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!
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
Independent educational and research work
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:
Independent educational and research work
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
No. Method Observation Chemical reaction equation(s)
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
Method. Apply a few drops of the chloroform extract on
a slide, evaporate to dryness at room temperature.
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
Method. Apply a few drops of the chloroform extract on
a slide, evaporate to dryness at room temperature.
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
Method. Apply a few drops of the chloroform extract on
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
Method. Place 5–6 drops of the chloroform extract
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
The reaction with iron (III) chloride
Method. Place a few drops of the chloroform extract
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
examined into a porcelain cup, evaporate it to dryness.
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)
Method. Place 2–5 drops of the chloroform extract
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
The reaction with iron (III) chloride
Method. Place a few drops of the chloroform extract
examined into a porcelain cup, evaporate it to dryness.
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)
Method. Place 4–5 drops of the chloroform extract
examined into a porcelain cup, evaporate it to dryness.
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
Independent educational and research work
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
Laboratory Practicals Assignments
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
Laboratory Practicals Assignments
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
No. Method Observation Chemical reaction equation(s)
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
crystals in 10–15 min, describe their form and draw them
Specificity:
sensitivity:
0.7 µg per
a sample
3.
Pachycarpine
The reaction with iodine in potassium iodide solution
(Bushard 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 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
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 1–2 drops
of 0.5 % picric acid solution to the residue. Observe the
crystals in 10–15 min, describe their form and draw them
Specificity:
sensitivity:
5 µg per
a sample
5.
The reaction with cobalt rhodanide 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 1–2 drops
of cobalt rodanide reagent to the residue. Observe the
crystals in 10–15 min, describe their form and draw them
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.
Observe appearance of colour
Specificity:
sensitivity:
1 µg per
a sample
2.
The confirmative reactions
Atropine
The reaction with 1 % Reinecke’s salt solution
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
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
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 1–2 drops
of 0.5 % picric acid solution to the residue. Observe the
crystals in 10–15 min, describe their form and draw them
Specificity:
sensitivity:
5 µg per
a sample
36
4.
Scopolamine
The reaction with 1 % Reinecke`s salt solution
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
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
Method. Place a few drops of the chloroform extract
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
Method. Place a few drops of the chloroform extract
examined in a porcelain cup, evaporate the solvent
without heating. Add a drop of Froehde reagent (add the
excess of ammonium molibdate to concentrated
sulphuric acid, prepare fresh) to the residue. Observe
appearance of colour
Specificity:
sensitivity:
0.05 µg per
a sample
(morphine)
3.
The reaction with Mandelin reagent
Method. Place a few drops of the chloroform extract
examined in a porcelain cup, evaporate the solvent
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
appearance of colour
Specificity:
4.
Morphine
The reaction with iron (III) chloride
Method. Place a few drops of the chloroform extract
examined in a porcelain cup, evaporate the solvent
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
Method. Place 4–5 drops of the chloroform extract
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
Method. Place a few drops of the chloroform extract
examined in a porcelain cup, evaporate the solvent
without heating. Add a drop of Mandelin reagent to the
residue. Observe appearance of colour
Specificity:
The conclusion:
39
Independent educational and research work
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
No. Method Observation Chemical reaction equation(s)
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
Method. Place 4–5 drops of the chloroform extract
examined in a porcelain cup, evaporate chloroform to
dryness. Add a drop of concentrated sulphuric acid.
Observe appearance of colour
Specificity:
2.
The reaction with concentrated nitric acid
Method. Place 4–5 drops of the chloroform extract
examined in a porcelain cup and evaporate chloroform to
dryness. Add a drop of concentrated nitric acid. Observe
appearance of colour
Specificity:
3.
The reaction with concentrated hydrochloric acid
Method. Place 4–5 drops of the chloroform extract
examined in a porcelain cup and evaporate chloroform to
dryness. Add a drop of concentrated hydrochloric acid.
Observe appearance of colour
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
Laboratory Practicals Assignments
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
Laboratory Practicals Assignments
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
Method. Place 5 ml of the body fluid into a separating
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
appearance of colour
3.
Amino-benzophenons (hydrolysis products of 1,4-
benzodiazepines)
Method. Place 5 ml of the body fluid into a separating
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.
Observe appearance of colour
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
Independent educational and research work
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)
No. Method Observation Chemical reaction equation(s)
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
appearance of colour
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
appearance of colour
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.
Observe appearance of colour
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.
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:
0.3 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
appearance of colour
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.
Observe appearance of colour
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)
No. Method Observation Chemical reaction equation(s)
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.
Observe appearance of colour
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
Independent educational and research work
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
Laboratory Practicals Assignments
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
Independent educational and research work
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
No. Method Observation Chemical reaction equation(s)
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.
Observe appearance of colour
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
appearance of colour
Specificity:
4.
The reaction with resorcinol alkaline solution
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:
Independent educational and research work
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
No. Method Observation Chemical reaction equation(s)
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
solution to the reextract obtained. Observe appearance
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
solution to the reextract obtained. Observe appearance
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
appearance of colour
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.
The reaction with dithizone
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
appearance of colour
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.
Observe appearance of colour
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.
The reaction with dithizone
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.
Observe appearance of colour
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
Independent educational and research work
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.
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.