CONTENTS

INTRODUCTION 4

Topic 1. Introduction into toxicological chemistry 5

Topic 2. Regularities of the poison behaviour in a body 13

Topic 3. Metabolism of toxic substances 27

Topic 4. The group of substances isolated from biological material

by mineralization (Metallic poisons). General and special

mineralization methods 39

Topic 5. Analysis of the mineralizate by the fractional method.

Quantitative determination of metallic poisons in the mineralizate 50

Topic 6. The group of substances isolated from the biological material by

steam distillation (Volatile poisons). The methods of isolation and

analysis of distillates by the chemical method 63

Topic 7. Gas chromatography detection and quantitative determination

of volatile poisons 89

Topic 8. The group of substances isolated from the biological material

by the organic solvent extraction (Pesticides) 96

Topic 9. The group of substances isolated from the biological material by

solvent extraction (Drugs). General and special methods of the drug

isolation from biological material 117

Topic 10. The general scheme of the chemical toxicological

analysis of drugs 136

Topic 11. Chemical toxicological analysis of acidic, neutral and weak

basic drugs 160

Topic 12. Chemical toxicological analysis of alkaloids 176

Topic 13. Chemical toxicological analysis of synthetic basic drugs 190

APPENDIX 206

GLOSSARY 217

LITERATURE 221

4

INTRODUCTION

According to the Standard Toxicological Chemistry Syllabus, which has

been written by Toxicological Chemistry Department of National University of

Pharmacy, this Lecture Course for the students of the fifth year comprises the

following topics: Introduction into Toxicological Chemistry; Regularities of the

poison behaviour in a body and metabolism; Metallic poisons; Volatile poisons;

Pesticides; Drugs. Topics learnt by students independently: Mineral acids, alkalis,

nitrate and nitrite; Fluoride and silicifluoride; Carbon monoxide are presented in

schemes and tables of the appendix.

The theoretical and practical aspects of isolation, identification, quantitative

determination for each group of poisons, as well as the problem of the biological

sample choice, toxicity, the distribution in a body, metabolism and excretion of

poisons are considered.

The Lecture Course presents concepts in logical progression from general

principles to specific topics and examples. The information is presented in an

easily accessible and understandable manner.

This Lecture Course is intended for the students of pharmaceutical

departments requiring a basic foundation in Toxicological Chemistry.

5

Topic 1: INTRODUCTION INTO TOXICOLOGICAL CHEMISTRY

Plan of the lecture

1. The content of the discipline, its tasks, sections and objects of the chemical

toxicological analysis.

2. Poisons and their classifications.

3. Intoxications and their classifications.

4. Relationship of toxicological chemistry with other disciplines.

5. Peculiarities of the chemical-toxicological analysis.

6. Organization of the forensic medical service and the forensic toxicological

examination in Ukraine.

7. The general stages of the forensic toxicological examination and making its

plan.

1. The content of the discipline, its tasks, sections and objects of the

chemical toxicological analysis

Toxicological chemistry is an applied science, which studies the methods of

isolation, purification, identification and quantification of poisonous substances, as

well as their metabolites in various samples such as the biological material of the

animal, human and plant origin; the air of industrial enterprises, water, soil, etc.

The tasks of toxicological chemistry are:

development of new isolation methods of poisons and their metabolites from

various samples, and also improvement of the existent methods;

development of effective purification methods of the extracts obtained from

samples during the chemical toxicological analysis;

introduction of new sensitive and specific reactions and identification

methods of toxic substances isolated from the samples (chromatography,

spectroscopy, etc.) into the chemical toxicological analysis practice;

development and introduction of sensitive quantification methods of toxic

substances into the chemical toxicological analysis practice;

6

research of the toxic substance metabolism in an organism and development

of methods for metabolite analysis.

The chemical toxicological analysis is a complex of the scientifically based

methods, which are used in practice for isolation, detection and quantification of

toxic substances. The results of the chemical toxicological analysis are used in:

court proceedings and criminal investigations into murder cases or accidents;

medical service in rescuing of a human life;

ecological service in protection of a human being and his environment from

pollution. So, at present foods (vegetables, milk) are examined for the presence

of pesticides.

The sections of toxicological chemistry. Depending on the samples

submitted and questions decided by the toxicological examination it is possible to

distinguish such sections as:

1. Forensic chemistry studies the methods of the biological material examination

for the presence of poisonous substances, which can be a cause of mortal

poisoning, and also different material proofs (instruments of murder, money,

values, clothes and other things, which are used in court proceedings or

criminal investigations);

2. Laboratory express-analysis of acute intoxications studies the methods of the

poisonous substance analysis in the biological fluids of living persons with the

purpose of helping a doctor in the rescue of the human life;

3. Analysis of remaining amounts of pesticides studies the methods of the

poisonous substance analysis in water, soil, food with the purpose of prevention

of pesticide intoxications;

4. Sanitary chemical analysis studies the methods of the poisonous substances

analysis in the air of industrial enterprises with the purpose of occupational

intoxication prevention.

Objects of chemical toxicological analysis can be divided into the following

groups:

7

The samples withdrawn from the dead body of persons who died as a result of

poisoning (tissues and organs of dead bodies, biological fluids – blood, urine,

vomit, etc.).

The samples withdrawn from living persons who used the non-lethal doses of

poisonous substances (blood, urine, stomach washings, vomit and excrement).

The samples of chemical toxicological analysis listed above are the biological

material.

The samples, which could be the reason of poisoning (food products, water,

drinks, parts of plants, medicines, various chemical substances, the air in

houses and working premises where a poisoned victim was found).

Parts of clothes, spots on the clothes, drug containers, crockery, from which

poisonous substances were accepted, and other things containing the traces of

crime.

2. Poisons and their classifications

Poisons are any chemical substances, which at certain terms (overdose,

change of an organism reactivity, etc.) can be harmful for living organisms, disturb

the vital important functions of an organism causing the pathological changes, and

sometimes death.

Classifications of poisons fall into two groups: general based on a general

principle of estimation, which is common for all chemical substances, and special

representing relations between some physicochemical or other properties of

substances and their possible toxicity.

General classifications of poisons

Chemical classification – according to the chemical structure (organic,

inorganic and metal-containing organic compounds).

Practical classification - according to application (industrial poisons,

pesticides, medicines, domestic chemicals, biological poisons of the plant and

animal origin, warfare agents).

8

Hygienic classification - according to the toxicity rate (extremely toxic, highly-

toxic, moderately toxic, little toxic substances).

Toxicological classification - according to non-organ-directed toxicity

(nervous-paralytic, skin-resorptive, general toxic, lacrimators and irritating,

psychotic substances).

Classification according to target organ toxicity (cardiac, nervous, hepatic,

renal, blood and stomach-intestinal poisons).

Special classifications of poisons

Pathophysiological classification - according to the type of developing

hypoxia.

Pathochemical classification -according to the mechanism of co-operation

with enzyme systems.

Biological classification - according to the character of the biological

consequence of poisoning.

Classification according to the rate of carcinogenic activity.

Classification of poisons used in toxicological chemistry

In toxicological chemistry poisons are divided into groups in accordance

with their isolation methods from the biological material:

1. Volatile poisons are the substances isolated by steam distillation (alcohols,

phenols, aldehydes, halocarbons);

2. Metallic poisons are the substances isolated by the method of mineralization

of the biological material (zinc, manganese, mercury);

3. Drug poisons are the substances isolated by extraction with polar solvents,

acidified water or acidified ethanol (barbiturates, alkaloids, etc.);

4. Pesticides are the substances isolated by extraction with organic solvents

(chlorinated and organophosphorus pesticides);

5. Substances isolated by water extraction (some salts, mineral acids, alkalis);

6. Substances isolated by some special methods (fluorides);

7. Substances determined without isolation (carbon monoxide, hydrogen

sulphide).

9

3. Intoxications and their classifications

Toxicology is the science, which studies the laws of a living organism

interaction with a poison. Intoxication is the disordes of vital important functions

of an organism under the action of a poison. The classification of intoxications as

diseases of chemical etiology has three main principles in its basis:

ethyopathogenetic, clinical and nosological.

Ethyopathogenetic classification of intoxications

According to the origin –casual (self-treatment, overdose, alcoholic or narcotic

intoxication, failure or accident in manufacture and daily life) and intentional

(suicidal and criminal) intoxications.

According to the place of origin – occupational, domestic, hospital intoxica-

tions.

According to the route of poison administration – peroral, inhalation, injection

intoxications, etc.

According to the origin of a poison – medicinal, industrial, alcoholic intoxica-

tions.

Clinical classification of intoxications

According to the signs of clinical manifestation (acute, chronic and subacute

intoxications).

According to the degree of disease.

According to the complications.

According to the eventual result of the disease.

Nosological classification of intoxications

According to the name of particular poisons, their groups and classes – intoxi-

cations by methyl alcohol, charcoal gas, barbiturates, alkaloids, pesticides, the

poisons of the plant and animal origin.

10

4. Relationship of toxicological chemistry with others disciplines

Toxicological chemistry is the special discipline, which is interlinked with

all courses of profession "pharmacy":

Methods of analytical and pharmaceutical chemistry are used for identification

and quantification of poisons in biological samples;

Chemical knowledge enable to realize the qualitative analysis of organic and

inorganic poisonous substances;

Physicochemical, biochemical methods, immunoassay are the most important in

the chemical toxicological analysis due to their high sensitivity and specificity;

The knowledge of botany and pharmacognosy are used in analysing different

parts of poisonous plants taking into account localization of poisons in them;

Forensic chemistry uses the forensic medicine and toxicology data, which are

important for the examination of the biological material because they allow to

choose samples for the analysis taking into account behaviour of poisons in a

human body;

Juridical knowledge are necessary to a forensic-medical toxicologist for

discharging the duties, because they are regulated by the legislation.

5. Special features of the chemical toxicological analysis

1. Variety of the samples examined. Biological fluids, internal organs of dead

bodies, food, food products, domestic chemicals, medicinal substances,

pesticides and utensils, air, ground, clothes can be the samples for the chemical

toxicological examination.

2. Relatively large amount of the biological sample examined (100 g) in compari-

son with little amount of the poisonous substance analysed (10-5 – 10-6 g). The

analysis of poison traces results in necessity of choosing the most sensitive

method.

3. The necessity of the poisonous substance analysis in the presence of admixtures.

Poisons isolated are not really separated from most of the endogenous material

and metabolites. Therefore, purification of the extracts obtained and separation

11

of parent substances and their metabolites are the important stages of chemical

toxicological examination.

4. Interpretation of the results obtained. A poison concentration may be measured

by a technique, which is not sufficiently selective to exclude all metabolites,

medicines, which may be present in therapeutic concentrations, and endogenous

material, which may interfere with the analysis. For example, phenobarbitone

concentrations measured by HPLC (High Pressure Liquid Chromatography)

may be only 10% of those measured by RIA (radioimmunoassay). Therefore,

an accurate interpretation of analytical results requires the use of a specific

immunoassay, or identification and quantification with a chromatographic

technique.

6. Organization of the forensic medical service and forensic toxicological

examinations in Ukraine

The forensic medical service is run by the chief forensic medical expert, who

is appointed by the Ministry of Public Health Care. The forensic toxicological

examinations are performed in the regional bureaus of the forensic medical

examination.

A bureau of forensic medical examination is included into the system of the

Ministry of Public Health Care and consists of the following departments:

forensic medical clinics. In this department the forensic medical exami-

nations of alive persons in the cases of accidents, domestic quarrels, etc.

take place;

morgue, in which forensic medical experts work; they give the biological

material to forensic-medical toxicologists for the analysis;

forensic medical laboratory is the department where material evidence is

examined.

Morgue has the forensic histological section. The forensic medical labora-

tory has the following sections: forensic biological, physicotechnical and forensic

toxicological ones.

12

Guiding principles of forensic toxicology determined by its legal aspect

1. All chemists who undertake this work must have toxicological experience.

2. The analyst must be given a complete case history that contains all the infor-

mation available.

3. All the material evidence, suitably labeled and sealed in clean containers,

must be submitted and examined.

4. All the known identification tests should be applied and adequate notes ma-

de at the time

5. All the necessary reagents used for these tests should be pure, and blank

tests should be performed to determine this fact.

6. All tests should be repeated, and compared with control samples, to which

the indicated poison has been added.

7. The general stages of the forensic toxicological examination and

making its plan

The general stages of the forensic toxicological examination are:

isolation – to separate a poisonous substance from the examined sample;

detection – to detect any medicines or poisons in the samples submitted by means

of screening procedures;

identification – to identify conclusively any medicines, metabolites or poisons

present by means of specific relevant physicochemical tests;

quantification – to quantify accurately those medicines, metabolites or poisons

present;

interpretation – to interpret the analytical findings in the context of the case, the

information given and the questions asked by the investigating officer.

The plan of the forensic toxicological examination is based on the informati-

on from the accompanying documents (the data from preliminary criminal

investigations or in subsequent court proceedings, conclusions of a medical

examiner, pathologist, etc.); results of the external examination (colour, odour,

consistence, presence or absence of suspended solids or sediments, pH); results of

13

preliminary tests (general drug screening, preliminary tests for heavy metals and

arsenic compounds, alcohols, volatile poisons, organophosphorus pesticides, etc.)

14

Topic 2: REGULARITIES OF THE POISON BEHAIVIOUR IN A BODY

Plan of the lecture

1. Routes of the poison administration into a body.

2. Absorption, distribution and elimination of poisons from a body.

3. Methods of poison detoxication.

In order to interpret the analytical results in a toxicological investigation it is

necessary to have knowledge of poison behaviour in an living organism.

Toxicology considers two aspects of the xenobiotic action, influence of a poison

on a body (Toxicodynamics) and influence of a body on a poison (Toxicokinetics).

A variety of questions may be put to a toxicologist, including “What poison was

taken?”, “What was its dose?”, “By which route and when was it taken?”, “Was

the observed pharmacological response a result of chronic or acute exposure to the

poison?”, and “Can the response be explained in terms of the poison concentration

found?” The knowledge of absorption, distribution and elimination of poisons

from the body, together with the understanding of poison the metabolism, will

allow these questions to be answered with a fair degree of confidence.

1. Routes of the poison administration into a body

Routes of the poison administration:

1. oral ingestion;

2. intravenous injection;

3. intramuscular injection;

4. inhalation;

5. dermal absorption.

To produce an effect, a poison must first enter the blood stream and be

carried to the target organ. With the exception of the intravenous injection, the first

15

process is that of absorption, either from the gastro-intestinal tract, from an

injection site or from the lungs.

Following intravenous administration, there is a rapid decrease in the plasma

poison concentration in the early period (-phase) (Fig. 1a) when distribution is

the major process, followed by a slower constant rate of decrease in the elimination

phase (-phase). After oral administration, the plasma concentration initially

increases while the poison is being absorbed and then decrease when elimination

becomes the major process (Fig. 1b).

a) b)

Fig.1. Typical semilogarithmic plots of the plasma concentration (c) versus time for

a poison given by intravenous injection (a) and orally (b)

The route of administration is an important factor in determining the rate and

extent of absorption. For example, absorption after an oral dose can be relatively

slow and erratic, while absorption from the lungs is usually fast because of the

good blood supply. Most poisons are administered orally, and the understanding of

the mechanism of absorption by this route is by far the most important for the

toxicologist.

2. Absorption, distribution and elimination of poisons from a body

Absorption from the Gastro-intestinal Tract. The traditional way of

describing poison absorption is by the pH-partition hypothesis. The hypothesis

regards the passage of a poison into the blood as being by passive diffusion of non-

lg c

time

α-phase β-phase lg c

time

16

ionized molecules across the lipoid barrier of the cells of the gut lining and into the

blood.

According to the hypothesis, an acid poison such as aspirin (pKa 3.5) is

absorbed in the stomach at about pH 2 when the poison is non-ionized. The poison

diffuses passively across a single barrier into the capillaries and is taken up by the

plasma at pH 7.4. Here, it ionizes and is, therefore, unable to return to the stomach

because there is a net concentration gradient of 25 000 to 1, stomach to plasma, of

the non-ionized species. In practice, aspirin is not totally absorbed in the stomach

due to the poor blood supply and small surface area. Basic poisons such as

ephedrine (pKa 9.6) are only slightly absorbed in the stomach, the major part being

absorbed from the upper section of the small intestine (pH from 5 to 7), the lower

section (pH from 7 to 8), or from the colon (pH from 7 to 8).

However, contrary to the hypothesis, the absorption of ionized species also

occurs. For example, paraquat is highly ionized but appears to be absorbed slowly

from the gastro-intestinal tract throughout its length and over a considerable period

of time from the moment of ingestion. Poisoning with such compounds can be

treated effectively by the prompt administration of an oral adsorbent, which

prevents further absorption of the poison.

Absorption generally depends very much on the large difference in the

poison concentration between the gastro-intestinal tract and the blood. The

knowledge of the blood flow to the different parts of the gastro-intestinal tract and

of the pH of its content is, therefore, important. Absorption is possible throughout

the gastro-intestinal tract, from stomach to rectum, although the major site is the

upper small intestine. This has intensive peristalsis, large surface area, high blood

flow, and the optimal pH for the absorption of most poisons, all of which result in

a high absorption rate.

Enterohepatic Circulation. Poisons and metabolites, which are eliminated

from the liver into the bile subsequently pass into the lumen of the gastro-intestinal

tract. Such compounds, usually of a relatively high molecular weight (>500), and

containing a hydrophilic residue in the molecule (e.g. digitoxin), may then be

17

reabsorbed either directly or indirectly following further metabolism. This recyc-

ling of poison is known as enterohepatic circulation. Glucuronide conjugates of

poison, which are excreted in significant amounts in the bile, such as morphine,

may be hydrolyzed by the intestinal microflora and reabsorbed. Enterohepatic

circulation thus prolongs the persistence of a poison in the body and may lead to

delayed toxicity, or to toxicity arising from the absorption of a poison metabolite

produced by intestinal bacteria. Poisons, which undergo enterohepatic circulation

may be detected in the feces in unchanged form even if they have been

administered by the parenteral route.

Distribution. After a poison has been absorbed, i.e. after it has passed from

the gastro-intestinal tract, through the liver, and into the systemic circulation, it is

then distributed throughout the body. Distribution depends on a number of factors.

These include the blood flow to the tissues, the partition coefficient of the poison

between blood and the tissue, the degree of ionization of the poison at the pH of

the plasma, the molecular size of the poison, and the extent of tissue and plasma

protein binding. For example, the distribution of plasma protein-bound poisons

such as the warfarin-type anticoagulants is restricted to the plasma and the

extracellular fluid, whereas alcohol distributes equally into the total body water.

The approximate volumes of the body water compartments for a person of

an average weight are: intracellular water 25 litres and extracellular water 17 litres

(of which 3 litres is plasma water). An intravenous dose of a poison which is

distributed immediately into the total body water (approximately 42 litres), would

give the initial plasma concentration (the dose is divided by 42) approximately

two-fifths of that obtained if the same dose was distributed only into the

extracellular water (the dose is divided by 17). If the poison is extensively bound

to tissue proteins, an even lower initial plasma concentration would be obtained.

The approximate proportions of weights and water content of the various body

tissues are given in Table 1 and Table 2.

18

Table 1

Comparison of weights of tissues and water

content in an average adult male

Tissues % Body weight % water

Muscle 40 76

Fat 10 10

Liver 2.5 68

Brain 2 75

Lungs 0.7 79

Kidneys 0.4 83

Table 2

Total body water

% Body weight

Male Female

10-18 59 57

18-40 61 51

40-60 55 47

>60 52 46

Distribution of Poisons to Tissues. The instantaneous equilibrium of the

poison concentration throughout the body as considered above does not necessarily

require that the concentrations are equal throughout the body. In fact, poison

concentrations in tissues are rarely equal to those in the plasma. For example, the

tissue plasma/concentration ratio will be very low immediately following

intravenous administration. As time progresses, the amount of the poison in the

tissue compartment will increase and, like that in the plasma compartment, will

eventually reach its plateau. If the poison is actively stored in a particular tissue-

compartment, then the ultimate concentration ratio between the tissue and plasma

will be relatively high. It should also be noted that the tissue/plasma concentration

19

ratio depends not only on the processes of distribution, but also to a large extent on

the route of the poison administration, and on whether single or multiple doses

have been given.

Elimination. Most poisons are eliminated from the body by metabolism in

the liver and/or by excretion of the poison and its metabolites by the kidneys.

However, metabolism and elimination by other tissues such as the lungs may

occur, as well as excretion of poisons and metabolites in the bile.

Poisons are removed from the liver as metabolites or an unchanged

substance. Some poisons and metabolites, e.g. morphine glucuronide, are excreted

into the bile in high concentrations.

A considerable amount of eliminated poisons by kidneys are water-soluble

ionized species. The principal factor, which determines the variability in the rate of

the poison excretion into the urine, other than the organ function and the blood

flow, is the pH of the urine. Thus, acidic poisons (e.g. barbiturates, salicylates) are

excreted more rapidly at high pH than basic poisons (e.g. amphetamines).

Conversely, basic poisons are excreted more rapidly at low pH. For example, about

85% of a dose of aspirin is excreted as free salicylic acid in the alkaline urine, but

only about 5% is excreted when the urine is acidic. Conversely, about 75% of a

dose of amphetamine is excreted unchanged in the acidic urine, but less than 5% if

the urine is alkaline.

The effect of varying urinary pH has been used in the treatment of poison

overdose by applying forced alkaline diuresis as an adjunct to the treatment of

salicylate or phenobarbitone poisoning. The success of the treatment is limited by

the extent to which these poisons are distributed, and by the presence of alternative

pathways of elimination.

Poison Accumulation. The problems of the poison accumulation are of

particular interest to the toxicologist because the resulting high poison

concentrations may lead to a progressive and insidious toxicity. Poisons

accumulate in the plasma or tissues if more than one dose is administered and the

interval between doses is less than the time taken to eliminate the preliminary dose.

20

3. Methods of poison detoxication

Detoxication is the process of decreasing toxicity and intensifying the poison

excretion from an organism. The methods of natural detoxication are gastric

emptying, irrigation of the bowel, forced diuresis, hyperventilation of the lungs.

The methods of artificial detoxication are hemodialysis, peritoneal dialysis, hemo-

perfusion, antidote therapy.

Gastric detoxication includes gastric emptying, adsorption of the toxin in

the gut and irrigation of the bowel.

Forced diuresis with furosemide is beneficial in phenobarbital poisoning.

Urinary alkalization promotes excretion of long-acting barbiturates. Sodium bicar-

bonate should be administrated.

Hemodialysis is performed with the help of the special apparatus known as

an “artifical kidney”.

Peritoneal dialysis consists of the removal of poisonous substances from the

systemic circulation in the result of poison diffusion across a semipermeable

membrane into a dialysis solution (dialysate).

Antidote therapy. Administration of antidotes is the effective method of

detoxication only during the early stages of poisoning. Activated charcoal (1g/kg)

should be administrated to bind any poisonous substances. Specific antidotes

chemically interact with poisons (or are their pharmacological antagonists), and as

a result, inactivation of poisons and their transformation to non-toxic substances

take place. Then the non-toxic products formed are excreted from the body in the

urine or in the bile. The antidotes most widely used are listed in Table 3.

21

Table 3

ANTIDOTES AND PHARMACOLOGY

No. Antidote Type Pharmacology

1 2 3 4

1. Activated charcoal

Non-

specific

chemical

antidote

Adsorbent. It by virtue of its large

surface area, adsorbs many

medicines and toxins. Highly ionic

salts (e.g., iron, lithium, and cya-

nide) or small polar molecules (e.g.,

alcohols) are poorly adsorbed

2. Lignin hydrolised (L.h.) Non-

specific

chemical

antidote

Adsorbent with a highly adsorbent

ability. It adsorbs alkaloids, heavy

metal salts, alcohols, etc.

3. Neohemodez

NO

CH

H CH

2

Hn

Non-

specific

chemical

antidote

Adsorbent. It adsorbs xenobiotics

and bacterial toxins, hydrolysis

products of the blood, barbiturates,

etc.

4. Ammonium hydrochloride

(3 % solution) NH4Cl

Specific

chemical

antidote

It forms non-toxic or little toxic

products with formaldehyde.

5. Sodium sulphate

Na2SO4

Specific

chemical

antidote

It precipitates the ingested barium

as an insoluble sulphate salt

6. Sodium chloride

NaCl

Specific

chemical

antidote

Administrating of chloride promotes

bromide excretion. Sodium chloride

also forms non-toxic and little toxic

products with silver nitrate

7. Dithioglycerol

CH

2

CH

CH2

SH

OH

SH

Specific

chemical

antidote

It binds ions of arsenic, antimony,

aurum, copper, nickel and mercury.

It is unacceptable in lead, iron,

selenium, tellurium compounds

intoxication, and in patients with li-

ver degenerative changes and

hypertension

22

Continuation of the table 3

1 2 3 4

8. Calcium gluconate

OH C

H2

CH

CH

CH

CH

COO-

OHOH

OH

OH 2

Ca2+

Specific

chemical

antidote

It forms non-toxic or little toxic

complexes with strontium, radium,

and fluoride-ion

9. Mercaptamine

SN

H

H

H HCl.

Specific

chemical

antidote

Dimercapto chelating agent is used

in the treatment of poisoning by se-

veral heavy metals, principally mer-

cury, arsenic, and lead

10. Penicillamine

N

H

H

OH

O

S

CH3

CH3

H

Specific

chemical

antidote

Penicillamine is a derivative of pe-

nicillin, that has no antimicrobial

activity but effectively chelates so-

me heavy metals such as lead, mer-

cury, and copper

11. Pentacine Specific

chemical

antidote

It is a chelating agent used in the

treatment of poisoning by lead, zinc,

plutonium, zirconium, cesium, yttri-

um

12. Unitiol

CH

2

CH

CH2

SH

SH

SO3Na

Specific

chemical

antidote

It (DMPS; 2,3 – dimercaptopropa-

nolsulfonic acid; Dimaval), a

immercapto chelating agent that is

water-soluble and used in the treat-

ment of poisoning by several heavy

metals (principally mercury, lead)

and arsenic

13. EDTA

NN

COOH

COONaNaOOC

HOOC

Specific

chemical

antidote

It is used as a chelating agent to

enhance elimination of certain toxic

metals, principally lead

23

Continuation of the table 3

1 2 3 4

14. Deferoxamine

NH2

N

N

O

O

CH3

N

N

N

O

OH

O

O

OH

OH H

H

Specific

chemical

antidote

It is a specific chelating agent for

iron. It binds free iron and, to some

extent, loosely bound iron (e.g.,

from ferritin or hemosiderin). Iron

bound to hemoglobin, transferring,

cytochrome enzymes, and all other

sites is unaffected. The red iron

deferoxamine (ferrioxamine) comp-

lex is water soluble and is excreted

renally, where it imparts an orange-

pink, or “vin rose” colour to the

urine

15. Sodium thiosulphate

Na2S2O3

Specific

chemical

antidote

It is a sulphur donor that promotes

the conversion of cyanide to less

toxic thiocyanate by sulphur

transferase enzyme rhodanese.

Unlike nitrites, thiosulphate is

essentially non-toxic and may be

given empirically in suspected cya-

nide poisoning. It also forms non-

toxic or little toxic products with

arsenic, antimony, lead, mercury,

thallium, bithmus ions

16. Urotropine (methenamine)

CH2

N

CH

2

N

CH2

N

CH2

NCH

2CH

2

Specific

chemical

antidote

It is used in the treatment of

phosgene poisoning

24

Continuation of the table 3

1 2 3 4

17. Acetylcisteine

(N-acetylcisteine; NAC)

SH

NH

OH

O

CH3

O

Specific

chemical

antidote

It is a mycolytic agent that acts as a

sulfhydryl group donor, substituting

for the liver’s usual sulfhydryl

donor, gluthathione. It rapidly binds

(detoxifies) the highly reactive

electrophilic intermediates of

metabolism or it may enhance the

reduction of the toxic intermediate

of Paracetamol. It is most effective

in preventing acetaminophen –

induced liver injury when given

early in the course of intoxication

(within 8 to 10 hours)

18. Fomepizole

(4-methylpyrazole; 4-MP)

NH

N

CH3

Biochem

ical/

pharmac

ological

antidote

It is a potent competitive inhibitor

of alcohol dehydrogenase.

Fomepizole can prevent the formati-

on of toxic metabolites after metha-

nol or ethylene glycol ingestion.

With its introduction most patients

with ethylene glycol or methanol

poisoning will probably be treated

with this medicine instead of etha-

nol, particularly in cases involving

small children, patients taking disul-

firam, patients with pancreatitis, and

in hospitals lacking laboratory sup-

port to perform rapid ethanol levels

(for monitoring treatment)

25

Continuation of the table 3

1 2 3 4

19. Ethanol

С2Н5ОН

Bioche

mical

antidote

It acts as a competitive substrate for

the enzyme alcohol dehydrogenase,

preventing metabolic formation of

toxic metabolites from methanol or

ethylene glycol

20. Cholestyramine Specific

chemical

antidote

It is used in the treatment of

digitoxin, digoxin and other cardiac

glycoside poisoning

21. Methylene blue

S

N

N(CH3)

2

(CH3)

2N

Cl+

. H2O3

Bioche-

mical

antidote

It is a thiazine dye, which increases

the conversion of methemoglobin to

hemoglobin. Methemoglobin is an

oxidized form of hemoglobin,

which is incapable of carrying

oxygen. Many oxidant chemicals

and medicines are capable of in-

ducing methemoglobenia: nitrites

and nitrates, bromates and chlorates,

aniline derivatives, antimalarial

agents, sulfonamides, local anesthe-

tics, carbon monoxide, cyanide,

hydrogen sulfide

22. Naloxone OHOO

OH

N

CH2

CHCH2

. HCl

Biochem

ical/

pharmac

ological

antidote

It is a pure opioid antagonist that

competitively block mu, kappa, and

delta opiate receptors within the

central nervous system. They have

no opioid agonist properties and can

be given safely in large doses with-

out producing the respiratory or

central nervous system depression

26

Continuation of the table 3

1 2 3 4

23. Naltrexone

N

OH

O

O

OH

Biochem

ical /

pharma-

cological

antidote

It is another potent competitive

opioid antagonist that is active

orally and is used to prevent

recurrence in patients detoxified

from opioid abuse. It is not used for

the acute reversal of opioid into-

xication

24. Flumazenil (Romazicon)

N

N

NFCH

3O

COOC2H

5

Biochem

ical/

pharmac

ological

antidote

It is a highly selective competitive

inhibitor of the central nervous

system benzodiazepine recaptors

25. Dimedrol

(Diphenhydramine)

C6H

5

C6H

5

ON

CH3

CH3

. HCl

Biochem

ical/

pharmac

ological

antidote

It is an antidote for Histamine

overdose.

26. Phentolamine

CH

3

NH

OH

NH

N

.HCl

Biochem

ical /

pharma-

cological

antidote

It is a competitive presynaptic and

postsynaptic alpha-adrenergic

receptor blocker that produces

peripheral vasodilation and is used

as an antidote for Adrenalin

27. Neostigmine

O N

O

CH3

CH3

N+

CH3

CH3CH

3

CH3SO

4

-

Biochem

ical /

pharma-

cological

antidote

It is a reversible inhibitor of

acetylcholinesterase (the enzyme

that degrades acetylcholine) and is

used to reverse the effect of non-de-

polarizing neuromuscular blocking

agents (Pipecuronium (Arduan),

Tubocurarine, etc.)

27

Continuation of the table 3

1 2 3 4

28. Vikasol O

O

SO3Na

CH3

. H2O3

Biochem

ical/

pharmac

ological

antidote

It is used in the treatment of

poisoning by coumarin derivatives

(Dicumarol, Warfarin) and other

anticoagulants

29. Phytomenadione O

O

CH3

C

H2

CH

C

CH3

C

H2

C

H2

C

H2

CH

CH3

CH3

3

Biochem

ical /

pharma-

cological

antidote

It is an essential cofactor in the

hepatic synthesis of coagulation

factors II, VII, IX and X. It reverses

the inhibitory effects of coumarin

and indanedione derivatives on the

synthesis of this factors

30

.

Botulinum Antitoxin Immuno-

antidote

It contains concentrated equine –

derived antibodies directed against

the toxins produced by various

strains of Clostridium botulinum (A,

B, E)

28

Topic 3: METABOLISM OF TOXIC SUBSTANCES

Plan of the lecture

1. Metabolism of xenobiotics and its role for an organism.

2. Classification of the processes of xenobiotic metabolism. Two phases of

metabolism.

3. Factors, which affect metabolism and toxic response.

1. Metabolism of xenobiotics and its role for an organism

Metabolism is an integral part of the poison elimination. As well as

facilitating excretion of a poison, it may affect the toxic response of a poison by

altering its potency and/or duration of the action. When a lipid-insoluble, polar, or

ionized poison is absorbed into the body, it is largely excreted unchanged by the

kidneys. However, the majority of poisons is lipid-soluble to some extent and is

reabsorbed into the blood from the glomerular filtrate. They need to be converted

to a more polar (water-soluble) form before they can be excreted in the urine. Such

compounds often undergo extensive metabolism. Metabolites are usually excreted

in the urine although large proportions of the glucuronides of some poisons (e.g.

morphine and codeine) are eliminated in the bile.

Thus, the metabolic process is usually regarded as one of detoxification, this

is not always so and metabolites, which are more toxic than the parent poison, are

known.

The majority of poisons are metabolized in the liver by the metabolic

enzyme systems (microsomal membrane-bound enzymes in hepatocytes).

However, some metabolic pathways employ non-microsomal enzymes and are

performed by enzyme systems found in the mitochondria or intracellular fluids.

Metabolism can occur in tissues other than the liver; the gastro-intestinal

tract, kidneys, and lungs may have a significant effect on the metabolism of a

poison depending on its route of administration. For example, many metabolic

reactions occur in the gastro-intestinal tract before an orally-administrated poison

29

is absorbed, carried out by enzymes in the mucosal lining or by microflora. Most

of these reactions involve reduction and hydrolysis because of the anaerobic

environment.

Pathways of poison metabolism are conventionally divided into two groups:

according to toxic or pharmacological properties of metabolites;

according to the type of chemical properties (non-synthetic and

synthetic).

2. Classification of the processes of xenobiotic metabolism. Two phases

of metabolism

Classification according to toxic or pharmacological properties of

metabolites.

1. Formation of less toxic metabolites than parent poisons:

O

O

N CH3

CH3

NH2

OH

O

NH2

N CH3

CH3

OH+

procaine p-aminobenzoic acid 2-(N,N-diethylamino)ethanol

2. Formation of more toxic metabolites than parent poisons:

CH3

OH OH

Hmethanol formaldehyde

[O]

3. Lethal synthesis:

OH

OH

O

F

OH

O

COOHF

OH

O

fluoroacetic acid (non toxic)

fluorocitric acid (poison)

30

4. Formation of metabolites with different pharmacological properties to

parent medicines:

NH

OCH3

O

CH3

CH3

H

ONH

OH

CH3

O

paracetamol (morepharmacologically active)

phenacetin

+

acetic aldehyde

Metabolites may be pharmacologically inactive (e.g. the hydroxylated deri-

vative of phenobarbitone) or they may be active with different pharmokinetic

properties but similar modes of action to the parent medicines. This is the case

with many psychotropic medicines. For example, hydroxylation and demethylation

of the tranquilliser diazepam gives the metabolite – oxazepam, which is also

marketed as a tranquilliser. Similarly, amitriptyline, a tricyclic antidepressant, is

demethylated to yield another antidepressant, nortriptyline. Active metabolites may

also have different modes of action or different potencies; thus, dealkylation of the

antidepressant iproniazid gives the tuberculostatic medicine isoniazid; the

antibacterial and fungistatic medicine primidone is metabolized to phenobarbital, a

sedative and hypnotic agent.

Classification according to type of chemical processes (non-synthetic and

synthetic)

The non-synthetic routes are principally oxidation (including hydro-

xylation, N- and O-dealkylation, and sulphoxide formation) and to a lesser extent

reduction and hydrolysis. These are generally referred to as Phase I reactions. The

synthetic routes mainly involve conjugation with glucuronic acid, but acetylation,

methylation, and conjugation with amino acids and sulphate also occur. These are

usually referred to as Phase II reactions. Phase II reactions remove or mask func-

tional groups (e.g. amino, carboxyl, hydroxyl, sulphydryl, etc.) on the medicine or

Phase I metabolite by the addition of an endogenous substrate. In this way Phase I

and Phase II reactions are frequently associated with each other, although this is by

no means a prerequisite. The major Phase II reactions is conjugation of glucuronic

31

acid with the phenolic or alcoholic hydroxyl groups, which are common products

of Phase I reactions. Conjugation reactions usually result in reduction of toxicity

and can, therefore, be referred to as genuine detoxification mechanisms.

A number of examples are given below to illustrate the variety of metabolic

routes, which can be followed in human.

Oxidation Reactions. Oxidation reactions are emphasised as those occuring

most frequently, yielding metabolites, which are commonly conjugated before

excretion.

a) oxidation of alcohols and aldehydes:

OHR OR

H

OR

OH

OR

H

alcohol aldehyde

[O]

carboxylic acidaldehyde

[O]

These processes take place in the liver, kidneys, lungs; oxidation of alcohols

occurs under the action of alcohol dehydrogenase.

b) hydroxylation of aromatic and cyclic compounds:

OH

COOH

OH

COOH

OH

OH

benzene phenol

salicylic acid 2,5-dihydroxybenzoic acid

32

OH

cyclohexane cyclohexanol

c) hydroxylation of radicals in aromatic compounds:

CH3

CH2OH COOH

benzyl alcoholtoluene benzoic acid

d) amino group hydroxylation of aromatic compounds:

NH2

NHOH

NO

phenylhydroxylamineaniline nitrozobenzene

e) N-, S-oxidation:

S

N

R

R

S

N

R

R

O

S

N

R

R

OO

derivatives of phenothiazine sulphonesulphoxyde

[O][O]

Reduction Reactions. Aldehydes and ketones undergo reduction to primary

and secondary alcohols; nitro and azocompounds are reduced to amines;

disulphides are reduced to thiols, etc.

Some examples are:

R R

O

R R

OH

R H

O

R OH

[H]

[H]

33

NH2N

+ OO

RS

R

O

RS

R

RN

+

O

O

RNH

2RN

O

anilinenitrobenzene

[H]

[H]

[H] [H]

Hydrolysis. Hydrolysis is an important route of metabolism for esters,

amides, carbamates and acyl hydrazines:

O

OH OH

OHN CH3

ON CH3

O OH

tropic acidatropine tropine

+

Dealkylation. N-, O-, S-dealkylation is more common:

O

OH

OCH

3

NCH

3

O

OH

OH

NCH

3

O-dealkylation

codeine morphine

O

OH

OH

NH

N-dealkylation

normorphine

34

SCH

3R OH

HSH

R

thiol

S-dealkylation

aldehyde

+

thioester

Dezamination:

RCH

3

NH2

RCH

3

O

amine ketone

+ NH3

Desulphonation:

NH

NH

S

O

O

C2H

5

C2H

5 NH

NH

O

O

O

C2H

5

C2H

5

thiobarbital barbital

Conjugation Reactions. Oxidation, reduction and hydrolysis usually

produce metabolites, which have a reactive functional group. Several of more com-

mon conjugation reactions mask these functional groups. Conjugation reactions are

usually referred to Phase II of metabolism processes. Glucoronides facilitated drug

elimination either by the kidney or the liver.

Glucuronide Formation. Glucuronic acid can be conjugated with a wide va-

riety of functional groups to form either an ester (as with carboxylic acids) or an

ether ( as with phenolic or alcoholic groups):

OH

H

OHH

OH

OH

HOH

H

COOH

ROH

OH

H

OHH

OH

OH

HO

H

COOH

R

glucuronic acid

+conjugation

35

OH

H

OHH

OH

OH

HOH

H

COOH

R OH

O OH

H

OHH

OH

OH

HO

H

COOH

O

R

OH

H

OHH

OH

OH

HOH

H

COOH

RNH

2

OH

H

OHH

OH

OH

H NH

H

COOH

R

+

+

conjugation

conjugation

Sulphate Formation. Sulphate conjugates are formed with hydroxy com-

pounds (e.g. alcohols and phenols) or aromatic amines:

OH OSO

3H

CH3

OH CH3

OSO

3H

phenylsulphatephenol

conjugation

conjugation

ethanol ethylsulphate

Conjugation with Amino Acids. Following the activation of drugs and meta-

bolites by acetyl-coenzyme A, conjugation with glutamine (in particular of aryl-

acetic acids) and glycine (of carboxylic and aromatic acids) is common.

Glutamine conjugation occurs less frequently than conjugation with glycine

(NH2–CH2–COOH):

COOHNH

O COOH

gipuric acidbenzoic acid

conjugation

36

COOH

OH

NH

O COOH

OH

salicyloric acidsalicylic acid

conjugation

Methylation. N-, S-, O-methylation is a common feature of the metabolism

of phenol; thiols and N-heterocyclic compounds:

N N+

CH3

SH SCH

3

N-methyl pyridiniumkation

pyridine

methylation

methylthiophenolthiophenol

methylation

Acetylation. Acetylation occurs frequently with primary and secondary ami-

nes, substituted hydrozines. The N-monoacetyl derivatives are usually produced:

NH2

NH CH3

O

N-acetylanilineaniline

The basic routes of benzodiazepines metabolism:

a) Oxidation and N-dezmethylation:

N

N

Cl

CH3 O

C6H

5

N

NH

Cl

O

C6H

5

OH

diazepam

[O]

oxazepam

37

b) N-dezalkylation:

N+

N

Cl

NH

C6H

5

CH3

ON

+

N

Cl

NH2

C6H

5

O

chlorodiazepoxide dezmethyl chlorodiazepoxide

c) reduction:

N

NH O

C6H

5

O2N N

NH

NH2

O

C6H

5

nitrazepam

[H]

d) hydrolysis:

NH2

Cl

C6H

5

ON

NH

Cl

O

C6H

5

OHNH

2

OH

COOH

2-amino-5-chlorobenzophenonoxazepam

H2O

+

e) glucuronide formation:

N

NH

Cl

O

C6H

5

OH

N

NH

Cl

O

C6H

5

O

C6H

9O

6

oxazepam

glucuronic acid

conjugate

NH2

Cl

C6H

5

O

NH

Cl

C6H

5

O

C6H

9O

6

2-amino-5-chlorobenzophenon

glucuronic acid

conjugate

Metabolic conversions by oxidation and reduction yield derivatives, which

have similar pharmacological activities to those of the parent compounds.

38

Metabolic processes by breaking of azepine cycle and glucuronide for-

mation yield pharmacologically inactive metabolites. They are these derivatives,

which account for the major fraction of the dose excreted in the urine.

3. Factors, which affect metabolism and toxic response

Physiological Factors

Age. Young children and elderly people generally have a lower metabolic

capacity compared with subjects between these extremes of age. The enhanced

sensitivity of the very young to poisons can be accounted for by the fact that

microsomal enzymes, which are responsible for metabolism (especially

conjugation with glucuronic acid), are not fully active until several months after

birth. In elderly subjects (over 60 years old) there appears to be a decreasing

capacity for drug metabolism as a consequence of a gradual decline in overall

physiological efficiency.

Disease. Diseases can affect all the processes, by which a medicine is

absorbed, distributed, and eliminated from the body. A medicine may be poorly

absorbed during gastro-intestinal diseases. Diseases, which affect the liver or

kidneys, probably have the greatest effect on drug concentrations because normal

functioning of these organs is essential for efficient metabolism and excretion.

Weight. The weight of an individual obviously affects the toxic response

because of the poison concentration in the blood is determined the volume, which

the medicine is distributed into.

Genetic Factors. The genetic control of the number of receptor sites, and

genetic variations in the extent of protein binding or the rate and extent of the

medicine metabolism, can make a marked contribution to variations in the poison

concentration and responses. Inefficient poison metabolism can lead to increased

bioavailability, accumulation of the poison, and there for to increased toxicity. For

example, with some hydrazine monoamine-oxidase inhibitors, the incidence of

side-effects and toxicity is increased in slow acetylates.

39

The route of Drug Administration. The route of drug administration

together with the nature of the dosage form determine the rate and extent of

absorption. Thus, a fatal drug dose given intravenously is often much smaller than

a fatal dose given by mouth because the injected medicine is able to reach the site

of action very rapidly. When administrating orally poisons may be unstable in the

acidic conditions of the stomach or in the alkaline conditions of the small intestine;

they may be metabolized by bacteria in the gastro-intestinal tract; they may

become adsorbed onto the stomach contents; a diet decreases the capacity of the

poison metabolism as a consequence of decreasing protein in the food that lowers

the enzyme activity.

Protein Binding and Distribution in the Blood. After absorption a medicine

is distributed non-uniformly around the body, including binding to plasma

proteins, distribution between plasma and erythrocytes, and binding to peripheral

tissues. Only the free (unbound) medicine can be cleared from the body by

metabolism and excretion. Further more, only the free fraction can diffuse across

biological membranes and equilibrate with the receptor site.

Drug Interaction. Drug interactions can be divided into two types: those,

which affect the drug concentration (i.e. which alter the processes of absorption,

distribution, and elimination), and those, which affect the response (by changing its

duration and severity).

Medicines with opposite pharmacological activities (e.g. barbiturates and

amphetamines) may have an antagonistic effect. Conversely, the additive effects or

side-effects of two medicines with the same pharmacological action (e.g. central

nervous system depressants) may prove to be fatal even though the individual

poison concentrations are not toxic themselves.

Further, a poison with a high affinity for tissue proteins might displace

second one from binding sites.

40

Topic 4: GROUP OF SUBSTANCES ISOLATED FROM THE

BIOLOGICAL MATERIAL BY MINERALIZATION (METALLIC

POISONS).

GENERAL AND SPECIAL MINERALIZATION METHODS

Plan of the lecture

1. General description of the metallic poison group.

2. The general rules of the metallic poison behaviour in a body.

3. The general scheme of the toxicological examination for metals.

4. Methods of mineralization.

5. Destructive mineralization method.

1. General description of the metallic poison group

The group of metallic poisons includes the compounds of barium, cobalt,

thallium, manganese, chromium, zinc, silver, lead, cadmium, copper, bismuth,

mercury, tin, arsenic and stibium.

Compounds of metals are widely distributed in the environment, occur

naturally in the living organisms (Mn, Cu, Co, etc., are biogenic). Metals and their

compounds are widely used in the national economy: manufacture of metallic

alloys, glass, ceramics, varnishes, paints, rubbers, chemical reagents, in agriculture

as pesticides (barium chloride, Granozan (ethyl mercuric chloride), Blue vitriol

(copper sulphate)), in medicine as pharmaceuticals (barium sulphate, potassium

permanganate, silver nitrate, Salvarsan, etc.), chemical warfare agents (Lewisite

(chlorovinyldichloroarsine)).

Causes of poisoning by metal poisons:

1 incorrect using of copper-, zinc- and cadmium-plated utensils for food

preparing and storage;

2 inhalation of highly dispersed metallic parts when treating metals;

3 incorrect using of pesticides and pharmaceuticals containing metallic com-

pounds.

41

2. The general rules of the metallic poison behaviour in a body

The study of poison behaviour in a living organism is necessary for the

correct choice of the samples for toxicological examination, the isolation method,

estimation of the poison storage time in the samples.

The routes of metallic poison administration:

gastro-intestinal tract (through a mouth);

breathing organs (such route of administration is characteristic for the

volatile compounds: antimoniated hydrogen, arsine, mercury and its

compounds, highly dispersed state of substances);

skin (compounds of mercury, thallium, cadmium which are soluble in

fats);

placenta and mucous (compounds of arsenic).

Absorption of metallic ions mostly takes place in the upper section of the

small intestine. Some compounds of metals are not absorbed from the gastro-

intestinal tract due to their insolubility, for example, barium sulphate. Metabolic

transformations of metal poisons in a living organism are oxidation, reduction,

conjugation and hydrolysis. Metals are distributed in the stomach, intestine, liver

and kidneys. They mostly accumulate in the liver and kidneys; in addition, silver

accumulates in the skin; lead, barium, cadmium, arsenic accumulate in flat bones.

The character of poisoning must be taken into account. So in acute arsenic and

mercury poisonings the metals are detected in the kidneys, liver; in chronic ones

they are detected in nails, hair, bones. Metals are mostly excreted by the kidneys

(with urine), the gastro-intestinal tract (through intestine), some metals (arsenic,

mercury) are excreted by sweat and mammary glands. The toxic action of metals

is due to the binding of the metallic cations with amino acids, peptides and proteins

by the reactive functional groups (–SH, –NH2, –COOH, –OH), which lead to the

formation of stable complexes and destruction of protein molecules.

The toxic action of some metal poisons:

42

Manganese is a protoplasmatic poison, affects the central nervous system,

kidneys, lungs, the circulatory system;

Chromium has the nephrotoxic, cauterizing action, blocks some enzymes;

Silver has the cauterizing action, affects some capillaries;

Copper produces the nervous-, hemo-, nephrotoxic, local cauterizing action;

Zinc has the enterotoxic action;

Bismuth forms methemoglobine; has the nervous-, hepatotoxic action;

Mercury has nervous-, nephrotoxic action;

Arsenic promotes penetration and causes the paralysis of capillaries; causes

hemolysis, blocks thiol enzymes;

Thallium is a protoplasmatic poison; nervous toxic action;

Barium promotes penetration of cellular membranes and capillaries (death

from heart-circulatory insufficiency) ;

Lead has a nephrotoxic action; blocks some enzymes.

3. The general scheme of the toxicological examination for metals

The group of metallic poisons should be checked if vomiting and diarrhoea

are noted as symptoms. In cases of the suspected poisoning a person should be

admitted to hospital, specimens of blood and urine should be taken. If possible,

vomit, stomach aspirate and washout fluid avaible from cases of acute poisoning

should be transported to the laboratory. Simple qualitative tests should be applied

first, for example the Reinsch test (Scheme 1). This test detects only seven metals,

but it can be applied to almost any materials (body fluids, homogenised tissue,

food and drinks) without any elaborate preparation. The Reinsch test is sensitive

enough to detect toxic concentrations of the most common poisonous metals in a

shout time. However, it misses many metals for it to be considered as a complete

group-exclusion test.

A satisfactory routine metal-screening method must be able to analyze any

biological material, without drastic modifications (drastic conditions take place in

43

Scheme 1

THE GENERAL SCHEME

OF THE TOXICOLOGICAL EXAMINATION FOR METALS

Clinical presentation: vomiting and diarrhoea

Blood, urine, serum, stomach washings, vomit, stomach

aspirate, tissue, skin, bone, food, drinks, etc.

Reinsch Test:

(add 3 ml of hydrochloric acid to a sample and immerse a

copper strip or spiral copper wire in the sample for 2 hours)

Silver deposit – mercury

Shiny black deposit insoluble in KCN solution –

bismuth Dull black deposit soluble in KCN solution – arsenic

Purple deposit insoluble in KCN solution – antimony

Dark deposit – selenium or tellurium

Speckled discoloration – sulphur high concentrations

Mineralization (biological tissue)

Extraction (food, stomach content, homogenised tissue,

blood)

Without isolation: filtration using a coarse-grade filter

paper or dilution with purified water may be used

(homogenised tissue, body fluid, food, drinks)

Chemical (colorimetric) method

Electrochemical method (anodic stripping voltametry

(ASV))

Atomic absorption spectrometry (AAS)

Electrothermal atomic absorption spectrometry (ETAAS)

Flame atomic emission spectrometry (AES)

Inductively coupled plasma – mass spectrometry (ICP-

MS)

“Internal” control using reference materials with trace

metal concentrations: normal, borderline toxic and toxic

levels (certified reference materials (blood, serum, urine,

bone) are available commercially)

Physicochemical methods

Case of the

toxicological

examination

Samples

Metal-screening

test (body fluid,

homogenized tissue

extract or some

another liquid

sample)

Confirmative

analysis of metals

in samples

Isolation

Identification

Quality assurance (for analysis of

trace metals)

Quantitative

analysis (generally

in medical legal

cases)

44

Scheme 2

THE EXTRACTION SCHEME OF METALS FROM A SAMPLE

Samples Homogenized tissue, food, stomach

contents, blood

Treating by nitric

acid for overnight

Centrifuge

Supernatant luquid Complexing agent:

ammonium pyrrolidine

dithiocarbamate, sodium diethyl

carbamate, potassium sodium

tartrate

Sb, As, Bi, Cd

Pb, Hg, Tl

Lithium Fluoride –

Graphite Mixture,

evaporate by infra-red

lamp

Extract by chlorophorm two times

(blank experience)

AAS, AES, ETAAS or ICP-MS

Ba

, Cr, etc.

To analysis

45

mineralization of the biological material) and with detection limits that provide

reliable exclusion of minimum quoted toxic concentrations.

The main stages of metal extraction from a sample are given in Scheme 2.

The most widely used methods for analysis of metals in biological fluids are

colorimetric, electrochemical (anodic stripping voltametry), electrothermal atomic

absorption spectrometry, flame atomic absorption spectrometry, inductively

coupled plasma emission spectrometry and inductively coupled plasma MS.

Specimens such as tissues, skin and bone may sometimes be collected at

postmortem as part of the investigation of complex medical legal cases.

The general scheme of toxicological examination for metals is given in Scheme. 1.

In addition, although for most metals the lower threshold of toxicity is at least an

order of magnitude higher than the normal level, interpretation of the results

without access to comparable reference specimens can be very difficult.

4. Methods of mineralization

Choice of the samples for the chemical toxicological examination of metallic

poisons depends on their distribution and accumulation in organs and tissues. In

undirected analysis for metallic poisons the stomach with its content, the intestine

with its content, liver, kidney, urine, spleen are analysed. In the directed analysis

for particular metals the following samples are analysed additionally:

rectum, hair (for mercury compounds) ;

flat bones (for lead compounds) ;

flat bones and hair (for thallium compounds) ;

hair, nails, flat bones (for arsenic compounds);

brain, lungs (for tetraethyl lead).

Preparation of the samples for mineralization. Samples are reduced to fine

particles separately. Volumes of the liquid samples, for example urine, are mea-

sured. When the sample is preserved with ethyl alcohol (using for this purpose

formaldehyde or phenol is forbidden), it is slightly alkalified with sodium

carbonate (for decomposition of volatile arsenic and mercury chlorides), put into a

46

porcelain cup and alcohol is evaporated with the help of water bath at the tem-

perature bellow 50ºC.

Mostly 100 g of the sample are examined. The rest can be used for the

examination after steam distillation when there is little amount of the sample. The

excess of water is removed by careful evaporation with the help of water bath. To

control the reagents the blank experiment is carried out.

Mineralization is the process of wet oxidation (ashing) of organic matter,

being the sample of research, with the purpose of destruction of metal complexes

with proteins.

There are “dry” and “wet” methods of mineralization. The liquid phase oxi-

dation by the mixtures of acids (sulphuric and nitric; sulphuric, nitric and

perchloric) belongs to the methods of “wet” mineralization. Ashing and alloying

with soda an saltpeter are “dry” mineralization methods.

The methods of mineralization are divided into general and specific ones.

Oxidation by means of acids are general methods of mineralization. The methods

of “dry” mineralization are used mostly as specific methods. Destructive

mineralization is an specific method too. It is used for the isolation of inorganic

mercury compounds.

The methods of mineralization by the mixture of sulphuric and nitric acids

and the mixture of sulphuric, nitric and perchloric acids are the most widely used.

Both methods are fast, allow to destruct organic matters completely; allow to

obtain quite small volumes of the mineralizate. The drawback of these methods is a

considerable loss of mercury that is determined by volatility of its compounds. In

addition, the mixture of sulphuric, nitric and perchloric acids is explosive, but this

method is faster.

The methods of “dry” mineralization are used as specific ones in analysis of

some metal poisons (silver, lead, manganese, zinc) in small amounts of such

samples as hair, skin, undestroyed precipitates, etc. The drawback of these methods

is the loss of mercury compounds.

47

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 destruct of hard lumps of the sample. Then increase the temperature

and add nitric acid (1:1) by drops into the reactionary retort until the retort content

will become colourless or slightly rather yellow.

At the beginning of mineralization concentrated sulphuric acid acts as a

water-removing agent, damages the structure of cells and tissues. When increasing

the temperature (above 110ºC) the concentration of sulphuric acid reaches 60–70%

and it acts as a strong oxidizer as it is destructed to sulphur (IV) oxide, water and

atomic oxygen:

SOH

OOH

O

SO2 + H2O2

O

H2O

At the beginning of mineralization nitric acid is a weak oxidizer. Due to

formation of nitrogen oxides and nitrogenous acid and also with increasing of the

temperature it acts as a strong oxidizer:

N+O

O

OH 2NO2 + H2O2

O

H2O

2

During the mineralization process some amount of nitrososulphuric acid

appears. It prevents to detect some metals.

OHS

O

O

ONO

When heating aromatic substances with the mixture of sulphuric and nitric

acids undesirable processes of nitration and sulphurization take place; they

complicate mineralization. Preliminary dilution of sulphuric and nitric acids before

mineralization considerably decreases the degree of nitration and sulphurization.

48

Mineralization passes two stages. At the first stage, which is called

destruction, destruction of the biological material under the action of acids, the

oxidizers (without the complete destruction of organic matters), as well as

destruction of complexes of metals with proteins take place, as a result metals pass

to the solution (destructate) as ions. The destruction lasts for 30–40 min. while

heating carefully. A destructate is a yellowish brown heavy transparent liquid.

At the second stage of the mineralization the complete destruction of

organic matters takes place. This stage is long. It is carried out with the intensive

heating and addition of an oxidizer, diluted nitric acid (1:1). The end of mine-

ralization is determined by heating of the mineralizate without adding nitric acid

until thick white steams of sulphur oxides appear. When the liquid does not

become dark, mineralization is considered to be over.

Denitration is the process of removing nitric, nitrogenous, nitrososulphuric

acids and nitrogen oxides from the mineralizate. These substances are oxidizers,

which prevent the analysis for metallic poisons.

Various methods of denitration have been developed. The hydrolysis method

(it is obsolete now) is based on dilution of the mineralizate with water followed by

heating of the mixture obtained. Thus, nitric, nitrogenous acids, nitrogen oxides are

removed; nitrososulphuric acid is subjected to hydrolysis:

OHS

O

O

ONO

OHS

O

O

OH

OHNO+

H2O

This method lasts about 15–17 hours.

Various reductants (urea, sodium sulphite, formaldehyde) were proposed

for denitration of mineralizate. Formaldehyde is the best reagent for this purpose,

since destruction of the oxidizers takes place quickly (for 1–2 min.); the excess of

the reductant is easily removed by boiling for a few minutes. The mechanism of

these processes is as follows:

2N2 + 5CO2 + 7H2O4HNO3 + 5CH2O

2NO + N2 + 2CO2 + 4H2O4HNO2 + 2CH2O

49

NO + O NO2

2NO2 + H2O HNO2 + HNO3 To destructn nitrososulphuric acid the mineralizate is diluted preliminary

with water and heated to 110ºC. Then formalin is added. Verification of denitration

completeness is performed with the help of the reaction with diphenylamine. A

dark blue colour appears in the presence of nitric, nitrogenous acids, nitrogen oxi-

des:

NH

NH

NH

N N

[O] [O]

The blank experiment is required because sulphuric acid, the solvent of di-

phenylamine for the experiment, can contain nitric acid as an admixture.

Mostly the mineralizate is a colourless, transparent, heavy liquid. It can be

yellowish due to ferric (III) cations, the tissue components, greenish (in the

presence of chromium (III) salts) or blue (due to copper (II) salts). The

mineralizate can contain a white sediment (due to lead, barium or calcium

sulphates) or dirty-green one (due to the cosedimantary of chromium (III)

sulphate).

5. Destructive mineralization method

Destructive mineralization (or Destruction) is the specific isolation method

for inorganic mercury compounds. The necessity of the specific method is

determined by the loss of mercury in the process of the general mineralization.

Therefore, complete destroying of organic matters is not carried out. It is replaced

with the partial one, during which bonds between mercury and proteins are broken.

Method. The samples examined are 20 g of the liver and 20 g of the kidney.

Destruction of the liver and kidneys is performed separately. The mixture of

50

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 water bath for 10–15 min. Thus, the destructate obtained contains

mercury cation and various biological admixtures (proteins, peptides, aminoacids,

lipids, etc.). The admixtures are removed by extraction with chloroform.

To remove oxidizers from the destructate urea is used:

2HNO2 + O=C(NH2)2 2N2 + CO2 + 3H2O;

2HNO2 + O=C(NH2)2 N2 + 2NO + CO2 + 3H2O.

The destructate obtained is examined for mercury cation.

51

Topic 5: ANALYSIS OF THE MINERALIZATE BY THE FRACTIONAL

METHOD. QUANTITATIVE DETERMINATION OF METALLIC

POISONS IN THE MINERALIZATE

Plan of the lecture

1. General principles of the fractional analysis method. Methods of «masking»

of interference ions.

2. The scheme of the mineralizate analysis for metallic poisons by A.N.

Krylova`s method. Detection of mercury in the destructate.

3. Quantitative determination of metallic poisons.

4. Analysis of metallic poisons by atomic absorption spectrometry.

1. General principles of the fractional analysis method. Methods of

«masking» of interference ions

In the chemical toxicological analysis of metallic poisons the systematic

method and the fractional one can be used. The systematic analysis method is

based on the consecutive distribution of the metallic cations according to analytical

groups and sub-groups followed by identification of the cation in the particular

sub-group. The systematic method takes a lot of time, it is dangerous when

hydrogen sulphide is used as a reagent and little sensitive. Nowadays it is almost

replaced by the fractional method. The fractional method is based on using the

reactions, which allow to detect metallic cations in separate small portions of the

solution examined. The fractional method is rapid, sensitive, allows to determine

metallic poisons without the preliminary separation them from each other. N. A.

Tananaev was the founder of fractional analysis method. A.N. Krylova has made a

great contribution in development of the fractional method and introduction it into

practice of chemical toxicological analysis.

The following techniques are used in the fractional analysis method:

Replacement of the sedimentary reactions by the liquid-phase complexati-

on reactions followed by extraction of the complexes obtained; using the most sen-

52

sitive and specific identification reactions (for example, for manganese – oxidi-

zation to permanganate ion, for chromium – formation of perchromic acids, etc.);

When a specific reaction is absent a preliminary test, the most sensitive

reaction, and then confirmative reactions, they are more specific ones, are per-

fomed;

«Masking » of ions, which interfere the analysis;

Methods of «masking» of interference ions are:

Complexation, the binding of interference ions into colourless stable

complexes. For masking of many trace metals cyanide, fluoride, phosphate,

thiosulphate, thiourea, trilon B, ascorbic acid, hydroxylamine are used.

For example, ferric (III) cations prevents the reaction for cobalt (II) cations

with thiocyanate, so fluorides or phosphates, which bind ferric (III) into colourless

complexes [FeF6]3- or [Fe(PO4)2]

3- are used.

The use of small volumes or large dilution of the mineralizate for

“masking” of endogenous metal ions; for this purpose the mineralizate is diluted at

once to 180 ml and particular small portions of the mineralizate are analysed: for

Mn2+ – 1 ml; Cu2+ – 3 ml; Bi3+ – 10 ml, etc.

Variation in the pH medium; so, lead dithizonate complex is formed only

in the alkaline medium, complexation of mercury and silver with dithizone takes

place in both the acidic and alkaline medium, silver dithizonate is destroyed by

addition of hydrochloric acid solution and mercury dithizonate is not destroyed in

these conditions.

The use of oxidization-reduction reactions (trace manganese (II) cation

interferes with the reaction for chromium (III) with diphenylcarbazide when both

Mn2+ and Cr3+ are oxidized by ammonium persulphate to permanganate-ion and

dichromate-ion; permanganate-ion is reduced to manganese (II) cation with the

help of sodium azide).

The use of diethyldithiocarbaminate (DDTC) activity range (lead is

replaced by copper from DDTC complex, copper is replaced by mercury). The

Tananaev’s activity range for DDTC at pH 5 is:

53

Hg > Ag > Cu > Ni > Co > Pb > Bi > Cd > Tl > Sb > Zn > Mn > Fe

Reagents most commonly used in the fractional method

Dithizone. Two tautomer forms of this reagent occur:

NH

S

NN

NH

C5H

6

C5H

6 N

SH

NN

NH

C5H

6

C5H

6

pH > 7

pH < 7

thion form thiol form

There are various points of view about the structures of dithizonates, the

most probable form is:

N

S

NNC5H

6

NH

C6H

5

Me/n

In the acidic medium singly substituted dithizonates are formed, in the

strong alkaline medium the substitution of the second hydrogen atom takes place.

Dithizonates can be coloured (the colour often depends on the pH medium),

and this fact is used in qualitative and quantitative analysis of metallic poisons.

Dithizonates are dissolved in organic solvents and destroyed by acids, and this fact

is used for separation of cations from the mineralizate.

Diethyldithiocarbaminate. As a reagent sodium diethyldithiocarbaminate

is the most commonly used. It forms inner-complex compounds with heavy metal

cations:

C2H

5

N

C2H

5

SNa

S

C2H

5

N

C2H

5

S

S

Me/nMen+

Diethyldithiocarbaminates are soluble in organic solvents, many of them are

colourless (zinc, cadmium diethyldithiocarbaminates), some are coloured (copper,

bismuth diethyldithiocarbaminates); they are destroyed by mineral acids. These

reactions are used as preliminary tests, for separation of cations from the

54

mineralizate, in quantitative analysis of metallic poisons by photocolourimetric

method.

Diphenylcarbazide. This reagent is used for identification of Cr3+ in the

mineralizate. Preliminary Cr3+ is oxidized to dichromate ion by ammonium

persulphate in the presence of silver nitrate, a catalyst. Then the dichromate ion

obtained oxidizes diphenylcarbazide (I) to diphenylcarbazone (II) and at the same

time Cr2O72- is reduced to Cr2+, which with diphenylcarbazone, as an enole form

(III), forms the inner-complex salt (IV) with a red-violet colour. The mechanism of

the reactions, which take place, is as follows:

2Cr3+ + 3S2O82- + 7H2O

Ag+

Cr2O72- + 6SO4

2- + 14H+

NH

O

NH

NH

NH

C5H

6

C5H

6

N

OH

NN

NH

C5H

6

C5H

6

NH

O

NN

NH

C5H

6

C5H

6

NH

O

NN

NH

C5H

6

C5H

6N

N

NH

H6C

5

N C6H

5

O

Cr

[O]

Cr2O72-

+ Cr22+

Cr22+

I II

III IVII

8-Oxyquinoline. This reagent is used for identification of bismuth Bi3+..

Preliminary bismuth ions are transformed into acidocomplex [BiJ4]-, which forms

an ionic associate with oxine in the acidic medium; the orange-red sediment is

observed:

Bi3+ + 4KI [BiI4]¯ + 4K+

N

OH

N+

OH H

H+ + [BiI4]-

[BiI4]-

Malachite or diamond green. These reagents are used for identification of

thallium and antimony. Preliminary antimony (it occurs in the mineralizate as

55

HSbO2) and thallium (it occurs in the mineralizate as Tl3+) are transformed to

acidocomplexes [SbCl6]- and [TlCl4]

-, respectively, which form the ionic associates

coloured dark blue or blue with malachite or diamond green and extracted by

toluene or xylene:

N

CH3

CH3

N+

CH3

CH3

[X]-

, [X]- = [SbCl6]- or [TlCl4]

-

2. The scheme of the mineralizate analysis for metallic poisons by

A.N. Krylova`s method. Detection of mercury in the destructate

Examination of the precipitate

Separation of PbSO4 and BaSO4 precipitates from the total volume of

the mineralizate (filtrate I).

he precipitate is washed by water acidified with sulphuric acid (for

removal of co-precipitated Fe3+; Cu2+; Zn2+; Cd2+, etc., cations). When the

precipitate is dirty-green, it is washed by ammonium persulphate (for washing from

Cr3+). To separate barium and lead sulphates from each other the precipitate is

washed by a hot solution of ammonium acetate; PbSO4 dissolves (filtrate II):

2PbSO4 + 2CH3COONH4 → [Pb(CH3COO)2 PbSO4] + (NH4)2SO4

Examination of the barium sulphate precipitate

Recrystallization of barium sulphate from the concentrated sulphuric acid is

performed; the reaction is sensitive, with the negative result of this reaction the

examination for barium compounds can be finished.

The reaction of formation of the barium iodate precipitate is as follows; the

first stage of the reaction is reduction of BaSO4 into the flame of a gas-ring:

56

BaSO4 + 2C BaS + 2CO2↑ a green colour of the gas-ring flame,

then BaS is placed on the glass slide and dissolved in a drop of 5М solution of

hydrohloric acid. Then a crystal of potassium iodate is placed into the solution

obtained. Ba(IO3)2 precipitate is examined with the help of microscope.

BaS + 2HCl BaCl2 + H2S (g)

Ba2+ + 2IO3- Ba(IO3)2 (s)

characteristic colorless crystals

The reaction is highly sensitive, confirmative.

Examination of the II filtrate for lead cation (Pb2+)

The reaction with chloroform solution of dithizone occurs in the alkaline

medium (pH 7.5–8.0); a red colour of the chloroform layer is observed.

The reaction is preliminary: with the negative result the research is finished,

with the positive result the confirmative reactions are carried out.

The re-extraction of Pb2+ from dithizonate obtained with the help of nitric

acid solution into the aqueous phase is carried out; then the reactions of PbS,

PbSO4; PbCrO4; PbI2 precipitate formation are performed; black, white, orange-

yellow and yellow precipitates are observed, respectively.

Examination of the filtrate I

Detection of manganese (II) cation (Mn2+)

The reactions of Mn2+ oxidization by means of potassium periodate or

ammonium persulphate to permanganate-ion are carried out, a violet colour is

observed. For the reaction with potassium periodate sodium dihydrophosphate is

added for masking interference Fe3+ cation:

2Mn2+ + 5IO4- + 3H2O

H2PO4-

2MnO4- + 5IO3

- + 6H+

The reaction is highly sensitive and specific, it detects endogenous manga-

nese, therefore, with the positive result the confirmative reaction is necessary:

2Mn2+ + 5S2O82- + 8H2O

Ag+

2MnO4- + 10SO4

2- + 16H+

57

The reaction takes place in the presence of silver nitrate as a catalyst, for

masking Fe3+ dihydrophosphate is added. The reaction is sensitive, but it does not

detect the endogenous manganese, and specific.

Detection of chromium (III) cation (Cr3+)

The reaction with diphenylcarbazide (chemism is given above) is highly

sensitive, but it is not specific. To masking the interference ions (ferric (III),

antimony) phosphate is added, the interference permanganate (II) cation is masking

by addition of sodium azide.

The reaction of Cr3+ oxidization to perchromic acids followed by the

extraction of the acids obtained with organic solvents (ethyl acetate), a dark blue

colour of the organic layer is observed:

Cr3+(NH4)2S2O8; AgNO3

Cr2O7

2- H2O2; H+

H2CrO6+ H3CrO8

This reaction is specific for chromium.

Detection of silver cation (Ag+)

The reaction of the silver dithizonate formation occurs in the sulphuric acid

medium (a gold-yellow colour of the chloroform layer is observed). In order to

differ silver dithizonate from mercury dithizonate (it is orange-yellow), it is

destroyed by 0.5 M solution of hydrochloric acid, while mercury dithizonate is not

destroyed in these conditions.

With the positive result of this reaction a silver cation is separated from the

total volume of the mineralizate (about 90 ml) by addition of sodium chloride (as

AgCl (s). Then the sediment obtained is dissolved in ammonia solution. The

confirmative reactions are carried out with the solution of the silver ammoniate

obtained:

AgCl + 2NH4OH [Ag(NH3)2Cl +2H2O

The reaction with nitric acid:

[Ag(NH3)2]Cl + 2HNO3 AgCl (s) + 2NH4NO3 a white precipitate

Reaction with potassium iodide:

[Ag(NH3)2]Cl + KI + H2O AgI (s) + KCl + NH4OН + NH3

58

a yellow precipitate

The reaction with thiourea and potassium picrate (yellow prismatic crystals

are observed).

Detection of copper (II ) cation (Cu2+)

The reaction with (DDTC)2Pb is specific for Cu2+ (according to the

Tananaev’s rule lead is replaced from its (DDTC) complex only by copper, silver

and mercury). The yellow-brown colour of the chloroform layer is observed. With

the positive result of this reaction a copper cation is re-extracted into the aqueous

layer by HgCl2 and confirmative research is carried out.

The reaction with pyridine-rhodanide (thiocyanate) reagent where esmerald-

green precipitate dissolved in chloroform is formed:

N

N

N

N

Cu2+ 2(SCN)-

The reaction with potassium hexacyanoferrate (II):

2Cu2 + K4[Fe(CN)6] → Cu2[Fe(CN)6] (s) + 4K

a brown precipitate

The reaction with ammonium mercuric thiocyanate:

Cu2 + (NH4)2[Hg(SCN)4] Cu[Hg(SCN)4] (s)+ 2NH4

a yellow-green crystalline precipitate

Detection of bithmuth (III ) cation (Bi3+)

The reactions with 8-oxyquinoline (the mechanism is given above) and

thiourea are carried out. When bismuth (III) cation interacts with thiourea, the

complexes of various structure having the lemon-yellow colour appear:

59

S

NH2

NH2

n

Bi

, n = 2, 3, 9

3+

These reactions are preliminary. With the positive result of these reactions

Bi3+ is separated from the mineralizate as (DDTC)3Bi, then re-extracted by nitric

acid and the re-extract is used for the confirmative research – the reaction with

thiourea; reduction of bismuth cations Bi3+ to metallic bismuth with the help of zinc

dust; crystalline precipitate formation with brucine (C23H26N2O4) and potassium

bromide, with cesium chloride and potassium iodide.

Detection of zinc cation (Zn2+)

The reaction with dithizone at pH 4.5-5.0 is carried out; it is preliminary;

with the positive result of this reaction a zinc cation is separated from the

mineralizate as diethyldithiocarbaminate (DDTC)2Zn and after re-extraction the

confirmative reactions are carried out.

The reaction of zinc sulphide formation:

Zn2+ + S2- ZnS (s); a white precipitate

the reaction with potassium ferrocyanide (II):

3Zn2+ + 2K4[Fe(CN)6] K2Zn3[Fe(CN)6]2 (s) + 6К;

a white precipitate

reaction with ammonium mercury thiocyanate:

Zn2+ + (NH4)2[Hg(SCN)4] Zn[Hg(SCN)4] (s) + 2NH4+

a colourless wedge-shaped precipitate

Detection of thallium (III ) cation (Tl3+ ) and antimony compounds

The preliminary reaction with malachite or diamond green (the mechanism

is given above) are carried out. With the positive result of this reaction the

confirmative reactions are carried out.

The formation of antimony sulphide sediment (for antimony):

2Sb3+ + 3Na2S2O3 + 3H2O Sb2S3 (s) + 3Na2SO4 + 6H+, an orange precipitate

60

the reaction with dithizone (for thallium after transformation of Tl3+ to Tl+).

Detection of cadmium cation (Cd2+)

A cadmium cation is separated from the mineralizate as (DDTC)2Cd, which

passes into the chloroform layer, then the complex is destroyed by hydrochloric

acid. In the re-extract obtained a cadmium cation is determined with sodium

sulphide:

Cd2+ + S2- → CdS (s)

a yellow precipitate

Then the reactions of crystalline precipitate formation with brucine and po-

tassium bromide, with pyridine and potassium bromide are performed.

Detection of arsenic compounds

Sanger-Bleck`s reaction is preliminary. This reaction is based on the trans-

formation of arsenite and arsenate obtained after mineralization to arsine and

determination of the latter with the help of test-paper treated with mercury

bromide:

AsO2- + 7H+ + 3Zn AsН3 (g)+ 3Zn2+ + 2H2O

AsН3 + HgBr2 AsH2(HgBr) + HBr

AsН3 + 2HgBr2 AsH(HgBr)2 + 2HBr

AsН3 + 3HgBr2 As(HgBr)3 + 3HBr

In the presence of arsenic compounds in the mineralizate examined the test-

paper turns yellow or brown. This reaction is highly sensitive (0.1 µg in the ana-

lysed test of the mineralizate), but not specific (PH3, SbH3 interfer with AsH3),

therefore, the confirmative research is required.

Marsh`s test is one of the most specific tests for arsenic in the mineralizate.

Blank experiment is necessary to confirm the absence of arsenic in sulphuric acid,

zinc and the glass of chemical vessels used for the test.

In according to the Marsh`s test arsenic compounds are reduced to arsine

followed by the detection with the help of such signs:

a garlic smell of arsine appearing from the tube of Marsh`s apparatus;

61

a blue colour of the flame when set on fire of H2 from the tube of Marsh`s

apparatus in the presence of AsH3 ;

turbidity of AgNO3 solution when AsH3 passing through it;

a thin coating on a porcelain plate brought into AsH3 flame.

The basic test is thermal AsH3 destruction in the narrow part of the Marsh`s

tube:

2AsH3 2As (s) + 3H2 (g)to

A thin coating, which appears at the place of heating, is analysed

additionally. For this purpose the tube is separated from the apparatus and then a

thin coating is heated:

3O2 + 4As 2As2O3

Arsenous anhydride appears as characteristic octahedrons crystals.

Advantages of the method are as follows:

repeated control of the mineralizate for arsenic;

it is obvious for the tests described above.

The disadvantage of the method is:

its duration, danger of explosion of H2 when igniting if oxygen is not

replaced completely from the Marsh`s apparatus.

Specificity of the method:

antimony, sulphur, carbon interfere with arsenic. However, antimonic

anhydride is an amorphous precipitate, carbon and sulphur oxides are volatile.

There are other methods, which allow distinguishing arsenic and antimony

compounds based on the microcristaloscopic reactions.

Detection of mercury (II) cation (Hg2+) in the destructate

The reaction with dithizone is used after preliminary back extraction of the

destructate (nonspecific reaction).

The reaction with copper (I) iodide suspension:

Hg2++ Cu2I2 + 2I → Cu2[HgI4] (s)

62

A pink or orange-red precipitate of copper (I) tetraiodomercurate (II)

appears, this reaction is sensitive and specific.

3. Quantitative determination of metallic poisons

After detection and identification of metallic poisons in the mineralizate

quantitative determination is carried out in most cases, it is conditioned by the

natural content of many elements (manganese, copper, zinc, chromium) in an

organism or accumulation of them in the process of life (arsenic, mercury, lead).

Such methods of quantitative determination as: gravimetry (for barium – bya

precipitate BaSO4); titrimetry: complexonometry (barium, lead, zinc, copper,

bismuth, cadmium), iodometry (lead), argentometry (arsenic), rhodanometry

(silver); photocolorimetry (manganese by the reaction with potassium periodate;

mercury, lead, silver, thallium, zinc by the reaction with dithizone; thallium,

antimony by the reaction with malachite or diamond green; chromium by the

reaction with diphenylcarbazide; copper by the reaction with lead

diethyldithiocarbaminate; bismuth by the reaction with thiourea); visual

colorimetry (mercury by the reaction with copper (I) iodide suspension, arsenic by

Sanger-Bleck`s test) are used; atomic-absorption spectrometry can be used both

for qualitative, and quantitative analysis of all metals.

4. Analysis of metallic poisons by atomic absorption spectrometry

Atomic absorption spectrometry (AAS) is one of the most widely used

physicochemical methods for the metallic compounds analysis. The fundamental

scheme of the atomic absorption spectrometer is given below (Fig. 2).

For analysis atomization of the sample examined is carried out. For this

purpose flame atomization or electrothermal one (ETA) can be used. For the flame

atomization mixtures of acetylene and air (t 2000ºC) or acetylene and nitrous

oxide (t 7000ºC) are the most widely used. The electrothermal atomization is

carried out with the help of the graphite furnace.

63

54321

Fig. 2. The fundamental cheme of the atomic absorption spectrometer:

1 – radiator; 2 – flame; 3 – monochromator; 4 – photoamplifier; 5 – recording device.

A lamp with a hollow cathode filled with neon is used as a radiator in

the range of 190–850 nm. As the result of the electromagnetic radiation absorption

by the atoms of the sample the atomic transitions with the energy ΔE occur. These

transitions are responsible for the resonance lines in the atomic spectrum, which are

characteristic for each element.

The energy (ΔE) can be expressed in terms of the principal parameters that

define electromagnetic radiation, namely frequency ν (Hz), wavelength λ (nm) and

wavenumber (cm–1):

∆E = h ν = h c

= h c ΰ λ

where h is the Planck’s constant; c is the velocity of radiation in vacuum.

According to the Bouguer-Lambert-Beer law (A = k c b, where A is absorption;

c is the concentration of the absorbing species; b is the path-length; k is

absorptivity of the system, the constant), the absorption (A) is measure of the

element’s concentration: A = lg(I◦ /I) where I◦ and I are intensities of radiation from

a source before and after passing the through atomized test, respectively.

Atomic-absorption spectroscopy is applied for determination the about 70

elements, mainly metals.

Sensitivity of the method is 1–100 µg/l (when atomization is in flame), 0.1–

100 µg/l (when atomization is in a graphite stove). The error of determination is

0.2–1.0 %. In the automatic mode a flaming spectrometer allows examining about

ν

ν

υ

64

500 samples in an hour, and the spectrometer with a graphite stove analyses about

30 tests / hour.

65

Topic 6: THE GROUP OF SUBSTANCES ISOLATED FROM THE

BIOLOGICAL MATERIAL BY STEAM DISTILLATION (VOLATILE

POISONS). THE METHODS OF ISOLATION AND ANALYSIS OF

DISTILLATES BY THE CHEMICAL METHOD

Plan of the lecture

1. General description of volatile poisons.

2. Methods of the volatile poison isolation from the biological material.

3. The scheme of the distillate analysis by the chemical method.

1. General description of volatile poisons

Volatile poisons are toxic substances, which are isolated from biological

material by steam distillation and other similar methods. Volatile poisons are acetic

acid; hydrocyanic acid; halocarbons (chloroform, tetrachloromethane, dichloroet-

hane, chloral hydrate); aldehydes and ketones (formaldehyde, acetone); alcohols

(methanol, ethanol, isopentanol, ethylene glycol); esters (ethyl acetate, isopentyl

acetate); aromatic hydrocarbons and their derivatives (benzene, toluene, xylene,

aniline); phenols and phenoloacids (phenol, salicylic acid); tetraethyl lead (TEL);

hydrogen sulfide; phosphorus and its products of oxidation (phosphoric acids) and

reduction (phosphine).

Lethal doses of some volatile poisons are as follows:

for hydrocyanic acid (HCN) the lethal dose is 0.05–0.1 g;

for potassium cyanide (KCN) the lethal dose is 0.15–0.25 g;

for methanol (CH3OH) the lethal dose is 25–100 g (7–8 g causes

blindness);

for chloroform (CHCl3) the lethal dose is 50–70 g;

for acetic acid (CH3COOH) the lethal dose is 15 g.

Substances of the group mentioned are widely used in industry,

agriculture, medicine. For example, chloroform, ethanol, phenol, diethyl ether

are used in medicine; cyclones A and B are used in agriculture (for pesticide

66

control), chloroform, ethanol, benzene, acetone, etc., are used as solvents in

various branches of industry.

The general property of poisons of this group is volatility and ability to

be distilled. The main isolation method of this group of poisons is steam dis-

tillation. This method allows creating softer conditions then, for example, usual

distillation at atmospheric pressure when the given substances are isolated from

the biological material. This advantage is important for those compounds,

which can be destroyed at high temperature.

Dependence of pressure of saturated vapour of the mixture against

temperature is in the theoretical basis of this method. The liquid begins boiling

and can be distilled when pressure of vapour above the liquid is equal to

atmospheric pressure or exceeds it:

P1 + P2 + P3... = Pn > Patm

where P1 + P2 + P3 +... is the sum of partion pressures of water vapour

(P1) and vapours of liquids analysed (P2, P3, …) in the mixture;

Pn is the pressure of vapour above the mixture.

Consequently, the mixture is distilled at the temperature, which is below

then boiling temperatures of pure substances. For example:

T boil (C6H6) = 80.2ºC;

T boil (H2O) = 100ºC.

The mixture of equal volumes of benzene and water boils at 69.2ºC.

2. Methods of the volatile poison isolation from the biological material

In chemical toxicological analysis for the isolation of volatile poisons the

following methods are used:

steam distillation at atmospheric pressure;

distillation at increased pressure;

distillation at reduced pressure;

microdiffusion;

dry distillation;

67

vapour-phase method.

The method of steam distillation is the most commonly used. In accordance

with this method 100 g of the biological material is reduced to fine particles, mixed

up with distilled water (Vmixture= 1/3 Vretort ) and placed into the retort placed into a

cold water-bath. Then the vaporization flask is heated until water vapour appears,

the biological material examined is acidified by the saturated water solution of

oxalic acid to pH 2.0–2.5 and all parts of the device (vaporization flask, retort,

refrigerator and receiver) are connected immediately and only then the water-bath

is heated.

Fig. 3. Apparatus for steam distillation:

1 – vaporization flask; 2 – retort with the sample; 3 – water refrigerator; 4 – receiver; 5 – water

bath

The choice of the pH value of the sample examined is due to the fact that

when pH is 2.0–2.5, the most complete destruction of bonds between proteins and

poisonous substances occurs.

The choice of the acids for acidification of the biological material is due to

the fact that mineral acids can decompose volatile poisons, for example,

hydrocyanic acid:

HCN + 2H2O + H+ HCOOH + NH4+

,

or hydrolyze the endogenous phenol, which as a result of protein consumption

appears in a living organism as phenol sulphate, a conjugate:

68

OSO

3H

OH

H2O

the conjugate is not volatile volatile

Weak organic acids do not destroy this conjugate, and only phenol coming

to a body is distilled.

In the case of acetic acid isolation the biological material is acidified with

the help of H2SO4 or H3PO4 solutions, in order to displace the acetic acid

dissociation equilibrium in the aqueous solution to the molecular form and increase

the volatility of the acid.

The distillates are collected into receivers. The first distillate, which contains

the most volatile poisons, for example HCN, is collected into the receiver with 2

ml of 2% NaOH solution. The total volume of the first distillate is (V1) 5 ml.

Then two more distillates (V2; V3) are collected:

V2 =V3 =25 ml.

When the positive result is obtained when examining the distillate sample

for a particular volatile poison, the distillation is continued out until the negative

result of the corresponding reaction.

Some basic substances are distillated from the alkalized biological material:

aniline, pyridine, nicotine, anabasine, etc. In this case after distillation of

substances from the acid medium, the biological material is alkalized by the

addition of 5 NaOH solution to pH 8–9 and is distilled again collecting 2–4

distillates with 10–15 ml each are collected in 0.1 M solution of hydrochloric acid.

All volatile poisons may be divided into groups according to their ability to

mix with water:

1. Substances, which are mixed with water in all ratios, but do not form

azeotropic mixtures (methanol, acetone, acetic acid, ethylene glycol, etc.);

2. Substances, which are not mixed or poorly mixed with water

(benzene, chloroform); in this case after distillation two clear layers appear, the

water layer and the organic solvent layer, which are easily separated.

69

3. Substances, which form azeotropic mixtures. Azeotropic mixture has

the identical composition of the vapour and liquid phases and cannot be divided

completely by distillation at atmospheric pressure (phenol, ethanol). For sepa-

ration of azeotropic mixtures distillation at increased or reduced pressure is

used. For example: the azeotropic mixture of ethanol with water (96 % C2H5OH

and 4 % H2O) is distilled at atmospheric pressure at 78ºC. When reducing the

pressure to 100 mm mer. col. (13.3 kPa) the distilled mixture has the following

composition, C2H5OH 99.62 % and H2O 0.4 % and it is distilled at 34ºC.

With the purpose of concentration of the isolated poisons in distillates and

their purification from biological impurities these distillates should be subjected to

fraction distillation.

The method of distillation at reduced pressure is carried out by means of

rotary vaporizers and used most commonly for thermally unsteady substances.

The method of distillation at increased pressure is used for the isolation of

thermally unsteady substances with a high boiling temperature.

The method of microdiffusion is used for analysis of the blood, urine,

homogenized biological samples. This isolation method is carried out with the help

of special chambers; the sample examined and a crucible with the reaction solution

are placed at the bottom of this chamber and it is closed. Evaporation of poison

into the reaction solution is performed in the presence (or without) of salting-out,

at the room temperature or while heating to 37–50ºC. Then the reaction solution is

examined for the presence of volatile poisons.

The method of dry distillation is similar to `microdiffusion and differs only

by dry air which, is skipped through the sample examined. This method is used for

the analysis of substances with a low boiling temperature.

The Vapour phase method (or gaseous extraction) uses the transfer of the

volatile poison examined to the vapour phase followed by the analysis of the

resulting phase with the help of the gas chromatography (GC) method. The vapour

phase method is very important when laboratory express-analysis of biological

fluids in acute intoxication takes place.

70

3. The scheme of the distillate analysis by the chemical method

The first distillate is analysed for the presence of hydrocyanic acid.

Hydrocyanic acid

Physical properties. Hydrocyanic acid is a volatile liquid with the boiling

temperature of 25.6ºC and the characteristic almond odour.

It is very weak acid (Kd = 4.8 · 10-10), salts are unsteady in water:

KCN + H2O + CO2 HCN + KHCO3

HCN + 2H2O HCOONH4

KCN + 2H2O NH3 (g) + HCOOK

Toxic action. Hydrocyanic acid produces hypoxia by inhibiting

cytochromeoxidase.

Metabolism of hydrocyanic acid. Two basic metabolism pathways take

place – hydrolysis with formation of ammonium formiate and conversion via

rhodanase, the liver enzyme, to thiocyanate, which is subsequently excreted in the

urine.

HCN

hydrolysis

rhodanase, S

HCOONH4

HSCN

Antidotes, which are used in hydrocyanic acid poisoning, are:

1. Substances containing sodium (potassium) thiosulphate:

CN + S2O32-

rhodanaseSCN + SO4

2-

2. Substances forming methemoglobin (salts and esters of nitrous acid):

NaNO2; KNO2; C5H11-O-NO; methylene blue:

Hb(Fe2+) MtHb(Fe3+) MtHb(Fe3+)-CN CN-NO2

-

cyanmethemoglobin(not toxic)

hemoglobin methemoglobin

But CN--ion can separate, therefore, it is necessary to give simultaneously

sulphur-contaning substances and carbohydrates to a patient.

71

3. Carbohydrates (e. g. glucose) bind hydrocyanic acid and its salts with

formation of glucose cyanhydrine.

OH

OH

H O

H

H

H OH

H OH

CH2OH

OH

OHH

H

H OH

H OH

CH2OH

H OH

CN

HCN

D-glucose cyanhydrineD-glucose

Peculiarities of hydrocyanic acid isolation from the biological material.

Taking into account that hydrocyanic acid is volatile well and dissociates poorly in

water solutions, it is transformed to salt when the first distillate is collected into

sodium hydroxide solution:

HCN + NaOH NaCN + H2O

The analysis of the distillate for hydrocyanic acid is started with the

reaction of Prussian blue formation; highly sensitive and specific:

2NaCN + FeSO4 Fe(CN)2+ Na2SO4

Fe(CN)2+ 4NaCN Na4[Fe(CN)6]

3Na4[Fe(CN)6]+ 4FeCl3 Fe4[Fe(CN6)3] s + 12NaCl

This reaction takes place in the alkaline medium; under this condition it is

possible to form Fe(OH)3 and Fe(OH)2, for their dissolution HCl is added:

Fe(OH)3+ 3HCl FeCl3 + 3H2O,

the excess of HCl can slow the precipitate formation.

Conclusion about the presence of cyanide is made in 24–48 hours, because

in the presence of HCN traces and protein admixtures the precipitate appears

slowly. For acceleration of the sedimentation solution of BaCl2 is added; Prussian

blue precipitate coprecipitates on the BaSO4 precipitate.

Prussian blue precipitate is considered to be a material proof for court and

investigation establishments.

72

Sensitivity of this reaction is 20 µg / ml.

In addition, for detecting hydrocyanic acid the following colour tests may be

used.

The reaction of ferric (III) thiocyanate formation; it is highly sensitive

(10 µg / ml), but non-specific:

KCN + (NH4)2 S2 KSCN + (NH4)2S ammonium polysulphide

H+

3KSCN + FeCl3 Fe(SCN)3+ 3KCl a red colour

The reaction of polymethine formation; it is highly sensitive (0.2 µg / ml),

non-specific:

HCN + Cl2 ClCN + HCl chlorocyane

N N

CN

Cl N

CN

OH

NH

CN

O NH

CN

NC

6H

5

ClCN H2O

-HCl

NH2-C6H5

pyridine N-cyanpyridine chloride

polymethine dye(an orange colour)

+ +

The reaction of benzidine blue formation;it is sensitive, non-specific:

2HCN + Cu(CH3COO)2 Cu(CN)2+ 2CH3COOH

2Cu(CN)2 (CN)2 + 2CuCN dicyane

(CN)2 + H2O O + 2HCN

73

NH2

NH2

NH2

NH2

NH NH

2HX +

H2O

2

. 2HX +

O

benzidine (a blue colour)

Paper moistened by copper salt solution and benzidine turns blue in the

presence of hydrocyanic acid or its salts.

The analysis of the second distillate

The analysis of the second distillate is started with the reactions for

halogenated hydrocarbons.

Halogenated hydrocarbons

Lethal doses for some halogenated hydrocarbons are as follows:

for chloroform – CHCl3 the lethal dose is 50–70 g;

for tetraclormethane – CCl4 the lethal dose is 20-50 ml;

for chloralhydrate – CCl3-COH · H2O the lethal dose is 10 g and less;

for 1,2-Dichloroetane – CH2Cl-CH2Cl the lethal dose is 15–25 ml.

Toxic action. Chloroform and chloral hydrate are narcotics. At first, they

excite and then depress the nervous system. Acute ingestion of as little as 10 ml of

chloroform may result in death due to the central nervous system depression.

Tetrachloromethane acts on an organism like chloroform, but slower and

causes more considerable disorders in organs; the liver and kidneys are subjected

to fatty degenerative changes.

Dichloroethane according to the narcotic action is the strongest poison

among halocarbons.

74

Halocarbon poisonings are accompanied by vomiting, diarrhea, swelling of

the stomach, increasing and sickliness of the liver.

Metabolism has not been studied completely, carbon dioxide and

hydrochloric acid are the eventual results of the metabolic processes:

CHCl3 CO2 + HCl

CCl4 CHCl3 and CO2

The feature of halocarbon isolation is distillation to the first portions of the

distillate. With a great amount of the poisons (less than 1 g) drops of organic

liquid, which does not mix with water are observed in the distillate.

The analysis is started with general (non-specific) and little-sensitive

reaction on Cl-

with AgNO3 after separation of the organic bonded chlorine. This

reaction is characteristic for all halocarbons:

CO

HCl

3C

C ONa

O

H

C ONa

O

H

CH2

CH2

OHOH

CH2

CH2

ClCl

NaOH

CHCl3

CCl4

NaCl + H2O +

NaCl + CO2 + H2O

CHCl3 + H2O +

NaCl +

NaOH

(the reaction mixture is heated in a soldered ampoule for 4 hours)

Cl- + Ag+ AgCl (s)

A white precipitate or white lees appear when Cl– -ion is present.

If the lees or the precipitate do not appear, Fujivara reaction (based on the

polymethine formation) is performed; it is sensitive, non-specific.

The reaction of isonitril formation; it is highly sensitive, non-specific

(among the halocarbons mentioned only dihloroethane does not give this reaction):

75

NH2

N+

CCHCl3OH-

isonitril (unpleasant odour appears)

With the positive result of this reaction the complete analysis for

halocarbons is performed.

The reaction with resorcinol in the alkaline medium; it is sensitive; non-

specific (aldehydes, formic acid give this reaction). The mechanism has been

studied incompletely:

OH

OH

O

OH

R

H

OO

OH

H

R

keto-form

A pink colour appears.

The reaction with Fehling’s reagent; it is little sensitive and non-specific

(for aldehydes), tetracloromethane and dichloroethane do not give this reaction.

CuSO4 + 2NaOH Cu(OH)2 + Na2SO4

COOK

CH

CH OH

OH

COONa

CH

CH

O

OH

O O

CH

CH

COOK

OH

O

OOCOONa

H

H

Cu2

Cu(OH)2

-KOH-NaOH

CH

CH

COOK

OH

OH

OO

CH

CH

OH

OH

O O

COONa

R C

O

H

R C

O

ONa

COOK

CH

CH

OH

OH

COONa

Cu+

3NaOH 2KOH

+ 2CuOH + 2H2O + 4

2

2CuOHt

Cu2O + H2O

76

The reaction with Nessler`s reagent gives only chloralhidrate in the group

of halogenated hydrocarbons:

Cl3C C

O

H

Cl3C C

O

K

+ K2[Hg I4] + 3KOH + 4KI + Hg + 2H2O

An orange precipitate is observed, after a while it becomes green.

Dichloroetane gives some general reactions for halogenated hydrocarbons

(Fujivara’s reaction, separation of organic bonded chlorine):

The reaction of separation of organic bonded chlorine:

Cl CH2

CH2

Cl OH CH2

CH2

OH+ NaOHto

+ 2NaCl

A white precipitate of silver chloride appears after addition of argentum

nitrate in the nitric acid medium.

Then the detection of ethylene glycol is carried out by oxidation to formal-

dehyde followed by identification of formaldehyde with the help of the reactions

described for formaldehyde above:

HO–CH2–CH2–OH + KIO4 2HCHO + KIO3 + H2O

The reaction of copper acetylenide formation:

Cl CH2

CH2

Cl CH CH

CH CH CuC CCu

C2H5OH

t + 2NaOH + 2NaCl + 2H2O

+ 2CuNO3 + 2NH4OH + 2NH4NO3 + 2H2O

a pink or red colour

Then the second distillate is examined for the presence of formaldehyde.

Formaldehyde

The lethal dose is 15–25 ml.

Toxic action. Formaldehyde vapour disturbs respiration, causes sharp cough,

lacrimation. The oral administration of formaldehyde is accompanied by nausea,

cramps, faint, circulatory collapse, damage of the stomach and small intestine,

kidneys.

Formaldehyde metabolism occurs according to the following mechanism:

77

O

H

H

O

H

OH

[O] [O]CO2 + H2O

The analysis of the distillate for formaldehyde is started with the following highly

sensitive reactions:

The reaction with chromotropic acid, a violet or red-violet colour indicates

the presence of formaldehyde. The reaction is non-specific because substances,

which in hydrolysis, dehydration or oxidization form formaldehyde, give this

reaction too.

SO3H

OH

OH

SO3H

H C

O

H

SO3H

OH

OH

SO3H

CH

2

HSO3

OH

OH

HSO3

SO3H

OH

OH

SO3H

CH

HSO3

O

OH

HSO3

+2- H2O

[O]

+ H2O

The reaction with codeine and sulphuric acid, a blue-violet or red-violet

colour appears:

O

OH

OCH

3

NCH

3

O

OH

OH

NCH

3

H

H

O

O

OH

OH

NCH

3

CH

2

O

OH

OH

NCH

3

codeine morphine

H2SO4 conc

- CH3OH

78

The reaction with fuchsin-sulphurous acid; it is non-specific because

aldehydes (furfurol, acetaldehyde, etc.) and even oxidants of the air (chlorine,

oxygen, nitrogen oxides) give this reaction. A dark blue colour of the solution can

appear in 10–15 min. However, when colour appears in half an hour, it is not the

positive result for aldehydes.

It should be noted that under certain conditions this reaction can be specific

for formaldehyde – only formaldehyde gives this reaction in the strong acidic

medium (pH 0.7) and a lot of aldehydes give it at pH 2.7

Reactions with resorcinol, Fehling reagent, reduction of silver are less

sensitive and non-specific, but they are used when the positive results of the highly

sensitive reactions described above are obtained.

With the negative results of the reactions for formaldehyde the analysis for

alcohols (methanol and ethanol) and then for ketones (acetone) is carried out.

Methanol, ethanol and isopentanol have the most toxicological value in the

group of monobasic alcohols.

Alcohols are used in medicine, manufacture, food industry.

Methanol

The lethal dose is 40–100 ml, when administering 7–8 ml of methanol

blindness occurs.

Toxic action. Methanol damages the nervous and circulatory systems, ocular

nerve, eye retina. The initial narcotic effects of methanol are much milder than

those of ethanol, and the characteristic toxic syndrome may not appear in 6–30

hours after ingestion.

Methanol toxicity is conditioned mostly by formaldehyde and formic acid,

the products of methanol metabolism are:

O

H

H

O

H

OHCH

3

OH[O] [O]

CO2 + H2O[O]

79

Formaldehyde strikes the ocular nerve and formic acid is responsible for the

severe metabolic acidosis.

Treatment may include administration of ethanol to inhibit methanol meta-

bolism.

Peculiarities of isolation. Methanol is volatile, therefore, it is collected in

the receiver cooled by ice or cold water for decreasing of the alcohol loss.

When examining wines the volatile acids are preliminary bound by addition

of sodium carbonate, and then methanol is distilled over.

When examining cheap eau-de-Colognes they are preliminary purified from

essential oils by their extraction with ether and then volatile acids are bound and

methanol is distilled over.

Analysis for methanol is started with the reaction of methylsalicylate

formation, non-specific because ethanol gives this reaction too, but the reaction of

methyl salicylate formation is in 40 times more sensitive (sensitivity is 0.3 mg in

the test), than the reaction of ethyl salicylate formation.

The reaction of methanol oxidation to formaldehyde, various oxidizers are

used in this reaction (e.g., potassium permanganate):

O

H

H

CH3

OH + 8H2O5 + 2KMnO4 + 3H2SO4 5 + 2MnSO4 + K2SO4

The excess of the oxidant is eliminated by addition of oxalic acid or sodium

sulphite. Then formaldehyde is detected by the reactions with codeine in sulphuric

acid and with fuchsin-sulphurous acid.

Ethanol

The lethal doses is 300 ml of 96 % ethanol for a non-drinker, 15–25 g for

children.

Toxic action. Ethanol is a narcotic, which excites and then depresses the

nervous system. Ethanol damages nervous and circulatory systems, causes

cirrhosis of the liver, psychoses.

Ethanol distributes eventually through the body water.

80

Metabolism. 90 % of the ethanol dose administered is oxidized to H2O and

CO2 under the action of alcohol dehydrogenase and 10 % is excreted unchanged by

lungs and kidneys. Ethanol can be present as an endogenous substance in the blood

(0.2 ‰ corresponds to the natural maintenance) probably produced in the intestinal

tract:

O

CH3

H

O

CH3

OHC

2H

5OH

C2H

5OH

[O]

CO2 + H2O

[O]

alcohol dehydrogenase

When administeraing disulfiram, the medicine, the acetaldehyde oxidization

to acetic acid slows down, and it results in accumulation of acetaldehyde in a body

that is accompanied by nausea, vomiting and headache.

The analysis for ethyl alcohol is started with the reaction of iodoform

formation, which is non-specific, because acetone, lactic acid gives this reaction

too, it is used as preliminary:

C2H

5OH CH

3COH

CH3

COH CI3

COH

CI3

COH CHI3

I2 + 2NaOH NaOI + NaI + H2O

+ NaOI + NaI + H2O

+ 3I2 + 3NaOH + 3NaI + 3H2O

+ NaOH + HCOONa

a specific odour, a yellow precipitate

With the positive result of this reaction the confirmative reactions for

ethanol and acetone are performed.

The reaction of ethyl acetate formation is sensitive (the sensitivity is 15 µg

/mL), specific – in the presence of ethanol a specific odour of ethyl acetate (“fresh

apple”) appears:

2CH3COONa + 2C2H5OH + H2SO4 2CH3COOC2H5 + Na2SO4 + 2H2O

The ester smell increases when applying ester on water surface.

Reaction of ethanol oxidation to acetaldehyde is specific in the presence of

ethanol a specific odour of ethylaldehyde appears:

81

CH3

C

H

O

3C2H5OH + K2Cr2O7 + 4H2SO4 + Cr2(SO4)3 + K2SO4 + 7H2O

The reaction of ethyl benzoate formation is specific in the presence of

ethanol a specific odour of ethyl benzoate appears:

C2H

5OH

Cl

O

O

O

C2H

5

+

benzoyl chloride

+ HCl

Acetone

The lethal dose is 25–50 ml. In healthy adults, endogenous acetone blood

levels is up to 10 mg/L. Acetone concentrations are markedly elevated during

diabetic or fasting ketoacidosis and may range from 100–700 mg/L.

Metabolism:

CH3

C CH3

O

CO2 + H2O

Peculiarities of isolation. Acetone is separated from the distillate, because it

mixes with water, alcohol, ether in any ratios. With this purpose the distillate is

saturated by various salts (sodium chloride, calcium chloride, potassium carbonate)

that results in formation of two layers, the acetone and water ones, which are easily

separated.

Analysis for acetone is started with the reaction of iodoform formation,

sensitive, non-specific:

CH3

C

O

CH3

CI3

C

O

CH3

CI3

C

O

CH3

+ 3I2 + 3NaOH + 3NaI + 3H2O

+ NaOH CHI3 + CH3COONa

The odour of CHI3 and a yellow precipitate specify the possibility of acetone

presence in the distillate.

82

The reaction with sodium nitroprusside is non-specific, it is characteristic

for ketones; it is used in the difference of acetone from ethanol:

CH3COCH3 + Na2[Fe(CN)5NO] + 2NaOHNa4[Fe(CN)5ON=CHCOCH3] + 2H2O a red colour

Reactions with some aldehydes, for example with furfural, are non-

specific, when combining with other reactions for acetone can confirm its presence

in the distillate:

O

CH3 CH

3

OH

O

O

O O2

a red colour

+ 2H2O

Analysis of the mixture of the second and the third distillates

The remainder of the second distillate is mixed with the third distillate, and

analysis for halocarbons and formaldehyde is repeated. Then the analysis for

phenol and isopentanol is performed.

Phenol

The lethal dose is 8–15 g.

Toxic action. Phenol causes severe irritation and corrosion on contact with

the skin or other tissues. Absorption of the chemical may produce cyanosis, shock,

weakness, cardiac arrhythmia, collapse, convulsions, liver and kidneys damage,

coma and death. Phenol is rapidly absorbed upon dermal contact.

Metabolism. An oral dose of phenol is efficiently eliminated in the 24-hour

urine as sulphate (77 % of the dose) and glucuronide conjugates (16 % of the

dose):

OSO

3H

OH

H

OHH

OH

OH

HO

H

COOH

83

An olive-green colour of the urine indicates the metabolic oxidation of

phenol according to the scheme:

OH OH

OH

O

O

O

O

HO

HOhydroquinonephenol

[O] [O]

n-quinone

[O]

quinhydrone

Peculiarities of isolation. The distillate is alkalified by sodium

hydrocarbonate solution to pH 8–9 for removing of weak acids (acetic, salicylic,

lactic), which can react with FeCl3. Then phenol is extracted by diethyl ether from

the alkaline distillate obtained, the ether extract is evaporated to dryness and the

remainder is examined for the presence of phenol.

In the directed analysis for phenol the biological sample is acidified with

diluted solution of acetic acid, the distillate obtained is alkalified by sodium carbo-

nate and phenol is extracted by ether.

Analysis for phenol is started with the reaction of tribromophenol forma-

tion; highly sensitive (endogeneous phenol is detected by this reaction), non-

specific (aniline, salicylic acid, aromatic amines, etc., give this reaction too):

OH OH

BrBr

Br

O

BrBr

Br Br

3Br2

a white precipitate

Br2

The reaction with ferric (III) chloride specifies the presence of phenolic

hydroxyl in the molecule of the detected substance:

OH OFeCl

2

OOFe

OFeCl3

-HCl

2C6H5OH

-2HCl

a blue colour

84

The indophenol reaction is non-specific:

NH2

OH

N

H

Cl

N OHO

a blue colour

HOCl

-H2O HOCl

Liberman`s reaction, the reaction of indophenol formation, is non-specific:

OH

OH

NOOH

NO OH

NO OH

HNO2

blue red green colour

The reaction with Millon`s reagent (mixture of mercury (I) and mercury (II)

nitrates, which contains nitric acid); the reaction is non-specific:

OH OH

NO

O

N OH

O

N O

O

NO

HNO2

Hg2+

Hg

o-quinonemonoxime

a red colour ё

Isopentanol

CH3

CH3

OH

The lethal dose is 10–15 g.

Isopentanol (C5H11OH) is the main component of fusel oils. They also con-

tain butanol, pentanol, aldehydes, ethers, ketones. Isopentanol is used in medicine

and manufacture of smokeless powder.

85

Toxic action. Isopentanol is a narcotic; it is more poisonous than ethanol,

because it is absorbed, metabolizes and is excreted slowly causing severe and long

intoxication accompanied by neurological and psychic disorders.

Metabolism. The part of the dose administrated is oxidized to isovaleric

aldehyde and isovaleric acid and the rest part is excreted unchanged by the kidneys

and lungs.

Peculiarities of isolation. Isopentanol is extracted from the distillate by

ether (or chloroform), then ether is evaporated to dryness and the residue is

examined for the presence of isopetanol.

When examining wine 40–50 ml of the sample is diluted with water to 10–

15 % ethanol concentration (ethanol is not extracted by ether or chloroform from

such diluted solutions) and then isopentanol is extracted by 15 ml of chloroform.

The analysis for isopentanol is started with the reaction of isoamylacetate

formation, which is specific:

2CH3COONa + H2SO4 2CH3COOH + Na2SO4

C5H11OH + CH3COOH H2SO4 conc.

CH3COOC5H11 + H2O

The pear essence odour appears, the sensitivity of the reaction can be

increased when applying the isoamylacetate layer on the water surface.

The reaction of isopentanol oxidation is specific:

CH3

CH3

H

O

CH3

CH3

OH

OC5H11OH

t, H2SO4 conc.

KMnO4 [O]

isovaleric aldehyde isovaleric asid (the odour of “rotten cheese”)

Reactions with aromatic aldehydes (reaction of Komarovsky) are sensitive,

non-specific. For example, isopentanol with p-(N,N-dimethylamino)benzaldehyde

gives a blue colour; with salicylic aldehyde a pink colour appears.

Higher alcohols give these reactions, methanol and ethanol do not give them.

86

Acetic acid

Forensic toxicological examination for acetic acid is carried out when assu-

ming this poison in the sample (directed analysis).

The lethal dose is 200 ml of vinegar, 10–20 g of the acetic essence.

The toxic action of acetic acid is burn of the gullet, uremia, hemolytical

anemia.

Metabolism:

CH3

C

O

H

CH3COOH

[O]

CH3COOH

[H] [H]C2H5OH

CO2 + H2O

Peculiarities of isolation. The biological material is acidified by sulphuric or

phosphate acids. Acetic acid is volatile, therefore, it is collected in the receiver

containing sodium hydroxide solution.

The analysis for acetic acid is started with the reaction with ferric (III)

chloride; it is sensitive and non-specific, – preliminary:

8CH3COO- + 3Fe3+ + 2H2O [Fe3(OH)2(CH3COO)6]+ + 2CH3COOH

a red colour

The reaction of ethyl acetate formation is sensitive and specific – confir-

mative:

CH3COOH + C2H5OHH2SO4 conc.

CH3COOC2H5 + H2O

In the presence of acetic acid in the distillate examined the odour of “fresh

apples” appears.

The reaction of indigo formation:

CH3

O

CH3

2CH3COOH + CaO (CH3COO)2Ca + H2O

to

(CH3COO)2Ca + + CaCO3 H2O

Then the acetone formed reacts with o-nitrobenzaldehyde forming indigo in

the alkaline medium:

87

CH3

O

CH3

H

O

NO2

OH

NO2

O

CH3

NH

NH

O

O

a blue colour

Indigo is extracted with chloroform. This reaction is non-specific, it can be

used in combination with other reactions when confirming acetic acid in the

distillate.

Ethylene glycol

CH2

CH2

OHOH

The lethal dose is about 100 ml.

Ethylene glycol is dihydric alcohol, it is colourless oily liquid, mixes with

water in any ratios.

Ethylene glycol is used as a principal component of many automobile anti-

freeze solutions; as a solvent in chemical industry; for carrying out of organic

synthesis.

Ethylene glycol administrates through the mouth, skin; its administration via

breathing is limited because of little volatility.

According to the target organ toxicity ethylene glycol is a vascular and

protoplasmatic poison, which causes degeneration of vessels and damage of the

kidneys due to transformation in them to oxalates.

Metabolism is difficult, includes the multistage oxidization:

CH2

CH2

OHOH

CH2

C

OH

O

HCH

O

OHCH

2C

OH

O

OH

CH2

CH2

OHOH

COOHHOOC

[O] [O] [O]

+ CO2

[O]

glycolic aldehyde glycolic acid

oxalic acid

88

Ethylene glycol and its metabolites are excreted from an organism with the

urine.

Peculiarities of isolation from the biological material by steam distillation

when carrying out the directed examination for ethylene glycol is the use of

benzene, as a selective carrier of ethylene glycol. The volume of the distillate

collected is not less than 500 ml. The distillate divides into two layers – upper one,

the benzene and lower one, the mixture of ethylene glycol with water.

The analysis of distillate for ethylene glycol is started with the reaction of

oxidation of ethylene glycol to formaldehyde, sensitive and non-specific –

preliminary:

CH2

CH2

OHOHCH

O

H

+ KIO4 + H+ 2 + HIO3 + H2O + K+

Then the formaldehyde obtained is detected by the reaction with fuchsin-

sulphurious acid (a lilac colour appears).

The reaction with copper sulphate is non-specific:

CH2

CH2

OH

OHO

CH2

Cu

CH2

HO

CH2

O

CH2

H

O2

Cu(OH)2

a blue colour

The reaction of ethylene glycol oxidation to oxalic acid is specific; it is

confirmative:

CH2

CH2

OH

OH

COOH

COOH

O

O

Ca

O

O

CaCl2[O]

The characteristic crystals of calcium oxalate appears.

Tetraethyl lead (TEL)

Tetraethyl lead – (C2H5)4Pb is a transparent colourless liquid, poorly soluble

in water, readily soluble in petrol ether, chloroform, ethanol, etc. It is easily

decomposed with formation of inorganic lead salts under the action of heating or

sunrays.

89

TEL is used to improve of the octane rating of automobile and aviation ga-

soline.

Toxic action. Acute exposure to tetraethyl lead causes symptoms of the

central nervous system toxicity, including insomnia, anxiety, lassitude, tremor,

hallucinations, psychotic behavior and convulsions. Absorption of only 1 g of the

chemical may be sufficient to cause death within 3–30 days due to slow

degradation of the compound to triethyl lead. Subjects with chronic exposure may

demonstrate symptoms of inorganic lead poisoning, as well as the central nervous

system effects of the organic lead intoxication.

Metabolism. Tetraethyl lead is slowly metabolized to triethyl lead, which is

attributed the toxic effects of the parent compound. Triethyl lead undergoes further

dealkylation. It is believed that eventually the alkyl compounds are converted to

inorganic lead and largely excreted in the urine.

Peculiarities of the isolation when carrying out the directed toxicological

examination for TEL. On steam distillation tetraethyl lead is collected in the rece-

iver containing ethanol solution of iodine:

(C2H5)4Pb + I2 = PbI2 (s)+ 2C4H10

Then the distillate is evaporated to dryness, the residue is dissolved in nitric

acid:

PbI2 + 2HNO3 = Pb(NO3)2+ 2HI

The solution obtained is evaporated again and the dry remainder is dissolved

in water and examined for the presence of Pb2+ (reactions of lead dithizonate

formation and formation of colour precipitates of PbSO4; PbI2; PbCrO4 ; PbS ).

When examining food products, clothes, vegetables tetraethyl lead is

extracted from the sample by organic solvents.

4. Quantitative analysis of volatile poisons

The majority of volatile poisons need quantitative determination in a sample

when forensic toxicological examination takes place. Some poisons can be admi-

nistered by a body as medicinal substances (chloroform, chloral-hydrate), others

90

can be endogenous (ethanol, acetone, phenol, acetic acid, traces of hydrocyanic

acid). In acute intoxications laboratory support is needed to perform rapid poison

levels (for monitoring treatment).

For quantitative determination of volatile poisons the following methods can

be used: gas-liquid chromatography, gravimetry, titrimetry, photocolorimetry.

Gravimetric method can be used for determination of the following substan-

ces – hydrocyanic acid (cyanides) according to silver cyanide, phenol according to

tribromophenol, halocarbons according to silver chloride.

Titrimetry can be used for determination of hydrocyanic acid and halocar-

bons (argentometry), phenol (bromatometry), formaldehyde and acetone (iodomet-

ry), acetic acid (alkalimetry).

Photocolorimetry can be used for determination of hydrocyanic acid by the

reaction of polymethine dye formation, formaldehyde and ethylene glycol (after

transforming them to formaldehyde) by the reaction with fuchsin-sulphurious acid,

ethanol by the reaction with potassium dichromate in the sulphuric acid medium,

halocarbons (chloroform, chloral hydrate, tetrachloromethane) by the Fujivar`s

reaction. Photocolorimetry is more sensitive than gravimetry and titrimetry, but it

is less sensitive than gas-liquid chromatography.

Gas chromatography is the only way of achieving the good general scree-

ning method of a wide variety of industrial solvents since all other methods require

the application of separate colour tests or chemical reactions for each volatile

substance.

91

Topic 7: GAS CHROMATOGRAPHY DETECTION AND QUANTITATIVE

DETERMINATION OF VOLATILE POISONS

Plan of the lecture

1. General principles of the gas chromatography method.

2. The arrangement of a gas chromatograph.

3. Identification and quantitative determination of volatile poisons by the gas-

liquid chromatography method.

1. General principles of the Gas Chromatography method

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) one.

Stationary phase can be an active solid (a 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 a stationary phase.

The method is called Gas-Liquid chromatography when using a gas as a

mobile phase and a liquid coated on a support material as an immobile phase.

Advantages of the Gas-Luquid Chromatography method are:

high quality of the substance’s separation;

possibility of performing both qualitative and quantitative analysis

simultaneously;

possibility of analysis of little amounts of poisons;

high speed of analysis (it takes minutes or seconds);

possibility of the complete automation of the analysis.

92

2. The arrangement of a gas chromatograph

The scheme of a gas chromatograph (Fig. 4)

1. Bulb with a carrier gas

2. Regulator of the carrier gas flow (digital flow controler)

3. Injector (the place of the sample injection)

4. Column

5. Detector

6. Amplifier

7. Recorder

1 3

4

5

6

72

Fig. 4. The scheme of a gas chromatograph

In order to perform accurate and reproducible gas chromatography, it is

necessary to maintain a constant carrier gas flow.

A carrier gas can be H2, N2, He2. The carrier gas should be:

Inert;

Accessible;

Clean;

Suitable for the type of the detector.

Column is the part of the chromatograph where substance separation takes

place. Columns, which are used in GLC, can be various in accordance with:

the form – straight one (the most effective), spiral and U-form

columns;

the length – 0.5– 20 m;

the diameter – 2–6 mm,

the material – glass, stainless steel columns.

93

Support material, which fills in a column (chromosorb, silica gel,

aluminium oxide, activated charcoal) should be:

inert;

with a large surface area;

with the uniform particle size.

Stationary phase should be the same with its chemical properties as the

separated substances. There is no faultless method of the stationary phase choice; it

is found during the analysis experimentally.

Example. Hydrocarbons are separated better when using hydrocarbons as a

stationary phase (vaseline oil); polar compound (e.g. alcohols) are separated better

when using polar stationary phases (polyglycerols).

The columns with a little amount of a stationary liquid phase (2–10 %)

provide high speed of analysis and are most frequently used now.

Example. Substances with a low volatility are separated better when using a

little amount of a stationary liquid phase – 4 % and less; for the compounds with a

high volatility it is necessary to use 20–40 % of the liquid phase because of their

little solubility in the stationary phase.

Detector detects substances exiting from a column. Generally detectors

measure some physicochemical property of exiting substances and convert the

signal obtained to electric one. Electric signal is reinforced by the amplifier and

written by the recorder.

Types of the detectors:

Thermal Conductivity Detector (TCD);

Ionization Detectors:

o Flame Ionization Detector (FID),

o Alkali Flame Ionization Detector (AFID)

o Electron Capture Detector (ECD)

o Flame Photometric Detector (FPD)

o Mass Spectrometer as a detector

94

TCD and FID are the general detectors. AFID, ECD, FPD are selective

detectors.

Thermal Conductivity Detector consists of a pair of heated filaments, each

as the arm of a Wheatstone bridge, over which the column effluent and a reference

gas steam flow. When a compound reaches the filament, the thermal conductivity

is changed and the resulting change in temperature of wire causes a change in

resistance. This change unbalances the bridge to provide the signal. This detector is

used for the analysis of permanent gases and in cases where the amount of sample

is not limited. The disadvantages include a rather low sensitivity (2×10–6 – 5×10–6

g) and a critical dependence on temperature stability and gas flow.

Flame Ionization Detector (FID) is probably the most widely used of all

detector since it responds to nearly all classes of compounds.

The effluent from the column is mixed with hydrogen and the mixture burnt

at a small jet in a flow of air. Above the jet is the collector electrode (a wire or ring

around the jet) and a polarizing potential of about volts is applied between the jet

and the electrode. When a component elutes from the column it is burnt in the

flame and the resulting ions carry a current between the electrodes, which provides

the signal.

Any of the usual carrier gases can be used and minor changes in gas flow

are without effect. Sensitivity is very high (10–8 – 10–10 g). The insensitivity of the

detector to water is the most useful feature, which allows the aqueous solution to

be used.

Selective detectors. Alkali Flame Ionization Detector marked by

introduction of alkali metal vapours into the flame of a flame ionization detector. It

confers an enchanced response to compounds containing phosphorus and nitrogen.

This detector is particulary useful for drug analysis since most medicines contain

nitrogen, while the solvent and the bulk of the co-extracted material from a

biological sample do not. Also this detector is especially useful for the detection of

pesticides containing phosphorus.

95

Electron Capture Detector is a selective detector, which is very highly

sensitive to compounds with a high affinity for electrons. So, compounds

containing a halogen, nitro group, or carbonyl group are detected at very low

concentrations. The selectivity of the ECD with its extreme sensitivity makes it

very useful for compounds like benzodiazepines or halogenated pesticides. For

some compounds the sensitivity of this detector may exceed that of the mass

spectrometer.

Flame Photometric Detector is selective to compounds containing

phosphorus and sulphur.

Selective detectors are characterized by high sensitivity (about 10–10 g and

less).

The guadrupole or dodecapole mass spectrometer is an ideal detector as the

chromatograph is an almost ideal inlet device for the mass spectrometer. Its

sensitivity is 10–11 –10–12g.

3. Identification and quantitative determination of volatile poisons by

the gas-luguid chromatography method

The vapour phase method is widely 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

Conditions of GLC analysis of monobasic alcohols are:

Stainless steel column, 1m length with 6mm internal diameter;

Carrier gas – nitrogen, 50–60 ml/min;

Support material – spherochrom;

Stationary liquid phase – 12 % triethylene glycol (M=1000–1500);

96

Column and detector temperature – 75ºC;

Injector temperature – 20–25ºC;

Detector – TCD.

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 (Fig. 5).

t, min

Fig. 5. The schematic chromatogram of separation of С1 – С5 alcohols mixture:

1 – methyl nitrite ; 2 – ethyl nitrite; 3 – propyl nitrite; 4 – butyl nitrite;

5 – isoamyl nitrite.

Identification is performed by comparison of the retention time of the

substance analysed and the retention time of the authentic substance.

Quantitati ve analysis. The peak height or peak area can be measured

either manually or with electronic devices. Peak height measurements have the

1

2

3

4 5

97

advantage of simplicity but are sensitive to changes in the peak shape; peak area

measurements should always be used where peaks are broad and tailing.

For the given system, a calibration curve (or equation) must be created for

each compound to be analysed because the detector response to each will be

different. This curve (or equation) of the peak height (or area) against the analysed

substance concentration can then be used to quantify the unknown sample. Such

external calibration requires careful control of injection volumes and the valve

injection should be used. However, external calibration is still susceptible to errors

arising from fluctuations in the column perfomance, and the internal standard

technique gives better precision. This involves the addition of a fixed amount of a

substance (internal standard) to the sample before injection. Quantification is

carried out using the peak height (or area) ratios of the substance analysed in

relation to the internal standard. In this case, calibration curves (or equations) are

prepared by injecting solutions containing the known amounts of substance

analysed together with the same fixed quantity of the internal standard. When

ethanol is quantitatively analysed, propanol or isopropanol are used as internal

standards.

98

Topic 8: THE GROUP OF SUBSTANCES ISOLATED FROM THE

BIOLOGICAL MATERIAL BY THE ORGANIC SOLVENT

EXTRACTION (PESTICIDES)

Plan of the lecture

1. General description of pesticides. Importance of the pesticide application

for the national economy. Negative pesticide influence on the environment

and human health.

2. Classification of pesticides.

3. Physical and chemical properties, reqularities of behaviour in the body,

toxicity of pesticides: chlorinated pesticides and organophosphorus

compounds.

4. Chemical toxicological examination of biological samples for the presence

of chlorinated pesticides and organophosphorus compounds.

5. Chemical toxicological examination of biological samples for the presence

of carbamic acid derivatives.

6. Chemical toxicological examination of biological samples for pyrethrins.

1. General description of pesticides. Importance of the pesticide

application for the national economy. Negative pesticide influence on the envi-

ronment and human health

The word "pesticide" originates with two Latin words: pestis – contagion,

plague; cido – to kill. So “pesticide” is a general definition, which covers a wide

variety of substances used for destroing undesirable life forms.

Application of pesticides in the agriculture allows enhancing of productivity

of the plants (2–3 centners of corns or about 5 centners of rice are stored additio-

nally).

Pesticide application makes a great contribution in the liquidation of such

diseases as malaria, typhoid, etc.

99

Besides positive aspects application of pesticides has negative sides – they are

toxic for a human being and animals, useful insects and plants; some of them

accumulate causing the environmental pollution.

In 1939 the Swiss biochemist P. Müller discovered the insecticide properties

of dichlorodiphenyl trichloromethy lmethane (DDT) that had been first synthesized

in 1874 by Zeidler. DDT has been employed as a contact insecticide since 1940.

DDT has come under severe restrictions in some countries since 1970 due to its

persistence and accumulation in the food chain.

In the world the cases of acute and chromic intoxications by organophos-

phorus pesticides occur. The cases of the mortal poisoning by chlorophos,

carbophos, trichlorometaphos, phosphamide and many other pesticides take place.

2. Classification of pesticides

There are various classifications of pesticides – in accordance with their

application; the way of their penetration into a body; the chemical structure; their

toxicity rating, the persistence in the environment.

By their application there are the following groups of pesticides:

insecticides, fungicides, zoocides (rodenticides), acaricides, nematocides, bac-

tericides, herbicides, molluscicides, plant growth regulators, defoliants,

repellentes, attractants.

By the way of their penetration into an organism there are such groups of

the insecticides: contact, respiratory (fumigants), intestinal and systemic.

Classes of pesticides by their toxicity rating are given in Table 4.

By the pesticide persistence they are: very persistent (the period of

decomposition is more than 2 years); persistent (the period of decomposition is 6

months – 1 year); moderately persistent (1 – 6 months); slightly persistent (1

month).

100

Table 4

Classification of pesticides by their toxicity rating

No Toxicity rating Estimated lethal dose

(for a 70 kg person) DL50

1 Non-toxic > 1000 g

2 Slightly toxic 500 to 1000 g 1000 mg/kg

3 Moderately toxic 28 to 500 g 200 – 1000

mg/kg

4 Very toxic 4 to 28 g 50 – 200 ml/kg

5 Extremely toxic 500 mg to 4 g <50 ml/kg

6 Super toxic <500 mg (<10 drops in a

sample) –

By their chemical structure there are inorganic and organic pesticides.

Organic pesticides are divided into such groups as chlorinated

hydrocarbons, organophosphorus compounds; carbaminates, organic metal

pesticides (e. g., organic mercurial compounds), pesticides obtained on the basis

of natural compounds (e.g., pyrethrins), etc.

The classification of the pesticides by their chemical structure is used in the

chemical toxicological analysis.

3. Physical and chemical properties, regularities of behaviour in the bo-

dy, toxicity of pesticides: chlorinated pesticides and organophosphorus com-

pounds

General description of chlorinated pesticides

Chemical structure. Chlorinated pesticides include chlorinated hydrocar-

bons (used as insecticides) and chlorinated phenoxy acids (used as herbicides).

Chlorinated hydrocarbons are:

a) DDT group (dicophane and its analogues, e.g. dicofol and methoxychlor):

101

CCl3

Cl Cl

DDT (dichlorodiphenyl trichloromethyl methane)

b) Hexachlorocyclohexane (benzene hexachloride, BHC) isomers group (hexa-

chlorane, is the mixture of 8 isomers of BHC; Lindane, is the γ–isomer of BHC):

Cl

Cl

Cl

Cl

Cl

Cl

c) Bridged polycyclic chlorinated compound group (heptachlor, chlordane, chlor-

dene, dieldrin, aldrin, endrin):

Cl

Cl

Cl

Cl Cl

CCl2

heptachlor

d) Chlorinated Phenoxy Acids:

O

R1

Cl

R2

[CH2]nCOOH

,

where n=1, 2, 3; R1 and R2: –CH3, –Cl, or –H

Physical and chemical properties. Chlorinated pesticides are solid, inso-

luble or slightly soluble in water, readily soluble in organic solvents and lipids, vo-

latile, very persistent – they are not destroyed when boiling even in soda solutions.

It has been found that 29 % of the initial amount of chlorinated pesticides

are saved in soil for 7 years after application, in another case it has been found 80

102

% of the initial amount. Such pesticides, as DDT, aldrin, dildrin, endrin have been

banned to use in some countries due to its persistence in the environment and its

potential for chronic toxicity.

Regularities of behaviour in the body. Chlorinated pesticides are readily

absorbed following inhalation, ingestion or dermal contact. Poisoning symptoms

are vomiting and convulsions, the principal features; the following additional

symptoms may be used as a guide: dizziness, headache, muscular weakness,

tremors (chlorinated hydrocarbons); burning sensation, low blood pressure (chlo-

rinated phenoxyacetic acids; convulsions are not the main feature for these sub-

stances).

A case of fatal poisoning of a worker following dermal exposure to lindane

was described.

The contamination of foodstuffs by endrin has resulted in several mass poi-

sonings, with multiple fatalities; the onset of symptoms, including vomiting,

convulsions and unconsciousness, ranged from 0.5–10 hours after ingestion of the

poison. Death often occurs within 1–2 hours after ingestion of 6 g of endrin.

Distribution in the body. Chlorinated pesticides accumulate in a human

organism, forming body depot for pesticides storage, mainly in the adipouse tissue.

Metabolism. Chlorinated pesticides are subjected to different metabolic

processes, for example, heptachlor is transformed to heptachlor epoxide, which is

more toxic, than the parent compound. The major detoxification pathway of DDT

is via dechlorination to DDD (dichlorodiphenyl dichloromethyl methane), an

active insecticide, which readily degradedes to DDA (dichlorodiphenyl acetic

acid), a water soluble, rapidly excreted decontamination product. DDT is converted

to a slight extent to the much less toxic DDE (dichlorodiphenyl dichloroethylene)

by dehydrochlorination. DDE apparently does not undergo further

biotransformation, but is stored for an indefinite period of time in adipose tissues.

103

The mechanism of some metabolic processes are:

Cl

Cl

Cl

Cl Cl

CCl2

Cl

Cl

Cl

Cl Cl

CCl2

O[O]

heptachlor epoxide of heptachlor

CCl3

Cl Cl Cl Cl

COOHDDT

[O]

DDA

Excretion. Chlorinated pesticides are excreted with the urine, in the feces

and the pectoral milk.

The toxicity mechanism has not been definitely found. It is believed that the

enzymic system depression is the initial factor of the toxic action. Cellular memb-

ranes are damaged because of lipid peroxidation causing disorder of metabolic pro-

cesses in the body.

Lethal doses of chlorinated pesticides are from 5 to 60 g.

Genaral description of organophosphorus pesticides

The organophosphorus insecticides are tending to replace the

organochlorine compounds.

Chemical structure. Organophosphorus compounds may be conveniently

divided into four main structural groups:

OP

S

O S

X

R

R OP

O

O S

X

R

R

OP

S

O O

X

R

R OP

O

O O

X

R

R

phosphorodithionates phosphorothionates

phosphorothiolates phosphates

(R = alkyl; X includes a wide variety of chemical structures)

104

Examples of some widely used organophosphorus pesticides are given in

Table 5.

Table 5

Organophosphorus pesticides

Compound Chemical formula Chemical group

Сhlorophos

CH

3O

P

CH3O O

OH

CCl3

phosphonate

Paraoxan C2H

5O

P

O

C2H

5O O

NO2

phosphate

Parathion C2H

5O

P

O

C2H

5O S

NO2

phosphorothionate

Carbophos CH3O

P

S

CH3O S

COOC2H

5

COOC2H

5

phosphorodithionate

Octamethyl N(CH3)

2P

O

N(CH3)

2O

(CH3)

2N

P

(CH3)

2N O

pirophosphate

Physical and chemical properties. The majority of organophosphorus

compounds are yellow oily liquids, in pure form are supplied as dusts, wettable

powders aerosols in the concentration of up to 50 %, readily soluble in organic

solvents, poorly soluble in water, volatile. Unrefined preparations have a strong

garlic smell. Some substances can be crystalline and dissolve in water, for

example, chlorophos.

Under the action of the organism’s enzymes organophosphorus compounds

can be transformed to more toxic products (e.g., when oxidizing):

105

C2H

5O

P

O

C2H

5O S

NO2 C

2H

5O

P

O

C2H

5O O

NO2

[O]

parathion paraoxan

Under severe conditions oxidation can occur with formation of inorganic

compounds.

Organophosphorus compounds easily hydrolyze especially in the alkaline

medium. For example, when heating with potassium hydroxide solution

trichlorometaphos is transformed in the following way:

CH3O

P

O

CH3O S

Cl

Cl

Cl

CH3O

P

OH

CH3O S

Cl

Cl

Cl

KO

Cl

Cl

Cl

OH

KOH+

KOH

H2SO4

+ K2SO4

CH3OH + K2S + K3PO4

H2SO4

CH3OH + H2S + H3PO4

Regularities of behaviour in the body. The compounds are well absorbed

after inhalation, dermal contact or ingestion.

Organophosphorus compounds are distributed in the liver, kidneys, brain,

lungs, but mostly in the liver and kidneys. They do not accumulate in the

organism.

Metabolism. Organophosphorus compounds are rapidly hydrolyzed by

plazma and tissue esterases. Non-toxic products appear in hydrolysis, more toxic

substances can appear in oxidization (for example, parathion oxidization).

Excretion. The metabolic products are largely excreted in the urine, partly in

feces, with pectoral milk.

106

Toxicty. Organophosphorus compounds are the cholinesterase inhibitors.

Cholinesterase is the catalyst of acethylholine hydrolysis leading to choline and

acetic acid formation:

[CH3COOCH2CH2N(CH3)3]+OH-

cholinesterase

H2OCH3COOH + [HOCH2CH2N(CH3)3]

+OH-

The organophosphorus pesticide poisoning is accompanied by accumulation

of acetylcholine, the transmitter of nervous impulses. The symptoms include

respiratory difficulty, excessive salivation, miosis, nausea, vomiting, muscle

weakness and paralysis. Atropine and 2-PAM (pralidoxime) are administered as

specific antidotes.

There are 4 degrees of organophosphorus compound poisoning: slight (when

50 % of cholinesterase is inhibited), moderate (when 60–70 % of cholinesterase is

inhibited), severe (when 80–90 % of cholinesterase is inhibited) and fatal (when

95–99 % of cholinesterase is inhibited).

Organophosphorus compounds have the functional type of accumulation –

repeated exposures lead to more severe intoxications than previous ones.

The fatal dose of parathion for an adult by ingestion or inhalation is 10–30

mg.

4. Chemical toxicological examination of biological samples for the

presence of chlorinated pesticides and organophosphorus compounds

Direction of the analysis. When indirected the chemical toxicological

analysis takes place, the examination for the presence of organophosphorus

compounds is required. The analysis for chlorinated pesticides is performed in

special cases.

Samples for the chemical toxicological examination for organophospho-

rus compounds:

Stomach with its content, liver with the gall bladder, kidney, small and large

intestine with the content, blood, urine.

Lungs and brain are examined additionally when inhaling the poison.

107

Samples for the chemical toxicological examination for chlorinated pesti-

cides:

Stomach with its content, liver, kidney, brain, adipose tissue.

Terms of the analysis. The analysis should be started in the day of the sam-

ples received. The samples can be stored in the refrigerator for 3 days, the extracts

containing organophosphorus compounds isolated can be stored for 5 days and

more, the extracts containing chlorinated pesticides can be stored for 10 days at the

temperature of 2–4ºC.

The samples can be preserved by ethanol.

Besides human and animal organs, biological fluids such samples as prepa-

rations of pesticides, food stuffs, water, air, feed-stuff can be examined for pesti-

cides.

General stages of the chemical toxicological examination for pesticides

include:

the сhoiсe and preparation of the samples;

isolation of the pesticides;

purification of the extracts obtained from admixtures;

identification and quantitative determination of pesticides.

Analysis methods should be highly sensitive and specific, of wide applicabi-

lity, simple, and rapid, with results, which can be easily interpreted. Unfortunately,

no technique combines all these attributes.

Methods of pesticide isolation. The general isolation method of pesticides

from the plant and animal samples is organic solvent extraction (hexane, ether,

chloroform). This method is suitable for all chemical groups of pesticides. Volatile

chlorinated pesticides, for example hexachlorocyclohexane, can be also isolated by

steam distillation.

When organophosphorus compounds are extracted from biological samples

the preliminary precipitation of albumins for destruction of protein-phospholipid

bonds and releasing of fat can be useful, and then the extraction of a pesticide from

the fat by an organic solvent is performed. For this purpose the sample is homoge-

108

nized with equal quantity of water, heated with saturated calcium chloride solution

and allowed to stand overnight and filtered. Then the homogenate filtered is

extracted with the equal volume of redistilled ether (or another organic solvent).

Methods of extract purification. For purification of pesticide extracts the

following methods are most commonly used:

Extraction;

Chromatography;

Combination of extraction and chromatography;

Freezing-out of fat.

Other methods (steam distillation, vacuum sublimation, dialysis) are used

rarely.

Jons and Ridic studied extraction of pesticides by acetonitrile and hexane

mixture and concluded that this solvent system was suitable for purification of

pesticides from concomitant materials. Besides this system for purification of

pesticides such pairs of solvents can be used: petrolyne ether – acetonitrile, hexa-

ne – dimethylformamide.

Chromatography methods of purification include adsorption column chro-

matography, paper chromatography, chromatography in a thin layer to sorbent

(TLC). TLC is the most widely applied. The mobile phase hexane-acetone in

various ratios is recommended for purification of extracts, which contain

organophosphorus compounds. Freezing-out of fats is based on insolubility of fats

and waxes in cold acetone. Freezing-out admixtures are separated by filtration, and

pesticides remain in the acetone filtrate.

Methods of pesticide identification

For the analysis of pesticide extracts the following research methods are

used:

chemical;

biochemical;

thin-layer chromatography;

gas-liquid chromatography.

109

Such methods as UV-and IR- spectroscopy are used rarely.

Identification of pesticides by the chemical method. Chlorinated pesticides

are detected after chlorine (after transformation of covalence bound chlorine to

ionic state), then the reaction on chloride-ion with argentum nitrate in the nitric

acid medium is performed:

CCl3

Cl Cl Cl Cl

CCl2

DDT

C2H5OH

+ KCl + H2O

AgNO3

AgCl + HNO3

KOH

Chlorinated pesticides are detected after the reaction of chinon aci-salt

formation. At first, dechlorination and then nitration of the benzene ring with the

following addition of acetone to trinitrobenzene obtained and then potassium

hydroxide solution drops are performed:

CH3

CH3

O

Cl

Cl

Cl

Cl

Cl

Cl

NO2

NO2

O2N

N+

NO2

O2N

O

CH3

OKO

C2H5OH

KOH NaNO3

H2SO4

KOH

a violet colour

Both reactions are not specific and not too sensitive to determine the trace

amounts of chlorinated pesticides, for example, in food stuffs.

Colour Test with Nitric – Sulphyric Acid. Method. Dissolve the sample in 1

ml of ethanol, add a pellet of potassium hydroxide, and evaporate to dryness at

100ºC in the water-bath. To the residue add 0.5 ml of water and 1 ml of carbon

tetrachloride, shake, allow to separate, decant the lower carbon tetrachloride layer

110

and shake it with 1 ml of the reagent (mix 1 ml of nitric acid with 30 ml of

sulphuric acid).

Indication. A red colour in the acid layer suggests the presence of dicophane

(DDT) or its metabolite DDE. The red colour changes to orange and then to green.

Weak pink colours are given by aldrin, dieldrin, and endrin. A red colour is also

given by DDD, but the colour does not change.

The substances should be tested to ensure that they do not give colour with

sulphuric acid alone.

For organophosphorus pesticide identification phosphorus test and hydrope-

roxide test are mostly used. Phosphorus test is performed after mineralization of

pesticide with nitric and sulphyric acid mixture to phosphate-ion. Then the reac-

tion with ammonium molybdate is performed, as the result a bright yellow

solution or precipitate appears.

In the presence of the reductant phosphoromolybdic blue appears. This

reaction is highly sensitive and common for organophosphorus compounds.

H7P(Mo2O7)6 – phosphoromolybdic acid

(NH4)3PO4 · 12 MoO3 · H2O – phosphoromolybdic blue

As the result of the hydroperoxide test an azodye is formed. This reaction is

common and sensible for organophosphorus compounds:

. peroxyphosphoric acid, which

has a high oxidizing potential

NH2NH

2

NH2

NH2NN

peroxyphosphoric acid

Biochemical method

The biochemical method (inhibition of cholinesterase test) is used for

determination of organophosphorus pesticides. It is based on the measurement of

111

degree of acetylcholine hydrolysis (other esters can be used, e. g. acetylthiocholine

iodide) in comparison with the blank experiment. Cholinesterase, the enzyme, in-

duces the acetylcholine hydrolysis, as a result, some amount of acetic acid appears

and pH value of the medium becomes acidic. The change of the pH medium can be

detected with the help of indicators, for example bromothymol blue in a buffer

solution can be used. Various modifications of this test are described. For example,

Hestrin’s method of the blood examination for organophosphorus pesticides is

based on the measurement of undestroyed acetylcholine. The mechanism of the

processes which take place, in Hestrin’s method, is as follows:

CH3

NHOH

O

CH3

NH

O

O

Fe/3

[CH3COOCH2CH2N(CH3)3]+OH- + NH2OH

a red-violet complex

CH3CONHOH + [HOCH2CH2N(CH3)3]+OH- + H2O

Fe3+

Absorbance of the red-violet solution obtained is measured at λ max 520 nm.

The biochemical test is highly sensitive but non-specific for

organophosphorus pesticides. Inhibition of cholinesterase takes place when taking

carbamates, in such diseases as cirrhosis of the liver, anaemia, etc. The research of

organophosphorus compounds should be started with the biochemical test because

of its high sensitivity.

Thin-Layer Chromatography

This technique represents a cheap, simple, and quick method for screening

pesticides, but it suffers from the disadvantage that there is no single system and

the location method to cover all compounds of interest. Nevertheless, when

combined with ultraviolet spectroscopy it can provide a useful first approach to

screening for pesticides.

For separation of chlorinated pesticides various mobile phases are used;

location reagent is water – acetone solution of silver ammoniate and then exposure

112

by UV-light; grey-black spots on the plate appear. Sensitivity of the detection is

0.5–10 μg in the test analysed.

For organophosphorus pesticides the mixture of hexane – acetone (41׃) or

other ratios of these solvents is most commonly used as a mobile phase. Location

reagents are different:

1. Allow the plate to dry in air, heat at 110ºC for 2 hours, allow to cool,

spray with molybdate-antimony reagent, and then lightly over-spray

with ascorbic acid reagent; dark blue spots on a white background appe-

ar;

2. 2,6-dibromo-N-chloro-n-quinoneimine cyclohexane solution; in the

presence of sulphur-containing pesticides yellow or brown spots

appear.

Gas-liquid chromatography

This method is the most useful technique for screening pesticides since it has

wide applicability and sensitivity. Over 95 % of all pesticides may be chromato-

graphed intact or as a simple derivative. The sensitivity of the method is high using

a flame ionisation detector; when specific detectors are used, e.g. electron capture,

alkali flame ionisation, or flame photometric detectors, even lower concentrations

in body fluids may be detected.

The following GLC-system, as preferable, may be used for screening

pesticides. The column is 3% OV-17 on 80–100 mesh Gas Chrom Q, 1 m × 6 mm

in the internal diameter. The column temperature is programmed at 10º per minute

from 100ºC to 260ºC. The carrier gas is nitrogen at 50 ml/min. The detector is dual

FID/AFID detection system. The reference compound is caffeine in ethyl acetate.

The relative retention times with respect to this internal standard are deter-

mined for pesticides.

Quantitative determination of organophosphorus and chlorinated

pesticides

When positive result for the presence of a pesticide in the sample is obtained

the quantitative determination is performed. The following methods are used:

113

photocolorimetry by the phosphorus test;

biochemical method;

gas-liquid chromatography determination based on the peak height (or

area) against the pesticide concentration (the external calibration); the

internal standard method (e. g., caffeine can be used as the internal

standard) is more accurate;

titrimetry (iodimetry, argentometry) can be used for quantification of

chlorinated pesticides.

The interpretation of analytical results

1. The biochemical test for organophosphorus pesticides followed by TLC or

GLC analysis enable to determine reliably not only the chemical group of the

pesticide but identify the particular substance.

2. The negative result of the analysis does not exclude the possibility of the

organophosphorus pesticide poisoning associated with such phenomena as natural

excretion of pesticides from the body, their destruction under the action of

moisture and heat, or by detoxication agents, etc.

5. Chemical toxicological examination of biological samples for the

presence of carbamic acid derivatives

The carbamate esters fall into two general classes according to their chemi-

cal structure and biological activity. N-methylcarbamates are insecticides while N-

arylcarbamates are manly herbicides. Some commonly used N-methylcarbamates

are:

114

OCONHCH

3

O

OCONHCH

3

CH3

CH3

OCONHCH

3

O CH3

CH3

N

CH3

CH3

S

S S

N

S

CH3

CH3

carbaryl (sevin) carbofuran

propoxur thiuram D

The widest used carbamate is carbaryl.

Physicochemical properties. Carbaryl is a carbamite derivative of 1-naphtol

(1-naphtyl-N-methylcarbamate) that is used as a short-acting insecticide. Carbaryl

is a white crystalline substance (M.p. 142ºC), slightly soluble in water (less then

0.1 % at 20ºC), soluble in the most organic solvents. At the room temperature

carbaryl is stable to oxygen of the air and light. In the alkaline medium carbaryl

quickly hydrolyzes:

OCONHCH

3 OK

H2O, KOH+ CH3NH2

Carbaryl behaviour in the body. Human exposure is usually via inhalation,

although the compound is also absorbed though the skin. Carbaryl is rapidly

absorbed from the gastro-intestinal tract. In 5 minutes after administration it appe-

ars in the blood, and in 30 minutes the maximal concentration in organs occurs. In

2–3 days after the exposure carbaryl is not detected in the biological sample.

Metabolism and Excretion. Carbaryl is known to be metabolized by ring

hydroxylation, hydrolysis and conjugation. The hydrolysis pathway results in the

urinary excretion of free and conjugated 1-naphtol, which accounts for over 20 %

of an ingested dose. Another 4 % of the dose is excreted as a conjugated p-hyd-

roxycarbaryl:

115

OCONHCH

3OH

OCONHCH

3

OH

carbaryl naphthol-1

p-hydroxycarbaryl

sulphate andglucuronideconjugation

Toxicity. The inactivation of cholinesterase by carbaryl produces symptoms

of intoxication, including blurred vision, salivation, sweating, nausea, vomiting

and convulsions. The effects of carbamate insecticides in general do not persist as

long as those of organophosphates. Volunteers who ingested doses of carbaryl of

up to 0.13 mg/kg daily for 6 weeks were asymptomatic.

Ingestion of 250 mg of carbaryl has caused severe poisoning in an adult who

recovered after administration of 3 mg atropine.

Isolation from the biological material. For this purpose the extraction of the

pesticide from a biological sample with benzene is used. Then the organic solvent

is evaporated to dryness. The dry remainder is dissolved in a small volume of ethyl

alcohol and the solution obtained is used for identification and quantitative deter-

mination of carbaryl.

Methods of carbaryl identification. For the identification of carbaryl some

colour tests and chromatography methods are used.

The reaction with picric acid. With 1 % solution of picric acid carbaryl

gives dark-yellow crystals collected in bunches.

The reaction with the mixture of copper (II) chloride and sodium bromide.

The sample is alkalified by sodium hydroxide solution followed by heating in the

water bath (55ºC) for 10 min, hydrochloric acid solution is added to pH 5–6 and

then the mixture of copper (II) chloride and potassium bromide are added (while

heating). A blue-violet colour appears at the presence of carbaryl.

116

TLC method. As a location reagent diazotized sulphanylic acid is used (after

alkaline hydrolysis of pesticides). The reaction of azodye formation takes place.

Red spots appear in the presence of carbarmates.

GLC method. A wide range of carbamates can be successfully chromatogra-

phed intact on a low-loaded Carbowax 20 M column using a modified support and

a moderate (185ºC) column and injection port temperatures. At higher

temperatures N-methylcarbamates undergoing thermal decomposition to produce a

substituted phenol by elimination of methyl isocyanate:

CH3 NHCOOR CH3NCO + ROH

The N-arylcarbamates are much more thermally stable and are amenable to

GLC analysis. FID and mass spectrometer as a detector are used.

6. Chemical toxicological examination of biological samples for

pyrethrins

General description of pyrethrins. Natural and synthetic pyrethrins are

derivatives of cyclopropancarboxylic acids, the esters.

The natural pyrethrin insecticides are derivatives of keto-alcohols –

cinerolone, jasmolone, pyrethrolone and chrysanthemum or pyrethrin acids.

The basic structure of natural pyrethrin insecticides:

CH3

CH3

R 1

CH3

O

O O

R 2

CH3

The structure of some alicyclic keto-alcohols:

O

R 2

CH3

OH

R2 = CH3 cinerolone

R2 = C2H5 jasmolone

R2 = CH=CH2 pyrethrolone

Extracts of pyrethrum, the dried flowerheads of Chrysanthemum cinerariae-

folium, contain a mixture of six esters given below (Table 6).

117

Variations on these pyrethrin basic structures have led to the introduction of

the closely related synthetic pyrethrin insecticides, the structures of the important

ones being shown in Table 7.

Pyrethrin insecticides fall into three groups:

the first generation pyrethrins, which are closely related to natural py-

rethrins (for example, allethrin);

the second generation pyrethrins (for example, resmethrin);

the third generation pyrethrins, which are the esters of 2-dichloro-

ethenyl-3,3-dimethylcyclopropancarboxylic, so called permethrin

acid, 2-dibromoethenyl-3, 3-dimethylcyclopropancarboxylic and aryl-

isovaleric acids (for example, permethrin, cypermethrin).

Table 6

The structure of natural pyrethrin insecticides *

Pyrethrin R1 R2

Pyrethrin I esters

Cinerin I CH3 CH3

Jasmolin I CH3 C2H5

Pyrethrin I CH3 CH=CH2

Pyrethrin II esters

Cinerin II COOCH3 CH3

Jasmolin II COOCH3 C2H5

Pyrethrin II COOCH3 CH=CH2

* To see the basic structure of natural pyrethrin insecticides mentioned above

Physicochemical properties. Pyrethrins are solid matters, soluble in organic

solvents (acetone, chloroform, hexane, benzene, etc), insoluble in water. Only low

photochemical stability of pyrethrins (especially the esters of chrysanthemum

acid), limits their usage. Under action of light the side chain of chrysanthemum

acid is easily oxidized with formation of products which have not insecticide

activity. The pyrethrins of the third generation are more photochemical stable. The

third generation pyrethrins expose high insecticide activity, that stimulates the low

118

norms of expense of these pesticides, and as a result moderate toxicity for warm-

blooded animals.

Regularities of behaviour in the body. In an animal organism pyrethrins are

distributed in the adipose tissue and brain. From the adipose tissue pyrethrins are

excreted for 3–4 weeks and from the brain much more rapidly.

Metabolism. The main metabolites of the majority of the third generation

pyrethrins are m-phenoxybenzyl alcohol and m-phenoxybenzoic acid:

C6H

5O

COOH

C6H

5O

CH2OH

m-phenoxybenzoic acidm-phenoxybenzyl alcohol

A molecular mechanism of pyrethrin toxicity has not been studied completely.

Identification and quantitative determination of pyrethrins. Pyrethrins can

be identified with the help of TLC; a location reagent is silver nitrate solution

followed by exposure with UV-light. Pyrethrins are detected as black spots.

Pyrethrins can be determined be GLC readily, but since they contain only C,

H and O, they do not show selective response to AFID and other specific detectors.

Table 7

The structure of synthetic pyrethrin insecticides

CH3

CH3

R 1

O

OR 2

R1 R2

Allethrin CH=C(CH3)2

CH2

CH2

CH3

Resmethrin CH = C(CH3)2 O

Permethrin CH=CCl2

O

119

Cypermethrin CH=CCl2

O

CN