Basic Analytical Toxicology


Monographs - analytical and toxicological data (6.1 - 6.11)

These monographs give practical information on detecting and identifying some common poisons or groups of poisons (see Table 1). In order to simplify presentation, no original references have been given, but further details on individual entries are available in the references listed in the Bibliography. Reference is made as appropriate to specific aspects of laboratory practice (section 4) and to the poisons screening procedure (section 5.2). As noted previously, this procedure should usually be followed when there is no clinical or circumstantial evidence as to the poison(s) involved in a particular case.

6.1 Amfetamine alpha-Methylphenethylamine; C9H13N; relative molecular mass, 135

Amfetamine and its N-methyl analogue, metamfetamine, are central nervous system stimulants and are widely abused.

There is no simple qualitative test for amfetamine, but this and other similar compounds can be detected and identified by thin-layer chromatography of a basic solvent extract of urine, stomach contents or scene residues. However, the extract must be acidified by addition of 0.5 ml of methanolic hydrochloric acid (2 ml/l) to prevent loss of volatile bases at the evaporation stage, as noted in section 5.2.3.

Clinical interpretation

Oral or intravenous amfetamine overdosage may cause hyperthermia, convulsions, coma, and respiratory and/or cardiac failure, but death from acute amfetamine poisoning is comparatively rare. Treatment is generally symptomatic and supportive. Quantitative measurements in blood are not normally required in management.

6.2 Aminophenazone

Amidopyrine, aminopyrine, 4-dimethylamino-1,5-dimethyl-2-phenyl- 4-pyrazolin-3-one; C13H17N3O; relative molecular mass, 231.

Aminophenazone is an analgesic and antipyretic which is now little used since agranulocytosis and renal tubular necrosis may occur after therapeutic dosage. Ingestion of about 10 g can cause severe acute poisoning in an adult.

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reagents

1. Aqueous sodium hydroxide (1 mol/l).

2. Aqueous silver nitrate solution (100 g/l).

3. Aqueous hydrochloric acid (5 mol/l).

4. Potassium nitrite (solid).

Method

1. Add 1 ml of sodium hydroxide solution to 5 ml of sample and add 10 ml of chloroform.

2. Extract on a mechanical shaker for 5 minutes, centrifuge and discard the upper aqueous phase.

3. Filter the chloroform extract through phase-separating filter- paper (see section 4.3.2) into a clean tube, evaporate to dryness under a stream of compressed air or nitrogen, and reconstitute the residue in 1 ml of purified water.

4. Add 0.5 ml of silver nitrate solution to 0.5 ml of the reconstituted extract.

5. Add 1 ml of hydrochloric acid and about 1 mg of solid potassium nitrite to the remaining portion of the extract.

Results

On addition of silver nitrate (step 4), a blue solution which turns black on standing indicates aminophenazone. In step 5, potassium nitrite imparts a blue-violet colour which quickly fades.

Aminophenazone can also be detected by thin-layer chromatography of a basic extract of urine, stomach contents or scene residues (section 5.2.3), and this should always be performed in addition to the test given above.

Sensitivity

Aminophenazone, 50 mg/l.

Clinical interpretation

Aminophenazone overdosage may cause hypotension, convulsions, and delirium. Treatment is symptomatic and supportive. Quantitative measurements in blood are not required in management.

6.3 Amitriptyline

3-(10,11-Dihydro-5 H-dibenzo [a,d]cyclohepten-5-ylidene)- N,N-dimethylpropylamine; C20H23N; relative molecular mass, 277

Amitriptyline is a widely used tricyclic antidepressant. It is metabolized by N-demethylation to nortriptyline, which is an antidepressant in its own right. Protriptyline is an analogue of amitriptyline.

There is no simple qualitative test for amitriptyline, but this compound and other tricyclic antidepressants can be easily detected and identified by thin-layer chromatography of a basic solvent extract of urine, stomach contents or scene residues (see section 5.2.3).

Clinical interpretation

Acute poisoning with amitriptyline and other tricyclic antidepressants may cause dilated pupils, hypotension, hypothermia, cardiac arrhythmias, depressed respiration, coma, convulsions and cardiorespiratory arrest. Urinary retention is also a feature of poisoning with these compounds, and this may delay procurement of an appropriate specimen for analysis.

Treatment is generally symptomatic and supportive. The use of antiarrhythmic agents should generally be avoided, but alkalinization using sodium bicarbonate may be employed. Quantitative measurements in blood are not normally required in management.

6.4 Aniline

Phenylamine; C6H5NH2; relative molecular mass, 93

Aniline is used mainly as an intermediate in the manufacture of dyes and other chemicals. It is metabolized to p-aminophenol and p-acetamidophenol, which are excreted in urine as sulfate and glucuronide conjugates. On hydrolysis of urine, p-aminophenol is reformed, and can be detected using the o-cresol/ammonia test. Aniline and other primary aromatic amines form diazo compounds with nitrous acid, which couple with 1-naphthylethylenediamine to form highly coloured derivatives. This reaction forms the basis of the confirmatory test described below.

Qualitative test

Applicable to urine. o-Cresol/ammonia test.

Reagents

1. Concentrated hydrochloric acid (relative density 1.18).

2. Aqueous o-cresol (10 g/l).

3. Aqueous ammonium hydroxide (4 mol/l).

Method

1. Add 0.5 ml of hydrochloric acid to 0.5 ml of sample, boil for 10 minutes and cool.

2. Add 1 ml of o-cresol solution to 0.2 ml of the hydrolysate.

3. Add 2 ml of ammonium hydroxide solution and mix for 5 seconds.

Results

A strong, royal blue colour developing rapidly indicates the presence of p-aminophenol. Metabolites of paracetamol (and thus phenacetin) and nitrobenzene also give p-aminophenol on hydrolysis and so interfere. Ethylenediamine (from aminophylline, for example; see section 6.105) gives a green colour in this test.

Sensitivity

p-Aminophenol, 10 mg/l.

Confirmatory test

Applicable to stomach contents and scene residues.

Reagents

1. Aqueous sodium nitrite solution (2 g/l, freshly prepared).

2. Aqueous hydrochloric acid (2 mol/l).

3. Aqueous ammonium sulfamate solution (10 g/l).

4. Aqueous N-(1-naphthyl)ethylenediamine dihydrochloride solution (2 g/l, freshly prepared).

Method

1. Mix 0.1 ml of sodium nitrite solution and 0.2 ml of dilute hydrochloric acid in a 5-ml test-tube.

2. Add 0.1 ml of sample, mix and allow to stand for 2 minutes.

3. Add 0.2 ml of ammonium sulfamate solution followed by 0.1 ml of N-(1-naphthyl)ethylenediamine dihydrochloride solution.

Results

A purple colour after 1 minute indicates the presence of aniline.

Sensitivity

Aniline, 10 mg/l.

Clinical interpretation

Poisoning with aniline usually results from inhalation or dermal absorption. Symptoms occur within 1-3 hours of exposure and include confusion, nausea, vomiting and diarrhoea, with convulsions, coma and hepatic and renal damage in severe cases. Haemolysis, red (wine)- coloured urine, and methaemoglobinaemia (dark chocolate-coloured blood) may also occur (section 3.2.2). Blood methaemoglobin can be measured but is unstable and the use of stored samples is unreliable. Hepatic and renal function tests are essential, however. Treatment may include intravenous methylene blue, but this is contraindicated in patients with glucose-6-phosphate dehydrogenase deficiency, since there is a high risk of inducing haemolysis.

6.5 Antimony

Trivalent and pentavalent salts of antimony (Sb) are used parenterally in the treatment of schistosomiasis and leishmaniasis. Antimony salts are also used in pigments and abrasives and for flame- proofing fabrics. As with arsenic, bismuth and mercury, antimony can be detected using the Reinsch test.

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reagents

1. Concentrated hydrochloric acid (relative density 1.18).

2. Aqueous hydrochloric acid (2 mol/l).

3. Copper foil or mesh (5 × 10 mm) or wire (2-3 cm).

4. Aqueous nitric acid (500 ml/l).

Method

1. Immediately before use, clean the foil, mesh or wire in nitric acid until the copper acquires a bright surface.

2. Rinse the copper with purified water and add 10 ml of concentrated hydrochloric acid and 20 ml of test solution in a 100-ml conical flask.

3. Heat on a boiling water-bath in a fume cupboard for 1 hour. Maintain the volume of the solution by adding dilute hydrochloric acid as necessary.

4. Cool and gently wash the copper with purified water.

Results

Staining on the copper can be interpreted as follows:

purple black - antimony

dull black - arsenic

shiny black - bismuth

silvery - mercury

Selenium and tellurium may also give dark deposits, while high concentrations of sulfur may give a speckled appearance to the copper.

An estimation of the concentration of antimony in the sample can be made by comparison of the deposit on the copper with that obtained from a solution containing a known concentration of the element.

Sensitivity

Antimony, about 2 mg/l.

Confirmatory test

Applicable to the stained (purple black) copper from the test above.

Reagents

1. Aqueous potassium cyanide solution (100 g/l). Take care when using concentrated cyanide solutions.

2. Aqueous sodium sulfite solution (50 g/l, freshly prepared).

3. Aqueous nitric acid (3 mol/l).

4. Quinine/potassium iodide reagent. Dissolve 1 g of quinine sulfate in 100 ml of purified water containing 0.5 ml of concentrated nitric acid (relative density 1.42). When the quinine has completely dissolved, add 2 g of potassium iodide.

Method

1. Place the copper in potassium cyanide solution and leave for 10 minutes.

2. Wash any undissolved stain with purified water and add 1 ml of sodium sulfite solution and 1 ml of aqueous nitric acid.

3. Agitate frequently for 5 minutes and add 1 ml of purified water and 1 ml of quinine/potassium iodide reagent.

Results

Stains due to arsenic dissolve in potassium cyanide solution, while stains due to bismuth and antimony do not. Bismuth slowly forms an orange/brown suspension with quinine/potassium iodide.

Sensitivity

Antimony, about 2 mg/l.

Clinical interpretation

Parenteral administration of antimony salts may lead to cardiotoxicity; collapse and death from anaphylactic shock may also occur. Industrial poisoning is usually due to inhalation of antimony compounds either as fumes or dusts. The symptoms of acute oral antimony poisoning resemble those of acute arsenic poisoning and include abdominal pain, vomiting and diarrhoea. The measurement of blood antimony concentrations is useful in diagnosing acute poisoning, but this requires atomic absorption spectrophotometry.

6.6 Arsenic

A number of pesticides contain arsenic (As) in the form of arsenic acid, dimethylarsenic acid, and arsenite, arsenate and methanearsonate salts. Arsenical compounds are also used in pharmaceuticals and in the manufacture of ceramics and glass. Arsine (AsH3) gas is used in certain industrial processes and may also be liberated accidentally from other arsenical products.

As with antimony, bismuth and mercury, arsenic can be detected and identified using the Reinsch test. The method described below for the quantitative assay to measure urinary arsenic concentrations is a modified Gutzeit procedure. In summary, arsine is generated by reaction of arsenic-containing compounds in the sample with nascent hydrogen. The arsine is carried in a stream of hydrogen through a lead acetate-impregnated filter (to remove sulfides), and arsenic is trapped in a bubbler by a solution of silver diethyldithiocarbamate in pyridine.

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reinsch test - see antimony monograph (section 6.5).

Results

Staining on the copper can be interpreted as follows:

purple black - antimony

dull black - arsenic

shiny black - bismuth

silvery - mercury

Selenium and tellurium may also give dark deposits, while high concentrations of sulfur may give a speckled appearance to the copper.

An estimation of the concentration of arsenic in the sample can be made by comparison of the deposit on the copper with that obtained from a solution containing a known concentration of the element.

Sensitivity

Arsenic, about 5 mg/l.

Confirmatory test

Applicable to the stained (dull black) copper from the test above.

Reagent

Aqueous potassium cyanide solution (100 g/l). Take care when using concentrated cyanide solutions.

Method

Place the copper in potassium cyanide solution and leave for 10 minutes.

Results

Arsenical stains dissolve in potassium cyanide solution while stains due to bismuth and antimony do not.

Sensitivity

Arsenic, about 5 mg/l.

Quantitative assay

Applicable to urine.

Reagents

1. Silver diethyldithiocarbamate solution (5 g/l) in pyridine.

2. Aqueous lead acetate solution (200 g/l).

3. Stannous chloride (330 g/l) in aqueous hydrochloric acid (200 ml/l).

4. Concentrated hydrochloric acid (relative density 1.18).

5. Potassium iodide (solid).

6. Granulated zinc.

Apparatus

Modified Gutzeit apparatus (Fig. 9).

Standards

Dissolve 2.4 g of arsenic trichloride in 1 litre of dilute hydrochloric acid (1 mol/l); this gives a solution containing an arsenic concentration of 1 g/l. Dilute with purified water to give solutions containing arsenic concentrations of 0.5, 2.0, 5.0 and 10.0 mg/l.

Method

1. Clean the modified Gutzeit apparatus with acetone and dry.

2. Soak a plug of glass wool in lead acetate solution and allow to dry at room temperature.

3. Insert the treated glass wool into the top (capillary) end of the guard tube.

4. Introduce 3.0 ml of silver diethyldithiocarbamate solution into the bubbler.

5. Add 2 g of potassium iodide and 50 ml of sample to the 100-ml conical flask, swirl until dissolved, and add 2 ml of stannous chloride solution and 10 ml of concentrated hydrochloric acid.

6. Mix well, add 10 g of granulated zinc and quickly position the bubbler and check the seals of all joints.

7. Allow the reaction to proceed for 45 minutes at room temperature.

8. Disconnect the bubbler and swirl gently to dissolve any complex formed on the walls and to mix the solution thoroughly.

Results

Measure the absorbance of the solution at 540 nm against a reagent blank (see section 4.5.2) and calculate the arsenic concentration using a previously prepared calibration graph. The calibration is linear for arsenic concentrations up to 10 mg/l. Germanium and antimony interfere in this assay.

Sensitivity

Arsenic, 0.5 mg/l.

Clinical interpretation

Acute ingestion of arsenical salts produces severe abdominal pain, vomiting and copious, bloody diarrhoea. Death from circulatory collapse often ensues. Inhalation of arsine produces massive haemolysis and renal failure. Treatment with chelating agents may be indicated.

6.7 Atenolol

2-[-4-(2-Hydroxy-3-isopropylaminopropoxy)phenyl] acetamide; C14H22N2O3; relative molecular mass, 266

Atenolol is a cardio-selective ß-adrenoceptor blocking agent (ß-blocker) used in the treatment of hypertension.

There is no simple qualitative test for atenolol, but it can be detected and identified by thin-layer chromatography of a basic solvent extract of urine, stomach contents or scene residues (see section 5.2.3).

Clinical interpretation

In acute overdose, atenolol may cause bronchoconstriction, hypotension and cardiac failure. Treatment is symptomatic and supportive, and may include the use of ß-agonists. Quantitative measurements in blood are not normally required in management.

6.8 Atropine

(1R,3r,5S)-Tropan-3-yl(±)-tropate, C17H23NO3; relative molecular mass 289

Atropine occurs in plants such as Atropa belladonna and Datura stramonium. It has potent anticholinergic activity and is used to reduce bronchial and salivary secretions before anaesthesia, to treat gastrointestinal spasm and to produce mydriasis in ophthalmic procedures. Atropine is also used as an antidote to poisoning with inhibitors of cholinesterase, such as some organophosphorus pesticides, carbamate pesticides and some chemical warfare agents. Atropine is very potent and doses of 10 mg or more can cause severe poisoning.

There is no simple qualitative test for atropine, but it can be detected and identified by thin-layer chromatography of a basic solvent extract of stomach contents or scene residues (see section 5.2.3). It may also be possible to detect atropine in urine using this procedure, but urinary concentrations are often very low even after overdosage.

Clinical interpretation

Acute atropine overdose may cause tachycardia, hypertension, pyrexia, delirium and hallucinations. Physostigmine is an effective antidote. Quantitative measurements in blood are of no value in management.

6.9 Barbiturates

Barbiturates are 5,5'-disubstituted derivatives of barbituric acid. In addition, the nitrogen atom at position 1 may be methylated as in methylphenobarbital, while substitution of sulfur for oxygen at position 2 gives thiobarbiturates such as thiopental.

The structure of barbituric acid is shown below:

Some commonly occurring barbiturates are listed in Table 18. Other barbiturates that may be encountered include cyclobarbital, cyclopentobarbital, heptabarbital, hexobarbital, methohexital and vinbarbital. Note that barbituric acid itself is no longer used as a drug.

Barbiturates are potent hypnotics and sedatives, but in many countries only phenobarbital and (intravenous) thiopental find wide application nowadays. Barbiturates may also be used for euthanasia in veterinary medicine, and barbital sodium is used as a laboratory chemical, especially in buffer solutions.

In acute poisoning it may be important to ascertain whether barbital or phenobarbital (so-called long-acting barbiturates), or a short- or medium-acting compound has been taken. This is because alkaline diuresis (see section 2.2.3) can enhance the excretion of barbital and phenobarbital, but not of other barbiturates.

There is no reliable simple test for these compounds and a qualitative analysis is best performed by thin-layer chromatography of a solvent extract of urine, stomach contents or scene residues (see section 5.2.3). This should also permit identification of the type of barbiturate present, if not the actual compound ingested.

The method given below will permit measurement of total barbiturate in a solvent extract of the specimen, and relies on the characteristic spectral shift shown by barbiturates on going from pH 11 to pH 2. However, ideally a double-beam spectrophotometer is required (see section 4.5). Accurate measurement of individual barbiturates normally requires gas-liquid or high-performance liquid chromatography.

Quantitative assay

Applicable to whole blood, plasma or serum (5 ml).

Reagents

1. Borate buffer, pH 8.4. Mix 22.4 g of disodium tetraborate with 76 ml of aqueous hydrochloric acid (1 mol/l) and dilute to 2 litres with purified water.

2. Aqueous hydrochloric acid (2 mol/l).

3. Concentrated sulfuric acid (relative density 1.83).

4. Concentrated ammonium hydroxide (relative density 0.88).

5. Sodium sulfate/charcoal mixture. Add 100 mg of activated charcoal to 100 g of anhydrous sodium sulfate, mix thoroughly and heat in an evaporating basin at 100°C for 8 hours. Allow to cool and store in a tightly stoppered bottle.

Standards

Solutions containing barbital at concentrations of 5, 10, 25 and 50 mg/l in blank human plasma, prepared by dilution from an aqueous solution of barbital sodium (1.12 g/l, equivalent to diethylbarbituric acid at a concentration of 1.00 g/l).

Method

1. Add 5 ml of sample, 2 ml of hydrochloric acid and 60 ml of diethyl ether (with care) to a 250-ml separating funnel.

2. Lubricate the stopper of the funnel with purified water, insert and shake gently for 2 minutes.

3. Allow to stand for 5 minutes, and then discard the lower, aqueous phase through the tap of the separating funnel.

4. Add the diethyl ether extract to 10 ml of borate buffer in a second separating funnel and mix for 1 minute.

5. Allow to stand for 5 minutes and again discard the lower, aqueous phase through the funnel tap.

6. Wash round the funnel with 5 ml of purified water, allow to stand for 5 minutes and again discard the lower, aqueous phase through the funnel tap.

7. Add about 4 g of sodium sulfate/charcoal mixture to the ether extract in the funnel, shake to disperse, and filter the extract through phase-separating filter-paper into a 150-ml conical flask.

8. Add a further 20 ml of diethyl ether to the separating funnel, shake and add to the extract in the flask through the filter funnel.

9. Evaporate the extract to dryness on a water-bath at 40°C under a stream of compressed air or nitrogen.

10. Add 5.0 ml of purified water to the dry extract in the flask, swirl gently and allow to stand for 5 minutes.

11. Filter the reconstituted extract through phase-separating filter- paper into a 12.5-cm test-tube.

12. Check the spectrophotometer zero at 240 nm using purified water in both sample and reference positions (1 × 1 × 4-cm fused silica cells, see section 4.5.2).

13. Add 4 ml of filtrate from the test-tube to a clean, dry cell, add 50 µl of concentrated ammonium hydroxide and mix using a plastic paddle. Check that the pH is about 10 (universal indicator paper).

14. Quickly measure the absorbance at 240 nm against a purified water blank (see section 4.5.2). If necessary, accurately dilute a portion of the extract with purified water to bring the reading on to the scale, and record the magnitude of the dilution. If a scanning spectrophotometer is available, scan in the region 200-450 nm.

15. Repeat the reading or scan after 5 minutes.

16. Add 0.1 ml of concentrated sulfuric acid to the cell, mix using the plastic paddle, and check that the pH is about 2 (universal indicator paper).

17. Repeat the reading (240 nm) or scan (200-450 nm).

Results

A number of compounds can interfere. Glutethimide is hydrolysed rapidly at alkaline pH values, so that the absorbance at 240 nm will markedly decrease after 5 minutes at pH 11 (step 15 above) if this compound is present. The presence of other compounds, such as methaqualone or phenazone (e.g., dichloralphenazone), can be revealed by scanning in the region 200-450 nm. Addition of 0.1 ml of aqueous sodium hydroxide (2 mol/l) to the ammoniacal extract (step 14 above) produces a further characteristic spectral change (Fig. 10) which can be useful in qualitative work.

To perform a quantitative measurement, measure the difference between absorbance at pH 10 and at pH 2, construct a calibration graph by analysis of the standard barbiturate solutions, and calculate the barbiturate concentration in the sample.

Alternatively, use the following formula:

((absorbance at pH 10) - (absorbance at pH 2)) × dilution factor (if any) × 25 = barbiturate (mg/l)

Sample volumes of less than 5 ml may be used, but there will be a corresponding loss of sensitivity unless "micro"-volume fused silica spectrophotometer cells are available.

Sensitivity

Barbiturate, 2 mg/l.

Clinical interpretation

Barbiturates are very toxic in overdose and may cause peripheral vasodilation, hypotension, shock, hypoventilation, hypothermia, coma, convulsions and acute renal failure. Death normally follows respiratory or cardiorespiratory arrest or respiratory complications.

Plasma barbiturate concentrations greater than 10 mg/l (50 mg/l barbital and phenobarbital) may be associated with serious toxicity. Repeated oral doses of activated charcoal and/or alkaline diuresis may be valuable in severe poisoning with barbital and phenobarbital. Charcoal haemoperfusion has been used to treat severe poisoning with short- and medium-acting barbiturates (see section 2.2.3).

6.10 Barium

The most important source of barium (Ba) is barium sulfate (barytes, BaSO4) which is extremely insoluble in water. More soluble salts of barium, such as barium nitrate (BaNO3) and barium chloride (BaCl2), have a number of industrial uses and are relatively toxic. Barium sulfide (BaS) has been employed as a depilatory agent.

There is no simple method for the measurement of barium in biological specimens. However, the tests described below can be used to indicate the presence of barium salts in stomach contents or other samples that contain relatively high concentrations of the element.

The confirmatory test relies on the fact that lead sulfate is relatively soluble in dilute acetic acid but is precipitated in the presence of soluble barium salts, thus effectively enhancing the sensitivity of the reaction between barium and sulfate ions.

Qualitative test

Applicable to stomach contents and scene residues.

Reagents

1. Concentrated hydrochloric acid (relative density 1.18).

2. Platinum wire.

Method

1. Dip the end of the platinum wire in the concentrated acid.

2. Dip the moistened end of the wire into the test material.

3. Place the material in the hot part of the flame of a spirit lamp or microburner.

Results

A yellow-green flame indicates the presence of barium salts. Copper and thallium salts give a green flame in this test.

If large amounts of sodium salts are present, an orange/yellow coloration will obscure everything else.

Sensitivity

Barium, 50 mg/l.

Confirmatory test

Applicable to stomach contents and scene residues.

Reagents

1. Aqueous sulfuric acid (1 mol/l).

2. Aqueous lead acetate solution (100 g/l).

3. Aqueous acetic acid (50 ml/l).

4. Ammonium acetate (solid).

Method

1. Mix 2 ml of lead acetate solution and 2 ml of dilute sulfuric acid, and add sufficient ammonium acetate to dissolve the lead sulfate precipitate.

2. Add 0.1 ml of dilute acetic acid to 1 ml of sample, add 1 ml of the lead sulfo-acetate solution (from step 1), and vortex-mix for 5 seconds.

3. Centrifuge for 2 minutes and view the tube against a black background.

Results

A white turbidity or a white precipitate indicates the presence of barium. Calcium and strontium interfere in this test.

Sensitivity

Barium, 100 mg/l.

Clinical interpretation

The ingestion of soluble barium salts may produce gastroenteritis, ventricular fibrillation and muscular paralysis. Life-threatening hypokalaemia is an important feature of severe barium poisoning (see section 3.1.2).

6.11 Benzodiazepines

Most of these compounds have the general structure shown below:

Some common benzodiazepines are listed in Table 19. Alprazolam, camazepam, clorazepate, flunitrazepam, ketazolam, loprazolam, lormetazepam, medazepam, midazolam, prazepam and triazolam are among the 60 or so members of this group.

Benzodiazepines are used as tranquillizers, and clobazam, clonazepam, and diazepam are also used as anticonvulsants. Temazepam especially has been abused, often together with other drugs. Most benzodiazepines are extensively metabolized and many members of this group are in fact metabolites of other compounds. Thus, diazepam gives nordazepam, oxazepam (3-hydroxynordazepam), and temazepam (3-hydroxydiazepam), which are excreted in urine as glucuronide or sulfate conjugates.

There is no reliable colour test for these compounds. However, on hydrolysis most benzodiazepines and their conjugates give rise to aminobenzophenones which can be extracted and analysed by thin-layer chromatography. Two different spray reagents are used to increase the discriminating power of the method, p-dimethylaminocinnamaldehyde and nitrous acid/ N-(1-naphthyl)ethylenediamine (the Bratton-Marshall reaction).

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reagents

1. Concentrated hydrochloric acid (relative density 1.18).

2. Petroleum ether (40-60°C boiling fraction).

3. Silica gel thin-layer chromatography plate (10 × 20 cm; 20 µm average particle size; see section 4.4.1).

4. Aqueous hydrochloric acid (1 mol/l).

5. Toluene:glacial acetic acid (97:3).

6. Aqueous p-dimethylaminocinnamaldehyde solution (5 g/l).

7. Aqueous trichloroacetic acid (500 g/l).

8. Aqueous sulfuric acid (500 ml/l).

9. Aqueous sodium nitrite solution (10 g/l, freshly prepared).

10. Aqueous ammonium sulfamate solution (50 g/l).

11. N-(1-Naphthyl)ethylenediamine hydrochloride (10 g/l) in acetone: water (4:1).

Standards

Flurazepam and nitrazepam, both at concentrations of 100 mg/l in dilute hydrochloric acid (1 mol/l).

Method

1. Mix 3 ml of concentrated hydrochloric acid and 10 ml of sample or standard in a 30-ml glass tube with a ground-glass stopper.

2. Place the unstoppered tube in a boiling water-bath in a fume cupboard for 30 minutes.

3. Cool, add 10 ml of petroleum ether, stopper the tube and rotary- mix for 10 minutes.

4. Centrifuge in a bench centrifuge for 5 minutes and transfer the upper, organic layer to a clean tube.

5. Evaporate the extract to dryness under a stream of compressed air or nitrogen at 60°C.

Thin-layer chromatography

1. Reconstitute the extract in 100 µl of petroleum ether.

2. Divide the plate into four (two pairs of two columns), and spot two 25-µl portions of the sample and standard extracts on to each pair of columns (sample extracts first, see section 4.4.2)

3. Develop the chromatogram (10-cm run) using toluene:acetic acid (saturated tank, see section 4.4.3).

4. Remove the plate and allow to dry.

Take care - all of the spray reagents used are toxic. Spraying must be performed in a fume cupboard or under an efficient fume hood.

5. Spray one pair of columns (A) with p-dimethylaminocinnamaldehyde solution followed by trichloroacetic acid.

6. Spray the remaining pair of columns (B) with the following reagents, drying between each stage: sulfuric acid, sodium nitrite solution, ammonium sulfamate solution and naphthylethylenediamine solution.

Results

hRf values and colour reactions of some common benzophenones are given in Table 20.

Interference from other hydrolysis products can be minimized by extracting the petroleum ether extract (step 4) with aqueous sodium hydroxide (2 mol/l) on a rotary mixer for 5 minutes. Subsequently, separate the phases by centrifugation for 5 minutes, and evaporate the petroleum ether extract to dryness as described above (step 5).

Interpretation of the results can be difficult since many compounds give methylaminochlorobenzophenone and/or aminochlorobenzophenone on hydrolysis of urine. For example, it may not be possible to differentiate between temazepam or oxazepam and diazepam or nordazepam since these compounds give a similar pattern of benzophenones.

The following compounds do not themselves give benzophenones on hydrolysis: medazepam, triazolam, clobazam, norclobazam, and midazolam.

Sensitivity

Nordazepam (as aminochlorobenzophenone), 1 mg/l.

Clinical interpretation

Acute poisoning with benzodiazepines is common but, in adults, usually causes only drowsiness, confusion, ataxia, slurred speech, incoordination and sometimes coma. Respiratory depression is unusual in adults except with flurazepam. However, respiratory depression may occur if respiratory disease is already present, and in young children and the elderly. Benzodiazepines also have a synergistic respiratory depressant effect when taken with ethanol or other central nervous system depressants.

Treatment is normally symptomatic and supportive, although flumazenil may be used as a specific antagonist (see Table 4). There is little need to measure plasma benzodiazepine concentrations in the management of acute poisoning.

Share