International Programme on Chemical Safety

Basic Analytical Toxicology


Monographs - analytical and toxicological data (6.46 - 6.78)

6.46 Ethanol

Ethyl alcohol; alcohol; C2H5OH; relative molecular mass, 46

Acute poisoning with ethanol is very frequently encountered in hospital admissions and is usually the result of ingestion of alcoholic drinks. Poisoning with industrial alcohol (methylated spirit) containing various denaturants, notably methanol, also occurs.

The qualitative test described below will detect volatile reducing agents, of which ethanol is the most common. The quantitative assay is based on the oxidation of ethanol to acetaldehyde by alcohol dehydrogenase (ADH) in the presence of nicotinamide adenine dinucleotide (NAD), and is applicable to whole blood; if plasma or serum is used, the protein precipitation step with perchloric acid can be omitted. A number of manufacturers supply ethanol assay kits based on this reaction; if available, such kits often prove more economical than separate purchase of the reagents.

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reagent

Potassium dichromate (25 g/l) in aqueous sulfuric acid (500 ml/l).

Method

1. Apply 50 µl of potassium dichromate solution to a strip of glass- fibre filter-paper and insert the paper in the neck of a test- tube containing 1 ml of sample.

2. Lightly stopper the tube and place in a boiling water-bath for 2 minutes.

Results

A change in colour from orange to green indicates the presence of volatile reducing agents such as ethanol (see plate 7); metaldehyde, methanol and paraldehyde also react.

Sensitivity

Ethanol, 0.5 g/l.

Quantitative assay

Applicable to whole blood, plasma, or serum (0.5 ml).

Reagents

1. Semicarbazide reagent. Dissolve 10 g of tetrasodium pyrophosphate decahydrate, 2.5 g of semicarbazide hydrochloride and 0.5 g of glycine in 250 ml of purified water. Add 10 ml of aqueous sodium hydroxide (2 mol/l) and dilute to 300 ml.

2. Aqueous nicotinamide adenine dinucleotide (NAD; also known as diphosphopyridine nucleotide, DPN) (13 g/l). This solution is stable for 2-3 months at 4°C, but can be decomposed by vigorous agitation.

3. Alcohol dehydrogenase (ADH) suspension. Mix 45.5 g of ammonium sulfate and 3 g of tetrasodium pyrophosphate decahydrate in 100 ml of purified water adjusted to pH 7.3 (using a pH meter) with either aqueous hydrochloric acid or sodium hydroxide (both 1 mol/l) and containing 2.5 g of suspended crystalline yeast ADH; this solution is stable for 2-3 months at 4°C.

4. Aqueous perchloric acid (2.9 ml of perchloric acid (700 ml/l) in 100 ml of purified water).

Standards

Solutions containing ethanol concentrations of 0.5, 1.0, 2.0 and 4.0 g/l prepared in heparinized whole blood to which 10 g/l sodium fluoride has been added. These solutions are stable for up to 1 month if stored at 4°C in well-sealed containers.

Method

1. Add 0.5 ml of blood to 2 ml of perchloric acid solution in a test-tube.

2. Vortex-mix for 30 seconds and then centrifuge for 5 minutes.

3. Add 0.1 ml of the supernatant (or 0.2 ml of an aqueous dilution (1:9) of plasma/serum) to a 10-ml tube containing 4.5 ml of semicarbazide reagent and vortex-mix for 10 seconds.

4. Add 0.1 ml of NAD solution and 0.02 ml of ADH suspension and mix gently so as not to cause foaming.

5. Allow to stand for 70 minutes at 20-25°C and measure the absorbance at 340 nm against a reagent blank (see section 4.5.3).

Results

Construct a calibration graph of absorbance against blood ethanol concentration by analysis of the standard ethanol solutions and calculate the concentration of ethanol in the sample.

If the specimen contains an ethanol concentration of more than 4.0 g/l, the analysis should be repeated using a dilution (1:1 or 1:3) of the sample in blank plasma. Methanol does not interfere, but propan-2-ol and some higher alcohols will reduce NAD under the conditions used in this assay.

In all cases where the analysis may be delayed, it is important to add 10 g/l sodium fluoride to the specimen to inhibit microbial metabolism (see section 5.1.6).

Sensitivity

Ethanol, 0.5 g/l.

Clinical interpretation

Ethanol is rapidly absorbed from the small intestine and can cause disinhibition, blurred vision, drowsiness, incoordination and confusion, with nausea, vomiting and coma in severe cases. Hypoglycaemia and convulsions may also occur, especially in young children. Treatment of acute poisoning is normally symptomatic and supportive, although dialysis may be considered in severe cases (see section 2.2.3).

A simple guide to the interpretation of blood ethanol results is given in Table 27. However, remember that (1) ethanol potentiates the depressant effects on the central nervous system of many other drugs, and (2) chronic alcoholics may show few features of intoxication even with blood ethanol concentrations of 4 g/l or more. Blood ethanol measurements are therefore rarely helpful in the management of acute poisoning with this compound.

Ethanol can be given to treat poisoning with ethylene glycol and methanol (see section 2.2.2), since it inhibits the production of toxic metabolites. Monitoring of the plasma ethanol concentrations attained is often useful in such cases, as noted in the appropriate monographs (sections 6.48 and 6.70).

6.47 Ethchlorvynol

1-Chloro-3-ethylpent-1-en-4-yn-3-ol; C7H9ClO; relative molecular mass, 145

Ethchlorvynol is a nonbarbiturate hypnotic with a pungent smell. The test described is based on the reaction between ethchlorvynol and diphenylamine in the presence of concentrated sulfuric acid.

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reagents

1. Diphenylamine sulfate (solid).

2. Concentrated sulfuric acid (relative density 1.83).

Method

1. Add about 20 mg of diphenylamine sulfate crystals to the surface of 2 ml of test solution in a glass test-tube.

2. Slowly add 1 ml of sulfuric acid down the side of the tube.

Results

A bright red colour on the surface of the crystals indicates the presence of ethchlorvynol. This test is specific and will detect a therapeutic dosage of ethchlorvynol if performed on urine.

Sensitivity

Ethchlorvynol, 1 mg/l.

Clinical interpretation

Ingestion of ethchlorvynol may cause fatigue, headache, confusion, nausea, vomiting, coma and respiratory depression. Treatment is symptomatic and supportive.

6.48 Ethylene glycol

Ethane-1,2-diol; glycol; CH2OH.CH2OH; relative molecular mass, 62

Ethylene glycol is used mainly in vehicle radiator antifreeze as a concentrated 200-500 ml/l aqueous solution, sometimes together with methanol. Ethylene glycol is itself relatively nontoxic, but is metabolized by alcohol dehydrogenase giving rise to glycolic and oxalic acids. Some of the later features observed in ethylene glycol poisoning are therefore characteristic of poisoning with oxalates. The potentially fatal dose of antifreeze containing ethylene glycol in an adult is 50-100 ml.

There is no simple method for the detection and identification of ethylene glycol in biological specimens. However, a rise in plasma osmolality is a useful but nonspecific indicator of poisoning with this compound (see section 3.1.3). Oxalic acid may be excreted in urine as calcium oxalate, and the tests for oxalates will detect this compound (see section 6.82). The crystalluria produced may also be diagnostic (see section 5.2.1).

Clinical interpretation

Ingestion of ethylene glycol may give rise initially to clinical features similar to those of ethanol intoxication, and inebriation, drowsiness, nausea, and vomiting may occur. Production of glycolate may give rise to a marked metabolic acidosis, which may help to indicate the diagnosis (see section 3.1.2). Oxalic acid itself sequesters calcium and hypocalcaemia, muscular twitching and tetany, convulsions, flank pain, acute renal failure and cardiac arrest are later features of severe ethylene glycol poisoning.

Plasma concentrations of ethylene glycol of 0.5 g/l or more are normally associated with serious poisoning, although the time of ingestion is important in interpreting results. Ethanol prevents metabolism of ethylene glycol by competitive inhibition of alcohol dehydrogenase. Treatment of ethylene glycol poisoning consists of correction of any metabolic acidosis and of hypocalcaemia, ethanol administration and peritoneal dialysis or haemodialysis to treat renal failure and remove unchanged ethylene glycol. Plasma ethanol concentrations of about 1 g/l should be attained and monitored to ensure they are maintained during treatment since dialysis removes this compound as well as ethylene glycol.

6.49 Fluoride

Hydrogen fluoride (hydrofluoric acid, HF) and inorganic fluoride salts are widely used in industry and in the fluoridation of water supplies. Sodium fluoride (NaF), sodium fluorosilicate (Na2SiF6), and cryolite (Na3AlF6) are employed as insecticides and rodenticides. Fluorides are also used as preservatives, and in dentifrices and toothpastes. The estimated lethal dose of sodium fluoride in an adult is 1-4 g. Young children may ingest up to 0.5 g of fluoride by swallowing fluoride toothpaste.

The simple tests for fluorine-containing compounds given below will also detect fluoroacetates such as fluoroacetamide and sodium fluoroacetate. Fluoride ion can be measured reliably in blood and urine by use of a fluoride-selective electrode. Alternatively, the microdiffusion technique (see section 4.3.3) described below can be used. This relies on the liberation of HF from the sample; polypropylene vessels must be used, since HF attacks glass.

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reagents

1. Aqueous sodium chloride solution (50 g/l).

2. Concentrated sulfuric acid (relative density 1.83).

3. Solid calcium hydroxide.

4. Powdered silica (silicon dioxide, SiO2).

Method

1. Place 5 ml of sample in a porcelain crucible (10-ml volume), add 100 mg of calcium hydroxide and evaporate gently to dryness over a microburner.

2. To destroy organic material, heat strongly until a white ash remains.

3. Add 200 mg of powdered silica and mix with the residue, stirring and scraping the sides of the crucible.

4. Spot 100 µl of sodium chloride solution on to a glass microscope slide. Add 1 ml of concentrated sulfuric acid to the crucible and quickly cover with the slide, inverted so that the sodium chloride solution is suspended over the crucible.

5. Place a small beaker containing ice on the slide and heat the crucible gently over the microburner for 5 minutes.

6. Remove the slide and examine the sodium chloride solution under a low-power microscope.

Results

Volatile silicon tetrafluoride dissolves in the suspended drop to form sodium silicon tetrafluoride. As the water evaporates from the slide this forms small hexagonal crystals, sometimes with a pinkish tinge, which appear at the edge of the drop and before any larger, cubic sodium chloride crystals. This is a general test for fluorine- containing compounds, but is relatively insensitive.

Sensitivity

Fluoride, 100 mg/l.

Confirmatory test

Applicable to urine, stomach contents and scene residues.

Reagents

1. Concentrated sulfuric acid (relative density 1.83).

2. Solid calcium hydroxide.

3. Paraffin wax.

Method

1. Place 5 ml of sample in a 10-ml porcelain crucible, add 100 mg of calcium hydroxide and evaporate gently to dryness over a micro- burner.

2. To destroy organic material, heat strongly until a white ash remains.

3. Smear paraffin wax on to a glass microscope slide and scratch out a symbol (X) to expose part of the glass.

4. Add 1 ml of concentrated sulfuric acid to the crucible and quickly cover with the slide, inverted so that the exposed glass is over the crucible.

5. Remove the slide after 20 minutes and clean off the remaining wax with a solvent such as toluene.

Results

If fluorinated compounds are present, hydrogen fluoride is generated, which etches the exposed glass. This is a general test for fluorine-containing compounds, but is relatively insensitive.

Sensitivity

Fluoride, 100 mg/l.

Quantitative assay

Applicable to whole blood, plasma or serum.

Reagents

1. Aqueous sulfuric acid (800 ml/l) containing 2.5 g/l tergitol.

2. Aqueous cerous nitrate solution (432 mg/l).

3. Alizarin reagent. Mix 38.5 mg of alizarin complexone, 4.2 ml of glacial acetic acid and 2.2 g of anhydrous sodium acetate with 100 ml of purified water, final pH 4.3.

4. Polypropylene microdiffusion vessels, Öbrink's modification, which has a sealing well, in addition to the inner and outer wells (size 68).a


aConway polypropylene diffusion cells, Bel-Art Products, Pequannock, NJ 07440, USA.

Standards

Aqueous solutions of sodium fluoride containing fluoride ion concentrations of 0.5, 1.0 and 5.0 mg/l.

Method

1. To each microdiffusion cell add:

(a) 1.5 ml of sulfuric acid solution to the sealing well;

(b) 1.0 ml of sample or standard and 1.0 ml of sulfuric acid solution, without mixing, to the outer well;

(c) 0.25 ml of cerous nitrate solution and 0.25 ml of alizarin reagent to the centre well.

2. Seal the cells, gently mix the contents of the outer wells and allow to stand for 3 hours at room temperature.

Results

A blue colour in the centre well indicates the presence of fluoride. The fluoride concentration in the sample can be estimated by comparison with the results obtained on analysis of the standard fluoride solutions.

Sensitivity

Fluoride, 0.5 mg/l.

Clinical interpretation

Inhalation of hydrogen fluoride may cause coughing, choking, fever, dyspnoea, cyanosis and pulmonary oedema. Ingestion of hydrofluoric acid may cause nausea, vomiting, diarrhoea and abdominal pain, while skin contact may give deep and painful ulceration. Systemic toxic effects include weakness, tetany, convulsions, respiratory depression and acute hepatorenal failure. Treatment is symptomatic and may include intensive supportive measures.

Ingestion of fluoride salts may give rise to a burning sensation in the mouth and throat, dysphagia, thirst, excessive salivation, vomiting and diarrhoea. In severe cases muscle cramps, weakness and tremor may be followed by respiratory and cardiac failure. Plasma fluoride concentrations are normally less than 0.2 mg/l; urine concentrations are usually less than 1 mg/l, but up to 4 mg/l is not considered harmful. Blood concentrations of 2.6 mg/l or more have been recorded in fatalities.

6.50 Fluoroacetate

Sodium fluoroacetate (CF3.COONa) and fluoroacetamide (CF3.CONH2) are used mainly as rodenticides. Fluoroacetic acid is the toxic principle of Dichapetalum cymosum (gifblaar), a plant endemic to southern Africa. Fluoroacetates block the tricarboxylic acid cycle and are extremely toxic - the approximate lethal oral dose for adults is 30 mg.

Both the qualitative tests described below rely on the generation and subsequent detection of volatile fluorine derivatives from fluoroacetates present in the sample.

Qualitative test

Applicable to stomach contents, and scene residues. See fluoride monograph (section 6.49).

Results

Volatile silicon tetrafluoride dissolves in the suspended drop to form sodium silicon tetrafluoride. As the water evaporates from the slide this forms small hexagonal crystals, sometimes with a pinkish tinge, which appear at the edge of the drop and before any larger, cubic sodium chloride crystals. This is a general test for fluorine- containing compounds, but is relatively insensitive.

Sensitivity

Fluoroacetate, 100 mg/l.

Confirmatory test

Applicable to stomach contents and scene residues.

See fluoride monograph (section 6.49).

Results

If fluorinated compounds are present, hydrogen fluoride is generated, which etches the exposed glass. This is a general test for fluorine-containing compounds, but is relatively insensitive.

Sensitivity

Fluoroacetate, 100 mg/l.

Clinical interpretation

Exposure to fluoroacetates may cause nausea and apprehension, which may be followed by tremor, cardiac arryhthmias, convulsions and coma. The onset of symptoms may be delayed by 0.5-2 hours. Death occurs from respiratory and cardiac failure, often associated with pulmonary oedema. Treatment is symptomatic and supportive.

6.51 Formaldehyde

Formaldehyde (HCHO; relative molecular mass, 30) is a colourless, inflammable gas and is normally encountered as an aqueous solution (formalin, 340-380 ml/l), which also contains methanol as a stabilizer. Formalin is used as a disinfectant, an antiseptic and a tissue-fixing and embalming fluid. Polymerized formaldehyde (paraformaldehyde) is used as a fumigant, and other polymeric forms are used as adhesives in chipboard and plywood, and in the preparation of insulation materials.

Formaldehyde is rapidly metabolized in vivo to formate and is itself a metabolite of methanol. Acute formaldehyde poisoning is uncommon, but 30 ml of formalin may be fatal in an adult.

Qualitative test

Applicable to stomach contents and scene residues.

Reagents

1. Concentrated sulfuric acid (relative density 1.83).

2. Chromotropic acid (solid).

Method

1. Add about 100 mg of chromotropic acid to 0.5 ml of test solution and vortex-mix for 5 seconds.

2. Carefully add 1.5 ml of concentrated sulfuric acid.

Results

A purple-violet colour indicates the presence of formaldehyde.

Sensitivity

Formaldehyde, 20 mg/l.

Clinical interpretation

Formaldehyde vapour is very irritating and inhalation may cause conjunctivitis, coughing and laryngeal and pulmonary oedema. Ingestion of formaldehyde solution may give rise to abdominal pain, vomiting, diarrhoea, hypotension, coma, metabolic acidosis and acute renal failure. Treatment is normally symptomatic and supportive.

6.52 Formic acid and formate

Formic acid (HCOOH; relative molecular mass, 46), a colourless, aqueous solution, is very corrosive. Many proprietary descaling agents contain 500-600 ml/l formic acid. Formates such as sodium formate (HCOONa) are used as synthetic intermediates and in the dying, printing and tanning industries. Formic acid is itself a metabolite of methanol and formaldehyde. The minimum lethal dose of formic acid in an adult is thought to be about 30 ml.

The initial test given below should be used if formic acid is suspected. In the confirmatory test both formic acid and formates are reduced to formaldehyde, which can then be detected by reaction with chromotropic acid.

Qualitative test

Applicable to stomach contents and scene residues.

Reagents

1. Citric acid/acetamide reagent. Citric acid (5 g/l) and acetamide (100 g/l) in propan-2-ol.

2. Aqueous sodium acetate (300 g/l).

3. Acetic anhydride.

Method

1. Add 0.5 ml of test solution to 1 ml of citric acid/acetamide reagent and then add 0.1 ml of sodium acetate solution and 3.5 ml of acetic anhydride.

2. Vortex-mix for 5 seconds and heat in a boiling water-bath for 10 minutes.

Results

A red colour indicates the presence of formic acid. Formaldehyde and formate salts do not react in this test.

Sensitivity

Formic acid, 50 mg/l.

Confirmatory test

Applicable to stomach contents and scene residues.

Reagents

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

2. Magnesium powder.

3. Chromotropic acid (solid).

4. Concentrated sulfuric acid (relative density 1.83).

Method

1. Add 0.1 ml of dilute hydrochloric acid to 0.1 ml of test solution and vortex-mix for 5 seconds.

2. Slowly add about 100 mg of magnesium powder until the evolution of gas ceases.

3. Add about 100 mg of chromotropic acid and vortex-mix for 5 seconds.

4. Carefully add 15 ml of concentrated sulfuric acid and heat in a water-bath at 60°C for 10 minutes.

Results

A purple-violet colour indicates the presence of formates or formic acid. Formaldehyde reacts without prior reduction.

Sensitivity

Formate, 50 mg/l.

Clinical interpretation

Formic acid is very corrosive to tissues, and ingestion may cause burning and ulceration of the mouth and throat, corrosion of the glottis, oesophagus and stomach, metabolic acidosis, intravascular haemolysis, disseminated intravascular coagulation, circulatory collapse and renal and respiratory failure. Treatment is symptomatic and supportive.

6.53 Glutethimide

2-Ethyl-2-phenylglutarimide; C13H15NO2; relative molecular mass, 217

Glutethimide is a nonbarbiturate hypnotic which is now little used because of the risk of toxicity. The estimated minimum lethal dose of glutethimide in an adult is 5 g. Glutethimide forms a number of metabolites in humans, many of which are excreted in urine. Less than 2% of a dose is excreted unchanged.

There is no simple qualitative test for glutethimide, but this compound and its metabolites can be detected and identified by thin- layer chromatography of an acidic solvent extract of urine (see section 5.2.3). Glutethimide is unstable at pH 11 and above, and the rapid decline in absorbance at 240 nm observed when a solution containing this compound is treated with concentrated ammonium hydroxide (see section 6.9) provides a further means of identification.

Clinical interpretation

Acute ingestion of glutethimide may cause dilated and unreactive pupils, hypotension, severe metabolic acidosis, coma, cerebral oedema, papilloedema and acute respiratory failure. Treatment is generally symptomatic and supportive. Charcoal haemoperfusion may be indicated in severe cases.

6.54 Glyceryl trinitrate

Trinitroglycerin; nitroglycerin; propane-1,2,3-triol trinitrate; C3H5N3O9; relative molecular mass, 227

Glyceryl trinitrate is used as a vasodilator in the treatment of angina and as an explosive in dynamite. Other organic nitro compounds (amyl nitrite, butyl nitrite) are also vasodilators and are commonly abused. The estimated minimum lethal dose of glyceryl trinitrate in an adult is 2 g. Glyceryl trinitrate is rapidly metabolized in vivo to dinitrates and mononitrates. About 20% of a sublingual dose is excreted in urine in 24 hours, mainly as the mononitrate.

Take care - glyceryl trinitrate explodes on rapid heating or impact and is unsafe in alcoholic solution.

Qualitative test

Applicable to stomach contents and scene residues.

Reagents

1. Diphenylamine (10 ml/l) in concentrated sulfuric acid (relative density 1.83).

2. Silica gel thin-layer chromatography plate (5 × 20 cm, 20 µm average particle size; see section 4.4.1).

Standard

Aqueous glyceryl trinitrate (20 mg/l).

Method

1. Add 4 ml of chloroform:propan-2-ol (9:1) to 1 ml of sample or standard in a glass-stoppered test-tube.

2. Vortex-mix for 1 minute, allow to stand for 1 minute and discard the upper (aqueous) layer.

3. Filter the chloroform extract through phase-separating filter- paper into a clean tube and evaporate to dryness without heating under a stream of compressed air or nitrogen.

Thin-layer chromatography

1. Reconstitute the extract in 100 µl of chloroform and spot 20 µl of the sample and standard extracts on adjacent columns of the plate.

2. Develop the chromatogram in chloroform:acetone (4:1) (10-cm run, saturated tanks, see section 4.4.3).

3. Allow to dry and spray with diphenylamine solution.

Results

Glyceryl trinitrate (hRf 0.71) gives a blue spot on a white background.

Sensitivity

Glyceryl trinitrate, 5 mg/l.

Clinical interpretation

Many of the signs and symptoms of poisoning with glyceryl trinitrate and other organic nitrates and nitrites are similar to those observed with inorganic nitrates and nitrites. Thus acute poisoning with organic nitrates or nitrites may cause headache, nausea, vomiting, diarrhoea, abdominal pain, flushing, dizziness, confusion, hypotension, collapse, coma and convulsions. Methaemoglobinaemia can be produced and this may be indicated by dark chocolate-coloured blood (see section 3.2.2). Blood methaemoglobin can be measured, but is unstable and the use of stored samples is unreliable. Treatment is symptomatic and supportive.

6.55 Haloperidol

4-[4-(4-Chlorophenyl)-4-hydroxypiperidino]-4'-fluorobutyrophenone; C21H23ClFNO2; relative molecular mass, 376

Haloperidol is a neuroleptic used orally or parenterally to treat schizophrenia and a variety of other disorders. Haloperidol is slowly excreted in urine following oral dosage, about 40% being eliminated within 5 days, about 1% as unchanged drug.

There is no simple qualitative test for haloperidol, 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). Urinary concentrations are often below the limit of detection of this method, even after overdosage.

Clinical interpretation

Acute overdosage with haloperidol and other butyrophenones may cause drowsiness, hypotension, dystonic reactions and akathisia. Treatment is largely symptomatic and supportive.

6.56 Hydroxybenzonitrile herbicides

The hydroxybenzonitriles encountered most commonly are bromoxynil (3,5-dibromo-4-hydroxybenzonitrile; C7H3Br2NO; relative molecular mass, 277) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile; C7H3I2NO; relative molecular mass, 371).

These compounds are contact herbicides with some systemic activity, and are widely used on cereal crops. Both bromoxynil and ioxynil uncouple oxidative phosphorylation so that poisoning with these compounds follows a similar course to that with other uncouplers such as the dinitrophenol pesticides and pentachlorophenol, and may follow occupational exposure as well as oral ingestion.

There are no simple tests for these compounds. However, both bromoxynil and ioxynil show high ultraviolet absorbance at 255 nm, and this forms the basis of the quantitative assay outlined below.

Quantitative assay

Applicable to plasma or serum (1.0 ml).

Reagent

Aqueous trichloroacetic acid (10 g/l).

Standards

Solutions containing either compound at concentrations of 20, 50, 100, 200 and 400 mg/l in blank human plasma.

Method

1. Add 1 ml of trichloroacetic acid solution to 1 ml of sample or standard in a 10-ml test-tube fitted with a ground-glass stopper.

2. Add 5 ml of methyl tertiary-butyl ether, vortex-mix for 30 seconds and centrifuge for 5 minutes.

3. Remove the upper (ether) layer and filter through phase- separating filter-paper into a clean tube.

4. Measure the absorbance at 255 nm against a blank plasma extract (see section 4.5.2).

Results

Construct a calibration graph of absorbance against hydroxybenzonitrile concentration in the calibration standards and calculate the hydroxybenzonitrile concentration in the sample. Care must be taken to minimize loss of methyl tertiary-butyl ether by evaporation before the absorption of the extract is measured.

Chlorophenoxy herbicides and other compounds that are highly soluble in water do not interfere. However, if a scanning spectrophotometer is available, comparison of the absorption spectra of sample and standard extracts at 220-300 nm may reveal the presence of other interfering compounds (see section 4.5.2).

Sensitivity

Bromoxynil or ioxynil, 20 mg/l.

Clinical interpretation

Absorption of bromoxynil and ioxynil may give rise to fatigue, irritability, excessive sweating, hyperthermia, tachycardia, vomiting and thirst, which may be followed by exhaustion and cardiorespiratory arrest. However, there is no characteristic staining of the skin as with the dinitrophenol pesticides. Treatment is largely symptomatic and supportive. As with the chlorophenoxy herbicides, alkalinization may protect against the systemic toxicity of these compounds. Plasma concentrations of either compound greater than 20 mg/l may be associated with clinical features of toxicity.

6.57 Hypochlorites

Hypochlorites such as sodium hypochlorite (NaOCl) and calcium hypochlorite (bleaching powder, chlorinated lime, Ca(OCl)2) are widely used in bleach and disinfectant solutions. Domestic bleach is a 30-60 g/l aqueous solution of sodium hypochlorite, but higher concentrations (200 g/l) may be used, for example, to chlorinate swimming-pools.

Hypochlorites are strong oxidizing agents, and the test given below will also detect compounds with similar properties, such as bromates, chlorates, iodates, nitrates, and nitrites.

Qualitative test

Applicable to stomach contents and scene residues.

Reagent

Diphenylamine (10 g/l) in concentrated sulfuric acid (relative density 1.83).

Method

1. Filter, if necessary, 5 ml of stomach contents into a 10-ml glass tube.

2. Place 0.5 ml of filtrate or scene residue in a clean tube and slowly add 0.5 ml of diphenylamine solution down the side of the tube so that it forms a layer under the sample.

Results

A true positive is indicated by a strong blue colour which develops immediately at the junction of the two layers. A light blue colour will be given by most samples of stomach contents owing to the presence of organic material. As all strong oxidizing agents are rapidly reduced in biological samples, the test should be performed as soon as possible after receipt of the sample.

In contrast to other strong oxidizing agents, hypochlorites tend to evolve noxious green chlorine gas ( take care) when treated with concentrated sulfuric acid, and this is a further diagnostic feature.

Sensitivity

Hypochlorite, 10 mg/l.

Confirmatory tests

Applicable to stomach contents and scene residues.

1. Lead acetate test

Reagents

1. Glacial acetic acid.

2. Aqueous lead acetate solution (50 g/l).

Method

1. Add acetic acid drop by drop to 1 ml of test solution to give a final pH of about 6 (universal indicator paper).

2. Add 0.5 ml of lead acetate solution and boil for 2-3 minutes.

Results

A brown precipitate confirms hypochlorite. Sulfides give an immediate brown/black precipitate with lead acetate solution.

Sensitivity

Hypochlorite, 10 mg/l.

2. Potassium iodide/starch test

Reagents

1. Aqueous potassium iodide solution (100 g/l).

2. Glacial acetic acid.

3. Starch (solid).

Method

1. Add 0.1 ml of test solution to 0.1 ml of acetic acid and then add 0.1 ml of potassium iodide solution.

2. Mix and add about 20 mg of starch.

Results

A blue colour confirms hypochlorite.

Sensitivity

Hypochlorite, 10 mg/l.

Clinical interpretation

Ingestion of hypochlorites may lead to the formation of hypochlorous acid by reaction with gastric acid, and this in turn may release free chlorine which may be inhaled. Features of poisoning with hypochlorites therefore include nausea, vomiting, diarrhoea, abdominal pain, confusion, hypotension, coma and pulmonary oedema. Irritation and corrosion of the mucous membranes, and oesophageal and gastric perforation may also occur, especially with more concentrated formulations. Treatment is symptomatic and supportive.

6.58 Imipramine

3-(10,11-Dihydro-5 H-dibenz [b,f]azepin-5-yl)- N,N-dimethylpropylamine; C19H24N2; relative molecular mass, 280

Imipramine is a widely used tricyclic antidepressant; it is metabolized by N-demethylation to desipramine, which is also used as an antidepressant in its own right. Trimipramine and clomipramine are analogues of imipramine.

The test described below (Forrest test) is based on the reaction of these compounds with acidified potassium dichromate solution.

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reagent

Forrest reagent. Mix 25 ml of aqueous potassium dichromate solution (2 g/l) with 25 ml of aqueous sulfuric acid (300 ml/l), 25 ml of aqueous perchloric acid (200 g/kg) and 25 ml of aqueous nitric acid (500 ml/l).

Method

Add 1 ml of Forrest reagent to 0.5 ml of urine and vortex-mix for 5 seconds.

Results

A yellow-green colour deepening through dark green to blue indicates the presence of imipramine, desipramine, trimipramine or clomipramine. Phenothiazines may interfere and the FPN test for these latter compounds should also be performed (see section 6.91).

When applied to urine, this test will detect only acute overdosage. As with other tricyclic antidepressants, such as amitriptyline, greater sensitivity and selectivity can be obtained by thin-layer chromatography of a basic urine extract (see section 5.2.3), which should always be performed if possible.

Sensitivity

Imipramine, 25 mg/l.

Clinical interpretation

Acute poisoning with tricyclic antidepressants, such as imipramine, may be associated with dilated pupils, hypothermia, cardiac arrhythmias, respiratory depression, convulsions, coma 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 is generally avoided, but alkalinization using sodium bicarbonate is sometimes employed. Quantitative measurements in blood are not normally required in management.

6.59 Iodates

Iodates such as potassium iodate (KIO3) and sodium iodate (NaIO3) are used as disinfectants, food additives, dietary supplements and chemical reagents. Iodates are strong oxidizing agents, and the test given below will also detect compounds with similar properties, such as bromates, chlorates, hypochlorites, nitrates and nitrites.

Qualitative test

Applicable to stomach contents and scene residues.

Reagent

Diphenylamine (10 g/l) in concentrated sulfuric acid (relative density 1.83).

Method

1. Filter, if necessary, 5 ml of stomach contents into a 10-ml glass tube.

2. Place 0.5 ml of filtrate or scene residue in a clean tube and slowly add 0.5 ml of diphenylamine solution down the side of the tube so that it forms a layer under the sample.

Results

A true positive is indicated by a strong blue colour which develops immediately at the junction of the two layers. A light blue colour will be given by most samples of stomach contents owing to the presence of organic material. As all strong oxidizing agents are rapidly reduced in biological samples, the test should be performed as soon as possible after receipt of the sample.

Sensitivity

Iodate, 1 mg/l.

Confirmatory test

Applicable to stomach contents and scene residues.

Reagents

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

2. Aqueous starch solution (10 g/l, freshly prepared).

3. Aqueous potassium thiocyanate solution (50 g/l).

Method

1. To 0.1 ml of starch solution add 0.3 ml of purified water and 0.1 ml of potassium thiocyanate solution.

2. Mix well and add 0.1 ml of test solution acidified with 0.1 ml of acetic acid solution.

Results

A blue colour is specific for iodate. Iodate also reacts like chloride in the confirmatory test for bromates (see section 6.14).

Sensitivity

Iodate, 100 mg/l.

Clinical interpretation

Acute poisoning with iodates may cause nausea, vomiting, diarrhoea, abdominal pain, confusion, coma and convulsions. Methaemoglobinaemia is often produced and this may be indicated by dark chocolate-coloured blood (see section 3.2.2). Blood methaemoglobin can be measured, but is unstable and the use of stored samples is unreliable. Treatment is symptomatic and supportive.

6.60 Iodine and iodide

Iodine (I2) is one of the oldest antiseptics in medicine. It is used topically as a solution in ethanol (tincture) containing elemental iodine together with potassium iodide (KI) or sodium iodide (NaI), which enhances the solubility of iodine itself by forming polyiodide ion. Potassium iodide and sodium iodide are themselves used as dietary supplements and in photography, but are relatively innocuous in comparison with iodine.

When iodine is applied to the skin or mucous membranes it is absorbed as iodide. The qualitative test given below serves to indicate the presence of inorganic iodides or bromides and the appropriate confirmatory tests must then be used.

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reagents

1. Aqueous nitric acid (2 mol/l).

2. Aqueous silver nitrate solution (10 g/l).

3. Concentrated ammonium hydroxide (relative density 0.88).

Method

1. Add 0.1 ml of nitric acid to 1 ml of clear test solution, mix and add 0.1 ml of silver nitrate solution.

2. Centrifuge to isolate any significant precipitate and treat with 0.1 ml of ammonium hydroxide solution.

Results

A white precipitate soluble in ammonium hydroxide indicates chloride, an off-white precipitate sparingly soluble in ammonium hydroxide indicates bromide, and a creamy-yellow, insoluble precipitate indicates iodide.

Sensitivity

Iodide, 100 mg/l.

Confirmatory test

Applicable to urine, stomach contents and scene residues.

Reagents

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

2. Starch (solid).

3. Sodium nitrite solution (100 g/l, freshly prepared).

Method

Vortex-mix 0.1 ml of test solution, about 20 mg of starch, 0.1 ml of dilute hydrochloric acid and 0.1 ml of sodium nitrite solution in a test-tube.

Results

A blue colour confirms iodide.

Sensitivity

Iodide, 100 mg/l.

Clinical interpretation

Acute poisoning with iodine solutions may cause corrosion of the mucous membranes of the mouth, oesophagus and stomach, vomiting, diarrhoea and abdominal pain. In severe cases, delirium, coma, circulatory collapse and acute renal failure may ensue. Absorption of as little as 2-4 g of free iodine may cause death. Treatment is generally symptomatic and supportive. Starch may be administered to adsorb orally ingested iodine.

Acute ingestion of iodide salts may cause angioedema, swelling of the larynx and cutaneous haemorrhages. However, as with bromides, signs of toxicity are more likely to occur with chronic poisoning, and include the presence of a burning sensation in the mouth and throat, metallic taste, sore teeth and gums, hypersalivation, headache, pulmonary oedema, enlargement of the parotid and submaxillary glands, anorexia, diarrhoea, fever and depression. Treatment is symptomatic and supportive.

6.61 Iron

Ferrous (iron II) salts are used in the treatment of iron deficiency anaemia and ferric (iron III) salts, which are more toxic, have been used as abortifacients. The minimum lethal dose of ferrous sulfate in an adult is of the order of 30 g, but 1 g may be dangerous in an infant. A green or blue colour in vomit or stomach contents suggests the presence of iron or copper salts.

The qualitative test given below can be used to differentiate between ferrous and ferric iron and other metals, while the quantitative assay can be used to measure serum iron. It is very important to avoid contamination when collecting blood for the measurement of serum iron concentrations; vigorous discharge of the sample through the syringe needle can cause sufficient haemolysis to invalidate the assay.

Qualitative test

Applicable to stomach contents and scene residues.

Reagents

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

2. Aqueous potassium ferricyanide solution (10 g/l).

3. Aqueous potassium ferrocyanide solution (10 g/l).

Method

1. To 0.1 ml of sample add 0.1 ml of dilute hydrochloric acid and 0.05 ml of potassium ferricyanide solution and vortex-mix for 5 seconds.

2. To a further 0.1 ml of sample add 0.1 ml of dilute hydrochloric acid and 0.05 ml of potassium ferrocyanide solution, and vortex- mix for 5 seconds.

3. Leave for 5 minutes at ambient temperature and centrifuge for 5 minutes.

Results

Deep blue precipitates with potassium ferricyanide (step 1) and potassium ferrocyanide (step 2) indicate the presence of ferrous and ferric iron, respectively.

Sensitivity

Ferrous or ferric iron, 10 mg/l.

Quantitative assay

Applicable to unhaemolysed serum (2 ml).

Reagents

1. Aqueous sodium sulfite solution (0.1 mol/l, freshly prepared).

2. 2,2'-Bipyridyl (1 g/l) in aqueous acetic acid (30 ml/l).

3. Aqueous hydrochloric acid (0.005 mol/l).

Standards

Prepare aqueous solutions containing ferrous ion concentrations of 1.0, 2.0, 5.0 and 10.0 mg/l by dilution of ferrous ammonium sulfate solution (1.00 g of ferrous iron per litre) with dilute hydrochloric acid.

Method

1. Mix 2 ml of sample, 2 ml of sodium sulfite solution and 2 ml of 2,2'-bipyridyl solution in a 10-ml centrifuge tube with a ground- glass neck.

2. Heat in a boiling water-bath for 5 minutes, cool and add 1 ml of chloroform.

3. Stopper, rotary-mix for 5 minutes and then centrifuge for 5 minutes.

4. If an emulsion forms, vortex-mix for 30 seconds and repeat the centrifugation.

5. Remove the chloroform extract, filter through phase-separating filter-paper and measure the absorbance of the extract at 520 nm against a reagent blank (see section 4.5.2).

Results

Construct a calibration graph of absorbance against ferrous iron concentration in the calibration standards and calculate the iron concentration in the sample.

This method should not be used if chelating agents such as deferoxamine have been given before the specimen was obtained, since the result may not be reliable.

Sensitivity

Iron, 0.5 mg/l.

Clinical interpretation

Acute poisoning with iron salts is extremely dangerous, especially in young children. The absorbed iron may rapidly exceed the binding capacity of transferrin so that free iron accumulates in the blood. Rapid necrosis of the gastrointestinal mucosa may occur with haemorrhage and loss of electrolytes and fluid. Treatment may include chelation therapy with deferoxamine, given intravenously or orally (see Table 6).

Normal serum iron concentrations are less than 1.8 mg/l (34 µmol/l). Serious toxicity may occur at concentrations above 5 mg/l (90 µmol/l) in children or above 8 mg/l (145 µmol/l) in adults. The serum iron concentration should be measured before and during chelation therapy, both to confirm the need for such treatment and to monitor efficacy.

6.62 Isoniazid

Isonicotinic acid hydrazide; INAH; INH; isonicotinohydrazide; C6H7N3O; relative molecular mass, 137

Isoniazid is used in the treatment of tuberculosis. The principal metabolic reaction is acetylation, but other reactions include hydrolysis, conjugation with glycine and N-methylation. Up to 70% of a dose is excreted in urine, largely as metabolites. Serious toxicity may occur in adults with doses of 3 g.

The colorimetric procedure given below can be used to measure the plasma isoniazid concentration if overdosage with this drug is suspected.

Quantitative assay

Applicable to plasma or serum (2 ml).

Reagents

1. Aqueous metaphosphoric acid (200 g/l).

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

3. Sodium nitroprusside reagent. Mix 25 ml of aqueous sodium nitroprusside (20 g/l) with 25 ml of aqueous sodium hydroxide (4 mol/l), freshly prepared.

Standards

Standard solutions containing isoniazid concentrations of 5, 10, 20 and 50 mg/l in blank plasma.

Method

1. Add 4 ml of purified water to 2 ml of sample and add 2 ml of dilute metaphosphoric acid.

2. Vortex-mix for 30 seconds and allow to stand for 10 minutes.

3. Centrifuge for 5 minutes and transfer 4 ml of the supernatant to a clean tube.

4. Add 2 ml of dilute acetic acid, 2 ml of sodium nitroprusside reagent and vortex-mix for 5 seconds.

5. Allow to stand for 2 minutes and measure the absorbance at 440 nm against a plasma blank (see section 4.5.2).

Results

Prepare a calibration graph by analysis of the standard isoniazid solutions and calculate the isoniazid concentration in the sample. Specimens containing isoniazid at concentrations greater than 50 mg/l should be diluted with blank plasma and re-analysed.

Related drugs such as iproniazid and pyrazinamide interfere in this test. 4-Aminosalicylate also interferes, but only if present at a high concentration.

Sensitivity

Isoniazid, 5 mg/l.

Clinical interpretation

Overdosage with isoniazid may cause nausea, vomiting, dilated pupils, hypotension, hyperglycaemia, oliguria, metabolic acidosis, coma, convulsions, and circulatory and respiratory failure. Treatment is generally symptomatic and supportive, although intravenous administration of pyridoxine (vitamin B6) is indicated in severe cases (see Table 4).

The plasma isoniazid concentrations attained during therapy are normally less than 10 mg/l. Toxicity has been associated with plasma concentrations greater than 20 mg/l, and blood concentrations of up to 150 mg/l have been reported in fatalities.

6.63 Laxatives

Laxatives are sometimes abused by patients with eating disorders such as bulimia or anorexia nervosa. Some commonly encountered laxatives are listed in Table 28. Bisacodyl, dantron and phenolphthalein are synthetic compounds, while rhein is a common constituent of many laxatives of vegetable origin (senna, cascara, frangula and rhubarb root).

The test given below, which involves thin-layer chromatography of a solvent extract of a hydrolysed urine specimen, is especially useful in differentiating between diarrhoea due to microbial infection or food allergy and that due to self-medication with laxatives.

Qualitative test

Applicable to urine.

Reagents

1. Aqueous sodium hydroxide solution (6 mol/l).

2. Sodium acetate buffer. Adjust 300 g/l of sodium acetate dihydrate to pH 5 with glacial acetic acid.

3. Ketodase solution (ß-glucuronidase 5000 units/ml).

4. Chloroform:propan-2-ol (9:1)

5. m-Xylene:isopropylacetone:methanol (10:10:1) (XIAM).

6. n-Hexane:toluene:glacial acetic acid (3:1:1) (HTAA).

7. Silica gel thin-layer chromatography plates (20 × 20 cm, 20 µm average particle size, see section 4.4.1).

Standards

1. Bisacodyl. Dissolve 2 mg in 10 ml of methanol. Add 100 µl of sodium hydroxide solution and heat on a water-bath for 30 minutes at 70°C to prepare the monohydroxy and dihydroxy analogues.

2. Dantron (2 mg in 10 ml of chloroform).

3. Phenolphthalein (2 mg in 200 µl of methanol and 10 ml of chloroform).

4. Rhein (2 mg in 10 ml of chloroform).

These solutions are stable for at least 2 months if stored in stoppered tubes at 4°C.

Method

1. To 20 ml of urine add 1 ml of ketodase and 2 ml of sodium acetate buffer.

2. Incubate in a water-bath for 2 hours at 60°C.

3. Cool and add 25 ml of chloroform:propan-2-ol (9:1).

4. Vortex-mix for 5 minutes, centrifuge for 5 minutes and remove the upper, aqueous layer by aspiration.

5. Evaporate the organic extract to dryness under a stream of compressed air or, preferably, nitrogen at 40°C.

Thin-layer chromatography

1. Reconstitute the extract in 100 µl of chloroform.

2. Spot 10 µl of the reference solutions and 3 µl and 10 µl of the reconstituted extract on to each of two plates.

3. Develop plate 1 in XIAM and plate 2 in HTAA (10-cm run, saturated tanks, see section 4.4.3).

4. Inspect under ultraviolet light at 366 nm and mark the outline of the spots with a pencil.

5. Spray each plate with sodium hydroxide solution.

Results

The thin-layer chromatography characteristics of the compounds studied are given in Table 29. This method will detect a single dose of the named laxatives up to about 36 hours after ingestion.

Note that bisacodyl is only detected after hydrolysis as a dihydroxy compound and as a hydroxylated metabolite at a lower hRf value on the XIAM plate. Both of these compounds give a purple colour with the sodium hydroxide spray.

Sensitivity

Each laxative, about 0.2 mg/l.

Clinical interpretation

Chronic ingestion of laxatives may lead to diarrhoea, hypokalaemia and large bowel dilatation, while additional problems, such as skin disorders (with phenolphthalein), may occur in certain patients.

6.64 Lead

Lead (Pb) and lead compounds have a number of industrial uses ranging from paint additives to solder, batteries and building materials. Well known insoluble lead compounds include red lead (Pb3O4) and white lead (basic lead carbonate, PbCO3.Pb(OH)2). The most important soluble salts of lead are lead nitrate (Pb(NO3)2) and lead acetate (Pb(CH3COO)2). This latter compound is also known as sugar of lead because of its sweet taste. Organic lead compounds, such as tetraethyl lead, are still used as antiknock agents in petrol.

There is no simple qualitative test for lead that can be carried out on biological samples. However, physical and chemical tests on materials suspected of containing lead may be useful. Lead compounds will usually sink to the bottom when sprinkled into a glass of water, and this may be useful in the examination of paint flakes or cosmetics, such as surma (an Asian preparation often containing antimony or lead). It should be borne in mind that finely divided lead compounds may float on the surface as a result of the surface tension of the water.

Qualitative test

Applicable to stomach contents and scene residues.

Reagents

1. Sodium tartrate buffer, pH 2.8. Mix sodium bitartate (19 g/l) and tartaric acid (15 g/l) in purified water.

2. Aqueous sodium rhodizonate solution (10 g/l).

Method

1. Add 0.1 ml of sodium tartrate buffer to 0.1 ml of test solution and vortex-mix for 5 seconds.

2. Spot 50 µl of acidified solution on to phase-separating filter- paper and add 50 µl of sodium rhodizonate solution.

Results

Lead salts give a purple colour in this test. However, the test is not specific: barium salts give a brown colour and a number of other metals also give coloured complexes.

Sensitivity

Lead, 2 mg/l.

Clinical interpretation

The acute ingestion of soluble lead salts may cause severe colicky pain with constipation or diarrhoea. Chronic lead poisoning is more common and additional symptoms include fatigue, anaemia and joint weakness and pain. Lead poisoning in young children may cause coma and encephalopathy. Treatment may include chelation therapy.

In the absence of facilities for the accurate measurement of lead concentration in blood, a diagnosis of lead poisoning is best made from a careful evaluation of the history and clinical presentation. Nonspecific signs that may indicate a diagnosis of chronic lead poisoning include basophilic stippling of red cells, a blue gum line and wrist drop. Specialized clinical chemical tests that may also assist in diagnosis include red-cell zinc protoporphyrin concentration and urinary delta-amino-laevulinic acid excretion, but again suitable facilities may not be available.

6.65 Lidocaine

Lignocaine; 2-diethylaminoaceto-2',6'-xylidide; C14H22N2O; relative molecular mass, 234

Lidocaine is used as a local anaesthetic and is commonly found in lubricant gels for use with urinary catheters. It is also used as an antiarrhythmic but is only effective when given intravenously, since it undergoes extensive first-pass metabolism. Metabolic pathways include N-dealkylation, hydroxylation, amide hydrolysis and glucuronide formation; only some 3% of an oral dose is excreted unchanged in urine. The fatal oral dose of lidocaine is about 25 g in an adult.

Lidocaine is often found in urine and other samples from oisoned patients, sometimes in very high concentrations. This usually results from topical use of a lubricant gel containing lidocaine. Metabolites may also occur in urine following topical use of lidocaine.

There is no simple qualitative test for lidocaine, 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

Acute poisoning with lidocaine may cause confusion, paraesthesia, hypotension, coma, convulsions and circulatory collapse. Treatment is symptomatic and supportive.

6.66 Lithium

Lithium (Li) salts have a number of industrial uses, and lithium carbonate (Li2CO3; relative molecular mass, 74) and lithium citrate (C6H5Li3O7Ê4H2O; relative molecular mass, 282) are widely used in the treatment of depression and mania. Lithium is excreted in urine, but the plasma half-life is dependent on the duration of therapy, among other factors. In therapy, 2 g of lithium carbonate may be given daily for 5-7 days, but this is normally reduced to 0.6-1.2 g/day thereafter. Survival has followed the acute ingestion of 22 g of lithium carbonate, although this is unusual.

There is no simple method for the measurement of lithium in biological specimens. The test given below can be used to indicate the presence of lithium salts in samples which contain relatively high concentrations of this element.

Qualitative test

Applicable to 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 microburner.

Results

A crimson red flame denotes the presence of lithium salts. However, calcium and strontium salts also give red flames, while high concentrations of sodium (yellow flame) can mask other colours.

Sensitivity

Lithium, 50 mg/l.

Clinical interpretation

Acute poisoning with lithium salts can cause nausea, vomiting, apathy, drowsiness, tremor, ataxia and muscular rigidity, with coma, convulsions and death in severe cases. Overdosage with lithium tends to be more serious in patients on chronic lithium therapy, since tissue sites are saturated and lithium accumulates in plasma. Treatment is symptomatic and supportive, but peritoneal dialysis or haemodialysis may be indicated in severe cases (see section 2.2.3).

6.67 Meprobamate

2-Methyl-2- iso-propylpropane-1,3-diol dicarbamate; C9H18N2O4; relative molecular mass, 218

Meprobamate is a sedative and tranquillizer. Metabolites include meprobamate N-oxide and 2-hydroxypropylmeprobamate. About 90% of a dose is excreted in urine, 15% as unchanged drug. The estimated minimum lethal dose in an adult is 12 g, but recovery has occurred after much larger doses.

The qualitative test described here is based on a general reaction of carbamates with furfuraldehyde in the presence of hydrogen chloride. The confirmatory test is also applicable to urine, and is based on solvent extraction followed by thin-layer chromatography of the concentrated extract.

Qualitative test

Applicable to stomach contents and scene residues.

Reagents

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

2. Furfuraldehyde solution (100 ml/l) in methanol, freshly prepared.

3. Concentrated hydrochloric acid (relative density 1.18).

Method

1. Acidify 1 ml of sample with 0.5 ml of dilute hydrochloric acid, and extract with 4 ml of chloroform on a rotary mixer for 5 minutes.

2. Centrifuge in a bench centrifuge for 5 minutes, discard the upper, aqueous phase and filter the chloroform extract through phase-separating filter-paper into a clean tube.

3. Evaporate the extract to dryness under a stream of compressed air or nitrogen at 40°C.

4. Dissolve the residue in 0.1 ml of methanol and apply a 10-mm diameter spot to filter-paper, and allow to dry.

5. Apply 0.1 ml of furfuraldehyde solution to the spot, and allow to dry.

6. Expose the paper to concentrated hydrochloric acid fumes for 5 minutes in a fume cupboard.

Results

Meprobamate gives a black spot. Other carbamates, such as the carbamate pesticides, interfere in this test.

Sensitivity

Meprobamate, 100 mg/l.

Confirmatory test

Applicable to urine, stomach contents and scene residues.

Reagents

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

2. Methanol: concentrated ammonium hydroxide (relative density 0.88) (100:1.5) (MA, see also section 6.73).

3. Furfuraldehyde (20 ml/l) in acetone, freshly prepared.

4. Concentrated sulfuric acid (relative density 1.83) solution (40 ml/l) in acetone, freshly prepared.

5. Van Urk reagent. Mix 1 g of p-dimethylaminobenzaldehyde in 100 ml of methanol with 10 ml of concentrated hydrochloric acid (relative density 1.18).

6. Silica gel thin-layer chromatography plates (10 × 20 cm, 20 µm average particle size, see section 4.4.1).

Standard

Meprobamate 1 g/l in chloroform.

Method

1. Add 1 ml of dilute hydrochloric acid and 10 ml of chloroform to 10 ml of urine in a 30-ml glass tube.

2. Rotary-mix for 5 minutes, centrifuge for 5 minutes and discard the upper, aqueous phase.

3. Transfer the lower, organic layer to a 15-ml tapered glass tube and evaporate to dryness in a water-bath at 60°C under a stream of compressed air or nitrogen.

Thin-layer chromatography

1. Reconstitute the extract in 100 µl of chloroform and spot two portions of 50 µl of the extract and two portions of 10 µl of the meprobamate standard on to separate columns of the plate.

2. Develop (10-cm run) using MA (saturated tank, see section 4.4.3) and dry until no smell of ammonia remains.

3. Spray one pair of columns (sample and standard) sequentially with (a) furfuraldehyde solution and (b) sulfuric acid solution, and allow to dry.

4. Spray the second pair of columns with van Urk reagent.

Results

Meprobamate (hRf, 10) gives a violet reaction with the furfuraldehyde/sulfuric acid spray, and a yellow colour with van Urk reagent. It may be necessary to redistill the furfuraldehyde, since this compound polymerizes on standing giving a brown discoloration. The sensitivity of the furfuraldehyde/sulfuric acid spray may be increased by gently heating the plate with a hair-drier after spraying.

Other carbamate drugs, such as carisoprodol, mebutamate and tybamate, may interfere in this test, but meprobamate is by far the most common compound encountered.

Sensitivity

Meprobamate, 10 mg/l.

Clinical interpretation

Acute meprobamate poisoning may cause hypotension, hypothermia, muscle weakness, nystagmus, acidosis, coma, respiratory depression, pulmonary oedema, acute renal failure and disseminated intravascular coagulation. Treatment is normally symptomatic and supportive.

6.68 Mercury

Mercury (Hg) and its inorganic salts are used in the manufacture of thermometers, felt, paints, explosives, lamps, electrical equipment and batteries. Diethyl mercury, dimethyl mercury and a variety of other mercury compounds, including inorganic mercurials, are used as fungicides, primarily on seeds and bulbs, and in lawn sands. Mercuric chloride (HgCl2) is extremely toxic and ingestion of 1 g may prove fatal in an adult. As with antimony, arsenic and bismuth, mercury can be detected using the Reinsch test.

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

silver - mercury

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

Confirmatory test

Applicable to silver-stained foil from the Reinsch test.

Reagent

Copper (I) iodide suspension. Dissolve 5 g of copper (II) sulfate and 3 g of ferrous sulfate in 10 ml of purified water with continuous stirring and add 7 g of potassium iodide in 50 ml of water. Allow the copper (I) iodide precipitate to form, filter and wash with water. Transfer to a brown glass bottle as a suspension with the aid of a little water. This suspension is quite stable.

Method

Add 0.1 ml of copper (I) iodide suspension to a filter-paper, place the foil on the suspension, cover and leave for 1-12 hours.

Results

A salmon-pink colour indicates the presence of mercury. Positive results may be obtained within 1 hour, but with low concentrations, colour development may take up to 12 hours.

Sensitivity

Mercury, 5 mg/l.

Clinical interpretation

Elemental mercury is poorly absorbed from the gastrointestinal tract and is not considered toxic by this route. Mercury vapour is absorbed through the skin and lungs and may give rise to stomatitis, increased salivation, a metallic taste, diarrhoea, pneumonitis and renal failure. The ingestion of mercuric salts may cause severe gastric pain, vomiting, bloody diarrhoea and also renal failure, which is usually the cause of death. Organomercurials are concentrated in the central nervous system and produce ataxia, chorea and convulsions.

Treatment is symptomatic and supportive and may include chelation therapy. Mercury concentrations in blood and urine are good indicators of exposure, but only atomic absorption spectrophotometric methods of determination are reliable.

6.69 Methadone

(±)-6-Dimethylamino-4,4-diphenylheptan-3-one; C21H27NO; relative molecular mass, 310

Methadone is a narcotic analgesic structurally related to dextropropoxyphene and is widely used in the treatment of opioid dependence. Methadone is metabolized largely by N-demethylation and hydroxylation. However, approximately 30% of an oral dose is excreted unchanged in urine. Plasma methadone concentrations attained on chronic (maintenance) therapy are very much higher than those associated with serious toxicity in patients not taking methadone chronically.

There is no simple qualitative test for methadone, but this compound and its metabolites can be detected and identified by thin- layer chromatography of a basic solvent extract of urine (see section 5.2.3).

Clinical interpretation

Acute overdosage with methadone may give rise to pin-point pupils, hypotension, hypothermia, coma, convulsions and pulmonary oedema. Death may ensue from profound respiratory depression. Naloxone rapidly reverses the central toxic effects of methadone (see section 2.2.2).

6.70 Methanol

Methyl alcohol; wood alcohol; CH3OH; relative molecular mass, 32

Methanol is used as a general and laboratory solvent, and in antifreeze (often with ethylene glycol), windscreen washer additives and duplicating fluids. In an adult, death may follow the ingestion of 20-50 ml of methanol. Poisoning with industrial grades of ethanol (methylated spirit), which often contain methanol as a denaturant, also occurs, but is generally less serious than with methanol alone. This is because methanol toxicity results from its metabolism to formaldehyde and formate by alcohol dehydrogenase, a reaction inhibited by ethanol.

Qualitative test

Applicable to urine, stomach contents and scene residues.

Reagent

Potassium dichromate reagent. Potassium dichromate (25 g/l) in purified water:concentrated sulfuric acid (relative density 1.83) (1:1).

Method

1. Apply 50 µl of potassium dichromate reagent to a strip of glass- fibre filter-paper and insert the paper in the neck of a test- tube containing 1 ml of urine.

2. Lightly stopper the tube and place in a boiling water-bath for 2 minutes.

Results

A change in colour from orange to green indicates the presence of volatile reducing agents such as methanol. However, other such compounds, for example ethanol, metaldehyde, and paraldehyde, also react.

Sensitivity

Methanol, 50 mg/l.

Confirmatory test

Applicable to urine, stomach contents and scene residues.

Reagents

1. Potassium dichromate reagent. Potassium dichromate (25 g/l) in purified water: concentrated sulfuric acid (relative density 1.83) (1:1).

2. Concentrated sulfuric acid (relative density 1.83).

3. Chromotropic acid (solid).

Method

1. Add 0.1 ml of potassium dichromate reagent to 1 ml of urine and allow to stand at room temperature for 5 minutes.

2. Add 0.1 ml of ethanol and about 10 mg of chromotropic acid and gently add sulfuric acid down the side of the tube so that it forms a separate layer at the bottom.

Results

A violet colour at the junction of the two layers indicates the presence of methanol. Formaldehyde also gives a positive result in this test.

Sensitivity

Methanol, 50 mg/l.

Clinical interpretation

Acute methanol poisoning is characterized by delayed onset of coma, cyanosis, respiratory failure, marked metabolic acidosis, electrolyte imbalance, hyperglycaemia and blindness, which may be permanent. Treatment is aimed at correcting metabolic abnormalities, inhibiting methanol metabolism by giving ethanol and removing unchanged methanol by peritoneal dialysis or haemodialysis. Measurement of plasma ethanol concentrations can be useful in monitoring therapy with this compound (see section 6.46).

6.71 Methaqualone

2-Methyl-3- o-tolylquinazolin-4(3H)-one; C16H14N2O; relative molecular mass, 250

Methaqualone is a nonbarbiturate hypnotic, but is now little used because of the risk of toxicity. Metabolic pathways include aromatic hydroxylation, N-oxidation, and conjugation. Less than 2% of a dose is excreted unchanged. The estimated minimum lethal dose of methaqualone in an adult is 5 g.

There is no simple qualitative test for methaqualone, 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

Acute methaqualone poisoning may cause hypertonia, myoclonus, papilloedema, tachycardia, pulmonary oedema, coma and convulsions. Treatment is generally symptomatic and supportive. Charcoal haemoperfusion may be indicated in severe cases.

6.72 Methyl bromide

Methyl bromide (CH3Br) is used as a fumigant in ships' holds, grain silos and other large enclosed areas. Methyl bromide undergoes partial metabolism to give inorganic bromide in vivo. Since the concentrations of this ion encountered in methyl bromide poisoning are much lower than in serious poisoning with inorganic bromides, it is thought that methyl bromide itself is the primary toxin.

The qualitative test given below serves to indicate the presence of inorganic bromides or iodides, and the appropriate confirmatory test must then be used or blood bromide measured. There is no simple method for measuring unchanged methyl bromide.

Qualitative test

Applicable to urine. Detects inorganic bromide.

Reagents

1. Aqueous nitric acid (2 mol/l).

2. Aqueous silver nitrate solution (10 g/l).

3. Concentrated ammonium hydroxide (relative density 0.88).

Method

1. Add 0.1 ml of nitric acid to 1 ml of clear test solution, mix for 5 seconds and add 0.1 ml of silver nitrate solution.

2. Centrifuge to isolate any significant precipitate and treat with 0.1 ml of concentrated ammonium hydroxide.

Results

A white precipitate soluble in ammonium hydroxide indicates chloride, an off-white precipitate sparingly soluble in ammonium hydroxide indicates the presence of bromide and a creamy-yellow, insoluble precipitate indicates iodide.

Sensitivity

Bromide, 50 mg/l.

Confirmatory test

Applicable to urine. Detects inorganic bromide.

Reagents

1. Saturated fluorescein solution in aqueous acetic acid (600 ml/l).

2. Concentrated sulfuric acid (relative density 1.83).

3. Potassium permanganate (solid)

Method

1. Soak a strip of filter-paper in fluorescein solution.

2. Add about 50 mg of potassium permanganate to 2 ml of test solution in a 10-ml test-tube.

3. Add 0.2 ml of concentrated sulfuric acid and hold the fluorescein-impregnated filter-paper in the mouth of the tube.

Results

The bromide is oxidized to free bromine. This reacts with the yellow dye fluorescein to give eosin (tetrabromofluorescein) which has a pink/red colour.

Sensitivity

Bromide, 50 mg/l.

Quantitative assay

Applicable to plasma or serum (2 ml).

Reagents

1. Aqueous chloroauric acid. Dissolve 0.5 g of chloroauric acid (gold chloride, HAuCl4ÊxH2O) in 100 ml of purified water.

2. Aqueous trichloroacetic acid (200 g/l).

Standards

Dissolve 1.288 g of sodium bromide in 500 ml of purified water (bromide ion 2 g/l). Prepare serial dilutions in purified water containing bromide ion concentrations of 0.2, 0.4, 0.6, 0.8, 1.2 and 1.6 g/l.

Method

1. Add 6 ml of trichloroacetic acid solution to 2 ml of sample in a 10-ml test-tube, vortex-mix for 30 seconds and allow to stand for 15 minutes.

2. Centrifuge in a bench centrifuge for 5 minutes and filter the supernatant through phase-separating filter-paper into a clean tube.

3. Add 1 ml of chloroauric acid solution to 4 ml of the clear supernatant and vortex-mix for 5 seconds.

4. Record the absorbance at 440 nm against a purified water blank (see section 4.5.2).

Results

Construct a calibration graph of bromide concentration against absorbance by analysis of the standard bromide solutions, and calculate the concentration of bromide ion in the sample. The calibration is linear for concentrations from 25 mg/l to 2.5 g/l. This method is not reliable with specimens that may give turbid supernatants, e.g. postmortem samples.

Sensitivity

Bromide, 25 mg/l.

Clinical interpretation

Symptoms of poisoning due to methyl bromide often develop several hours after exposure and include confusion, dizziness, headache, nausea, vomiting, abdominal pain, blurred vision, hyporeflexia and paraesthesia. Coma and convulsions may occur in severe cases, and pulmonary oedema, jaundice and oliguria have also been described. Treatment is symptomatic and supportive.

Normal serum bromide concentrations are less than 10 mg/l. After the therapeutic administration of inorganic bromides, concentrations of up to 80 mg/l may be attained; toxicity is usually associated with concentrations greater than 500 mg/l. On the other hand, blood bromide concentrations of 90-400 mg/l have been reported in fatal methyl bromide poisoning.

6.73 Morphine

(4a R,5 S,7a R,SR,9c S)-4a,5,7a,8,9,9c-Hexahydro-12-methyl-8,9c- imino-ethanophenanthro[4,5-bcd]furan-3,5-diol monohydrate; C17H19NO3ÊH2O; relative molecular mass, 303

Morphine is the principal alkaloid of opium and is a potent narcotic analgesic. Diamorphine (heroin, 3,6- O-diacetylmorphine) has two to three times the potency of morphine and is obtained by treating morphine (or opium in the case of illicit preparations) with acetic anhydride.

Diamorphine is rapidly hydrolysed in vivo to 6-acetylmorphine and thence to morphine. Morphine is also a metabolite of codeine. Approximately 5% of a dose of morphine is metabolized to normorphine, but conjugation with glucuronic acid is the major pathway. The principal product is morphine-3-glucuronide, but morphine-6- glucuronide is also formed. Free morphine in urine accounts for about 10% of a dose, while morphine-3-glucuronide accounts for 75%. The estimated minimum fatal dose of morphine or diamorphine in an adult unaccustomed to taking these compounds is 100-200 mg.

Morphine can be detected by thin-layer chromatography of an extract of urine, stomach contents, or scene residues at pH 8.5-9. Since a large proportion of a dose of diamorphine or morphine is excreted as glucuronides, prior hydrolysis of urine can increase the sensitivity of the procedure.

Qualitative test

Applicable to urine.

Reagents

1. Concentrated hydrochloric acid (relative density 1.18).

2. Sodium bicarbonate (solid).

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

4. Methanol:concentrated ammonium hydroxide (relative density 0.88) (99:1.5) (MA).

5. Ethyl acetate:methanol:concentrated ammonium hydroxide (relative density 0.88) (85:10:5) (EMA).

6. Iodoplatinate reagent. Mix 0.25 g of platinic chloride and 5 g of potassium iodide in 100 ml of purified water.

Standards

1. Hydrolysis standards: codeine, dihydrocodeine and morphine-3- glucuronide (all 1 mg/l) in blank urine.

2. Thin-layer chromatography standards: cocaine, codeine, dihydrocodeine, methadone and morphine (all 1 g/l) in chloroform.

Method

1. Add 2 ml of concentrated hydrochloric acid to 10 ml of urine or hydrolysis standard in a test-tube.

2. Heat on a boiling water-bath for 30 minutes.

3. Allow to cool, decant into a 250 ml beaker and slowly add sodium bicarbonate until effervescence ceases and solid sodium bicarbonate remains in the beaker. Take care - this reaction can be violent.

4. Decant the hydrolysate into a clean tube, add 10 ml of ethyl acetate:propan-2-ol (9:1) and vortex-mix for 3 minutes.

5. Centrifuge for 5 minutes, filter the upper organic phase through phase-separating filter-paper into a clean tube and discard the lower, aqueous layer.

6. Evaporate the extract to dryness under a stream of compressed air.

Thin-layer chromatography

1. Reconstitute the extracts in 50 µl of ethyl acetate:propan-2-ol (9:1) and spot equal portions of the sample and standard extracts, and 10 µl of the chromatography standard on three columns of two plates.

2. Develop one plate in MA and the second in EMA (10-cm run, saturated tanks, see section 4.4.3).

3. Allow to dry and ensure that no smell of ammonia remains before spraying both plates with iodoplatinate reagent.

Results

The hRf values and colour reactions of the drugs in the standard mixtures and some additional compounds of interest are given in Table 30. This procedure gives no information as to the presence of diamorphine since this compound and both 3- O-acetylmorphine and 6- O-acetylmorphine, as well as morphine glucuronides, are hydrolysed to morphine.

The hydrolysis standard extract must be analysed as well as the chromatography standard, since compounds extracted from hydrolysed urine tend to migrate more slowly on thin-layer chromatography owing to the influence of co-extracted compounds, and this must be allowed for in the interpretation of results.

It is important to ensure that only concentrated ammonium hydroxide (relative density 0.88) is used to prepare the mobile phases (MA and EMA) and that a given batch of eluent is used only three times for MA and five times for EMA, as discussed in section 5.2.3.

Sensitivity

Morphine (free + conjugated), 1 mg/l.

Clinical interpretation

Acute poisoning with morphine and diamorphine gives rise to pin-point pupils, hypotension, hypothermia, coma, convulsions and pulmonary oedema. Death may ensue from profound respiratory depression. Naloxone rapidly reverses the central toxic effects of morphine (see section 2.2.2).

6.74 Nicotine

3-(1-Methylpyrrolidin-2-yl)pyridine; C10H14N2; relative molecular mass, 162

Nicotine is an alkaloid derived from the leaves of Nicotiana tabacum. As little as 40 mg of nicotine can prove fatal in an adult. Nicotine is commonly encountered in tobacco although usually in concentrations insufficient to cause acute poisoning, except when ingested by young children. Nicotine occurs in higher concentrations in some herbal medicines, and is also used as a fumigant in horticulture. Nicotine can be absorbed rapidly through the skin, and is metabolized principally by N-demethylation to give cotinine.

There is no simple qualitative test for nicotine, but this compound and cotinine can be detected and identified by thin-layer chromatography of a basic solvent extract of urine (see section 5.2.3).

Clinical interpretation

Initially nausea, dizziness, vomiting, respiratory stimulation, headache, tachycardia, sweating and excessive salivation occur followed by collapse, convulsions, cardiac arrhythmias and coma in severe cases. Death may supervene rapidly or be delayed by several hours. Treatment is symptomatic and supportive.

6.75 Nitrates

Nitrates such as sodium nitrate (NaNO3) are most commonly found in inorganic fertilizers, but are also used as antiseptics, food preservatives and explosives. Death in an adult may follow the ingestion of about 15 g of the sodium or potassium salt. Organic nitrates, such as glyceryl trinitrate, are used as vasodilators. Nitrates are metabolized to nitrites in the gastrointestinal tract. Nitrates are strong oxidizing agents and the test given below will also detect compounds with similar properties, such as bromates, chlorates, hypochlorites, iodates and nitrites.

Qualitative test

Applicable to stomach contents and scene residues.

Reagent

Diphenylamine (10 g/l) in concentrated sulfuric acid (relative density 1.83).

Method

1. Filter, if necessary, 5 ml of stomach contents into a 10-ml glass tube.

2. Add 0.5 ml of filtrate or scene residue to a clean tube and slowly add 0.5 ml of diphenylamine solution down the side of the tube so that it forms a layer under the sample.

Results

A true positive is indicated by a strong blue colour which develops immediately at the junction of the two layers. A light blue colour will be given by most samples of stomach contents owing to the presence of organic material. Since all strong oxidizing agents are rapidly reduced in biological samples, the test should be performed as soon as possible after receipt of the sample.

Sensitivity

Nitrate, 10 mg/l.

Confirmatory test

Applicable to urine, stomach contents and scene residues.

Reagents

1. Sulfamic acid reagent. Mix 1 ml of ammonium sulfamate (150 g/l) and 1 ml of aqueous hydrochloric acid (2 mol/l), freshly prepared.

2. Aqueous imipramine hydrochloride solution (20 g/l).

3. Concentrated sulfuric acid (relative density 1.83).

Method

1. Mix 0.1 ml of test solution and 0.1 ml of sulfamic acid reagent.

2. Add 0.1 ml of imipramine solution, and carefully add 0.2 ml of concentrated sulfuric acid down the side of the tube so that it forms a layer underneath the test mixture.

Results

An intense blue colour at the junction of the two layers confirms nitrate. Sulfamic acid treatment is used to remove any nitrite present prior to the test.

Sensitivity

Nitrate, 50 mg/l.

Clinical interpretation

Acute poisoning with nitrates can cause nausea, vomiting, diarrhoea, abdominal pain, confusion, coma and convulsions. In addition, nitrates may give rise to headache, flushing, dizziness, hypotension and collapse. Treatment is symptomatic and supportive. Methaemoglobinaemia is often produced and this may be indicated by dark chocolate-coloured blood (see section 3.2.2). Blood methaemoglobin can be measured but is unstable and the use of stored samples is unreliable.

6.76 Nitrites

Nitrites such as sodium nitrite (NaNO2) were formerly used as vasodilators, and are used to prevent rusting, as food preservatives and in explosives. Nitrites may also arise from the metabolism of nitrates. The fatal dose of sodium nitrite is about 10 g, although ingestion of as little as 2 g has caused death in an adult. Nitrites are strong oxidizing agents and the test given below will also detect compounds with similar properties such as bromates, chlorates, hypochlorites, iodates and nitrates.

Qualitative test

Applicable to stomach contents and scene residues.

Reagent

Diphenylamine (10 g/l) in concentrated sulfuric acid (relative density 1.83).

Method

1. Filter, if necessary, 5 ml of stomach contents into a 10-ml glass tube.

2. Add 0.5 ml of filtrate or scene residue to a clean tube and slowly add 0.5 ml of diphenylamine solution down the side of the tube so that it forms a layer under the sample.

Results

A true positive is indicated by a strong blue colour which develops immediately at the junction of the two layers. A light blue colour will be given by most samples of stomach contents owing to the presence of organic material. Since all strong oxidizing agents are rapidly reduced in biological samples, the test should be performed as soon as possible after receipt of the sample.

Sensitivity

Nitrite, 10 mg/l.

Confirmatory tests

Applicable to urine, stomach contents and scene residues.

1. Imipramine/hydrochloric acid test

Reagents

1. Aqueous imipramine hydrochloride (20 g/l).

2. Concentrated hydrochloric acid (relative density 1.18).

Method

To 0.1 ml of imipramine solution add 0.1 ml of test solution and 0.2 ml of hydrochloric acid.

Results

A blue colour is specific for nitrite.

Sensitivity

Nitrite, 1 mg/l.

2. Sulfanilic acid/1-aminonaphthalene test

Reagents

1. Sulfanilic acid (10 g/l) in aqueous acetic acid (300 ml/l).

2. 1-Aminonaphthalene (1 g/l) in aqueous acetic acid (300 ml/l).

Method

1. Add 0.1 ml of test solution to 0.1 ml of sulfanilic acid solution.

2. Mix and add 0.1 ml of 1-aminonaphthalene solution.

Results

A purple/red colour is specific for nitrite. If the test solution is strongly acidic or basic the pH should be adjusted beforehand to about 7 (universal indicator paper) by carefully adding 2 mol/l aqueous hydrochloric acid or sodium hydroxide.

Sensitivity

Nitrite, 0.2 mg/l.

Quantitative assay

Applicable to urine.

Reagents

1. Aqueous sodium acetate solution (164 g/l).

2. Sulfanilic acid (6 g/l) in concentrated hydrochloric acid (relative density 1.18):purified water (1:4).

3. 1-Aminonaphthalene (4.8 g/l) in concentrated hydrochloric acid (relative density 1.18):methanol (1:4).

Standards

Solutions containing nitrite ion at concentrations of 0, 10, 20, 50 and 100 mg/l in purified water, prepared by dilution from aqueous sodium nitrite solution (1.50 g/l, equivalent to a nitrite ion concentration of 1.00 g/l).

Method

1. Mix 0.5 ml of sample or standard and 0.5 ml of sulfanilic acid solution in a 25-ml volumetric flask.

2. Allow to stand for 10 minutes, add 0.5 ml of 1-aminonaphthalene solution and 0.5 ml of sodium acetate solution and dilute to 25 ml with purified water.

3. Allow to stand for 10 minutes and measure the absorbance at 510 nm against a water blank carried through the procedure (see section 4.5.2).

Results

Construct a graph of absorbance against nitrite concentration by analysis of the standard solutions and calculate the nitrite concentration in the sample.

Sensitivity

Nitrite, 5 mg/l.

Clinical interpretation

Acute poisoning with nitrites may cause nausea, vomiting, diarrhoea, abdominal pain, confusion, coma and convulsions. In addition, nitrites may give rise to headache, flushing, dizziness, hypotension and collapse. Methaemoglobinaemia is often produced and this may be indicated by dark chocolate-coloured blood (see section 3.2.2). Blood methaemoglobin can be measured but is unstable and the use of stored samples is unreliable. Treatment is symptomatic and supportive. Urinary nitrite ion concentrations of 10 mg/l and above have been reported in fatalities.

6.77 Nitrobenzene

Nitrobenzene (nitrobenzol; C6H5NO2; relative molecular mass, 123) has a characteristic odour of bitter almonds and is used in the manufacture of aniline, as a solvent for cellulose ethers, in metal and shoe polishes, perfumes, dyes and soaps, and as a synthetic intermediate. The acute toxicity of nitrobenzene is very similar to that of aniline, probably because of metabolic conversion. Nitrobenzene is metabolized to p-aminophenol and N-acetyl- p-aminophenol (paracetamol), which are both 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.

Qualitative test

Applicable to urine. o-Cresol/ammonia test - see paracetamol monograph (section 6.83).

Results

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

Sensitivity

p-Aminophenol, 1 mg/l.

Clinical interpretation

Poisoning with nitrobenzene may arise from inhalation or dermal absorption as well as ingestion. Symptoms occur within 1-3 hours of exposure and include confusion, nausea, vomiting and diarrhoea, with convulsions, coma and hepatorenal damage in severe cases. Haemolysis, red (wine-coloured) urine, and methaemoglobinaemia (dark chocolate- coloured blood) are characteristic features of poisoning with nitrobenzene, as with aniline (see section 3.2.2).

Blood methaemoglobin can be measured, but is unstable and the use of stored samples is unreliable. However, hepatic and renal function tests are essential. 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.

6.78 Nortriptyline

3-(10,11-Dihydro-5 H-dibenzo [a,d]cyclohepten-5-ylidene)- N-methylpropylamine; C19H21N; relative molecular mass, 263

Nortriptyline is the N-demethylated metabolite of amitriptyline and is also a tricyclic antidepressant in its own right.

There is no simple test for nortriptyline, 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 nortriptyline and other tricyclic antidepressants may be associated with 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 is generally avoided, but alkalinization using sodium bicarbonate is sometimes employed. Quantitative measurements in blood are not normally required for management.