOLOGICAL ANALYSIS OF DRUGS. EXAMINATION OF THE ACID CHLOROFORM

EXTRACT FOR DERIVATIVES OF BARBITURIC AND SALICYLIC ACIDS, PYRAZOLONE, PURINE

GENERAL ISOLATION METHODS OF DRUGS FROM BIOLOGICAL MATERIAL

Isolation of drugs by water acidified with oxalic acid

(A.A. Vasilyeva`s method). Place a biological sample reduced to

fine particles in a retort or glass, add distilled water, the volume is

equivalent to twice the volume of the sample taken, acidify the

mixture by 10 % oxalic acid solution to adjust pH to 2–3 with the

universal indicator paper. Retain the mixture with constant mixing

for two hours; filter through a double layer of gauze. Repeat the

extraction by one-half volume of the acidified water for an hour.

Combine the aqueous extracts and extract the drug by three portions

of chloroform, 10 ml each. Combine the chloroform extracts,

remove residual moisture from the extract by filtrartion and collect

the filtrate in a dry conical test-tube labeled “Acid chloroform

extract”. Add 25 % ammonium hydroxide solution to the aqueous

residue to adjust pH to 8–9 by the universal indicator paper, extract

the drug by three portions of chloroform, 10 ml each. Combine the

chloroform extracts; filter them as described above, in a dry conical

test-tube labeled “Alkaline chloroform extract”. When extracting

poisons by chloroform from alkaline aqueous solutions the stable

emulsions can appear, they can be destroyed by centrifugation or

addition of anhydrous sodium sulphate.

Isolation of drugs by ethanol acidified with oxalic acid

(Stas-Otto method). Place a biological sample reduced to fine

particles in a glass, add 96 % ethanol to form the mirror surface,

acidify the mixture with 10 % ethanol solution of oxalic acid to

adjust pH to 2–3 by the universal indicator paper and retain the

mixture for one day mixing periodically. Then filter the ethanol

extract through the paper filter moistened by ethanol. Repeat the

extraction 2–3 times. Combine the acidic ethanol extracts and

evaporate ethanol in the water bath at 40–50 ºC to obtain the

solution with the syrup consistence. Add 96 % ethanol by drops to

the solution obtained while protein admixtures being precipitated.

Retain the extract for precipitation of the admixtures and then filter

through a small filter with 5–6 cm in the diameter preliminary

moistened by ethanol. Wash the filter by ethanol. Then repeat the

evaporation to obtain the solution of the syrup consistence and

filtrate. Repeat these operations until protein admixtures have not

been precipitated by ethanol. Then evaporate the extract obtained in

the water bath to the syrup consistence, dissolve the residue in 25–30

ml of warm distilled water and filter the turbid solution through a

small smooth filter moistened by water in a separating funnel.

Extract drugs from the water-ethanol solution by three portions of

chloroform, 10 ml each. Filter the chloroform extract through a filter

moistened by chloroform in a dry conical test-tube labeled “Acid

chloroform extract”.

Alkalify the water-ethanol residue by 25 % ammonium

hydroxide solution to adjust pH to 8 – 9 by the universal indicator

paper and extract the poison by chloroform as described above.

Place the chloroform extract in a dry conical test-tube labeled

“Alkaline chloroform extract”.

22

TLC-SCREENING OF ACIDIC, NEUTRAL AND WEAK BASIC DRUGS

TLC-screening is the preliminary stage of the toxicological study. The negative result of TLC-screening gives the evidence of the absence

of the toxic drug dosage. The positive result of TLC-screening assumes performing of the confirmatory analysis, which conclude the complex of

chemical, physicochemical and pharmacological tests.

A TLC system is characterized by the following conditions: choice of stationary and mobile phases, a set of reference substances, as well

as a method for detecting toxicant. Now various variants of TLC-screening of drugs have been proposed.

Figure 2 – Arrangement for TLC

The basic chromatographic measurement of a

substance in TLC is the Rf value, determined

as:

Rf = The distance the solventtravels from the origin

The distance the substancetravels from the origin

11 chromatographic systems have been recommended by the Committee for Systematic

Toxicological Analysis of The International Association of Forensic Toxicologists

(TIAFT) for which the database includes the values of chromatographic mobility fof about

1,600 toxicologically relevant substances.

Mobile phase recommended by TIAFT:

1. Chloroform – acetone (4:1)

2. Ethyl acetate

3. Chloroform – methanol (9:1)

4. Ethyl acetate – methanol – 25 % ammonia (17:2:1) (the reference compounds are

acidic, neutral and weak basic drugs)

5. Ethyl acetate – methanol – 25 % ammonia (17:2:1) (the reference compounds are

basic drugs)

6. Methanol

7. Methanol–n-butanol (3:2) containing 0.1 mol/L sodium bromide

8. Methanol – 25 % ammonia (100:1.5)

9. Cyclohexane – toluene –diethylamine (15:3:2)

10. Chloroform – methanol (9:1)

11. Acetone

1 – 4 chromatographic systems for acidic, neutral and weak basic drugs.

5 – 11 chromatographic systems for basic drugs.

23

Figure 3 – Chromatographic plate

Cover the columns 3 and 4 by a a glass plate and spray columns 1 and 2 by 5 % mercury sulphate solution and then 0.1 %

diphenylcarbazone solution in chloroform. Blue and red-violet sports indicate the presence of barbiturates.

Cover columns 1, 2 and 4 by a glass plate and spray column 3 by 10 % iron (III) chloride solution. Blue, dark blue, blue-violet, red-violet spots

indicate the presence of pyrazolone derivatives; blue-violet spots are given by salicylic acid.

Cover columns 1, 2 and 3 by glass plate and spray column 4 by Dragendorff reagent and then 10 % sulphuric acid solution. Orange,

orange-brown, yellow-orange spots indicate the presence of weak basic drugs (caffeine, amidopyrine, antipyrine, diazepam, nitrazepam).

Location reagents for acidic drugs

UV-light (λ254 and λ366 nm).

Van Urk reagent. Spray the plate with the reagent and then heat in an

oven at 100° for 5 min. Yellow spots are given by sulfonamides and by

meprobamate

Iron (III) chloride solution. Blue or violet spots are given by phenols

(salicylic acid). Blue, dark-blue, blue-violet, red-violet spots indicate the

presence of pyrazolones. This solution can be used to overspray the plate,

which has been previously sprayed by Van Urk reagent.

Mercurous sulphate spray, then diphenylcarbazone in chloroform.

Blue and red-violet spots indicate the presence of barbiturates.

Acidified potassium permanganate solution. Yellow-brown spots on a

violet background are given by drugs with unsaturated aliphatic bonds, e.g.

quinalbarbitone. This solution can be used to overspray a plate, which has

been previously sprayed by mercurous sulphate spray.

Location reagents for neutral

drugs

Furfuraldehyde reagent. Violet to

blue-black spots are given by some

neutral compounds, e.g. carbamates.

Acidified iodoplatinate solution.

The method of TLC-screening of

acid chlotoform extract (a variant)

Plates. Silica gel G, 250 µm

Mobile Phase. Acetone–chloroform

(1:9)

Standard compound. Cyclobarbital

chloroform solution (10 µg/ml).

Divide a TLC plate into 4-equal columns and

draw a horizontal line in 10 cm distance from

the origin. Apply 10 µl aliquots of the

reference solution and 0.1–0.2 ml of the

extract aliquots (this is equivalent to 1–2 g of

the biological sample) to the columns on the

plate.

24

Evaluation of the results. When colour spots do not appear, the examination of the acid chloroform extract is over, in the presence of

coloured spots the analysis proceeds in TLC-systems for specific groups of drugs or the confirmative examinations are performed with the help of

chemical reactions, UV-spectroscopy, Gas-Luquid chromatography, High Pressure chromatography, etc.

Laboratory Practicals Assignments

To perform Toxicological screening of acid chloroform extract.

Samples: model chloroform solutions containing salicylic acid, antipyrine, aminazine; chloroform extract containing unknown drug.

Reagents and equipment: chromatographic plates; mobile phases: chloroform–acetone (9:1), location reagents: Dragendorff reagent, 5 %

iron (III) chloride solution, UV lamp, chromatography tank, capillaries.

Method. Apply the extracts examined and the authentic solutions of salicylic acid, antipyrine, aminazine on a chromatographic plate.

Place the chromatographic plate in a tank containing the mobile phase chloroform–acetone (9:1) and develop the chromatogram. Dry the

chromatographic plate, spray it with 5 % iron (III) chloride solution (spots with the characteristic colour appear in the presence of the drugs).

Calculate the Rf values.

Record the results obtained in Table 6 and draw schematically the chromatograms (Fig. 4).

Table 6 – TLC screening of the acid chloroform extract

Sample examined Mobile phase Location reagent The spot

colour Rf value

The conclusion:

Figure 4 – The scheme of TLC screening of

the acidic chloroform extract

25

Table 7 – Detection of derivatives of barbituric and salicylic acids, pyrazolone, purine by chemical tests


Specificity

and sensitivity

of the reaction

Detection of salicylates (salicylic acid)

1.

The reaction of tribromophenol formation

Method. Add a few drops of distilled water to the

remainder obtained after evaporation of the chloroform

extract, mix and add 2–3 drops of bromine water

saturated solution. Observe appearance of a precipitate

Specificity:

sensitivity:

1:40000, the

limiting

dilution

2.

The reaction with iron (III) chloride

Method (the first variant of the reaction performing).

Add 1 drop of iron (III) chloride solution to the residue

obtained after evaporation of the chloroform extract

placed in a porcelain cup. Observe appearance of colour.

Method (the second variant of the reaction performing).

Apply 1 drop of iron (III) chloride solution (prepare

fresh) on the filtration paper and dry. Apply 1–2 drops of

the chloroform extract examined at the same place.

Observe appearance of colour

Specificity:

3.

The reaction of methylsalycilate formation (study

theoretically)

Method. Place a few drops of the chloroform extract

examined into a test tube, evaporate to dryness in a water

bath and add to the remainder 2–3 drops of concentrated

sulphuric acid, 2–3 drops of methanol and heat in a water

bath. Methyl salicylate shows a characteristic odour

Specificity:

26

Detection of barbituric acid derivatives (barbital, phenobarbital)

1.

The model solutions containing some substances

imitaiting barbiturates are used

The general reaction for barbiturates

The reaction with cobalt nitrate or acetate and

ammonia

Method. Apply a drop of the chloroform extract on a

filter paper previously treated with 1 % cobalt nitrate

ethanol solution, dry out. Then fumigate the paper by

ammonia vapours (leave it over the surface of 25 %

ammonium hydroxide solution). Observe appearance of

colour

Specificity:

2.

The formation of barbiturate characteristic crystals

Method. Apply a few drops of the chloroform extract on

a slide, evaporate to dryness at room temperature.

Repeat this procedure. Dissolve the remainder obtained

in a drop of concentrated sulphuric acid. In 3–5 min.

place one drop of distilled water next to this drop,

combine carefully the contents of the drops with the

help of glass rod. Characteristic crystalline precipitates

are observed for particular barbiturates in 10–20 min.,

the crystals can appear in 1–2 hours when little amounts

of barbiturates are analysed. Describe the form of the

crystals obtained and draw them

Specificity:

sensitivity:

800 µg per

a sample

(barbital)

3.

The reaction with chlorine-zinc-iodine reagent



Repeat this procedure. Apply a drop of chlorine-zinc-

iodine reagent on the remainder. In 10–15 min. the

characteristic crystals appear. When the crystals do not

appear for a long time, add 1–2 crystals of iodine to the

sample and in 10–15 min. observe them again with the

help of a microscope. Describe the form of the crystals

obtained and draw them

Specificity:

sensitivity:

4 µg per

a sample

(barbital)

27

4.

The reaction with ferric-iodine reagent



Repeat this procedure. Apply one drop of ferric-iodine

reagent to the dry remainder. In 10–15 min.

characteristic crystals appear. With abundance of

crystals evaporate the reaction mixture placed on a slide

using an alcohol burner, then add a drop of distilled

water to the dry remainder. In 10–15 min. examine the

crystals obtained with the help of a microscope.

Describe the form of the crystals obtained and draw

them

Specificity:

sensitivity:

1.8 µg per

a sample

(barbamyl)

5.

The reaction with copper-iodine reagent


a slide, evaporate to dryness at room temperature. Repeat

this procedure. Add one drop of copper-iodine reagent to

the dry remainder. In 10–15 min. examine the crystals

obtained with the help of a microscope. Describe the

form of the crystals obtained and draw them

Specificity:

sensitivity:

2.1 µg per

a sample

(barbamyl)

Detection of purine alkaloids (caffeine)

1.

The general reaction

The reaction of murexide formation

Method. Place 5–6 drops of the chloroform extract

examined in a porcelain cup and evaporate the solvent

without heating. Add 0.5–1 ml of bromine water to the

dry remainder and evaporate using a water bath to

dryness. Apply a drop of 25 % ammonium hydroxide

solution on the remainder coloured in brown. Observe

appearance of colour

Specificity:

sensitivity:

0.05 mg per

a sample

(barbital)

28

2.

The reaction with Nessler`s reagent


examined in a porcelain cup and evaporate the solvent

without heating. Add Nessler`s reagent to the remainder

and heat the mixture to 100 °C in a water bath for 1–

2 min. Observe appearance of a coloured precipitate.

The blank experiment should be performed

Specificity:

Detection of pyrazolone derivatives (antipyrine, analgin)

1.

Antipyrine



examined into a porcelain cup, evaporate it to dryness.

Add a drop of 5 % iron (III) chloride solution to the dry

remainder. Observe appearance of colour

Specificity:

2.

The reaction of nitrosoantipyrine formation

Method. Place 3–5 ml of the chloroform extract


Dissolve the dry remainder in 3–5 drops of water, add

2–4 drops of 10 % sulphuric acid solution and 2–3

drops of sodium nitrite saturated solution to the solution

obtained. Observe appearance of colour

Specificity:

3.

The reaction of azo dye formation (study theoretically)


examined into a test tube, evaporate it in an water bath

to dryness. Add 1–2 drops of water to the residue. Then

add a drop of acetic acid and a drop of 5 % potassium

nitrite solution to the solution obtained. Shake

periodically for 5 min. Then add a bit of sodium azide

to the mixture. When gas liberation stops, add 3–4

crystals of β-naphtylamine and heat the test tube in a

water bath for 1–2 min. Observe appearance of colour

Specificity:

29

1.

Analgin




Add a drop of 5 % iron (III) chloride solution to the

residue. Observe appearance of colour

Specificity:

2.

The lignin test

Method. Apply 2–3 drops of the chloroform extract in

the same place of a newspaper piece. Observe

appearance of colour. It becomes more intensive after

treatment of the spot with hydrochloric acid

diluted solution

Specificity:

sensitivity:

5 µg per

a sample

Detection of cannabinoids

1.

The reaction with Duquenois-Levine reagent

the model solutions containing some substance

imitaiting cannabinoids is used)



Add a drop of Duquenois-Levine reagent (2 g of

vaniline in 100 ml of 1 % acetaldehyde ethanol

solution) and a drop of concentrated hydrochloric acid

to the residue. Observe appearance of colour

Specificity:

sensitivity:

1 µg per

a sample

30


1. Physicochemical property of drugs (solubility, ionization constant, lipophilicity).

2. Theoretical bases of liquid-liquid extraction.

3. Theoretical bases of salting out, gel-chromatography, sublimation, electrophoresis.

4. Physico-chemical properties of barbiturates.

5. Chemical-toxicological analysis of p-aminophenol derivatives (paracetamol).

31

Practical lesson 4 _____

Date

Topic: EXAMINATION OF THE BASIC CHLOROFORM EXTRACT FOR ALKALOIDS

TLC-SCREENING OF BASIC DRUGS

Location Reagents for basic drugs

1. UV-light (λ254 and λ366 nm).

2. Ninhydrin spray. Spray the plate by the reagent and then heat it an oven at 100ºC for 5 minutes. Violet or pink spots are given by primary

amines and yel¬low colours by secondary amines.

3. FPN reagent. Red or brown-red spots are given by phenothiazines.

4. Dragendorff’s spray. Yellow, orange, red-orange, or brown-orange spots are given by tertiary alkaloids. This reagent can be used to overspray

a plate, which has been previously sprayed by ninhydrin spray.

5. Acidified iodoplatinate solution. Violet, blue-violet, grey-violet, or brown-violet spots on a pink background are given by tertiary amines and

quaternary ammonium compounds.

6. Mandelin’s reagent. Many different colours are given with a variety of drugs.

7. Marquis reagent. Black or violet spots are given by alkaloids related to morphine. Many different colours are given with a variety of drugs.

8. Acidified potassium permanganate solution. Yellow-brown spots on a vi¬o¬let background are given by drugs with unsaturated aliphatic

bonds.

The method of TLC-screening of basic chlotoform extract (a variant) Plates. Silica gel G, 250 µm. Mobile Phase. Chloroform–dioxane–acetone–25 % ammonium hydroxide solution (47.5:45:5:2.5). Standard compound. Etapyrazine chloroform solution (10 µg / ml). Method. Divide a TLC-plate into 4 equal columns and draw a horizontal line in 10 cm distance from the origin. Apply 10 µl aliquot of the

reference solution and 0.1–0.2 ml aliquots of the extract examined (equivalent to 1–2 g of the biological sample) in 4 points of the origin line according to the columns marked. Place the plate in a tank containing the mobile phase. After development of the chromatogram take out the plate from the tank and dry in a stream of a cold air until the plate will not smell of ammonia. (Should not dry the plate in a hot air because of some volatile drugs could be lost).

Location Reagents. Examine the plate under UV light. Cover columns 3 and 4 with a glass plate and spray columns 1 and 2 by Dragendorff reagent. Orange and orange-brown sports are given by

most basic drugs. Cover columns 1, 2 and 4 with a glass plate and spray column 3 by 50 % sulphuric acid ethanol solution. Most phenothiazines are

extensively metabolized, and extracts yield a number of spots on the chromatogram with colours ranging from pink to blue. Cover columns 1, 2 and 3 with a glass plate and spray column 4 by 10 % iron (III) chloride solution. Red, dark blue, blue sports indicate the

presence of pyrazolone and phenothiazine derivatives. Avaluation of the results. If colour spots do not appear on the chromatogram, the examination can be over. When colour sports appear,

determine the TLC-systems for the specific groups of drugs according to the Rf and Rs values obtained in the general screen.

32


To perform Toxicological screening of basic chloroform extract.

Samples: model chloroform solutions containing quinine, aminazine, procaine; chloroform extract containing unknown drug.

Reagents and equipment: chromatographic plates; mobile phase ethyl acetate – methanol – 25 % ammonia (17:2:1), location reagents:

Dragendorff reagent, 50 % sulphuric acid ethanol solution, 1 % sodium nitrite solution, 2 M hydrochloric acid solution, -naphthol alkaline

solution, UV lamp, chromatography tank, capillaries.

Method. Apply the extract examined and the authentic solutions of quinine, aminazine, procaine on a chromatographic plate. Place the

chromatographic plate in a tank containing the mobile phase acetate – methanol – 25 % ammonia (17:2:1) and develop the chromatogram. Dry

the chromatographic plate, spray it Dragendorff reagent (the first variant). Spray columns 1, 2 and 4 by 50 % sulphuric acid ethanol solution,

examine the plate under UV light. Spray columns 3 and 4 with 1 % sodium nitrite solution, 2 M hydrochloric acid solution, -naphthol alkaline

solution (the second variant). Spots with the characteristic colour appear in the presence of the drugs. Calculate the Rf values.

Record the results obtained in Table 7 and draw schematically the chromatograms (Fig. 5).

Table 7 – TLC screening of the basic chloroform extract

Sample examined Mobile phase Location reagent The spot

colour Rf value

The conclusion:

Figure 5 –The scheme of TLC screening

of the acidic chloroform extract

33


To perform Toxicological screening of basic chloroform extract.

Samples: model chloroform solutions containing quinine, procaine, aminazine, extract contained unknown drug.

Reagents and equipment: chromatographic plates; mobile phase: chloroform – acetone – isopropanol – 25 % ammonium hydroxide

solution (7:7:7:2); location reagents: Dragendorff reagent, 10 % sulphuric acid solution, 2 M hydrochloric acid solution, 1 % sodium nitrite

solution, -naphthol alkaline solution, UV lamp, chromatography tank, capillaries.

Method. Apply the extracts examined and the authentic solutions of aminazine, quinine and procaine on a chromatographic plate. Place

the chromatographic plate in a tank containing the mobile phase chloroform – acetone – isopropanol – 25 % ammonium hydroxide solution

(7:7:7:2) and develop the chromatogram. Dry the chromatographic plate, spray it with Dragendorff reagent (orange-brown spots appear in the

presence of basic drugs) (another variant of detection: aminazine is detected with 10 % sulphuric acid solution, quinine is detected with 10 %

sulphuric acid solution and UV light, procaine is detected by the diazothization reaction). Calculate the Rf values.

Record the results obtained in Tables 12; 13 and draw schematically the chromatograms (Fig. 1; 2).

Table 8 – Chemical tests for alkaloids


Specificity and

sensitivity of

the reaction

Pyridine and piperidine alkaloids (anabazine, pachycarpine)

1.

Anabazine

The reaction with Dragendorff reagent

Method. Apply 2–3 drops of the chloroform extract

examined on a slide and evaporate to dryness. Add a

drop of 0.1 М hydrochloric acid solution and a drop of

Dragendorff`s reagent to the residue. Observe the

crystals in 10–15 min, describe their form and draw them

Specificity:

sensitivity:

1 µg per

a sample

34

2.

The reaction with 1 % Reinecke’s salt

Method. Apply on a slide 2–3 drops of the chloroform

extract examined and evaporate to dryness. Add a drop

of 0.1 М hydrochloric acid solution and a drop of 1 %

Reineke’s salt (prepare fresh) to the residue. Observe the


Specificity:

sensitivity:

0.7 µg per

a sample

3.

Pachycarpine

The reaction with iodine in potassium iodide solution

(Bushard reagent)



drop of 0.1 М hydrochloric acid solution and 1–2 drops

of iodine in potassium iodide solution to the residue.

Observe the crystals in 10–15 min, describe their form

and draw them

Specificity:

sensitivity:

3.5–4.2 µg per

a sample

4.

The reaction with picric acid




of 0.5 % picric acid solution to the residue. Observe the


Specificity:

sensitivity:

5 µg per

a sample

5.

The reaction with cobalt rhodanide reagent Method. Apply 2–3 drops of the chloroform extract



of cobalt rodanide reagent to the residue. Observe the


Specificity:

sensitivity:

1.5 µg per

a sample

35

Tropane alkaloids (atropine, scopolamine, cocaine)

1.

The general reaction

(with the exception of cocaine)

The Vitali-Morin reaction

Method. Add a few drops of concentrated nitric acid to

the dry residue obtained after evaporation of the

chloroform extract in a porcelain cup and evaporate

carefully to a dry residue using a water bath. Repeat this

procedure not less than 3 times, cool. Then add

potassium hydroxide ethanol solution to the residue.


Specificity:

sensitivity:

1 µg per

a sample

2.

The confirmative reactions

Atropine

The reaction with 1 % Reinecke’s salt solution




1 % Reinecke`s salt (prepare fresh) to the residue.

Observe the crystals in 10–15 min, describe their form

and draw them

Specificity:

sensitivity:

0.1 µg per

a sample

3.

The reaction with picric acid




of 0.5 % picric acid solution to the residue. Observe the


Specificity:

sensitivity:

5 µg per

a sample

36

4.

Scopolamine

The reaction with 1 % Reinecke`s salt solution




1 % Reinecke`s salt solution (prepare fresh) to the

residue. Observe the crystals in 10–15 min, describe their

form and draw them

Specificity:

5.

The reaction of cocaine permanganate formation (the

model solution containing some substance imitaiting

cocaine is used)

Method. Apply a few drops of the chloroform extract

examined on a slide, evaporate the organic solvent

without heating, add a drop of 10 % hydrochloric acid

solution to the residue. Evaporate the liquid at room

temperature. Repeat this procedure 2–3 times. Then add

a drop of 1 % potassium permanganate solution to the

residue. Observe the crystals in 10–15 min, describe their

form and draw them

Specificity:

sensitivity:

4 µg per

a sample

Isoquinoline alkaloids (morphine, codeine)

1.

The general reactions (the model solutions containing some substances

imitaiting opiates are used)

The reaction Marquis reagent


examined in a porcelain cup, evaporate the solvent

without heating. Add a drop of Marquis reagent (1 ml of

concentrated sulphuric acid, 1 drop of formaline, prepare

fresh) to the residue. Observe appearance of colour

Specificity:

sensitivity:

0.05 µg per

a sample

(morphine)

37

2.

The reaction with Froehde reagent



without heating. Add a drop of Froehde reagent (add the

excess of ammonium molibdate to concentrated

sulphuric acid, prepare fresh) to the residue. Observe


Specificity:

sensitivity:

0.05 µg per

a sample

(morphine)

3.

The reaction with Mandelin reagent



without heating. Add a drop of Mandelin reagent (add

0.01 g of ammonium vanadate to 2 ml of concentrated

sulphuric acid, prepare fresh) to the residue. Observe


Specificity:

4.

Morphine




without heating. Add a drop of iron (III) chloride

solution (prepare fresh) to the residue.

Specificity:

Quinoline alkaloids (quinine)

1.

Fluorescence of quinine sulphate aqueous solution

Method. Place the chloroform extract examined in a test

tube, evaporate chloroform using a water bath. Add 1 ml

of distilled water and 1 ml of 10 % sulphuric acid

solution to the residue. Observe fluorescence in UV light

Specificity:

2.

The thaleoquine test (modification by L.V. Peysahovich)

Method. Apply 5–10 drops of the chloroform extract on a

filter paper, moisten the spot with a drop of water and

treat it with bromine vapour until the yellow colour

appearance and then treat with 25 % ammonium

hydroxide solution. Observe appearance of colour

Specificity:

38

Acyclic alkaloids (ephedrine)

1.

The reaction with ninhydrin

(the model solution containing some substance imitaiting

ephedrine is used)

Method. Apply a few drops of the chloroform extract

examined on a piece of chromatographic plate, evaporate

the solvent without heating. Treat the plate by ninhydrin

solution in n-butanol. Heat the plate on a water bath at

100 °C for 5 min. Observe appearance of colour

Specificity:

Indole alkaloids (strychnine)

1.

Derivatives of (the model solution containing some

substance imitaiting strychnine is used)

The reaction with potassium bichromate in

concentrated sulphuric acid


examined in a porcelain cup and evaporate chloroform to

dryness. Add a drop of concentrated sulphuric acid and a

crystal of potassium bichromate. Observe appearance of

colour

Specificity:

sensitivity:

1 µg per

a sample

2.

The reaction with Mandelin reagent



without heating. Add a drop of Mandelin reagent to the

residue. Observe appearance of colour

Specificity:

The conclusion:

39


1. Toxicity, special symptoms of intoxication, first aid and detoxification in poisoning by alkaloids of various chemical groups.

2. Chemical-toxicological analysis of opium alkaloids: narcotine, papaverine, heroin, a semi-synthetic opiate and opium components,

meconic acid, meconin.

3. Drug addiction – social problem of society. Give definitions of terms "narcotic substance", "psychoactive substance", "drug addiction".

40

Practical lesson 5

_____

Date

Topic: EXAMINATION OF THE BASIC CHLOROFORM EXTRACT FOR DERIVATIVES OF

1,4-BENZODIAZEPINE, PHENOTHIAZINE, p-AMINOBENZOIC ACID

Table 9 – Chemical reactions for basic drugs


Specificity

and

sensitivity of

the reaction

Derivatives of 1,4-benzodiazepine

1.

2.

Acidic hydrolysis

Method. Evaporate 2 ml of the chloroform extract to dryness

in a water bath. Add 5 ml of 6 M hydrochloric acid solution to

the residue and reflux the solution at 100 °C for 60 min. Cool

the hydrolyzate obtained, neutralize it with saturated sodium

hydroxide solution to adjust the pH to 7–9. Place the solution

into a separating funnel and extract the products of 1,4-

benzodiazepine hydrolysis (aminobenzophenones) with the

equal volume of chloroform. Filter the organic phase through

anhydrous sodium sulphate. Evaporate the extract to the

volume of 0.2 ml and examine.

Examination in UV light

Place a few drops of the extract on a chromatographic plate

and examine in UV light. Observe fluorescence.

Azo dye coupling reaction

Method. Apply a few drops of the extract on a piece of

chromatographic plate and treat the spot with 1 % sodium

nitrite solution, then with 2 M hydrochloric acid solution and

-naphthol alkaline solution.

Specificity:

41

Derivatives of phenothiazine (aminazine, levomepromazine)

1.

The reaction with concentrated sulphuric acid


examined in a porcelain cup, evaporate chloroform to

dryness. Add a drop of concentrated sulphuric acid.


Specificity:

2.

The reaction with concentrated nitric acid



dryness. Add a drop of concentrated nitric acid. Observe


Specificity:

3.

The reaction with concentrated hydrochloric acid



dryness. Add a drop of concentrated hydrochloric acid.


Specificity:

Derivative of p-aminobenzoic acid (procaine)

1.

Azo dye coupling reaction

Method. Apply the chloroform extract examined on a

piece of chromatographic plate, evaporate to dryness,

apply a drop of 2 M hydrochloric acid and a drop of 1 %

sodium nitrite solution, then treat the spot with a drop of

-naphthol alkaline solution. Observe appearance of

colour.

Specificity:

42

Topic: QUANTITATIVE DETERMINATION OF DRUGS IN THE EXTRACTS

Figure 6 –Photocolorimeter


Perform quantitative determination of aminazine by the photocolorimetric method by the reaction with iron (ІІІ) chloride in the model

extract from the biological sample

Reagents:

1. 5 % iron (III) chloride solution.

2. Distilled water.

3. The model extract from the biological sample examined for aminazine.

Method

Place 5 ml of the extract from biological sample in a porcelain cup and evaporate the organ ic solvent to dryness, dissolve the remainder

in 5 ml of distilled water, place the aqueous solution obtained into a test tube, add 1 ml of 5 % iron (III) chloride solution and mix. Place the

coloured solution obtained into a 10 mm path-length cell and measure the absorbance (A) value using a photoelectrocolorimeter at 540 nm.

Prepare the reference solution: place 5 ml of the extract from the biological sample (blank experiment) in a porcelain cup and evaporate the

organic solvent to dryness, dissolve the remainder in 5 ml of distilled water, place the aqueous solution obtained into a test tube, add 1 ml of 5 %

iron (III) chloride solution and mix. Measure the absorbance of the solution obtained, calculate the aminazine concentration with the help of the

equation of the calibration dependence given below and determine the aminazine content in 1 g of the biological material (V0 = 30 ml, V1 = 5 ml,

m = 10 g).

Colorimetric analysis is a method of determining the concentration of a

chemical element or chemical compound in a solution with the aid of a color reagent.

The measured analytical signal is Absorbance (A):

Absorbance is linearly related to the concentration (c) and path-length (b) (the

Bouguer-Lambert-Beer’s Law): A = k c b

The validity of Bouguer-Lambert-Beer’s Law should be established for each

drug under the measurement conditions to be used over the appropriate concentration

range. The curve of absorbance (A) versus concentration (c), a calibration curve, is

established usind five or more standard solutions. A matched pair of cells are used,

absorbance of analysed solution is measured against the solvent as a reference

43

Lethal concentrations of aminazine are: 240-280 µg/g (liver); 126–148 µg/g (brain); 134–162 µg/g (kidney). [Clark’s analysis of Drugs

and Poisons: Third edition / Laurent Y. Galichet. – 80 Min / 700 MB.– Pharmaceutical Press, 2005. – 1 electron. opt. disc (CD-ROM); 12 cm].

Formula for the calculation:

,1

0

mV

CVX

where Х – is the content of aminazine in 1 g of the biological sample, mg;

C – is the content of aminazine detrmined by the equation, mg;

Vo – is the total volume of the extract from the biological sample, ml;

V1 – is the volume of the extract taken for the photocolorimetric determination, ml;

m – is the mass of the biological sample, g.

Table 10 – Data for construction of calibration curve: y=а + вx

С, mg 0.1 0.2 0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0 1.1 1.2

А 0.05 0.13 0.20 0.27 0.34 0.41 0.48 0.56 0.62 0.69 0.77 0.84

Table 11 – Metrological characteristics of the calibration curve

r b a S2 Δb Δa

0.995 0.712 -0.016 2·10-5 0.007 0.005

The calibration curve was determined by the linear regression method: y= 0.712·x – 0.016.

Make the conclusion about the concentration determined in the biological sample (therapeutic, toxic or lethal).

The conclusion:

44

Topic: EXAMINATION OF BODY FLUIDS FOR DRUGS IN ACUTE INTOXICATIONS

Specimens analysied for presence of drugs in acute intoxications: body fluids, they are whole blood, plasma, serum, urine sometime

saliva.

The sample pretreatment methods:

Deproteinization (or protein removal) techniques include chemical precipitation (using trichloroacetic acid, perchloric acid, hydrochloric

acid, phosphotungstic acid, acetonitrile, etc) and ultrafiltration.

Breaking the drug-protein bonds in plasma by adding acetonitrile, methanol, ethanol, acetone, so that the volume of the solvent exceeds

the volume of the plasma sample more than 10 times.

The hydrolysis of conjugates (the products of the second stage of drug biotransformation) in urine using acidic hydrolysis (for this, add

0.1 M hydrochloric acid to the sample being tested and insert the mixture for 10 – 12 hours) or enzymatic hydrolysis.

Methods of drug solation of from the body fluids:

Liquid-liquid extraction (LLE)

Solid-phase extraction (SPE)

Liquid-liquid extraction. Acidic substances are extracted with organic solvents from acidic medium (pH of 1 – 3), basic substances are

extracted with an organic solvent from an alkaline medium at an appropriate pH value. Chloroform, diethyl ether, methylene chloride, sometimes

dichloroethane, benzene or a mixture of organic solvents in the presence or without the salting-out agents are used. Optimization of the isolation

conditions is based on the previous study in the degree of drug extraction with a range of typical organic solvents depending on pH of aqueous

solution. A general approach is also used:

it is necessary to create a solution pH = pKa – 2 for the extraction of acidic substances,

it is necessary to create a solution pH = pKa + 2.for the extraction of basic substances.

Solid-phase extraction. The method consists in sorption of a drug with polymer resins modified by silica gel, activated carbon, etc.

followed by the elution of a toxic substance with a suitable solvent.

SPE technique involves the following stages (after sample pretreatment): 1 – column conditioning, the column is made ready for the sample

being loaded; 2 – sample loading, the sample is loaded onto the cartridge; 3 – column washing, interference admixtures are washed off from the

column; 4 – sample elution, the sample is eluted by a suitable solvent and collected (Fig. 7).

1 2 3 4

Figure 7 – The scheme of separation of the biological matrix components and substance being testedt using SPE cartridge

45

Methods of the extract purification from concomitant admixtures;

back extraction

solid-phase extraction,

thin layer chromatography (TLC-purification)

Methods of drug screening (detection of drugs in the extracts):

Immunochromatographic screening

Colour tests

HPLC-screening

GLC-screening

Figure 9 – Scheme of immunochromatographic strip

tests

Immunochromatographic screening. Other names are ”Express” urine drug testing”,

“Immunochromatographic strip tests”. Most widely accepted method for drug screening

urine uses the principle of specific immunochemical reactions.

Immunochromatographic method is implemented in the format of test strips or

test cassettes. The indicated test strips have three working zones: the immersion site in

the biological fluid, the test zone “T” and the control zone “C”. Antibodies specific to

defined antigen (drug) are applied to the test strip. Antibodies are conjugated with the

dye, a colloidal gold.

The tests are performed and interpreted according to the manufacturer’s

instructions. When using immunochromatographic tests “SNIPER” a single colored

band appears in the control zone “C” in the presence of psychotropic substances (or their

metabolites). And two colored strips appear on the test strip, in zones “T” and “C” in the

absence of a psychotropic substance or its metabolites (Fig. 8). The opposite is possible

(Fig. 89.

Features of the drug screening method:

Easy-to-read and accurate results in 3 to 8 minutes.

Able to detect as low as 300 ng/ml (morphine, codeine), 780 ng/ml (cocaine), 50

ng/ml cannabinoids, 75–300 ng/ml (barbiturates), 195 ng/ml (diazepam).

Amphetamines, cocaine, opiates can be determined over the last 2 or 3 days after

drug use. Marijuana and its metabolites, cannabinoids, may be detectable for

several weeks.

Accuracy is of 99.5%.

Figure 8 – Immunochromatographic tests “SNIPER”

46


Perform drug screening in the model body fluids using colour tests.

Samples: the model body fluids containing salicylic acid, paracetamol, aminazine, quinine, barbiturates, codeine, derivatives of 1,4-

benzodiazepines expected to be found (model solutions).

Table 12 – Chemical reactions used for body fluid examination for the presence of drugs

No. Method Observations Chemical reaction equation(s)

Specificity and

sensitivity of

the reaction

1.

Tests, which can be performed directly with the body

fluids (urine, plasma) without the preliminary extraction

of a drug

Salicylic acid and salicylates

Method. Add 3 drops of 5 % iron (III) chloride solution

to 1 ml of the body fluid. Observe appearance of colour

2.

Paracetamol

Method. Add 2–3 drops of 10 % hydrochloric acid

solution to 1 ml of the body fluid and cool. Then add 2–3

drops of 1 % sodium nitrite solution and 2–3 drops of

1 % β-naphtol solution in 10 % sodium hydroxide

solution (prepare fresh) to the cooled mixture. Remember

that a great excess of β-naphtol solution interferes with

this reaction. Observe appearance of colour

3

Aminazine

Method. Add 1 ml of FPN reagent (the mixture of iron

(III) chloride, perchloric acid, nitric acid) to 1 ml of the

body fluid. Observe appearance of colour

4.

Quinine

Method. Add 3 ml of 10 % sulphuric acid solution to

2 ml of the body fluid. Examine in UV light. Observe

appearance of fluorescence

47

1.

Tests, which should be performed after the preliminary

extraction of a drug from the body fluids (urine, plasma)

Barbiturates

Method. Place 5 ml of the body fluid into a separating

funnel, add dropwise 10 % sulphuric acid solution to

adjust the pH to 4–5, extract the drug with 5 ml of

chloroform. Filter the chloroform extract in the presence

of anhydrous sodium sulphate. Examine the chloroform

extract obtained for the presence of barbiturates. Apply a

drop of the chloroform extract on a piece of a filter paper

pre-treated with 1 % cobalt nitrate ethanol solution, dry

out. Then fumigate the paper by ammonia vapours (place

it on top of the vessel containing 25 % ammonium

hydroxide solution). Observe appearance of colour

2.

Codeine


funnel, add dropwise 25 % ammonium hydroxide

solution to adjust the pH to 9–10, extract the drug with

5 ml of chloroform. Separate the chloroform extract and

filter it in the presence of anhydrous sodium sulphate.

Examine the chloroform extract obtained for the presence

of codeine. Place it in a porcelain cup, evaporate to

dryness using a water bath. Apply a drop of Marquis

reagent (prepare fresh) on the remainder. Observe


3.

Amino-benzophenons (hydrolysis products of 1,4-

benzodiazepines)


funnel, add dropwise 25 % ammonium hydroxide

solution to adjust the pH to 9–10, extract the substance

with 5 ml of chloroform. Separate the chloroform extract

and filter it in the presence of anhydrous sodium

sulphate. Examine the chloroform extract obtained for

48

the presence of aminobenzophenones. Place it in a

porcelain cup, evaporate to dryness using a water bath,

treat the remainder with a drop of 2 M hydrochloric acid

solution and a drop of 1 % sodium nitrite solution, apply

a drop of -naphthol alkaline solution in 2–3 min.


Record the results obtained in Table 13 and make the conclusion about the presence or absence of drugs suspected in the body fluids

Table 13 – Results of of body fluids examination for the presence of drugs

Series

No.

Drugs, which are detected directly in the body fluids Drugs, which are detected after extraction from the body fluids

1 2 3 4 barbiturates codeine amino-

benzophenones

The conclusion:

49


1. The chemical toxicological significance and methods of toxicological screening of some narcotic, psychotropic and potent medicinal

substances (synthetic opioids, amphetamines, dimedrol, clonidine, amitriptyline).

2. Methods of quantitative determination of drugs in the extracts from the biological material.

3. Methods of clinical diagnosis of acute poisonings.

4. The main directions of laboratory express analysis of acute intoxication. The role of analytical diagnostics in the express analysis of

acute intoxications.

5. Use of immunochemical and immunochromatographic screening, TLC-screening, HPLC and GLC screening for analytical diagnostics

of acute poisoning.

50

CONTENT MODULE 3 CHEMICOTOXICOLOGICAL ANALYSIS OF VOLATILE SUBSTANCES AND HEAVY METAL COMPOUNDS.

ANALYTICAL DIAGNOSTICS OF TOXIC GAS POISONINGS

Practical lesson 6

_____

Date

Topic: CHEMICOTOXICOLOGICAL ANALYSIS OF VOLATILE SUBSTANCES. DETECTION OF VOLATILE SUBSTANCES

IN THE DISTILLATES BY CHEMICAL METHOD

Isolation of volatile toxic substances from biological material

The method of steam distillation. Prepare the device for steam distillation (Fig. 10). For this

purpose, pour water in a vaporization flask and heat it to boiling. Reduce 100 g of the biological

material to fine particles, mix up with distilled water and place into a retort (the total volume of the

mixture examined should be not more than 1/3 part of the retort volume in order to avoid the ejection

of the biological material during distillation) placed into a cold water bath. Then heat the

vaporization flask until water vapour appears, add oxalic acid saturated aqueous solution to adjust the

pH to 2.0–2.5 to the biological material examined, close the retort by a cork quickly and connect all

parts of the device (vaporization flask, retort, refrigerator and receiver) immediately (be careful with

the liberating steam) and only then heat the water bath.

Collect the first portion of the distillate (3 ml the first distillate) in the receiver containing

2 ml of 5 % sodium hydroxide solution for binding of hydrocyanic acid.

Prepare 50 ml empty flasks and collect the subsequent two portions (the second and the third

distillates) of the distillate 25 ml each. Carry out the distillation with such rate, which gives the

possiblity to count the drops of the distillate. When distillating too quickly reduce the intensity of

heating of the vaporization flask by a gas-ring, when distillating too slowly do vice versa.

In the directed examination for ethylene glycol carry out the distillation process in the

presence of benzene and collect 500 ml of the distillate (a high temperature boiling substance).

In the directed examination for acetic acid acidify the biological material by 10 % sulphuric

or phosphoric acid solution and collect the distillate into the flask containing 0.1 М sodium

hydroxide solution.

Figure 10 – Apparatus for stem

distillation

1. Steam generator;

2. The bulb with the sample;

3. Water bath;

4. Bulb reflux condenser;

5. Receiver vessel.

51

In the directed examination for methanol collect the distillate into the coolled flask.

In the quantitative determination of the volatile poisons carry out the distillation process to the negative result of the reactions for the

substance analysed.

Table 14 –Detection of volatile toxic substances in the distillates with chemical method (cyanide, formaldehyde, halocarbons)


Specificity and

sensitivity of

the reaction

Examination of the first distillate for the presence of cyanide

1.

The reaction of Prussian blue formation

(The model solution containing some substance

imitaiting cyanide is used)

Method. Add 2–3 drops of 40 % iron (II) sulphate

solution containing traces of iron (III) cation to 1 ml of

the alkaline distillate. Shake up the mixture, heat to

boiling and then cool at the room temperature and add

10 % hydrochloric acid solution adjust pH to the weak

acidic value. Observe appearance of colour

Specificity:

sensitivity:

20 µg/ml

Examination of the second distillate

Detection of formaldehyde

1.

The reaction with resorcinol alkaline solution

Method. Add 1 ml of the prepared fresh reagent (mix up

1 % resorcinol solution with the equal volume of 10 %

sodium hydroxide solution) to 1 ml of the distillate

examined placed into a test tube. Carry out the blank

experiment at the same time. Heat the content of both test

tubes for 3–5 min. on a water bath. Observe appearance

of colour

Specificity:

sensitivity:

0.03 µg per

a sample

52

2.

The reaction with Fehling’s reagent (reduction of

copper (II) hydroxide

Method. Add 2 drops of sodium hydroxide solution to

adjust to an alkaline pH and 2–3 drops of the prepared

fresh Fehling’s reagent (mix Fehling’s reagent 1 and

Fehling’s reagent 2 (1:1)) to 1 ml of the distillate

exmined, mix and heat on a water bath. Observe


Specificity:

3.

The reaction with codeine and sulphuric acid (study

theoretically)

Method. Mix 1 ml of the distillate exmined with 5 ml of

concentrated sulphuric acid in a porcelain cup. Add a

crystal of codeine to the solution obtained. Observe


Specificity:

sensitivity:

0.02 µg per

a sample

4.

The reaction with chromotropic acid

Method. Place 1 ml the distillate exmined in a porcelain

cup, add 3–4 drops of concentrated sulfuric acid and a

few crystals of chromotropic acid and mix carefully.


Specificity:

sensitivity:

1 µg per

a sample

53

5.

The reaction with fuchsin-sulphurous acid

Method. Add 2–3 drops of concentrated sulphuric acid to

the distillate exmined, mix carefully, cool and add 1 ml

of fuchsin-sulphurous acid. Observe appearance of

colour

Specificity:

sensitivity:

0.03 µg per

a sample

Detection of halocarbons (chloroform, chloral hydrate, tetrachloromethane)

1.

The determination of organically bound chlorine

Method. Add 1 ml of 10 % sodium hydroxide ethanol

solution to 1–2 ml of the distillate examined placed into a

test-tube. Heat the test tube with the content to boiling on

a water bath for 30 min. or a fire ring for 3–5 min. Cool

the solution, add 10 % nitric acid solution adjust pH to

the acidic value and 0.5 ml of 1 % silver nitrate solution.

Observe appearance of a precipitate

Specificity:

sensitivity:

0.2 mg/ml

(chloroform);

0.15 mg/ml

(chloral

hydrate);

6.8 mg/ml

(tetrachlorome-

thane);

2.5 mg/ml (1,2-

dichloro-ethane)

54

2.

Formation of isonitrile (study theoretically) Method. Add 10 drops of 10 % sodium hydroxide ethanol solution and 1 drop of aniline aqueous solution to 1 ml of the distillate exmined. Heat the mixture on a water bath for 1–2 min. Examine the odour of the mixture. Then add 10 % sulphuric acid solution to the reaction mixture and boil to disappearance of the odour for decomposition of the isonitrile formed

Specificity:

sensitivity:

0.01 mg/ml

(chloroform,

chloral

hydrate);

2.3 mg/ml

(tetrachlorome

-thane)

3.


Method. Add 1 ml of the prepared fresh reagent (mix up

1 % resorcinol solution with the equal volume of 10 %

sodium hydroxide solution) to 1 ml of the distillate

examined placed into a test tube. Carry out the blank

experiment at the same time. Heat the content of both test

tubes for 3–5 min. on a water bath. Observe appearance

of colour

Specificity:

sensitivity:

0.3 mg/ml

(chloroform);

0.25 mg/ml

(chloral

hydrate);

4.5 mg/ml

(tetrachlorome

-thane)

4.

The reaction with Fehling’s reagent

Method. Add 2 drops of sodium hydroxide solution to

adjust to an alkaline pH and 2–3 drops of the prepared

fresh Fehling’s reagent (mix Fehling’s reagent 1 and

Fehling’s reagent 2 (1:1)) to 1 ml of the distillate

examined, mix and heat on a water bath. Observe


Specificity:

sensitivity:

3.0 mg/ml

(chloroform);

2.0 mg/ml

(chloral

hydrate)

5.

The reaction with Nessler’s reagent

Method. Add 2–3 drops of Nessler’s reagent to 0.5 ml of

the distillate examined and mix. Observe appearance of

colour

Specificity:

55

Detection of 1,2-dichloroethane (study theoretically)

1.

The reaction with fuchsin-sulphurous acid (it is

performed after the transformation of 1,2-dichloroethane

to formaldehyde)

Method. Place 0.5 ml of the distilate examined and

0.5 ml of 10 % sodium carbonate solution into a 1 ml

ampoule. Solder the ampoule and heat it on the boiling

water bath for 1–2 hours. Then cool the content of the

ampoule, place it in a test tube, add 10 % sulphuric acid

solution to adjust to the acidic pH, then add 2 drops of

5 % potassium peroxide solution in 2 М sulphuric acid

solution. Determine the presence of formaldehyde in the

solution obtained by the reaction with fuchsin-sulphurous

acid in 5 min. For this purpose add 2–3 drops of

concentrated sulphuric or hydrochloric acid to 1 ml of the

solution. Mix the content of the test tube, cool and then

add 1 ml of fuchsin-sulphurous acid. Observe appearance

of colour

Specificity:

2

The reaction of copper acetylenide formation

Method. Place 0.5 ml of the distillate examined into a 1 ml

ampoule, add 0.5 ml of 30 % sodium hydroxide solution.

Solder the ampoule and heat it for a hour. Then cool the

ampoule, unseal it and place its content into a test tube, add

30 % acetic acid solution to adjust to the acidic pH by litmus,

2 drops of copper (I) salt ammoniac solution prepared fresh.


Specificity:

56

Table 15 – Reactions for identification of halocarbons

No. Reaction

Substance

chloroform chloral hydrate tetrachloromethane 1,2-dichloro-

ethane 1. The determination of organically

bound chlorine

2. Formation of isonitrile 3. Reaction with resorcinol alkaline

solution

4. Reaction with Fehling’s reagent 5. Reaction with Nessler’s reagent

Table 16 –Detection of volatile toxic substances in the distillates with chemical method (alcohols, acetone, phenol, acetic acid)


Specificity and

sensitivity of

the reaction

Detection of monobasic alcohols

Detection of methanol

1.

The reaction of methyl salicylate formation

Method. Place 1 ml of the distillate into a test tube, add

0.03–0.05 g of salicylic acid and 2 ml of concentrated

sulphuric acid, heat the mixture carefully. Examine the

odour

Specificity:

sensitivity:

0.3 mg per

a sample

57

2.

Oxidation of methanol to formaldehyde

Method. Add 2–3 ml of 10 % sulphuric acid solution to

5 ml of the distillate examined, cool, add 1 % potassium

permanganate solution until a faint pink colour appears

avoiding the excess of the reagent. The excess of the

oxidant is destroyed by addition of 15 % oxalic acid

solution or 15 % sodium sulphite solution in 15–20 min.

Perform the reactions for formaldehyde given above (in

the section “Identification of formaldehyde”) with the

colourless solution obtained

Specificity:

Detection of ethanol

1.

The reaction of iodoform formation

Method. Place 1 ml of the distillate examined into a test

tube. Add 2 ml of 5 % sodium hydroxide or potassium

carbonate solution and 1 % iodine solution in 2 % of

potassium iodide solution by drops until a yellow colour

persists. Heat the mixture on a water bath at 40–50 °C.

Observe appearance of a precipitate and the odour

Specificity:

sensitivity:

0.04 mg/ml

58

2.

Formation of ethylacetate

Method. Place 1 ml of the distillate examined into a test

tube. Add 0.1 g of sodium acetate dried and a double

volume of concentrated sulphuric acid. Heat the mixture

on a boiling water bath until the gas bubbles appear.

Examine the odour

Specificity:

sensitivity:

15 µg/ml

3.

The reaction of ethylbenzoate formation (study

theoretically)

Method. Mix up 1 ml of the distillate examined with 1–2

drops of benzoyl chloride and when shaking intensively

add 10 % sodium hydroxide solution by drops. Examine

the odour

Specificity:

sensitivity: 2–

3 mg/ml

Detection of isopentanol

1.

Identification of (study theoretically)

The reaction with salicylic aldehyde

Method. Extract isoamyl acetate from a part of the third

distillate examined by diethyl ether. Evaporate the

extract obtained to dryness. Add 1 ml of 1 % salicylic

aldehyde solution in concentrated sulphuric acid and

3 ml of concentrated sulphuric acid to the remainder.

Cool the mixture placed into a test tube and then heat it

on a boiling water bath for 3 min. Observe appearance of

colour

Specificity:

2.

The reaction with p-dimethylamino-benzaldehyde

solution in concentrated sulphuric acid (study

theoretically)

Method. Add 5–10 drops of the reagent (p-

dimethylaminobenzaldehyde in concentrated sulphuric

acid) to the remainder obtained after evaporation of the

ether extract. Observe appearance of colour

Specificity:

59

3.

Formation of isoamyl acetate

Method. Add two drops of concentrated sulfuric acid and

a little amount (about 0.03 g) of anhydrous sodium

acetate to the remainder obtained after evaporation of the

ether extract. Heat the mixture on a water bath. Examine

the odour.

Specificity:

Detection of acetone

1.

Identification of (the model solution containing some

substance imitaiting acetone is used)

The reaction of iodoform formation

Method. Add 1 ml of 10 % ammonium hydroxide

solution and a few drops of 1 % iodine solution in 2 % of

potassium iodide solution to 1 ml of the distillate

examined. Observe appearance of a precipitate and the

odour

Specificity:

sensitivity:

0.1 mg per

a sample

2.

The reaction with sodium nitroprusside

Method. Add 1 ml of 10 % sodium hydroxide solution

and a few drops of 1 % sodium nitroprusside solution

(prepare fresh) to 1 ml of the distillate examined.


Specificity:

Detection of phenol

Preparation of the distillate for analysis

Method. Add sodium dihydrocarbonate solution to a part

of the third distillate to adjust to the alkaline pH, place it

into a separating funnel and extract phenol by 10 ml

portion of diethyl ether (2–3 times). Combine the ether

extracts and evaporate them at the room temperature to

dryness. Dissolve the remainder in 1–2 ml of water and

examine

60

1.

The reaction of tribromophenol formation

Method. Add 2–4 drops of bromine water (saturated) to

0.5–1.0 ml of the solution examined. Observe appearance

of a precipitate

Specificity:

sensitivity:

1:50000, the

limiting

dilution

2.

The reaction with iron () chloride

Method. Place 1 drop of the solution examined in a

porcelain cup, add 1 drop of iron () chloride solution

(fresh prepare). Observe appearance of colour. Carry out

the blank experiment

Specificity:

sensitivity:

1:1000, the

limiting

dilution

Detection of acetic acid

Identification of

The reaction with iron () chloride

Method. Add 1 drop of 5 % iron () chloride solution

(prepare fresh) to 2–3 ml of the distillate examined.

Observe appearance of colour. Carry out the blank

experiment

Specificity:

sensitivity:

1.25 mg/ml

The reaction of ethyl acetate formation

Method. Add 1 ml of ethanol and 2 ml of concentrated

sulphuric acid to 1 ml of the distillate examined, mix

carefully, heat the mixture placed into a test tube on a

boiling water bath. Examine the odour

Specificity:

The conclusion:

61


1. Theoretical bases of the steam distillation method.

2. Microdiffusion and dry-air distillation in isolation of volatile substances from the biological samples.

3. Chemical-toxicological analysis of ethylene glycol and tetraethyl lead.

4. The chemical equations of the reactions used for detection of volatile substances in the distillate.

62

Practical lesson 7

_____

Date

Topic: EXAMINATION OF BODY FLUIDS FOR THE PRESENCE OF ALCOHOLS BY GAS-LIQUID

CHROMATOGRAPHY METHOD

Figure 11 – Chart of a gas chromatograph

Gas chromatography (GC) is a method of the substance’s separation using different distributive

properties of substances separated between two phases, mobile phase (carrier gas) and immobile (or stationary)

phase.

Stationary phase is an active solid (sorbent with a large surface area) or liquid, which is coated on a

support material

The method is called Gas-Solid chromatography when using gas as a mobile phase and an active sorbent as an

immobile phase.

The method is called Gas-Liquid chromatography when using gas as a mobile phase and a liquid coated

on a support material as an immobile phase.

Qualitative analysis. Each substance passing through a column will have a characteristic retention time

(tR), which is defined as the time from injection to the peak maximum at the detector.

Quantitative analysis. The calibration curve (or equation), the peak height (or area) against the analysed

substance concentration, is used to quantify the unknown substance.

Gas cylinder

Regulator of carrier gas flow

Injector

Column

Detector

Amplifier

Recorder

Figure 12 – Chart of flame ionization

detector (FID)

A − eluent exits the GC column

B − FID detector’s oven

C − hydrogen fuel

D − oxygen

E − burner nozzle head

where a positive bias voltage

exists

F − flame

H,G − Collector Electrode

J − exhaust port

63


IDENTIFICATION AND QUANTITATIVE DETERMINATION OF ETHANOL IN BIOLOGICAL LIQUIDS BY THE GLC METHOD

Identification of alcohols in the samples examined (blood, urine, distillates)

Method. Place 0.5 ml of 50 % thrichloroacetic acid solution and 0.5 ml of the sample examined into a small penicillin bottle. Close the

bottle by cork and fix the cork with the help of the fixedness. Act further as described above in the verification of the separing capacity of the

column and determination of the detector sensitivity section. Compare the retention times of alcohols in the standard test and in the test analysed.

The quantitative determination of ethanol is necessary if the alcohol has been detected.

The vapour phase method is used for analysis of volatile poisons in biological fluids and distillates. It uses

the transfer of volatile poison examined to the vapour phase followed by analysis of the resulting phase with the

help of the GLC method. Alcohols can be transfered to the vapour phase with the help of the reaction of alkylnitrite

formation:

C2H5OH + NaNO2 + CCl3COOH C2H5ONO + CCl3COONa + H2O

ethylnitrite

Verification of the separating capacity of the column and determination of the detector sensitivity

Method. Place 0.5 ml of 50 % thrichloroacetic acid solution and 0.5 ml of the standard mixture of the

alcohols into a small penicillin bottle. Close the bottle by a rubber cork and fix the cork. Mix the content of the

bottle and bring 0.5 ml of 30 % sodium nitrite solution into it with the help of syringe piercing the cork. Mix the

content again (about 30 pendulum or circular motions along the surface of the table) and wait for 1 min. Then

collect 0.5–3.0 ml of the gaseous phase from the bottle with the help of syringe and inject it into the chromatograph

(the volume of the gaseous phase examined depends on the sensitivity of the detector, collect the same amount of

the gaseous phase in future for each experiment). Determine the absolute and relative (in relation to propanol)

retention times of the alcohols in the standard mixture. Use the time from the peak maximum of air to the peak

maximum of the substance analysed to measure the absolute retention time (this is the corrected retention time –

t`R). This retention parameter gives better accuracy than one obtained by measurement of the time period from the

moment of the test injection into chromatograph to the maximum of the substance analysed.

Figure 13 – Laboratory

equipment for sample

preparation in body fluid

study by GLC

64

Quantitative determination of ethanol

Determination of the calibration dependence. Prepare series of the standard solutions of ethanol (the concentration range is 0.2 ‰–4 ‰)

and 2 ‰ solution of propanol, the internal standard. Place 2 ml of the internal standard solution and 2 ml of ethanol standard solutions with

different ethanol concentrations (0.2 ‰–4 ‰) into penicillin bottles. Mix the content of the bottles, collect 1 ml of the mixtures and bring them

into other penicillin bottles. Add 0.5 ml of 50 % thrichloroacetic acid solution in each bottle and act further as described above in the verification

of the separing capacity of the column and determination of the detector sensitivity section. Measure the peak heights of ethylnitrite and

isopropylnitrite and calculate their retios. The data obtained for determination of the calibration dependence are adduced in Table 17.

Table 17 – Data for construction of the calibration curve

y=а + вx

No. ‰ h ethanol /

h propanol ethanol propanol

1. 0.25 2 0.239

2. 0.40 2 0.378

3. 0.50 2 0.421

4. 0.80 2 0.784

5. 1.0 2 0.915

6. 1.5 2 1.400

7. 2.0 2 1.820

8. 4.0 2 3.338

The determination is performed by the linear regression method,

the equations of the dependences for two concentration ranges are

y= 0.93x (0.25 ‰–1.0 ‰) and y= 0.86x (1.0 ‰–4.0 ‰).

Table 18 – Metrological characteristics of the calibration dependence

The calibration

dependence

r b S2 Δb

y= 0.93x 0.995 0.93 1·10-3 0.06

y= 0.86x 0.994 0.86 1·10-2 0.1

Calculate the concentration of ethanol in the blood sample using the

typical chromatogram. Interpret the results using the reference data.

65

REFERANCE DATA

Table 19 – The rates of alcohol intoxication

Blood Alcohol Concentration, ‰ Rate of intoxication

0.3–0.5 Insignificant intoxication

0.5–1.5 Easy rate of intoxication

1.5–2.5 Middle rate of intoxication

2.5–3.0 Hard intoxication

3.0–5.0 Severe intoxication

more than 5.0 Lethal intoxication

Fig. 14 – Chromatogram of real blood sample with added internal standard

International Blood Alcohol Limits (‘Blood Alcohol Concentration’ (BAC), g/l, ‰)

Australia – 0.5; Austria – 0.5; Brazil – 0.8; Canada –0.8; Chile – 0.8; China – 0.3; Ecuador – 0.8; France – 0.5; Germany – 0.5; Greece –

0.5; India – 0.3; Israel – 0.5; Italy – 0.5; Jamaica – 0.8; Japan – 0.3; Jordan –0.0; Malaysia – 0.8; Mali – 0.0; Mauritius – 0.8; Namibia –

0.5; Pakistan – 0.0; Russia – 0.0; Saudi Arabia – 0.0; South Africa – 0.5; Spain – 0.5; Sudan – 0.2; Turkey – 0.5; Ukraine – 0.3; Uganda –

0.8; United Arab Emirates – 0.0; United Kingdom – 0.8; USA – 0.8.

The conclusion:

66


1. Theoretical basis of qualitative and quantitative analysis of chemical substances by gas-liquid chromatography. Advantages and

disadvantages of the GHG method in the analysis of volatile substances (relative to the chemical method and GC), assessment of the

method for sensitivity and specificity. The value of the GHG method for forensic toxicological analysis of volatile substances.

2. Charcoal gas (carbon monoxide). Toxicological significance, physical and chemical properties, toxicity, analysis of blood for presence

of charcoal gas by chemical and spectrophotometry methods.

3. Hydrogen sulphide. Physical and chemical properties, toxicity, reasons of poisonings, detection of hydrogen sulphide in biological

samples.

67

Practical lesson 8

_____

Date

Topic: CHEMICOTOXICOLOGICAL ANALYSIS OF ORGANOPHOSPHORUS PESTICIDES

“Pesticide” is a general definition, which covers a wide variety of substances used for destroing undesirable life forms.

Methods of pesticide isolation. The general isolation method of pesticides from the plant and animal samples is organic solvent extraction

(hexane, ether, chloroform). Other isolation methods are steam distillation, extraction with water acidified by sulphuric acd for chlorophos.

Table 20 – Detecton of chlorophos by cholinesterase test and chemical reactions


Specificity and

sensitivity of

the reaction

1. Cholinesterase test (or biochemical test)

Method. Prepare two test tubes. Place 1 ml of the

solution analysed into the first test tube and 1 ml of

twice distilled water into the second one. Add

acetylcholinesterase solution, enzyme, into both test

tubes by drops, mix, add acetylcholine solution, mix

again and add one drop of bromothymol blue in the

buffer solution, the indicator. Compare the colour in

both test tubes

Specificity:

sensitivity: 1–

2 µg per

a sample

1.

Identification of chlorophos

General reactions

Test for phosphorus

Method. Place 3–5 drops of the mineralizate obtained

Specificity:

68

after mineralization of the extract contaning the

organophosphoprus pesticide into a test tube and add 5

drops of molibdate ammonium solution. Acidify the

mixture by 10 % of nitric acid solution. Add 3–5 drops

of benzidine hydrochloride saturated solution to this

mixture. Then add 10 % ammonium hydroxide solution

dropwise until the mixture is just alkaline to litmus.


2.

Hydroperoxide test (study theoretically)

Method. Add the mixture containing 0.5 ml of 2.5 %

benzidine acetone solution and 2 ml of hydrogen

peroxide alkaline solution (prepared fresh) to 2 ml of

the solution examined. Observe appearance of colour

Specificity:

sensitivity:

10 µg/ml

3.

Confirmatory reactions for chlorophos

The reaction with Nessler’s reagent

Method. Add 2–3 drops of Nessler’s reagent to 0.5 ml

of the solution examined, mix the liquids. Observe


Specificity:

4.


Method. Add the reagent containing equal amounts of

1 % resorcinol solution and 10 % sodium hydroxide

solution to 0.5–1 ml of the solution examined. Prepare

the reagent directly before use. Observe appearance of

colour

Specificity:

sensitivity:

40 µg per

a sample

69

The conlusion:


1. Usage of pesticides in agriculture, industry and medicine.

2. Theoretical bases of Thin Layer Chromatography method.

3. Toxicity, clinical presentation, antidote therapy in the poisoning of acetyl choline inhibitors.

4. Application of organic solvents (halogenated hydrocarbons, alcohols), cyanides, formaldehyde, phenol, acetic acid in industry,

agriculture and medicine.

70

Practical lesson 9

_____

Date

Topic: CHEMICOTOXICOLOGICAL ANALYSIS OF HEAVY METAL COMPOUNDS

Mineralization of the biological material by the mixture of sulphuric and

nitric acids. Place the ground biological material into the Celdal retort. Add 75 ml

of the mixture containing equal volumes (25 ml) of concentrated sulphuric acid,

concentrated nitric acid and water to 100 g of the sample. Fasten the retort with the

help of the stand and heat it above the asbestos net (in the distance of 1–2 cm) on a

gas ring to break firm lumps of the sample. This stage, the destruction, lasts for

30–40 min. while heating carefully. A destructate is a yellowish-brown heavy

transparent liquid. Then increase the temperature and add nitric acid diluted with

water (1:1) by drops into the retort until the retort content becomes colourless or

yellowish. In order to determine the end of mineralization heat the mineralizate

without adding nitric acid until a white steam of sulphur (VI) oxide appears. If the

liquid does not become dark, mineralization will be over. Cool the mineralizate

obtained, add 10–15 ml of water and heat to 110–130 °C and then add formaline

carefully by drops – the denitration process. Brown or orange vapours are

liberated. When finishing vapour liberation heat the mineralizate again for 5–

10 min. Then place 1–2 drops of the mineralizate on a white tile and add a drop of

diphenylamine in concentrated sulphuric acid. A dark blue colour appears in the

presence of nitric, nitrogenous acids, nitrogen oxides. The negative result of the

reaction indicates the end of the denitration process. If there is a positive result of

the reaction with diphenylamine, repeat the denitration. The blank experiment is

required because sulphuric acid used for disolving of diphenylamine for the

experiment may contain nitric acid as an admixture.

Fig. 15 – Device for mineralization

of biological sample by “wet”

mineralization method:

1. Kjeldahl's flask with the sample

2. A separating funnel with diluted

nitric acid

71

Table 21 – Detecton and identification of heavy metal cations in the mineralisate with the chemical reactions according to the

fractional method of the analysis


Specificity and

sensitivity of

the reaction

Examination of the precipitate

Separate PbSO4 and BaSO4 precipitates from the total

volume of the mineralizate by filtration (filtrate I). Wash

the precipitate by water acidified with sulphuric acid

(for removal of co-precipitated Fe3+; Cu2+; Zn2+; Cd2+,

etc., cations). If the precipitate is dirty-green, it should

be washed by ammonium persulphate (for washing

from Cr3+). Wash the precipitate by a hot solution of

ammonium acetate to separate barium and lead sulphates

from each other; PbSO4 dissolves (filtrate II)

Identification of barium sulphate

1.

Recrystallization of barium sulphate from

concentrated sulphuric acid

Method. Place a grain of the precipitate on a subject

glass slide, dry it on the air, put 2–3 drops of

concentrated sulphuric acid on the precipitate and heat

the mixture in the flame till appearance of white steams

avoiding evaporation to dryness. Wait for 10–20 min.

and examine the crystals obtained using a microscope.

Specificity:

sensitivity:

0.05 µg per

a sample

2.

Formation of barium iodate (study theoretically)

Method. Take a grain of the precipitate with the help of

platinum loop or needle and bring it into the reducing

part of the gas ring flame, then place the precipitate into

a drop of 5 М solution of hydrohloric acid placed on a

subject glass slide. Repeat this twice. Then bring a

crystal of potassium iodate into the drop and examine.

Specificity:

sensitivity:

0.03 µg per

a sample

72

Examination of the filtrate I

Identification of lead (II) cation

1.

The reaction with dithizone

Method. Place the solution examined in a test tube, add

1 ml of 10 % solution of hydroxylamine chloride (but

do not use hydroxylamine sulphate) to adjust the pH to

7.5–8.0 in accordance with the universal indicator paper

by addition of 10 % ammonium hydroxide solution, add

2 ml of chloroform and a few drops of 0.01 % dithizone

chloroform solution, shake up the mixture vigorously.

Repeat this until the chloroform layer would not change

its green colour to the red one. Separate the chloroform

layer with the help of a separating funnel. Then

reextract the lead cation by addition of 1 М nitric acid

solution to the chloroform layer, divide the reextract

obtained into four test tubes and carry out the

confirmative reactions for lead cation

Specificity:

sensitivity:

0.05 µg/ml;

0.02 mg in the

biological

material

2.

The reaction with potassium iodide

Method. Add some drops of 5 % potassium iodide

solution to the reextract obtained. Observe appearance

of a precipitate

Specificity:

sensitivity:

60 µg per

a sample

3.

The reaction with sulphuric acid

Method. Add some drops of 10 % sulphuric acid


of a precipitate

Specificity:

sensitivity:

0.2 mg per

a sample

4.

The reaction with potassium dichromate

Method. Add some drops of 5 % potassium dichromate


of a precipitate

Specificity:

sensitivity:

2 µg per

a sample

73

5.

The reaction with hydrosulphur water (study

theoretically)

Method. Add some drops of hydrogen sulphite aqueous

solution (prepare fresh) to the reextract obtained.

Observe appearance of the precipitate

Specificity:

sensitivity:

6 µg per

a sample

Examination of the filtrate II

Identification of manganese (II) cation

1.

Oxidation of manganese (II) cation by potassium

iodate

Method. Add 4 ml of water, 1 ml of saturated solution of

sodium dihydrophosphate, 0.2 g of potassium periodate

to 1 ml of the mineralizate and heat the mixture

obtained in the boiling water bath for 20 min. Observe


Specificity:

sensitivity:

0.1 µg/ml;

0.02 mg in 100

g of the

biological

material

2.

Oxidation of manganese (II )cation by ammonium

persulphate

Method. Add 4 ml of water, 1 ml of sodium

dihydrophosphate saturated solution to 1 ml of the

mineralizate and heat the mixture obtained in the boiling

water bath for 5–6 min. Add 1 drop of 10 % silver

nitrate solution of 0.5 g of ammonium persulphate to the

hot solution and heat the mixture again until persulphate

is brokencompletely. Observe appearance of colour

Specificity:

sensitivity:

0.1 µg/ml;

0.1 mg in 100

g of the

biological

material

Identification of chromium (III) cation

1.

The reaction with diphenyl carbazide

Method. Add 4 ml of water, a drop of 10 % silver nitrate

solution, 0.5 g of ammonium persulphate to 1 ml of the

mineralizate examined, heat the mixture in the boiling

water bath for 20 min. Then add 1 ml of sodium

dihydrophosphate saturated solution to adjust the pH to

2.0 in accordance with the universal indicator paper by

addition of 10 % potassium hydroxide solution (by

Specificity:

sensitivity:

0.002 µg/ml;

0.1 mg in 100

g of the

74

drops) and 1 ml of 0.25 % diphenyl carbazide solution

to the mixture. Observe appearance of colour

biological

material

2.

Formation of perchromic acids

Method. Place 5 ml of the mineralizate examined in a

test tube. Add 30 % sodium hydroxide or potassium

hydroxide solution (by drops) to adjust the pH to 7.0, 1–

2 drops of 10 % silver nitrate solution, 0.5 g of

persulphate ammonium crystalline to the mineralizate

and heat the mixture in the boiling water bath for

20 min. Cool the test tube with the solution obtained in

the bath containing ice for 10–15 min. Add 1 ml of

potassium dihydrophosphate saturated solution to the

cooled liquid adjusting pH to 2.0. Then add ethyl acetate

or other organic solvent making the thickness of the

organic layer 0.5–1.0 cm. Then add 2–3 drops of 25 %

hydrogen peroxide solution to the mixture examined and

shake immediately and vigorously the content of the test

tube. Observe appearance of colour

Specificity:

sensitivity:

2 µg/ml;

0.2 mg in 100

g of the

biological

material

Identification of silver (I) cation (study theoretically)

1.


Method. Add 1 ml of 4 М sulphuric acid solution and

3 ml of 0.01 % dithizone chloroform solution to 5 ml of

the mineralizate. Shake up the mixture. Observe


Specificity:

sensitivity:

0.04 µg/ml;

0.05 mg in 100

g of the

biological

material

2.

Separation of a silver cation from the total volume of

the mineralizate

Method. Add 0.5 g of sodium chloride to the rest

volume of the mineralizate (about 90–100 ml), heat the

mixture to 80 °C and wait for the formation of the

75

precipitate for 2–24 hours (the sensitivity of the reaction

is 2.5·10-2

mg / 100 ml, the reaction is specific). Filter

off the precipitate, wash it by 0.5 М hydrochloric acid

solution and then dissolve it in 0.5–4.0 ml (or more) of

8 М ammonium hydroxide solution. Examine the filtrate

obtained

3.

Formation of silver iodide

Method. Add potassium iodide saturated solution to the

1\3 part of the filtrate obtained (volume of the filtrate

examined should be more than 2 ml). Observe

appearance of a precipitate

Specificity:

sensitivity:

1.5 mg per

a sample

Identification of copper (II) cation

1.

The reaction with lead diehyl dithiocarbaminate

Method. Place 10 ml of the mineralizate in a test tube,

adjust the pH to 3.0 in accordance with the universal

indicator paper by addition of 10 % ammonium

hydroxide solution, add 5 ml of lead diehyl

dithiocarbaminate chloroform solution and shake to

colouration of the chloroform layer. Separate the

chloroform layer with the help of a separation funnel,

add 6 М of hydrochloric acid solution destroying the

excess of lead diehyl dithiocarbaminate, shake and

separate the aqueous layer. Then add 1 % mercury (II)

chloride solution to the chloroform layer by drops to

discolouration of the chloroform layer, add 1.5–2.0 ml

of water into the separation funnel, shake and separate

the aqueous layer. Perform the confirmative reactions

with the reextract

Specificity:

sensitivity:

0.5 µg/ml

76

2.

The reaction with zinc sulphate and ammonium

mercuric thiocyanate (study theoretically)

Method. Add 0.2 g of zinc sulphate and some drops of

ammonium mercuric thiocyanate to the 1\3 part of the

reextract. Observe appearance of a precipitate

Specificity:

sensitivity:

0.1 mg per

a sample

3.

The reaction with potassium hexacyanoferrate (II)

Method. Add 10 drops of 2 % cadmium chloride

solution and 1–2 drops of 5 % potassium

hexacyanoferrate (II) solution to the 1/3 part of the

reextract. Observe appearance of a precipitate.

Specificity:

sensitivity:

0.1 µg per

a sample

4.

The reaction with the pyridine rhodanide reagent

Method. Add 1–2 ml of the pyridine rhodanide reagent

to the 1/3 part of the reextract by drops to formation of

lees or precipitate, then add 1–2 ml of chloroform.


Specificity:

sensitivity:

1 µg/ml;

0.4 mg in 100

g of the

biological

material

Identification of bismuth (III) cation

1.

The reaction with thiourea

Method. Add 3–5 ml of thiourea saturated solution to 5

ml of the mineralizate. Observe appearance of colour

Specificity:

sensitivity:

0.4 µg per

a sample;

0.1 mg in 100

g of the

biological

material

2. The reaction with 8-oxyquinoline

Method Add 20–30 drops of 20 % sodium thiosulphate

Specificity:

77

solution, 0.5 g of ascorbic acid, 10 drops of 10 %

potassium and sodium tartrate solution and the excess of

crystalline potassium iodide till appearance of a yellow

or orange colour to 10 ml of the mineralizate examined,

then add 1–2 ml of 2 % 8-oxyquinoline solution in 5%

hydrochloric acid solution carefully down the test tube’s

wall. Observe appearance colour

sensitivity:

5 µg per

a sample;

0.1 mg in 100

g of the

biological

material

Identification of zinc (II) cation

1.


Method. Add 0.25 ml of saturated sodium thiosulphate

solution and 1 ml of acetate buffer to adjust the pH to

5.0–5.5 to 0.5 ml of the mineralizate, then add 2 drops

of 0.01 % dithizone chloroform solution and 1 ml of

chloroform. Observe appearance of colour

Specificity:

sensitivity:

0.25 µg/ml;

5 mg in 100 g

of the

biological

material

2.

The separation of a zinc cation from the mineralizate

Method. Place 10 ml of the mineralizate in a test tube,

add 4 ml of 10 % potassium and sodium tartrate or 20 %

citric acid solution, 1 ml of saturated sodium

thiosulphate solution, some drops of 0.1 % nilotic blue

solution and 2.5 М potassium hydroxide solution by

drops till appearance of a pink colour, then add 1 М

sulphuric acid solution to adjust the pH to 8.5 in

accordance with the universal indicator paper, 3 ml of

1 % potassium diethyl dithiocarbaminate solution and

5 ml of chloroform and shake the mixture. Separate the

chloroform layer, wash it by water and shake for 3 ml of

1 М hydrochloric acid solution during 30 min. Perform

the confirmative reactions for a zinc cation with the

aqueous layer (the reextract)

78

3.

The reaction with potassium hexacyanoferrate (II)

Method. Place 1 ml of the reextract in a test tube, add

5 % potassium hydroxide solution to adjust the pH to

5.0 in accordance with the universal indicator paper and

then 3–4 drops of 5 % potassium hexacyanoferrate (II)

solution. Observe appearance of a precipitate

Specificity:

sensitivity:

3 µg/ml

4.

The reaction with hydrogen or ammoniun sulphide

Method. Place 1 ml of the reextract in a test tube, add

5 % potassium hydroxide solution to adjust the pH to

5.0 in accordance with the universal indicator paper and

then 2–3 drops of 5 % sodium or ammoniun sulfide

solution (prepare fresh). Observe appearance of a

precipitate.

Specificity:

sensitivity:

1.5 µg/ml

5.

The reaction with ammonium mercuric thiocyanate

(study theoretically)

Method. Place 3–4 drops of the reextract on a subject

glass slide and dry, dissolve the remainder in 10 %

acetic acid solution and add 1 drop of ammonium

mercuric thiocyanate solution (mix 5 g of mercury (II)

chloride and 5 g of ammonium thiocyanate; dissolve the

mixture in 60 ml of water) to the solution obtained.

Observe appearance of a precipitate

Specificity:

sensitivity:

0.2 µg/ml;

0.5 mg in 100

g of the

biological

material

Identification of antimony compounds

1.

The reaction with malachite or diamond green

Method. Place 5 ml of the mineralizate in a separating

funnel, add 1 ml of concentrated sulphuric acid, 3 ml of

5 М hydrochloric acid solution, 2 drops of 5 % sodium

nitrite solution, wait for 5 min. and add 1 ml of saturated

urea solution, 7 drops of 0.5 % malachite or diamond

Specificity:

sensitivity:

79

green solution, 2 g of anhydrous sodium sulphate and

5 ml of toluene. Shake the mixture for 10–15 seс.


0.05 µg/ml;

0.1 mg in 100

g of the

biological

material

2.

The reaction with the sodium thiosulphate

Method. Add 5 drops of sodium thiosulphate saturated

solution to 5 ml of the mineralizate and boil the solution

obtained for 1–2 min. Observe appearance of precipitate

Specificity:

sensitivity:

10 µg per

a sample;

0.4 mg in 100

g of the

biological

material

Identification of arsenic compounds

1.

Zanger-Black test

Method. Place the following reagents into the retort of

the Zanger-Black’s device in such a sequence: 2 ml of

the mineralizate, 10 ml of 2 М sulphuric acid solution,

5 ml of water, 1 ml of 10 % solution of tin (II) chloride

in sulphuric or hydrochloric acid solution, 2 g of

copper-plated zinc. Close the retort by attachment

containing the reagent paper treated by mercury

bromide or chloride solution and below the cotton wool

tampon treated by lead acetate solution. The yellow or

brown spots appear on the paper at once or in 45 min. in

the presence of arsenic. If the spots appear in 45 min.,

treat the paper by 3 % potassium iodide solution to

redness, then by potassium iodide saturated solution to

disappearance of the red colour. Brown spots appear in

Specificity:

sensitivity:

0.1 µg per

a sample;

0.01 mg in 100

g of the

biological

material

Fig. 16 – Device for Zanger – Black test

1 – flask;

2 – a cotton, impregnated

with lead acetate solution

3 – paper treated with

mercury (II) chloride

solution

4 – cotton swab

5 – reactive paper

80

the presence of arsenic

2.

The Marsh test (study theoretically)

Method. Place the following reagents into the retort of

the Marsh’s device in such a sequence: 10 g of copper-

plated zinc, 10–20 ml of 2 М sulphuric acid solution.

The Marsh’s device must be hermetically closed. Check

up the completeness of the air displacement by the

liberating hydrogen every 4–5 min. For this purpose

collect the gas liberating from the Marsh’s device into

the test tube and set fire (the mixture of air and

hydrogen produces crackle sound). Heat the narrow part

of the tube of the March’s device with help of a gas

ring, then cool the tube after the place of heating. Place

20 ml of the mineralizate and 2 ml of 10 % tin (II)

chloride solution in 50 % sulphuric acid solution into a

separating funnel. Add this mixture to the retort with the

help of the separating funnel for 30–40 min. Cool the

tube after the place of heating. In the presence of arsenic

the brilliant brown thin coating (like a grey-black

mirror) is observed in the place of cooling of the tube

at once or in 60 min. Then cool the device, disconnect

the tube and heat the place of thin coating location in the

air. In the presence of arsenic the characteristic crystals

(octahedrons, tetrahedrons) of arsenous anhydride are

observed

Specificity:

sensitivity:

0.05 mg per

a sample

Fig. 17 – Marsh’s apparatus

1 – flask

2 – droplet funnel

3 – calcium chloride tube

4 – reducing tube

81

Destruction of the biological material and examination of the destructate for mercury (II) cation

Destruction of the biological material by the mixture of sulphuric and nitric acids Method. The samples examined are 20 g of the liver and 20 g of the kidney. Destruction of the liver and kidney is performed separately. The

mixture of sulphuric and nitric (or sulphuric, nitric and perchloric) acids is used as an oxidizer. Ethyl alcohol is added as a catalyst of this process.

Heating is performed with the help of a water bath for 10–15 min. Thus, the destructate obtained contains a mercury cation and various biological

admixtures (proteins, peptides, aminoacids, lipids, etc.). The admixtures are removed by extraction with chloroform.

Table 22 – Detecton and identification of mercury (II) cation

No. Method Observa-

tion Chemical reaction equation(s)

Specificity and

sensitivity of the

reaction

1. The reaction with dithizone

Method. Shake the half of the destructate obtained with 10 ml

of chloroform in a separating funnel. Throw aside the yellow

chloroform layer. Repeat this procedure until the chloroform

layer becomes colourless. Separate the aqueous layer, add

10 ml of 10 % hydroxylamine sulphate solution or ascorbic

acid, 5 ml of chloroform and 0.5 ml or more of 0.01 %

dithizone chloroform solution (prepare fresh). Shake the

mixture intensively for 30 min. Observe appearance of colour

Specificity:

2. The reaction with copper (I) iodide

Method. Add 3 ml of copper (І) iodide suspension to 3 ml of

the destructate. Observe appearance of a precipitate

Specificity:

sensitivity: 0.5 µg

per a sample;

0.01 mg in 100 g of

the biological

material

82

Topic: QUANTITATIVE DETERMINATION OF LEAD COMPOUNDS IN THE MINERALIZATE

WITH THE PHOTOELECTROCOLORIMETRIC METHOD

Quantitative determination of lead compounds with the photocolorimetric method by the reaction with dithizone

Method. Place the certain volume (5 ml or more) of the minaralizate examined for a lead compound into a separating funnel. Perform the reaction

with dithizone as described above in the section “Identification of a lead cation”. The volume of the chloroform added is the same as the volume

of the aqueous layer. Add chloroform to the extract obtained to adjust the volume to 10 ml and measure the absorbance (A) of the lead

dithizonate solution obtained by a photoelectrocolorimeter at 520 nm, the thickness of the layer is 10 mm. The reference solution is chloroform.

Determine the concentration with the help of the calibration dependence given below.

Determination of the calibration dependence. Prepare the initial standard solution containing 1 mg/ml lead using recrystallized lead nitrate

(standard solution I). Dilute the standard solution I to obtaine the solution containing 0.01 mg/ml lead (the standard solution II). Using the

standard solution II prepare solutions containing 0.001; 0.002; 0.003; 0.004; 0.005; 0.006; 0.007; 0.008; 0.009 mg/ml of lead. Place 5 ml of the

standard solutions obtained into a separating funnel and perform the reaction with dithizone as described above. The volume of the chloroform

added is the same as the volume of the aqueous layer. Add chloroform to the extracts obtained to adjust the volume to 10 ml. Measure the

absorbance.

Formula for the calculation:

,100

1

0

VV

CV

where – is the content of lead compound in 1 dl (100 ml) of the biological fluid, mg/dl;

C – is the concentration of lead (II) cation in the mineralizate determined with the help of the calibration curve, mg;

Vo – is the total volume of the mineralizate obtained, ml;

V1 – is the volume of the mineralizate taken for the photoelectrocolorimetric determination, ml;

V – is the volume of the biological fluid examined, ml.

83

Table 23– Data for construction of calibration curve: у = вх + а

Table 24 – Metrological

characteristics of the calibration curve

С, mg 0.001 0.002 0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

А 0.07 0.15 0.23 0.31 0.38 0.46 0.54 0.62 0.69 0.77

Equestion of the calibration curve was determined by the linear regression method: y = 76.9x.

r b S2 Δb

0.9999 76.9 9·10-5 0.4

Calculate the lead (II) cation concentration in the model biological fluids (after mineralization) with the help of the equestion of

calibration curve. Then calculate the content of lead in the biological fluid (mg/dl) assuming that V = 5 ml; V0 = 10 ml; V1 = 5 ml. Interpret the

results obtained (normal, toxic or lethal content of ledd in the biological fluid) using the reference data.

THE REFERENCE DATA

Serious intoxication is usually associated with blood levels over 100 µg/dl [Poisoning &Drug Overdose. Fourth Edition / Ed. K.R. Olson.

– Zange Medical Books, Mc Graw-Hill, 2004. – P. 88-93]

The conclusion:

84


1. Usage of heavy metal compounds in industry, agriculture and medicine.

2. Theoretical bases of titrimetric methods of quantitative analysis (complexometry, iodometry, dichromatometry, rhodanometry).

3. Theoretical bases of photoelectrocolorimetric method of quantitative analysis.

4. Spectral methods of quantitative determination of heavy metal compounds: atomic absorbtioin spectrometry (AAS), atomic emission

spectrometry (AES), mass spectrometry with inductively coupled plasma (ICP-MS).

85

LITERATURE

Basic

1. Bondar, V. S. Toxicological chemistry. Schemes and Tables: Handbook for students of higher schools / V. S. Bondar, S. A. Karpushina. –

Kharkiv : NUPh : Golden Pages, 2009. – 120 p.

2. Karpushina, S. A. Toxicological chemistry. Lecture course / S. A. Karpushina, V. S. Bondar, I. A. Zhuravel. – Kharkiv : NUPh : Golden

pages, 2011. – 208 p.

3. Toxicological Chemistry. Laboratory workbook / S. A. Karpushina, I. A. Zhuravel, V. S. Bondar, S. V. Bayurka. – Kharkiv : NUPh, 2012.

– 63 p.

Additional

1. Baselt, C. R. Disposition of Toxic Drugs and Chemicals in Man: 9-th edition /

R. C. Baselt. – California : Biomedical Publications, 2011. – 1900 p.

2. Basic Analytical Toxicology / R. J. Flanagan [et al.]. – Geneva : World Health organization, 1995. – 363 p.

3. Bell, S. Forensic Chemistry / S. Bell. – New Jersey : Pearson Prentice Hall. – 671 p.

4. Clarke's analysis of drugs and poisons in pharmaceuticals, body fluids and postmortem material: 4-th edition / A. C. Moffat [et al.]. –

London ; Chicago : Pharmaceutical Press, 2011. – 2736 p.

5. Clarke’s Analytical Forensic Toxicology / ed. by Sue Jickells, Adam Negrusz. – London : Pharmaceutical Press, 2008. – 648 p.

6. Flanagan, R. J. Developing Analytical Toxicology Services: Principles and Guidance [Electronic resource] / R. J. Flanagan. – Geneva :

World Health Organization, 2005. – 36 p. – Available at : http://www.who.int/ipcs/publications

/training_poisons/hospital_analytical_toxicology.pdf (date of the application : (07.09.2017). – Developing Analytical Toxicology

Services: Principles and Guidance.

7. Poisoning & Drug Overdose. Fourth Edition / ed. by Kent R. Olson. – Zange Medical Books, Mc Graw-Hill, 2004. – 718 p.

http://www.who.int/ipcs/publications

86

СONTENTS

Chemistry laboratory safety rules……………………………………………………………………………………………………………… 3

CONTENT MODULE 1 DRUG TOXICOLOGY

Practical lesson 1

Toxicology of opioid analgesics, non-narcotic analgesics and non-steroidal anti-inflammatory drugs………………………………………….. 5

Practical lesson 2

Toxicology of neuroleptics, tranquilizers and hypnotic drugs. toxicological characteristics of medicines stimulating CNS and local

anesthetics………………………………………………………………………………………………………………………………………… 9

CONTENT MODULE 2 ANALYTICAL TOXICOLOGY. CHEMICOTOXICOLOGICAL ANALYSIS OF DRUGS

Practical lesson 3

Analytical testing scheme design. Specimen testing, external examination and preliminary tests………………………………………………. 13

Chemicotoxicological analysis of inorganic acids, alkalis, nitrates and nitrites………………………………………………………………….. 16

Chemicotoxicological analysis of drugs. Examination of the acid chloroform extract for derivatives of barbituric and salicylic acids,

pyrazolone, purine………………………………………………………………………………………………………………………………… 21

Practical lesson 4

Examination of the basic chloroform extract for alkaloids……………………………………………………………………………………….. 31

Practical lesson 5

Examination of the basic chloroform extract for derivatives of 1,4-benzodiazepine, phenothiazine, p-aminobenzoic acid…………………….. 40

Quantitative determination of drugs in the extracts……………………………………………………………………………………………… 42

Examination of body fluids for drugs in acute intoxications…………………………………………………………………………………….. 44

CONTENT MODULE 3 CHEMICOTOXICOLOGICAL ANALYSIS OF VOLATILE SUBSTANCES AND HEAVY METAL COMPOUNDS.

ANALYTICAL DIAGNOSTICS OF TOXIC GAS POISONINGS

Practical lesson 6

Сhemicotoxicological analysis of volatile substances. Detection of volatile substances in the distillates by chemical method………………… 50

Practical lesson 7

Examination of body fluids for the presence of alcohols by gas-liquid chromatography method……………………………………………….. 62

Practical lesson 8

Chemicotoxicological analysis of organophosphorus pesticides…………………………………………………………………………………. 67

Practical lesson 9

Сhemicotoxicological analysis of heavy metal compounds……………………………………………………………………………………… 70

LITERATURE…………………………………………………………………………………………………………………………………… 85

87

Робочий журнал для практичних занять з лікарської та аналітичної токсикології призначений для використання іноземними

студентами спеціальності 8.12020101 «Фармація», що навчаються англійською мовою, під час аудиторних практичних занять та

самостійної внеаудиторної роботи. Видання рекомендується для фармацевтичних факультетів та фармацевтичних університетів III-IV

рівнів акредитації.

Навчальне видання

Карпушина Світлана Анатолівна, Баюрка Сергій Васильович

РОБОЧИЙ ЖУРНАЛ

для практичних занять з лікарської та аналітичної токсикології

Англійською мовою

Формат 60х84/8. Ум. друк. арк. 7,2. Тираж 80 пр.

Національний фармацевтичний університет

вул. Пушкінська, 53, м. Харків, 61002

Свідоцтво суб’єкта видавничої справи ДК №3420 від 11.03.2009 р.

workbook for practical lessons on drug and analytical...

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