broadsheet 119: september · s2 salicylates aspirin itself will not react with trinder's...

L-p Association of Clinical Pathologists

Broadsheet 119: September 1988

Simple tests to detect poisoning

This broadsheet is based on the original version of Curry' and the revision published as technicalbulletin No 24 in the Annals ofClinical Biochemistry.2 As before, it is intended as a practical guide foruse in the laboratory rather than as a review of the symptoms and treatment of poisoning. Many ofthe tests previously described have been retained or are presented in modified form. This new versionalso takes into account recent advances in immunological tests, and cites, where relevant,recommended quantitative assays.

General strategy

Many poisons cause fluid or electrolyte imbalance anddamage the kidneys or the liver. Metabolic acidosis isanother common feature of severe poisoning dueeither to a direct action of the toxin itself or to cellularanoxia. Symptomatic treatment forms the basis ofmanagement and therefore biochemical tests musttake precedence over any toxicological investigations.Indeed, tests for poisons should be performed onlywhere the results are likely to affect treatment or havemedicolegal importance. Moreover, they should notbe undertaken without a full discussion between theclinician and the chemical pathologist or clinicalchemist, so that the order in which the tests are carriedout and the extent ofthe investigations required can bedecided. Thus if the circumstantial and clinicalevidence point to the ingestion of a specific poison, testfor this first. In more difficult cases- in unconsciouspatients where the evidence is equivocal-the schemeshould be followed systematically beginning with thespot tests on urine samples. Remember always to test aknown negative sample and an authentic positivealongside the unknown. Finally, strict attention mustbe given to health and safety precautions whenhandling the more dangerous reagents. Eye protectionand the use of a fume cupboard are essential, par-ticularly when spraying thin layer chromatographyplates.

INTERPRETATION OF RESULTSPositive qualitative results in urine samples orstomach contents must be interpreted with caution.Some ofthe tests are sensitive to therapeutic doses anddefinitive evidence for overdose is obtained only byquantitative analysis of the blood sample. This appliesparticularly to the spot tests for aspirin, paracetamol,and chloral hydrate (or dichloralphenazone) inges-

Accepted for publication 18 February 1988

tion, and to TLC procedures for tricyclic antidepres-sants, benzodiazepines, and barbiturates. Bear inmind also that a positive result does not rule outcategorically the presence of concurrent illness and inparticular a cerebrovascular accident.

Negative findings in no way preclude a diagnosis ofpoisoning. This scheme is not intended to be com-pletely comprehensive and if sound clinical reasons tosuspect poisoning persist, samples should be referredto a specialist toxicology laboratory where moreelaborate screening techniques can be applied.

UNITS OF MEASUREMENTGreat care should be taken to ensure that the clinicianunderstands fully the importance of quantitativeresults for commonly requested assays such assalicylate, paracetamol, and ethanol. Variation in theunits adopted still exists between hospitals, some usingmmol/l and others mg/l or mg/dl. It is advisable,therefore, always to speak directly to the clinician andto quote the "normal" or therapeutic range and thatassociated with toxic symptoms. These ranges shouldalso be included on report forms.

SPECIMEN COLLECTIONGastric contents (vomit, aspirate, first portion ofstomach washings)A 50 ml sample is sufficient. After prior examination(for undegraded tablets or capsules) remove any solidmaterial by centrifugation and conserve it. Carry outtests on the supernatant.

UrineObtain 50 ml of the first sample voided after admissionand preferably before any drugs are administered intreatment which might interfere with some of the tests.

BloodCollect 10 ml of lithium heparinised blood, 2 ml offluorided blood (for ethanol assay), and 10 ml of blood

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Association of Clinical Pathologists: Broadsheet 119. September 1988without anticoagulant. Avoid the use of swabs con-taining alcohols and heparin containing phenolicpreservatives such as cresol and chlorbutanol.

OthersAny materials (tablets, powders, liquids, bottles,syringes) found with the patient should be retained forsubsequent examination.For necropsy cases where putrefaction has begun,

specimens of cerebrospinal fluid and aqueous humourfrom the eye should be taken in the event of ethanoland glucose determinations being required.

Preliminary tests

URINEU, Glucose and ketones Check the urine with acommercial test strip.

Correlate with a blood glucose assay. A positiveketone result can indicate isopropanol or acetonepoisoning. Ketosis is often a feature of salicylatepoisoning, particularly in young children. The testmay also be positive in cases of starvation or diabeticketosis.

U2 Salicylate Add five drops of Trinder's reagent toI ml of urine.A violet colour indicates salicylate or salicylamide.

Diflunisal gives a very weak reaction even aftermassive overdose. Pink or blue colours are occasion-ally produced after ingestion of phenothiazines.

Therapeutic ingestion of aspirin can give a positivereaction. Tests for salicylate are best carried out bymeasuring the plasma salicylate concentration.

U3 Paracetamol Add 0 5 ml of concentrated hydro-chloric acid to 0-5 ml of urine and heat in a boilingwater bath for 10 minutes. Dilute 0- 1 ml ofthe mixturewith 10 ml of water and add 1 ml of o-cresol solutionand 4 ml of 2 M ammonium hydroxide.The paracetamol hydrolysis product (p-amino-

phenol) couples with o-cresol to give a deep blueindophenol dye.The test is very sensitive and can detect a therapeutic

dose. It is useful when several days have elapsedfollowing an overdose. If positive, the plasmaparacetamol concentration should be measuredimmediately.

U4 Ethanol and other volatile substances Place 1 ml ofurine in a test tube; apply one drop of potassiumdichromate reagent to a strip of Whatman glass fibrefilter paper. Insert the paper into the neck of the testtube and lightly stopper the tube. Put the tube in aboiling water bath for two minutes.A colour change to green indicates a positive result.

Other volatile reducing agents such as methanol,paraldehyde, and metaldehyde also react. Thepresence of methanol can be confirmed as follows:Add one drop of potassium dichromate reagent to1 ml of urine and leave at room temperature for fiveminutes. Add one drop of ethanol and a fewmilligrams of chromotropic acid. Carefully add 1 mlof concentrated sulphuric acid so that it forms a layerat the bottom of the tube. A purple colour at theinterface indicates methanol. Formaldehyde alsoreacts in this test.Measure the blood ethanol or methanol concentra-

tion, or both.

Us Phenothiazines Add 1 ml ofFPN reagent to 1 mlof urine.

Phenothiazines give pink, red, orange or bluecolours. Tricyclic antidepressants may also react togive green or blue colours. The presence of phenylketonuria or liver impairment can give false positivereactions.

U6 Imipramine and desipramine Add 1 ml of Forrestreagent to 1 ml of urine.Green or blue colours suggest imipramine,

desipramine, or trimipramine ingestion. Phenoth-iazines may also give a positive result, but the test ismore sensitive towards imipramine than FPN reagent.

There are no simple colour tests for other tricyclic(or quadracyclic) antidepressant drugs, but these canbe detected by thin layer chromatography.

U, Trichloro-compounds Add I ml of 5M sodiumhydroxide solution and 1 ml of pyridine to 1 ml ofurine. Heat in a boiling water bath for two minutes.A deep red colour developing in the pyridine layer

indicates intake of compounds such as chloralhydrate, dichloralphenazone, chloroform, chlorbutoland trichloroethylene. The test is very sensitive andwill detect chloroform in the laboratory atmosphere.Carry a blank urine sample simultaneously throughthe procedure together with 1 ml of an aqueoussolution of trichloroacetic acid (10 mg/l) as an authen-tic standard.Carbon tetrachloride is only partially metabolised

to trichloromethyl compounds and the test may fail todetect this compound. If carbon tetrachloride poison-ing is suspected appropriate serum enzyme assaysshould be carried out to test for liver damage.

U8 Paraquat Dissolve 100mg ofsodium dithionite in10 ml of M sodium hydroxide. Add 1 ml to I ml ofurine. An authentic solution of paraquat dichloride (5mg/l) must be tested at the same time as sodiumdithionite loses potency on storage.A blue colour indicates paraquat ingestion. Diquat

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Association of Clinical Pathologists: Broadsheet 119. September 1988

gives a green colour, but paraquat may also be present.The reducing' mixture can be prepared in an encap-

sulated form by mixing thoroughly sodium dithionite(10 g), disodium tetraborate (6 g), and sodium bicar-bonate (25 g). Pack the mixture into 1 g gelatinecapsules to produce about 30 capsules. To test forparaquat, tip the contents of one capsule into 5 ml ofurine and shake to dissolve. Sodium dithionite isslowly oxidised and a capsule from the same batchmust be tested on an authentic solution of paraquatdichloride (5 mg/I).A strong blue colour detected more than four hours

after ingestion suggests a poor prognosis. The severityof poisoning is more clearly assessed by measuring theplasma paraquat concentration.

STOMACH CONTENTS

S, Odour, colour, andpH Note the smell, colour andgeneral appearance of the sample. Test the pH.

Characteristic smells such as those due to phenolicdisinfectants, camphor, methyl salicylate, cyanide,ethanol and other organic solvents should be noted.Undegraded tablets or capsules may be present andcolours may derive from these. A green or blue coloursuggests the presence of iron salts. A high pH canindicate the ingestion of alkalies.

S2 Salicylates Aspirin itself will not react withTrinder's reagent as preliminary hydrolysis tosalicylate is required. Boil a 2 ml portion of the samplefor 10 minutes with 2 ml of 0.1 M hydrochloric acid.Filter if necessary, neutralise the filtrate with 0-1 Msodium hydroxide, and add three drops of Trinder'sreagent. A violet colour develops in the presence ofsalicylate.

This test is preferentially carried out on urine orblood.Measure the plasma salicylate concentration.

S3 Phenothiazines Carry out the test described underU5.

S4 Imipramine and desipramine Carry out the testdescribed under U6.

S5 Trichloro-compounds Carry out the test describedunder U7.

S6 Ferrous andferric iron (i) Add three drops of 2 Mhydrochloric acid and one drop of 1% (w/v) potas-sium ferricyanide solution to two drops of sample.(ii) Repeat test (i) but add one drop of 1% (w/v)potassium ferrocyanide solution.

(i) A deep blue precipitate indicates ferrous iron.(ii) A deep blue precipitate indicates ferric iron.Measure the serum iron concentration (see

Appendix).

S7 Arsenic, antimony, bismuth, mercury Clean' a 0-5cm square of copper foil with sand paper until shiny.Add this to a mixture of 15 ml of the sample and 3 mlconcentrated hydrochloric acid in a conical flask andheat gently for two hours. Remove the foil and rinsegently with distilled water.Mercury imparts a silvery deposit on the foil

surface. Arsenic, antimony, and bismuth give blackstains.

Confirmatory tests are essential. The clinicalfeatures should suggest the type of metal involved andquantitative assays on blood or urine, or both, shouldbe carried out, preferably by atomic absorption spec-trophotometry.

S8 Oxidising agents Add two drops of the filteredsample to I ml of diphenylamine reagent.A deep blue colour appearing immediately indicates

the presence of an oxidising agent. The test will detecthypochlorite (from domestic bleach), bromates,chlorates, dichromates, iodates, nitrates, nitrites andpermanganates.A light blue colour caused by organic material

should be ignored. Nitrate and nitrite from meatproducts will give a positive result.

S9 Cyanide To 5 ml of the filtered sample add I ml of4 M sodium hydroxide solution followed by five dropsof freshly prepared 10% (w/v) ferrous sulphate solu-tion. Add sufficient M hydrochloric acid to dissolvethe brown precipitate of ferrous hydroxide.A blue colour (Prussian blue) indicates the presence

of cyanide.Death usually ensues rapidly after exposure to

cyanide, although a small proportion ofpatients reachhospital and respond to antidotes and supportivetreatment.

BLOODB, Appearance Note the colour of the blood sample.A cherry red colour may suggest carbon monoxide

or cyanide poisoning. Confirmatory tests are given inthe section on quantitative assays. A chocolate browncolour may indicate methaemoglobinaemia followingpoisoning with chlorates, nitrates, nitrites or otheroxidising agents.

Confirmatory tests'are essential.

B2 Blood glucose Carry out a blood glucose deter-mination.

Paracetamol overdose results in high readings withreduction methods which can mask hypoglycaemia. Afalse decrease may occur with some glucose oxidasemethods. No interference occurs with the hexokinasemethod.

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Hypoglycaemia may indicate overdose with insulinor other hypoglycaemic agents in a diabetic patient.Acute ethanol poisoning often precipitateshypoglycaemia (especially in small children). It canalso occur in the early stages of liver damage followingparacetamol poisoning.

B3 Blood urea Measure the blood urea to excludeuraemia.

B4 Plasma osmolality andserum electrolytes Measurethe osmolality, preferably by freezing point depres-sion. Calculate the osmolar gap and the anion gap.An osmolar gap of greater than 10 mosmol/kg with

the freezing point depression technique suggests thepresence of ethanol, isopropranol, methanol or ethan-ediol (ethylene glycol). The presence (or subsequentdevelopment) ofa metabolic acidosis and an increasedanion gap suggests methanol or ethanediol intoxica-tion, although ethanol should first be excluded.

B5 Salicylates Add 5 ml of Trinder's reagent to 1 mlof plasma or serum; shake well and centrifuge.A violet colour in the supernatant indicates

salicylate or salicylamide ingestion.Measure the plasma salicylate concentration. An

alkaline diuresis is indicated if the plasma salicylateconcentration exceeds 500 mg/l (3-6 mmol/l). Severesalicylate poisoning is characterised by respiratoryalkalosis followed by metabolic acidosis; blood gasanalyses are essential. Impaired consciousness occursonly in very severe poisoning with acidaemia.

B6 Ethanol and related compounds Carry out the testdescribed for urine. If positive, measure the bloodethanol concentration, preferably by gaschromatography.

Ethanol can intensify the action of other depressantdrugs. Useful information is provided in unconsciouspatients admitted with head injuries and smelling ofdrink. Children are particularly at risk fromhypoglycaemia after swallowing ethanol. Methanolpoisoning can cause delayed coma with severemetabolic acidosis, an increased anion gap, and insome instances, raised blood glucose and serumamylase. Isopropanol resembles ethanol in toxicity,but has twice the potency. This substance is metabol-ised to acetone and gives rise to ketonuria. Thesevolatile compounds are best differentiated by gaschromatography.

B7 Organophosphate compounds Add 3 ml of dith-iobisnitrobenzoic acid solution and 0 1 ml of5% (w/v)acetylthiocholine iodide solution to each of two tubes.Add 20 Ul ofnormal serum to one tube and 20 p1 ofthe

sample serum to the other. Allow to stand for twominutes.

This test detects the inhibition of serum cholines-terase as denoted by the development of a much lowercolour intensity in the tube containing the test sample.

Measure the serum cholinesterase activity (seeAppendix).

Solvent extraction techniques

Liquid-liquid extraction methods applicable to bothurine and stomach contents have been described.3Commercially prepared columns containing gran-

ules of diatomaceous earth (Tox Elut, Extrelute-seeAppendix) offer a convenient means of extractingdrugs from urine. By using columns prebuffered to pH9, many of the weakly acidic and basic drugs can beextracted from the same portion ofa urine sample. Theneed to centrifuge and filter extracts is eliminated.1 Add 10-20 ml of urine to a ready buffered column(pH 9) and allow the sample to soak into the packingmaterial for two minutes.2 Place a 50 ml test tube under the column outletand pour three x 10 ml aliquots of a mixture ofchloroform and isopropanol (9:1) on to the column.3 Collect the eluates, place the test tube in a beaker ofhot water, and evaporate the extract to dryness undera stream of air or nitrogen.4 Dissolve the residue in 01l ml of the solventmixture.

Thin layer chromatography

ACIDIC AND NEUTRAL DRUGSReference solutions: Prepare solutions in chloroformof authentic samples of the drugs listed in table I atconcentrations of 1 mg/l.Method: Divide a ready made TLC plate (silica gel G,250 pm) into eight equal columns by scoring lines witha spatula and draw a horizontal line 10 cm from theorigin. Apply 10 p1 aliquots ofthe standards and oftheextract in the sequence shown in table 1.

Develop the plate in 100 ml of ethyl acetate:methanol: concentrated ammonia (85:10:5) and drythoroughly under a stream of cold air. Spray thecolumns or lanes in the order a to c, covering the rest ofthe plate while doing so.

Reference data for these and similar drugs are givenin table 1. Note that reference values are a useful guideonly; authentic compounds must be analysed along-side the sample extracts to obtain more reliableevidence of identity.

BASIC DRUGSReference solutions: Prepare solutions in chloroformof authentic samples of the drugs listed in table 3 at

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Association of Clinical Pathologists: Broadsheet 119. September 1988Table I Sequence ofapplying acidic and neutral drugsolutions to TLC plate

Column number Solution

2

3

45

678

Columnllanea 1,2

Amylobarbitone and phenobarbitoneSample extractPhenobarbitone and phenytoinSample extractGlutethimideSample extractMeprobamateSample extract

Spray reagentMercuric chloride-

diphenylcarbazone

b 3-6 Mercurous nitrate

c 7, 8 Furfuraldehyde

CommentsBarbiturates, phenytoin, and

glutethimide give white spotson a violet background

Barbiturates give immediateblack spots which turnchalky white as sprayingcontinues

Meprobamate gives a violetspot

Table 2 TLC datafor acidic and neutral drugs

Compound Reference value

Primidone 39Meprobamate 60Phenytoin 36Phenobarbitone 28Butobarbitone 38Amylobarbitone 36Quinalbarbitone 44Glutethimide 78

concentrations of 1 mg/ml.Method: Prepare a ready made TLC plate as describedabove and apply 10 p1 aliquots of the standards andthe extract in the order given in table 3.

Develop the plate in a mixture of methanol andconcentrated ammonia (100:1-5). Dry the plate in astream ofcold air until it no longer smells ofammonia(avoid using hot air which can volatilise certain drugs).Spray the columns or lanes in the order a to d (table 3),covering the rest of the plate while doing so.

Reference data and colour reactions for severalcommon basic drugs are given in table 4. Note thatreference data values are a useful guide only; authenticcompounds must be analysed alongside the sampleextracts to obtain more reliable evidence of identity.

DETECTION OF BENZODIAZEPINES IN URINE

These drugs are currently the most common cause ofdrug induced coma. The following TLC procedurerelies on the hydrolysis of benzodiazepines or theirmetabolites to aminobenzophenones which are detec-ted by the formation of an azo-dye (Bratton-Marshallreaction). Alternatively, a commercial immunoassay

system can be used, although caution is required (seenote iv below).Reference solution: Prepare a solution of 10 mg ofoxazepam and 10 mg of nitrazepam in 100 ml of Mhydrochloric acid.Method: Take two 20 ml screw capped glass bottles(McCartney bottles) and half fill one with the sampleand the other with blank urine. Add I ml of thereference solution to the blank bottle and 3 ml ofconcentrated hydrochloric acid to both bottles. Screwon the caps and heat the bottles in a boiling water bathfor 15 minutes. Cool the solutions, add 10 ml ofpetroleum spirit (boiling point 40-600) to each bottle,shake (five minutes), and centrifuge. Transfer thesolvent layers to 10 ml conical test tubes, evaporate todryness under a stream of air with heating, anddissolve the residues in 0 1 ml of methanol.Apply 50 p1 ofeach extract to a TLC plate (silica gel

G, 250 pm) and develop the plate in a mixture oftoluene and acetic acid (97:3). Dry the plate and spraywith the following reagents in sequence, drying theplate under warm air after each application:1 9 M sulphuric acid (spray lightly)2 Freshly prepared 1% sodium nitrite solution3 5% ammonium sulphamate solution4 A 1% solution of N-1-naphthylethylenediaminehydrochloride in 80 ml of acetone and 20 ml of water.A violet spot is formed if chlordiazepoxide,

diazepam, oxazepam, temazepam, medazepam andother benzodiazepines hydrolysed to 2-amino-5-chlorobenzophenone are present. Nitrazepam(hydrolysed to 2-amino-5-nitrobenzophenone) gives adiscrete purple band.Table 3 Sequence ofapplying basic drug solutions to TLCplate

Column number Solution

2345

678

CodeineSample extractAmitriptyline and nortriptylineSample extractChlorpromazineSample extractSuspected drug*Sample extract

*(Column 7 is reserved for an authentic solution of any basic drugwhich may be suggested by clinical or circumstantial evidence)

Columnllane Spray reagent Commenta 1, 2 Acidified iodoplatinate Most basic drugs give violet or

blue coloursb 3, 4 Mandelin's reagent Note any colours in visible and

(spray lightly) under uv (350 nm) lightc 5, 6 9 M sulphuric acid A series of spots with colours

(spray lightly) ranging from pink to blueindicates phenothiazinemetabolites

d 7, 8 Reagent with whichsuspected drug isknown to react

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Table 4 TLC datafor basic drugs

Mandelin's reagentReference Acidified Metabolite

Compound value idoplatinate Visible uv (350 nm) patterns

Desipramine 26 Violet BlueDihydrocodeine 26 Blue White One at Rf 16Codeine 33 BlueNortriptyline 34 Violet Violet Yellow (violet centre)Morphine 37 BlueImipramine 48 Violet Blue Quenches DesipramineMethadone 48 Pink (grey rim) Degradation product at Rf 15Chlorpromazine 49 Violet Pink Yellow (weak) ManyQuinine 50 Violet Blue (strong) Two above and below parent drugAmitriptyline 51 Violet Violet Yellow (violet centre) NortriptylineDothiepin 51 Red (blue rim) White Blue (weak) One or two below parent drugDiphenhydramine 55 Violet One or two below parent drugOrphenadrine 55 Violet Yellow Blue One below parent drugDextropropoxyphene 68 Violet Grey Blue streak at Rf 80; parent drug rarely

seen in urine extract

Notes(i) The information presented here is of necessitylimited and further data on alternative developingsolvents, additional spray reagents, and TLC charac-teristics should be sought in standard text books.45(ii) The procedures must be used on a regular basis toallow sufficient experience to be gained in interpretingthe plates.(iii) A standardised commercial TLC system (Toxi-Lab) is available which offers the advantage of a seriesof colour photographs of authentic urine extracts forcomparison with test chromatograms. This should beused only after undergoing preliminary training by thecompany as considerable experience is needed forreliable interpretation of the results.(iv) Several commercial immunoassay systems arenow available to detect drugs commonly abused ortaken in overdose. The selectivity of the antibodiesshould be carefully appraised when these are used. Forexample, tests for opiates may give positive resultswith codeine, dihydrocodeine, pholcodeine and othermorphine analogues in addition to morphine itself.Tests for amphetamines can also detect ephedrine,pseudoephedrine, phenylpropanolamine and othersympathomimetic amines. When used with discretion,however, these tests are useful adjuncts to TLCanalyses.

Quantitative assays

For some drugs and poisons measurement of theblood, plasma, or serum concentration can be an

essential requirement to the immediate managementof the patient. These are listed below, together withrecommendations on appropriate methods.

ALCOHOLS AND ETHANEDIOL

Commercial kits for ethanol determination are avail-able based either on the enzymic alcohol dehydrogen-

ase (ADH) method or radiative energy attenuationwhich is also based on ADH. These systems cross reactwith isopropanol, but show little cross reactivity withmethanol or ethanediol. Gas chromatography offers asimple and rapid means of identifying and measuringethanol, methanol, and isopropanol and is the methodof choice.6 Ethanediol can also be measured in bloodby gas-chromatography.7

BARBITURATESBlood barbiturate assays are only justified on anemergency basis if the clinician is considering activetreatment such as forced alkaline diuresis (FAD),haemodialysis, or haemoperfusion. It is essential todifferentiate between the intermediate acting bar-biturates (amylobarbitone, quinalbarbitone, pen-tobarbitone, butobarbitone, etc) and the long actingphenobarbitone ifFAD is contemplated as this treat-ment removes only the latter in large quantities.Homogenous immunoassay systems are ideal foremergency work, especially if antiserum selectivetowards phenobarbitone is available. Alternatively, anon-selective system can be used, coupled with adifferential TLC screen on urine or stomach contentsas described above. Ideally, gas chromatography,which permits simultaneous identification and quan-titation of barbiturates should be used.8

CARBON MONOXIDEDeliberate self-poisoning with carbon monoxide isalmost always evident when the patient is found.Accidental poisoning does occur, however, and maybe undiagnosed in the absence oflaboratory tests. Thegas disappears from the blood with a halflife offour tofive hours and in a much shorter time if oxygen isadministered. The spectrophotometric method ofBeutler and West is recommended.9 Alternatively,carboxyhaemoglobin can be quantified very rapidlyusing an IL Co-oximeter. Note that spectro-

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Index ofsubstances

AcetoneAlcoholsAmitriptylineAntimonyArsenicAspirinBarbituratesBenzodiazepinesBismuthBromateCamphorCarbon monoxideCarbon tetrachlorideChlorateChlorbutolChloralhydrateChoroformChlorpromazineCholinesterase inhibitorsCodeineCyanideDesipramineDextropropoxypheneDothiepinDichloralphenazoneDichromateDihydrocodeineDiphenhydramineDiquatEthanediolEthanolGlutethimideHypochloriteImipramineInsulin (overdose of)IodateIronIsopropanolKetonesMeprobamateMercuryMetaldehydeMethadoneMethanolMethyl salicylateMorphineNitrateNitriteNortriptylineOrganophosphateOrphenadrineParacetamolParaldehydeParaquatPermanganatePhenothiazinesPhenytoinPrimidoneQuinineSalicylamideSalicylateSurgical spiritTrichloroethyleneTrimipramine

U,U4, B6TLC (U, S)5757U2, S2, B5TLC (U, S)TLC (U)S7S8SIB,U7S8U7,S55U7, S5U7, S5US, S3B7TLC (U, S)Si, Sq, B,U6, S4, TLC (U, S)TLC (U, S)TLC(U,S)U7, S5S8TLC(U,S)TLC (U, S)U8B4U4, B6TLC (U, S)S8U6, S4,TLC (U, S)B2S8Si, S6B6U,TLC (U, S)S7U4TLC (U, S)U4, B6U2, SI,S2, B5TLC (U, S)S8

TLC (U, S)B7TLC (U, S)U3U4U8S8US, S3, TLC (U, S)TLC (U, S)TLC(U,S)TLC (U, S)U2, S2, B5U2, S2, B5U4, B6U7, SSU6, S4

Diphenylamine reagent:Dissolve 0 5 g of diphenylamine in 100 ml of concen-trated sulphuric acid.

Dithiobisnitrobenzoic acid solution:Dissolve 10 mg of 5, 5'-dithiobis-2-nitrobenzoic acidin 100 ml of phosphate buffer (pH 7 4).

Forrest reagent:Dissolve 0-2 g of potassium dichromate in 100 ml ofwater; carefully add 30 ml of concentrated sulphuricacid to 70 ml of water; add 20 ml of 72% perchloricacid to 80 ml of water; add 50 ml of concentratednitric acid to 50 ml of water. Mix all four solutions.

FPN reagent:Add 36 ml of 72% perchloric acid to 144 ml of water;add 100 ml of concentrated nitric acid to 100 ml ofwater; dissolve I g of ferric chloride in 100 ml ofwater. Mix all three solutions.

Furfuraldehyde spray:(a) Dilute 2 ml of redistilled furfuraldehyde to 100 mlwith acetone; (b) dilute 4 ml of concentrated sul-phuric acid to 100 ml with acetone. Spray with (a)first, followed by (b). The solutions must be preparedimmediately before use.

lodoplatinate spray (acidified):Dissolve 025 g of platinic chloride and 5 g ofpotassium iodide in 100 ml of water. Add 5 ml ofconcentrated hydrochloric acid.

Mandelin's reagent:Dissolve 0-5 g of ammonium vanadate in 1 5 ml ofwater and dilute to 100 ml with concentrated sul-phuric acid. Filter the solution through glass wool.

Mercuric chloride-diphenylcarbazone spray:(a) Dissolve 0-1 g of diphenylcarbazone in 50 ml ofethanol; (b) dissolve 1 g of mercuric chloride in 50 mlof ethanol. Mix (a) and (b) just before spraying. Thesolutions should be prepared daily.

Mercurous nitrate spray:A saturated solution of mercurous nitrate in water.

Ortho-cresol reagent:Dissolve I g ortho-cresol in 100 ml of water.

Potassium dichromate reagent:Cautiously add 60 ml concentrated sulphuric acid to40 ml of water and add a solution of 0-5 g potassiumdichromate in 5 ml of water.

Trinder's reagent:Dissolve 10gm of mercuric chloride in 200 ml ofwater and add 12 ml ofM hydrochloric acid and 4 g offerric nitrate (Fe (NO3)3 9H20). Make up to 250 mlwith water.

*Except where stated these reagents are suitable at roomtemperature.

Index ofreagents*

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Association ofClinical Pathologists: Broadsheet 119. September 1988photometric reference standards are now available(see Appendix).

IRONThe chelating agent desferrioxamine can precipitateprofound hypotension and shock when givenintravenously and the serum iron concentrationshould be measured before treating children with theantidote. The first blood sample should be takenbefore giving the antidote; subsequent samples takento monitor the progress of treatment must be treatedwith dithionite before analysis. About 20 mg of solidsodium dithionite is dissolved in 1 ml of serum.Haemolysed serum must not be used. The spectro-photometric method of Horak and Sunderman issuitable for emergency assays.'" Commercial kits alsoproduce rapid and reliable results (see Appendix).

PARACETAMOLThe colorimetric method of Glynn and Kendal iscumbersome and produces spurious results with ictericplasma or if salicylates are present." Kits based onhomogeneous immunoassay or the enzymaticcleavage of paracetamol to para-aminophenol withsubsequent colorimetric assay avoid these problemsand offer greater sensitivity (see Appendix).

PARAQUATMeasures used to treat paraquat poisoning are drasticand a plasma paraquat estimation can prevent theover treatment of patients not at risk. Colorimetric,'2and radioimmunoassay methods'3 have been devisedwhich give reliable results, although training andregular practice are required. Several laboratories aredesignated centres for paraquat assay and can becontacted through the National Poisons InformationService.

SALICYLATESThe method of Trinder'4 remains a rapid and simplemeans of measuring serum salicylate concentrations.Alternatively, enzymatic kits are now being marketedwhich are amenable to automation (see Appendix).

THEOPHYLLINETheophylline overdose can provoke serious hypoten-sion, cardiac arrhythmias, and convulsions which canbe delayed for several hours, particularly if slowrelease preparations have been taken. Severelypoisoned patients may require charcoal haemo-perfusion. Commercial immunoassay systems suitablefor rapid toxicological assays are available fromcompanies such as Abbott Laboratories and Syva.Alternatively, HPLC offers a rapid and simple conven-tional means of quantifying this drug.'5

Appendix

PROPRIETARY MATERIALSBarbiturate assayImmunoassay kit (Emit-tox serum barbiturate assay),Syva UK, Syntax House, St Ives Road, Maidenhead,Berkshire SL6 1 RD.

Carbon monoxideSpectrophotometric reference standards, TravenolLaboratories Limited, Wallingford Road, Compton,near Newbury, Berkshire.Co-oximeter, IL 282 Allied Laboratories (formallyInstrumentation Lab UK Ltd), Kelvin Close, Birch-wood Science Park, Warrington, Lancashire).

Cholinesterase activity assay kitSigma (London) Chemical Co Limited, Fancy Road,Poole, Dorset.

EthanolEnzymatic kit; Sigma (London) Chemical Co Limited,Fancy Road, Poole, Dorset.Radiative Energy Attenuation kit; Abbott Diagnos-tics Division, The Business Centre, Molly MillarsLane, Wokingham, Berkshire.

Extraction columnsExtrelut; BDH Chemicals Limited, Broom Road,Poole, Dorset.Tox Elut; Jones Chromatography Limited, NewRoad, Hengoed, Mid Glamorgan, Wales.

Iron assay kitLorne Laboratories Limited, PO Box 6, Twyford,Reading, Berkshire.

Labstix multiple urine test stripsAmes Division of Miles Laboratories Limited,PO Box 37, Stoke Court, Stoke Poges, Slough,Buckinghamshire. Boehringer Corporation (London),Bell Lane, Lewes, East Sussex.

Paracetamol assayImmunoassay kit; Abbott Diagnostics Division, TheBusiness Centre, Molly Millars Lane, Wokingham,Berkshire.Enzymatic kits; Cambridge Life Sciences plc,Cambridge Science Park, Milton Road, Cambridge;Porton Products Limited, CAMR, Porton Down,Salisbury, Wiltshire.

Salicylate assayEnzymatic kit; Porton Products Limited, CAMR,Porton Down, Salisbury, Wiltshire.

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1004 Association of Clinical Pathologists: Broadsheet 119. September 1988Toxi-LabMercia Diagnostics Limited, Mercia House, Broad-ford Park, Shalford, Guildford, Surrey.

B WIDDOPConsultant Biochemist,

Poisons Unit,New Cross Hospital,

Avonley Road,London SE14 5ER

Referes

1 Curry AS. Simple tests to detect poisoning. Association of ClinicalPathologists Broadsheet No 52. London: ACP/BMA, 1966.

2 Meade BW, Widdop B, Blackmore DJ, et al. Simple tests to detectpoisons. Ann Clin Biochem 1972,9:35-46.

3 Berry DJ, Grove J. Emergency toxicological screening for drugscommonly taken in overdose. J Chromatogr 1973;80:205-19.

4 Clarke's isolation and identification of drugs. Second ed. London:The Pharmaceutical Press, 1986.

5 Baselt RC. Disposition oftoxic drugs and chemicals in man. Seconded. Davis, California: Biomedical Publications, 1982.

6 Manno BR, Manno JE. A simple approach to gas chromato-graphic microanalysis of alcohols in blood and urine by a directinjection technique. J Anal Toxicol 1978;2:257-61.

7 Flanagan RJ, Dawling S, Buckley BM. Measurement of ethyleneglycol(ethane-1, 2-diol) in biological specimens using derivatisa-tion and gas-liquid chromatography with flame ionisationdetection. Ann Clin Biochem 1987;24:80-4.

8 Flanagan RJ, Berry DJ. Routine analysis ofbarbiturates and someother hypnotic drugs in the blood plasma as an aid to thediagnosis of poisoning. J Chromatogr 1977;131:131-46.

9 Beutler E, West C. Simplified determination of carboxy-haemoglobin. Clin Chem 1984;39:871-4.

10 Horak E, Sunderman FW Jr. Direct spectrophotometric methodsof measurements of serum iron and latent iron-bindingcapacity. Am J Clin Pathol 1974;62:133-4.

11 Glynn JP, Kendal SE. Paracetamol measurement. Lancet 1975;i:1147-8.

12 Jarvie DR, Stewart MJ. The rapid extraction of paraquat fromplasma using an ion-pairing technique. Clin Chim Acta 1979;94:241-51.

13 Levitt T. Determination of paraquat in clinical practice usingradioimmunoassay. Proc Anal Div Chem Soc 1979;16:72-6.

14 Trinder P. Rapid determination of salicylate in biological fluids.Biochem J 1954;57:301-3.

15 Orcutt JJ, Kozak PO, Gillman SA, Cummins LH. Micro-scalemethod for theophylline in body fluids by reversed-phase high-pressure liquid chromatography. Clin Chem 1977;23:599-601.

Requests for reprints to: Dr PD Dawkins, Musgrove ParkBranch, Taunton and Somerset Hospital, Taunton, SomersetTAI 5DA, England.

This Broadsheet has been prepared by the authors at theinvitation ofthe Committee on Technical Methods. Copyrightreserved by the Association of Clinical Pathologists. Furthercopies ofthis Broadsheet may be obtainedfrom the PublishingManager, Journal of Clinical Pathology, BMA House, Tavis-tock Square, WCIH 9JR.

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broadsheet 119: september · s2 salicylates aspirin itself will not react with trinder's...

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