Basic Analytical Toxicology
Monographs - analytical and toxicological data (6.24 - 6.45)
6.24 Chloral hydrate
Chloral; 2,2,2-trichloroethane-1,1-diol; C2H3O2Cl3; relative molecular mass, 165
Cl OH ' ' ' ' Cl---C---C---H ' ' ' ' Cl OH
Chloral hydrate is a mild sedative and hypnotic agent. The pharmacological activity of chloral, and of the related compound dichloralphenazone, is thought to derive largely from a metabolite, 2,2,2-trichloroethanol, which is in turn metabolized to trichloroacetic acid. This latter compound can be detected in urine using the Fujiwara test.
Qualitative test
Applicable to urine. Fujiwara test - see carbon tetrachloride monograph (section 6.23). This test must be performed in a fume cupboard.
Results
An intense red/purple colour in the upper, pyridine layer indicates the presence of trichloro compounds. The blank (purified water) analysis excludes contamination with compounds such as chloroform from the laboratory atmosphere. Other trichloro compounds react in this test, but trichloroacetic acid is by far the most common compound encountered.
This test is very sensitive and will detect a therapeutic dose of chloral hydrate 12-24 hours after ingestion. However, other compounds, notably the solvent trichloroethylene, also give rise to trichloroacetic acid in vivo and caution must be exercised in reporting results.
Sensitivity
Trichloroacetate, 1 mg/l.
Clinical interpretation
Acute poisoning with chloral hydrate can cause vomiting, excitement, ataxia, confusion, drowsiness, stupor, hypotension, coma, cardiac arrhythmias, respiratory depression and pulmonary oedema. Treatment is normally symptomatic and supportive.
6.25 Chloralose
alpha-Chloralose; (R)-1,2- O-(2,2,2-trichloroethylidene)-alpha-D- glucofuranose; C8H11Cl3O6; relative molecular mass, 310
Chloralose is a hypnotic drug and has been used as a surgical anaesthetic in laboratory animals. It is also used as a bird repellent on seed grain and as a rodenticide, especially against mice, in cooler climates. The dose associated with toxicity in adults is about 1 g.
Chloralose may be oxidized with periodic acid to trichloroacetic acid, which can be detected using the Fujiwara test as for carbon tetrachloride. It was thought that chloralose undergoes hydrolysis in vivo, and that urine from patients who had ingested chloralose would give a positive reaction without periodate oxidation. However, recent work suggests that this is not the case.
Qualitative test
Applicable to plasma or serum, urine, stomach contents and scene residues. This test must be performed in a fume cupboard.
Reagents
1. Periodic acid reagent. Mix 3 g of sodium periodate and 3 ml of aqueous sulfuric acid (0.5 mol/l) and dilute to 100 ml with water.
2. Aqueous sodium hydroxide solution (5 mol/l, i.e., 200 g/l).
3. Aqueous trichloroacetic acid (10 mg/l).
Method
1. Add 1 ml of periodic acid reagent to 1 ml of test solution (or a portion of a solid residue extracted with 1 ml of water) in a 10-ml glass test-tube.
2. Mix and allow to stand for 5 minutes.
3. To separate 10-ml tubes add 2-ml portions of:
(a) test solution;
(b) purified water;
(c) trichloroacetic acid solution.
4. Add 1 ml of sodium hydroxide solution and 1 ml of pyridine to all four tubes, mix gently and fit with a loose stopper.
5. Heat in a boiling water-bath for 2 minutes.
Results
An intense red/purple colour in the upper, pyridine layer of the periodate-treated tube indicates the presence of chloralose. The sample analysis without periodate is to exclude the presence of compounds, such as chloral hydrate, that give rise to trichloroacetic acid in vivo. The blank analysis excludes contamination with chloroform from the laboratory atmosphere.
Sensitivity
Trichloroacetate, 1 mg/l.
Clinical interpretation
Ingestion of chloralose may cause drowsiness, hypotonia and coma. Treatment is generally symptomatic and supportive.
6.26 Chlorates
Sodium chlorate (NaClO3) is used as a weedkiller and in matches and fireworks. Chlorates are also used in small amounts in throat gargles and toothpastes. In an adult, serious poisoning may follow the ingestion of 15 g of sodium chlorate. Chlorates are strong oxidizing agents, and the test given below will also detect compounds with similar properties, such as bromates, hypochlorites, 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 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 reduced rapidly in biological samples, the test should be performed as soon as possible after receipt of the sample.
Sensitivity
Chlorate, 10 mg/l.
Confirmatory test
Applicable to stomach contents and scene residues.
Reagents
1. Manganous sulfate reagent. Mix saturated aqueous manganous sulfate with o-phosphoric acid (1:1).
2. Diphenylcarbazide (10 g/l) in methanol.
Method
1. To 0.1 ml of test solution add 0.2 ml of manganous sulfate reagent and warm briefly over a spirit lamp or microburner.
2. Cool and add 0.1 ml of diphenylcarbazide solution.
Results
A purple/violet colour, which is intensified after cooling and adding diphenylcarbazide, indicates chlorate.
Persulfates and periodates give a similar reaction; persulfates can be eliminated by evaporating the test solution with 0.1 ml of concentrated sulfuric acid (relative density 1.83) and 0.1 ml of aqueous silver nitrate (10 g/l).
Sensitivity
Chlorate, 100 mg/l.
Clinical interpretation
Acute poisoning with chlorates 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.
6.27 Chloroform
Trichloromethane; CHCl3; relative molecular mass, 119
Chloroform was used as an anaesthetic and general solvent, but is relatively toxic since it is partly metabolized to phosgene (COCl2) which is a potent hepatorenal toxin.
Chloroform can be detected readily using the Fujiwara test. However, this test will also detect ingestion or exposure to compounds that are extensively metabolized to trichloroacetic acid, such as chloral hydrate, dichloralphenazone and trichloroethylene.
Qualitative test
Applicable to urine. Fujiwara test - see carbon tetrachloride monograph (section 6.23). This test must be performed in a fume cupboard.
Results
An intense red/purple colour in the upper, pyridine layer indicates the presence of trichloro compounds. The blank analysis excludes contamination with compounds such as chloroform from the laboratory atmosphere. Trichloroacetic acid is by far the most common compound encountered in this test.
Sensitivity
Trichloroacetate, 1 mg/l.
Clinical interpretation
Acute poisoning with chloroform is rare. Clinical features include ataxia, nausea, vomiting, coma, convulsions, respiratory depression, cardiac arrhythmias and hepatorenal damage. Treatment is symptomatic and supportive. Acetylcysteine may protect against hepatorenal damage (see Table 4).
6.28 Chlorophenoxy herbicides
These compounds have the general formula shown below. Some common chlorophenoxy herbicides are listed in Table 23.
2,4-D (not to be confused with DNOC, i.e. dinitro- o-cresol, see section 6.42) and related compounds are used to control broad-leaved weeds in lawns and in cereal crops and, at higher application rates, for total vegetation control. They are frequently encountered as mixtures, both with other members of the group and with other pesticides.
Qualitative test
Applicable to stomach contents and scene residues.
Reagents
1. Aqueous hydrochloric acid (1 mol/l).
2. Sodium nitrite (100 g/l) in concentrated sulfuric acid (relative density 1.83), freshly prepared. Take care - brown nitrogen dioxide fumes may be evolved.
3. Chromotropic acid (2,5-dihydroxynaphthalene-2,7-disulfonic acid) (2 g/l) in concentrated sulfuric acid (relative density 1.83).
Method
1. Add 1 ml of dilute hydrochloric acid to 10 ml of sample, and extract with 20 ml of toluene on a rotary mixer for 5 minutes.
2. Centrifuge for 5 minutes, remove the upper, toluene layer, and extract the residue with a second 20-ml portion of toluene.
3. Combine the toluene extracts and evaporate to dryness under a stream of compressed air or nitrogen in a water-bath at 60°C.
4. Dissolve the residue in 0.2 ml of concentrated sulfuric acid and divide between two wells of a porcelain spotting tile.
5. Add 0.1 ml of sodium nitrite solution to one well and 0.1 ml of chromotropic acid solution to the other.
6. Heat the tile in a beaker over a boiling water-bath or on a hot plate at 80°C.
Results
The colours given by some common chlorophenoxy compounds are given in Table 24. These tests are not specific and can only be used to indicate the presence of chlorophenoxy compounds.
Chlorophenoxy compounds, 500 mg/l.
Clinical interpretation
Absorption of chlorophenoxy herbicides may lead to vomiting, diarrhoea, areflexia, muscle weakness, pulmonary oedema and coma, with death in severe cases. Alkalinization may increase the renal excretion of 2,4-D and other chlorophenoxy compounds, and also protect against systemic toxicity (see section 2.2.3).
6.29 Chloroquine
7-Chloro-4-(4-diethylamino-1-methylbutylamino)quinoline; C18H26ClN3; relative molecular mass, 320
Chloroquine is a derivative of 4-aminoquinoline and is commonly used to treat malaria. Chloroquine has a long half-life in the body (25-60 days) and several metabolic products are formed, initially by N-dealkylation and deamination. As little as 1 g of chloroquine may cause death in a young child, and fatalities in adults have occurred after the ingestion of between 3 and 44 g.
There is no simple test for chloroquine in biological fluids, but this compound and its metabolites can be detected by thin-layer chromatography of a basic extract of urine (see section 5.2.3). Like quinine, chloroquine fluoresces under ultraviolet light (254 nm and 366 nm), and this provides an additional feature to aid identification.
Clinical interpretation
Acute chloroquine poisoning can develop within 30 minutes of ingestion. Clinical features include nausea, vomiting, abdominal pain, diarrhoea, tinnitus, blurred vision, dizziness, agitation, hypotension, coma, convulsions and respiratory depression. Sudden cardiorespiratory arrest may occur in severe cases. Treatment is generally symptomatic and supportive, but the specific combination of diazepam and epinephrine has proved particularly effective.
6.30 Cholinesterase activity
Many insecticides, such as carbamate and organophosphorus compounds, interfere with nerve transmission by inhibiting acetylcholinesterase. Semi-quantitative measurement of plasma cholinesterase activity provides a simple method of assessing exposure to these compounds (see section 3.1.5).
Qualitative test
Applicable to plasma or serum.
Reagents
1. Dithiobisnitrobenzoate reagent. 5,5'-Dithiobis(2-nitrobenzoic add) (0.2 g/l) in sodium dihydrogen orthophosphate buffer (0.1 mol/l, pH 7.4).
2. Aqueous acetylthiocholine iodide solution (5 g/l).
3. Aqueous pralidoxime chloride solution (200 g/l).
4. Plasma or serum from an unexposed individual (control plasma).
Method
1. Add 2.0 ml of dithiobisnitrobenzoate reagent and 1.0 ml of acetylthiocholine iodide solution to each of three 10-ml test- tubes.
2. Add 20 µl of control plasma to one tube and 20 µl of test plasma to a second.
3. Add 20 µl of pralidoxime solution and 20 µl of test plasma to the third tube.
4. Vortex-mix the contents of all three tubes and allow to stand at room temperature for 2 minutes.
Results
The presence of an acetylcholinesterase inhibitor is indicated if the yellow colour in the control tube is deeper than in the test tube. If the colour in the tube containing pralidoxime is similar to that in the control tube, this provides further confirmation that an inhibitor of acetylcholinesterase is present in the sample (see section 3.1.5).
Clinical interpretation
Exposure to organophosphorus pesticides may cause bronchorrhoea, respiratory distress, excessive salivation, nausea, muscle weakness and eventually paralysis. Treatment is supportive, but should also include the administration of atropine and pralidoxime.
Exposure to carbamate pesticides may cause anorexia, abdominal pain, nausea, vomiting, diarrhoea, lacrimation, increased salivation, sweating, anxiety, ataxia and acute pulmonary oedema. Antidotal therapy with atropine may be indicated, but pralidoxime should not be used.
6.31 Clomethiazole
Chlormethiazole; 5-(2-chloroethyl)-4-methylthiazole; C6H8ClNS; relative molecular mass, 162
Clomethiazole is used as a hypnotic in elderly patients, as an anticonvulsant, and in the treatment of alcohol dependence and drug withdrawal. Less than 5% of an oral dose is excreted unchanged in urine, and a large number of metabolites have been identified. Clomethiazole has a characteristic smell on the breath and in stomach contents.
There is no simple qualitative test for clomethiazole, 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 poisoning with clomethiazole may cause sneezing, increased salivation, conjunctival irritation, hypotension, hypothermia, coma and respiratory depression. Ethanol potentiates the depressant effects of clomethiazole on the central nervous system, and these compounds are often encountered together in fatal cases. Treatment is symptomatic and supportive.
6.32 Cocaine
Methyl benzoylecgonine; (1 R,2 R,3 s,5 S)-2-methoxycarbonyltropan- 3-yl benzoate; C17H21NO4; relative molecular mass, 303
Cocaine is an alkaloid obtained from coca, the dried leaves of Erythroxylon coca and other species of Erythroxylon, or by synthesis from ecgonine. The hydrochloride salt is an effective local anaesthetic when used at concentrations of 10-200 g/l, but is normally only applied topically because of the risk of systemic toxicity if given by other routes.
Cocaine is frequently abused by injection or inhalation (sniffing, snorting) into the nasal passages; ingested cocaine has less effect owing to hydrolysis in the gastrointestinal tract. Cocaine free-base (crack) is very rapidly absorbed when inhaled into the nasal passages or smoked. The estimated minimum fatal dose in an adult is 1-2 g, but addicts may tolerate up to 5 g/day. The principal metabolites are benzoylecgonine, ecgonine and ecgonine methyl ester. Only 1-9% of an intravenous dose is excreted in urine as cocaine, while 35-55% is excreted as benzoylecgonine.
There is no simple qualitative test for cocaine, 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 cocaine poisoning may cause euphoria, restlessness, vomiting, pyrexia, mydriasis, delirium, tremor, hyperreflexia, hypertension, hyperventilation, convulsions and cardiorespiratory failure. Treatment is symptomatic and supportive.
6.33 Codeine
Morphine methyl ether; 3- O-methylmorphine monohydrate; C18H21NO3ÊH2O; relative molecular mass, 317
Codeine is a narcotic analgesic obtained either from opium or by methylation of morphine. Codeine is metabolized by O-demethylation and N-demethylation to give morphine and norcodeine, respectively, and by conjugation to form glucuronides and sulfates of both parent drug and metabolites. The estimated fatal dose of codeine in an adult is 800 mg. However, codeine is much less toxic than morphine, and death directly attributable to codeine is rare.
There is no simple qualitative test for codeine, but this compound and norcodeine 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 codeine gives rise to pinpoint pupils, hypotension, hypothermia, coma, convulsions, pulmonary oedema and cardiac arrhythmias. Death may ensue from profound respiratory depression. Naloxone rapidly reverses the central toxic effects of codeine (see section 2.2.2).
6.34 Copper
Copper salts such as copper(II) sulfate, chloride, and carbonate and cuprammonium salts such as cuprammonium carbonate (Cu(NH3)2CO3) are used as insecticides and fungicides. As with iron salts, copper salts often impart a blue or green colour to stomach contents. Cuprammonium salts are much more toxic than copper salts owing to rapid absorption and the intrinsic toxicity of the cuprammonium ion. Acute poisoning may also follow inhalation of metallic copper fumes or powder.
Qualitative test
Applicable to stomach contents and scene residues.
Reagents
1. Dithiooxamide in methanol (10 g/l).
2. Concentrated ammonium hydroxide (relative density 0.88).
Method
1. Slowly place 0.1 ml of sample on a filter-paper to give a spot no greater than 1 cm in diameter, drying with a hairdrier if necessary.
2. Expose the spot to ammonia fumes from concentrated ammonium hydroxide in a fume cupboard, and add 0.1 ml of dithiooxamide solution to the spot.
Results
Copper salts give an olive-green stain. Chromium salts also give a green stain, which is normally visible before the dithiooxamide is added. A number of other metals give yellow-brown or red-brown colours with this reagent.
Sensitivity
Copper, 1 mg/l.
Confirmatory test
Applicable to stomach contents and scene residues.
Reagents
1. Aqueous zinc acetate solution (10 g/l).
2. Ammonium mercurithiocyanate reagent. Mix 8 g of mercuric chloride and 9 g of ammonium thiocyanate in 100 ml of purified water.
3. Aqueous hydrochloric acid (0.01 mol/l).
Method
1. Place 0.1 ml of sample in a well of a porcelain spotting tile and add 0.05 ml of dilute hydrochloric acid.
2. Mix 0.1 ml of ammonium mercurithiocyanate reagent with 0.1 ml of zinc acetate solution and add to the sample in the well.
Result
A violet precipitate of zinc mercurithiocyanate forms in the presence of copper salts.
Sensitivity
Copper, 50 mg/l.
Quantitative assay
Applicable to plasma or serum (1 ml).
Reagents
1. Oxalyl dihydrazide reagent. Mix 8 ml of saturated aqueous oxalyl dihydrazide, 12 ml of concentrated ammonium hydroxide (relative density 0.88), 20 ml of aqueous acetaldehyde (400 ml/l) and 20 ml of purified water.
2. Aqueous trichloroacetic acid (200 g/l).
3. Aqueous hydrochloric acid (2 mol/l).
Standards
Dissolve 1.00 g of copper foil, mesh or wire in a minimum volume of nitric acid (500 ml/l) and make up to 1 litre with dilute nitric acid (10 ml/l). Dilute portions of this solution with water to give solutions containing copper ion concentrations of 1.0, 2.0 and 5.0 mg/l.
Method
1. Add 0.7 ml of dilute hydrochloric acid to 1 ml of sample or standard in a plastic centrifuge tube, mix and allow to stand for 15 minutes.
2. Add 1 ml of trichloroacetic acid solution and mix thoroughly.
3. Allow to stand for 15 minutes and then centrifuge for 5 minutes.
4. Add 3 ml of oxalyl dihydrazide reagent to 1 ml of supernatant, mix and allow to stand for 20 minutes.
Results
Read the absorbance of the solution at 542 nm against an aqueous blank (see section 4.5.2) carried through the procedure. Plot the absorbance of the standard solutions against copper concentration, and calculate the copper concentration in the sample. The calibration graph is linear for copper concentrations of 1-25 mg/l.
Sensitivity
Copper, 1 mg/l.
Clinical interpretation
Ingestion of copper or cuprammonium salts leads initially to gastrointestinal symptoms (metallic taste, nausea, vomiting, epigastric pain and diarrhoea). In severe cases, hepatic damage (particularly in children), renal damage, haemolysis, coma and circulatory collapse may ensue. Normal serum copper concentrations are 0.7-1.6 mg/l, but in severe acute poisoning concentrations greater than 5 mg/l may be attained. Treatment is symptomatic and supportive, but may also include chelation therapy.
6.35 Coumarin anticoagulants
Phenprocoumon (4-hydroxy-3-(1-phenylpropyl)coumarin; C18H16O3; relative molecular mass, 280) and warfarin (4-hydroxy-3-(3-oxo-1- phenylbutyl)coumarin; C19H16O4; relative molecular mass, 308) are substituted 4-hydroxycoumarins.
These compounds are widely used therapeutic agents; warfarin is also used as a rodenticide. Both inhibit blood coagulation by interfering with the synthesis of vitamin-K-dependent clotting factors. Their action is cumulative so that toxicity normally results from chronic administration. In contrast, severe toxicity may occur following a single large dose of a "superwarfarin" rodenticide such as difenacoum or brodifacoum.
The prothrombin time (see section 3.2.1) provides a simple, but nonspecific, means of measuring the severity of acute anticoagulant poisoning and of monitoring treatment. The simple method given below can be used to assess plasma phenprocoumon and warfarin concentrations.
Qualitative test
Applicable to plasma or serum (1.0 ml).
Reagents
1. Aqueous hydrochloric acid (1 mol/l).
2. n-Butyl acetate:chloroform:aqueous formic acid (850 ml/l) (60:40: 10).
3. Triethylamine (50 g/l) in n-hexane.
4. Silica gel thin-layer chromatography plate (20 × 20 cm, 20 µm average particle size; see section 4.4.1).
Standards
Serum containing phenprocoumon or warfarin concentrations of 0, 1, 5 and 10 mg/l.
Method
1. To 1.0 ml of sample or standard add 0.9 ml of dilute hydrochloric acid, 0.1 ml of acetone and 5 ml of chloroform.
2. Mix for 2 minutes on a mechanical shaker and then centrifuge for 10 minutes.
3. Remove the upper (aqueous) layer, filter the extract through phase-separating filter-paper and evaporate to dryness under a stream of compressed air or nitrogen.
Thin-layer chromatography
1. Dissolve the residues in 50 µl of chloroform, spot on the plate, and develop (10-cm run) in n-butyl acetate:chloroform:formic acid (saturated tank; see section 4.4.3).
2. Allow the solvent to evaporate completely, develop again in triethylamine: n-hexane, and inspect under ultraviolet light (366 nm).
Results
Warfarin (hRf about 87) shows a dark purple fluorescence, phenprocoumon (hRf about 95) a brighter purple. The plasma concentrations of either compound can be assessed by comparison with the results obtained from the standards.
Sensitivity
Warfarin or phenprocoumon, 0.5 mg/l.
Clinical interpretation
Features of acute poisoning with anticoagulants include the occurrence of petechiae, spontaneous bruising, haematoma formation and frank haemorrhage, especially from the genitourinary and gastrointestinal tracts. Serum concentrations of either compound greater than 5 mg/l are often accompanied by haemorrhagic complications. Phenprocoumon and warfarin have long plasma half-lives (6-7 days and 0.5-3 days, respectively), and patients with high serum concentrations should be treated promptly. Therapy consists of vitamin K supplementation until a prothrombin time in the normal range is obtained. In very serious cases, intravenous administration of fresh frozen plasma or purified clotting factors may be considered.
6.36 Cyanide
Cyanide (CN-) poisoning may be encountered after the inhalation of hydrogen cyanide (HCN) or after the ingestion of hydrocyanic acid or potassium or sodium cyanide. Complex cyanide solutions are used in metal electroplating and acidification of such solutions often leads to the release of hydrogen cyanide. Cyanogenic glycosides and other nitrile-containing compounds, such as amygdalin, which release cyanide in vivo occur in a number of plant tissues, including peach and apricot kernels, cassava root and lima beans.
Thiocyanate insecticides (ethyl thiocyanate, methyl thiocyanate) are also metabolized to cyanide ion in vivo and can cause serious toxicity. Cyanide is also a metabolite of sodium nitroprusside (used as a vasodilator) and some other nitrile-containing compounds, but cyanide poisoning is unusual in such cases. Inorganic thiocyanates and ferricyanide and ferrocyanide salts do not give rise to cyanide in vivo and are relatively nontoxic.
The qualitative test described below is based on the formation of a blue ferriferrocyanide complex (Prussian blue) with ferrous ions. Two microdiffusion methods (section 4.3.3) applicable to blood specimens are also given, both based on the liberation of hydrogen cyanide and subsequent formation of a coloured complex. The first, the p-nitrobenzaldehyde/ o-dinitrobenzene method, can be used to give a rapid semiquantitative result, while the pyridine/barbituric acid method should be used for a full quantitative analysis.
Qualitative test
Applicable to stomach contents and scene residues. Take care - specimens containing cyanides often evolve hydrogen cyanide if acidified.
Reagents
1. Aqueous sodium hydroxide solution (100 g/l).
2. Aqueous ferrous sulfate solution (100 g/l, freshly prepared in freshly boiled and cooled water).
3. Aqueous hydrochloric acid (100 ml/l).
Method
1. Dilute 1 ml of sample with 2 ml of sodium hydroxide solution.
2. Add 2 ml of ferrous sulfate solution.
3. Add sufficient hydrochloric acid to dissolve the ferrous hydroxide precipitate.
Result
A blue colour indicates the presence of cyanide. There are no common sources of interference.
Sensitivity
Cyanide, 10 mg/l.
Quantitative assays
Applicable to heparinized whole blood (0.1-1.0 ml), which can be stored at 4°C for 1-2 days if the analysis is delayed for any reason. (Cyanide in blood is less stable if stored at room temperature or at -20°C.)
1. p-Nitrobenzaldehyde/o-dinitrobenzene method
Reagents
1. Aqueous sodium hydroxide (0.5 mol/l).
2. Aqueous sulfuric acid (3.6 mol/l).
3. p-Nitrobenzaldehyde (0.05 mol/l) in 2-methoxyethanol.
4. o-Dinitrobenzene (0.05 mol/l) in 2-methoxyethanol.
Standard
Aqueous potassium cyanide (10 mg/l, i.e., cyanide ion concentration, 4 mg/l).
Method
1. Take three microdiffusion cells (see section 4.3.3) and add to each of the centre wells:
(a) 0.5 ml of p-nitrobenzaldehyde solution;
(b) 0.5 ml of o-dinitrobenzene solution;
(c) 0.1 ml of sodium hydroxide solution.
2. To the outer wells add 0.1 ml of:
- purified water (cell 1);
- potassium cyanide solution (cell 2);
- test blood specimen (cell 3).
3. To each outer well add 0.5 ml of purified water and, on the opposite side of the outer well, 1.0 ml of dilute sulfuric acid.
4. Seal each well using silicone grease, and carefully mix the components of the outer wells.
5. Incubate at room temperature for 20 minutes and then add 1 ml of aqueous methanol (1:1) to the centre wells.
6. Transfer the contents of the centre wells to 5.0-ml volumetric flasks and make up to volume with aqueous methanol (1:1).
Results
The red coloration obtained with cyanide-containing solutions is stable for about 15 minutes. Measure the absorbance of the solutions from cells 2 and 3 at 560 nm against the purified water blank (cell 1; see section 4.5.2). Assess the cyanide ion concentration in the sample by comparison with the reading obtained from the standard.
Sensitivity
Cyanide, 0.5 mg/l.
2. Pyridine/barbituric acid method
Reagents
1. Aqueous sodium hydroxide (0.1 mol/l).
2. Aqueous chloramine T solution (2.5 g/l). (N.B. Solid chloramine T is not stable, and fresh supplies should be obtained frequently.)
3. Aqueous sodium hydrogen orthophosphate (1 mol/l).
4. Pyridine-barbituric acid reagent. Stir 6 g of barbituric acid ( not diethyl barbituric acid, see section 6.9) into 6 ml of concentrated hydrochloric acid (relative density 1.18), and dilute with 30 ml of pyridine. Dilute the resulting solution to 100 ml with purified water. This solution must be freshly prepared.
5. Aqueous sulfuric acid (1 mol/l).
Standards
1. Cyanide stock solution. Dissolve 50 mg of potassium cyanide in 100 ml of aqueous sodium hydroxide (0.1 mol/l); cyanide ion concentration, 200 mg/l. Take care when using concentrated cyanide solutions.
2. Cyanide calibration solution. Dilute (1:99) the standard cyanide ion solution (200 mg/l) in aqueous sodium hydroxide (0.1 mol/l); final cyanide ion concentration 2 mg/l.
Method
1. Label seven microdiffusion cells (a) to (g) and add the reagents shown in Table 25 to the outer wells, reagent 5 (dilute sulfuric acid) being placed at the opposite side of the well to the others.
2. Add 2 ml of sodium hydroxide solution to each inner well, seal the cells with silicone grease, carefully mix the contents of the outer wells and incubate at room temperature for 4 hours.
3. Pipette 1.0 ml of the sodium hydroxide solution from each of the inner wells into prelabelled, stoppered test-tubes.
4. Add the following reagents sequentially and shake to mix:
(a) 2 ml of phosphate buffer;
(b) 1 ml of chloramine T solution;
(c) 3 ml of pyridine-barbituric acid reagent.
5. Allow to stand for 10 minutes at room temperature.
Results
The presence of cyanide is indicated by a red/blue colour. Measure the absorbance at 587 nm of each solution against the purified water blank (section 4.5.2), diluting if necessary to bring the test reading on to the scale. Construct a calibration graph using the results obtained from the standard cyanide solutions and calculate the cyanide concentration in the sample.
Sensitivity
Cyanide, 0.2 mg/l.
Clinical interpretation
Acute cyanide poisoning is characterized by ataxia, headache, anxiety, dyspnoea, confusion, coma, collapse, metabolic acidosis, pulmonary oedema and respiratory arrest. Cyanosis may not be present. Supportive treatment includes the administration of oxygen. A number of antidotes have been used, including dicobalt edetate, hydroxocobalamin, sodium nitrite and sodium thiosulfate.
In serious poisoning with cyanide salts or hydrocyanic acid, blood cyanide ion concentrations are usually of the order of 2-10 mg/l. The qualitative test described above has insufficient sensitivity to detect these concentrations and should only be used for stomach contents and scene residues. Quantitative cyanide measurements have little immediate relevance to the treatment of acute poisoning since, to have any hope of success, therapy must be commenced as soon as possible.
Cyanide may also be present in the blood of fire victims owing to inhalation of hydrogen cyanide from the partial combustion of wool, silk and synthetic polymers such as polyurethanes and polyacrylonitriles. In such cases, blood cyanide concentrations may range from 0.2 to 1.0 mg/l. Carbon monoxide is usually also present. Blood cyanide concentrations in heavy cigarette smokers may be as high as 0.3 mg/l.
6.37 Dapsone
Bis(4-aminophenyl)sulfone; C12H12N2O2S; relative molecular mass, 248
Dapsone is a structural analogue of the sulfonamide antibacterials and is used in the treatment of leprosy and dermatitis herpetiformis. Dapsone is metabolized to monoacetyldapsone, which also occurs in plasma, and to a variety of other products which are largely excreted in urine. Enterohepatic recirculation also occurs. In an adult, death may ensue 4-6 days after the ingestion of 1.5-5 g of dapsone.
The qualitative test described below can be used to give an estimate of the plasma dapsone concentration, if used with the appropriate standard solutions.
Qualitative test
Applicable to plasma or serum (0.5 ml).
Reagents
1. Aqueous sodium hydroxide (1 mol/l).
2. Chloroform:ethanol:glacial acetic acid (90:10:5).
3. Silica gel thin-layer chromatography plate (10 × 20 cm, 20 µm average particle size, see section 4.4.1).
Standards
Solutions containing dapsone concentrations of 5, 10, 20 and 50 mg/l in blank plasma, prepared by dilution from a methanolic stock solution (dapsone 1.00 g/l).
Method
1. Add 0.5 ml of sample or standard to 0.2 ml of sodium hydroxide solution and 6 ml of chloroform in a test-tube with a ground- glass stopper.
2. Stopper the tube, vortex-mix for 30 seconds and centrifuge for 5 minutes.
3. Discard the upper, aqueous layer, transfer 5 ml of the chloroform extract to a second tube and evaporate to dryness under a stream of compressed air or nitrogen.
Thin-layer chromatography
1. Reconstitute the extracts in 50 µl of methanol and spot on the plate. Spot extracts of the standard dapsone solutions on adjacent columns on the plate.
2. Develop the chromatogram (10-cm run) with chloroform:ethanol: glacial acetic acid (saturated tank, section 4.4.3).
3. Remove the plate, allow to dry and inspect under ultraviolet light (254 nm).
Results
Estimate the dapsone concentration in the sample by comparison with the results from the standard solutions, hRf values: dapsone, 57; monoacetyldapsone, 40.
Sensitivity
Dapsone, 2 mg/l.
Clinical interpretation
Ingestion of dapsone may cause anorexia, nausea, vomiting, abdominal pain, headache, tinnitus and blurred vision, with dizziness, agitation, coma and convulsions in severe cases. Haemolytic anaemia, methaemoglobinaemia, haematuria, jaundice and acute renal failure are further complications.
Plasma dapsone concentrations of 10 mg/l or more may be associated with toxicity. Repeat-dose oral activated charcoal is probably the most effective method of treatment, but methylene blue and exchange transfusion may be needed to treat methaemoglobinaemia and haemolytic anaemia, respectively.
6.38 Dextropropoxyphene
(+)-Propoxyphene; (+)-(1S,2R)-1-benzyl-3-dimethylamino-2-methyl-1- phenylpropyl propionate; C22H29NO2; relative molecular mass, 340
Dextropropoxyphene is a narcotic analgesic structurally related to methadone, and is often formulated together with paracetamol. Dextropropoxyphene is extensively metabolized to N-desmethyldextropropoxyphene (nordextropropoxyphene), which is the principal urinary metabolite.
There is no simple qualitative test for dextropropoxyphene, 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). In addition, paracetamol can be detected in urine using the o-cresol/ammonia test (see section 6.83).
Clinical interpretation
Acute overdosage with dextropropoxyphene gives rise to pin-point pupils, hypotension, hypothermia, coma, pulmonary oedema, convulsions and cardiac arrhythmias. Death may ensue rapidly from profound respiratory depression, especially if ethanol is also present. Naloxone rapidly reverses the central toxic effects of dextropropoxyphene (see section 2.2.2).
6.39 Dichloralphenazone
A stoichiometric complex of chloral hydrate and phenazone, C15H18Cl6N2O5; relative molecular mass, 519
Dichloralphenazone is a mild hypnotic and acts as a mixture of each of its components, chloral hydrate and phenazone, in vivo. Dichloralphenazone ingestion can therefore be detected in urine using the Fujiwara test for the chloral metabolite trichloroacetic acid. Phenazone does not possess hypnotic activity and can be detected by thin-layer chromatography of a basic/neutral extract of urine (see section 5.2.3), using acidified iodoplatinate reagent only.
Qualitative test
Applicable to urine. Fujiwara test - see carbon tetrachloride monograph (section 6.23).
Results
An intense red/purple colour in the upper, pyridine layer indicates the presence of trichloro compounds. The blank analysis excludes contamination with chloroform from the laboratory atmosphere.
This test is very sensitive and will detect ingestion of a therapeutic dose of dichloralphenazone or chloral hydrate 12-24 hours later. However, other compounds, notably the solvent trichloroethylene, also give rise to trichloroacetic acid in vivo, so that caution must be exercised in reporting results.
Sensitivity
Trichloroacetate, 1 mg/l.
Clinical interpretation
Acute poisoning with dichloralphenazone can cause vomiting, excitement, ataxia, confusion, drowsiness, stupor, hypotension, coma, cardiac arrhythmias, respiratory depression and pulmonary oedema. Treatment is normally symptomatic and supportive.
6.40 Dichloromethane
Methylene chloride; CH2Cl2; relative molecular mass, 85
Dichloromethane is widely used in paint strippers, sometimes together with toluene, and as a laboratory and industrial solvent. Its acute toxicity is largely due to direct depressant effects on the central nervous system. Dichloromethane is partially metabolized to carbon monoxide, which may contribute to chronic toxicity.
There is no simple test for dichloromethane in biological samples. However, measurement of carboxyhaemoglobin saturation is important in the assessment of chronic exposure to dichloromethane. Urine samples from patients exposed to dichloromethane do not give a positive result with the Fujiwara test.
Clinical interpretation
Exposure to dichloromethane may cause dizziness, numbness, irritability, fatigue, nausea, hypoventilation, pulmonary oedema and respiratory arrest. Recovery is normally rapid once the patient is removed from the contaminated atmosphere. Supplemental oxygen may be indicated, especially if features of carbon monoxide poisoning are present.
6.41 Digoxin and digitoxin
Digoxin (C41H64O14; relative molecular mass, 781) and digitoxin (C41H64O13; relative molecular mass, 765) are cardioactive glycosides obtained from the leaves of certain species of Digitalis (e.g. foxglove). Digoxin is widely used as an antiarrhythmic. Cardiac glycosides are also used as euthanasia agents in veterinary practice in certain countries. These compounds are very potent and there is no simple method for detecting them in blood or urine.
Qualitative test
Applicable to stomach contents and scene residues.
Reagents
1. Silica gel thin-layer chromatography plate (10 × 20 cm, 20 µm average particle size; see section 4.4.1).
2. Toluene:ethanol (7:3).
3. Chloramine T reagent. Mix 10 ml of aqueous chloramine T (30 g/l) and 40 ml of methanol containing 250 g/l trichloroacetic acid.
4. Aqueous perchloric acid (150 g/l).
Standards
Digoxin and digitoxin (both 100 mg/l) in chloroform.
Method
1. Add 5 ml of chloroform to 1 ml of sample, vortex-mix for 30 seconds and centrifuge for 5 minutes.
2. Discard the upper, aqueous layer 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 and reconstitute in 50 µl of chloroform.
Thin-layer chromatography
1. Divide the plate into two halves and spot 20 µl of the reconstituted extract and 10 µl of the standard solutions on to columns on both halves of the plate.
2. Develop the chromatogram (10-cm run) using toluene:ethanol (saturated tank, see section 4.4.3).
3. Allow the plate to dry, and spray one half of the plate with chloramine T reagent and the other half with perchloric acid solution.
4. Heat the plate in an oven at 100°C for 10 minutes.
Results
hRf values and colour reactions with the spray reagents are listed in Table 26.
Sensitivity
Digoxin or digitoxin, 10 mg/l.
Clinical interpretation
Digoxin and digitoxin are potent cardiotoxins and can give rise to fatal arrhythmias. Nausea, vomiting, diarrhoea, drowsiness and confusion occur in the early stages of poisoning with these compounds.
Hyperkalaemia and tachyarrhythmias are characteristic of severe poisoning. Treatment is generally supportive. Antigen-binding (Fab) antibody fragments will reverse toxicity in digoxin poisoning (see section 2.2.2), but are indicated only in very severe cases.
6.42 Dinitrophenol pesticides
These compounds have the general structure:
The dinitrophenols most commonly encountered are DNOC (2-methyl- 4,6-dinitrophenol; 4,6,-dinitro- o-cresol; C7H6N2O5; relative molecular mass, 198) and dinoseb (2-sec-butyl-4,6-dinitrophenol; C10H12N2O5; relative molecular mass, 240). DNOC is used as an insecticide and as a herbicide on fruit trees, while dinoseb is used mostly as a herbicide. Severe dinitrophenol poisoning may follow occupational exposure as well as ingestion. Skin is a common route of absorption and intense yellow staining may be diagnostic.
Both DNOC and dinoseb can be measured easily in whole blood since they show strong absorbance at 430 nm and the concentrations associated with toxicity are relatively high.
Quantitative assay
Applicable to whole blood (1 ml).
Reagents
1. Concentrated hydrochloric acid (relative density 1.18).
2. Aqueous sodium chloride (270 g/l) containing sodium carbonate (30 g/l).
Standards
Solutions containing dinitrophenol concentrations of 10, 20 and 50 mg/l in whole blood.
Method
1. Add 5 ml of butanone (methyl ethyl ketone) to 1 ml of sample or standard in a conical tube, and add 1 ml of the sodium chloride/ sodium carbonate solution.
2. Vortex-mix for 30 seconds, centrifuge for 5 minutes and transfer 2-ml portions of the extract to two clean tubes.
3. Add 50 µl of hydrochloric acid to one tube, vortex-mix for 10 seconds and centrifuge for 5 minutes.
4. Measure the difference in absorbance between the solutions at 430 nm (1-cm path-length cells).
Results
Construct a graph of the difference in absorbance against dinitrophenol concentration in the calibration solutions and calculate the dinitrophenol concentration in the sample.
Sensitivity
Dinitrophenol, 10 mg/l
Clinical interpretation
Dinitrophenols uncouple oxidative phosphorylation, and fatigue, excessive sweating, hyperthermia and thirst may be followed by exhaustion and death in severe cases. Toxic effects often appear at blood concentrations greater than 30 mg/l while concentrations greater than 60 mg/l are associated with severe toxicity.
6.43 Diphenhydramine
2-Benzhydryloxy- N,N-dimethylethylamine; C17H21NO; relative molecular mass, 255
Diphenhydramine is a widely used antihistamine. Less than 1% of the dose is excreted unchanged but N-dealkylation, oxidative deamination and conjugation give rise to a number of compounds which are excreted in urine.
There is no simple qualitative test for diphenhydramine and its metabolites, but this compound 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
Overdosage with diphenhydramine and other antihistamines may cause drowsiness, dizziness, dry mouth, headache, nausea, tachycardia, fever, hallucinations and tremor. In more severe cases, this may be followed by coma, convulsions and death. Treatment is symptomatic and supportive.
6.44 Diquat
1,1'-Ethylene-2,2'-bipyridylium ion; C12H12N2; relative molecular mass, 184
Diquat is a contact herbicide structurally related to paraquat, with which it is often formulated. Diquat is often encountered as the dibromide salt and death has been reported following the ingestion of as little as 2 g of diquat. Diquat and paraquat give highly coloured products with sodium dithionite, and this reaction forms the basis of the test described.
Qualitative test
Applicable to urine, stomach contents and scene residues.
Reagents
1. Sodium dithionite (solid, stored in a desiccator).
2. Aqueous ammonium hydroxide (2 mol/l).
3. Blank urine.
4. Urine specimen containing diquat ion (10 mg/l).
Method
1. Add 0.5 ml of ammonium hydroxide solution to the test solution and to the blank and standard urines (1-ml volumes) in separate test-tubes.
2. Add about 20 mg of sodium dithionite to each tube and mix.
3. If a colour forms in the test solution, agitate in air for several minutes.
Results
A yellow-green colour indicates diquat. Paraquat gives a blue/blue-black colour. This test cannot detect diquat in the presence of paraquat.
If the colour fades on continued agitation in air, diquat/ paraquat is confirmed - the original colour can be restored by adding more sodium dithionite.
Sensitivity
Diquat, 5 mg/l.
Clinical interpretation
Ingestion of diquat may cause irritation of the mouth and throat, epigastric pain, vomiting, diarrhoea, intestinal paralysis, malaise, excitement, convulsions, coma and hepatorenal failure. Unlike paraquat, diquat does not cause progressive pulmonary fibrosis. Treatment is largely symptomatic and supportive.
6.45 Ephedrine
(1 R,2 S)-2-Methylamino-1-phenylpropan-1-ol hemihydrate; C10H15NOÊ(H2O)1/2; relative molecular mass, 174
Ephedrine is a sympathomimetic agent. It is metabolized by N-demethylation to norephedrine (phenylpropanolamine), and by oxidative deamination and conjugation. Ephedrine is itself a metabolite of methylephedrine. The estimated minimum lethal dose of ephedrine in an adult is 4 g, but fatalities are rare.
There is no simple qualitative test for ephedrine, 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
Ephedrine overdosage may cause nausea, vomiting, headache, thirst, irritability, fever, tachycardia, sweating, dilated pupils, convulsions, coma and respiratory depression. Treatment is symptomatic and supportive.