Ann. clin. Biochem.

9 (1972) 35

TECHNICAL BULLETIN No. 24

Simple Tests to Detect Poisons

Prepared by a Joint Working Party 0/ the Association 0/ Clinical Biochemists and the Association 0/ Clinical
Pathologists

B. W. MEADE B. Wmoop D. J. BLACKMORE

(Chairf1/Q1/) Kingstonand Long Grove (Secretary) New Cross Hospital, Home Office Central Research
Group Hospitals, Kingston upon London, S£14 SER Establishment, Aldermaston, Berks.
Thames, Surrey
S. S. BROWN A. S. CuRRy R. GOULDING
Royal Infirmary, Edinburgh, Home Office Central Research New Cross Hospital,
EH39YW Establishment, Aldermaston, Berks. London, SE14 SER
G. HIGGINS H. J. S. MATIHEW M. G. RINSLER
Radcliffe Infirmary, Oxford, Royal Infirmary, Edinburgh, St. Stephen's Hospital, London,
OX26HE EH3 9YW SWI09TH

This review is a revised version of the Association clinician may well use it without any toxicological
of Clinical Pathologists' Broadsheet No. 52 (Curry, investigation. By contrast, it is rarely justifiable to
1966); it is intended to be a practical guide for use resort to forced diuresis, or to peritoneal- or haemo-
in the hospital laboratory, and not a survey of the dialysis unless qualitative and quantitative inform-
symptomatology and treatment of poisoning. Tests ation about the poison is available.
requiring elaborate apparatus have not been included,
nor have details of analyses (e.g, iron, glucose) with Interpretation ofresults
which the clinical chemist must already be familiar.
Comprehensive schemes of poison detection have Occasionally the detection of a foreign substance,
been published (Curry, 1969; Sunshine, 1969) but such as arsenic in gastric aspirate or paraquat in
complete identification and estimation is not always urine, is diagnostically definitive. Today, however,
practicable-or necessary-if results are to be ob- most cases of poisoning involve drug overdosage
tained quickly enough to have clinical relevance. The and caution is needed in interpreting qualitative
tests described here are intended to provide the maxi- analytical results. A patient may have taken a
mum of useful information in the minimum time, and therapeutic dose of a drug quite legitimately, and its
represent a compromise between speed, sensitivity detection in a urine or blood specimen will not in
and specificity. Whether the findings are sufficient itself indicate poisoning; this is most likely to
to suggest or confirm a diagnosis of poisoning in a happen with analgesic drugs such as aspirin, codeine
particular patient depends on the circumstances. A or paracetamol which can be obtained without
history of chronic drug ingestion or exposure to a prescription. Alternatively, patients may have been
toxic substance should be considered in relation to on long-standing regimes of sedative, tranquillising
the clinical features. In any event, discussion between or anti-epileptic drugs; poisoning may then occur on
the clinician and the clinical chemist is essential if account of chronic toxicity or an acute overdose. or
limited laboratory facilities are to be used to the best because of the ingestion of some quite unrelated
effect. substance. All of these possibilities should be con-
With the severely poisoned patient, biochemical sidered if the tests yield a positive result.
investigations such as blood gases, plasma electro- Several other aspects of the problem are also
lytes or serum enzymes may have greater bearing on important. There is a host of less common toxic
the immediate problems of management than toxi- substances whose detection requires more elaborate
cological screening, and these aspects have been analytical methods than those described here.
referred to in the tables below. This is because Furthermore, a positive finding does not automati-
symptomatic therapy, which is not specific for the cally rule out the possibility ofconcurrent disease, and
poison in question, must form the basis of treatment in particular a cerebrovascular accident. Negative
(Matthew and Lawson, 1970). In the few types of findings do not necessarily exclude a diagnosis of
poisoning, viz. carbon monoxide, cyanide, opiate poisoning. Much depends on the amount of active
and iron, where truly specific therapy is available, the substance absorbed, on individual sensitivity and on
35
36

FLOW-SHEET

I
Barbiturates B3
I
BLOOD

..-----:-
Solvent extraction ----
Glucose HI
I
Urea B2
I
URINE
I
Glucose, pH, Ketones UI
I
Salicylates U2
I
STOMACH ASPIRATE

I
I
Smell, colour, pH SI
Salicylates 82
I
Phenytoin SalicylatesB4 Phenothiazines U3 Phenothiazines S3
I I I I
Glutethimide Alcohols B5 Imipramine, Desipramine U4 Imipramine; Desipramine S4
I I I I
Methaqualone Carbon B6 Chloroform & related Chloroform & related
~ monoxide compounds US compounds S5
I I
Identification Measurement Paracetamol, Phenacetin U6 Arsenic, Antimony.
I Bismuth, Mercury S6
Paraquat, Diquat U7 I
I Bromate, Chlorate,
Meprobamate US Nitrate, Nitrite 87
I
Meprobamate S8 a
I
Carbromal S8 b
Solvent extraction

Barbizturates
--- mphetamines
----A .
I 1
Phenytoin Antihistamines
I I
Glutethimide Narcotics
I I
Methaqualone Phenothiazines
I I
Identification Tricyclicantidepressants
I
Other basic drugs
I
Identification

the lapse of time before the specimen is taken. Thus may be important to try to establish their chemical
gastric aspirate may have been taken too late after nature.
ingestion, or urine too soon. Careful enquiry should The following specimens should be sent for analysis,
be made about therapy administered before the each in a separate container, clearly marked with the
patient reaches hospital, or before the first specimen patient's full name, the date and time of collection,
is taken, because this may confuse or even invalidate and the nature of the specimen. A properly com-
toxicological screening. pleted and signed request form should of course
also be submitted.
Collection ofspecimens 1. Vomit, stomach aspirate and the first portion
of the lavage.
It is essential to obtain those specimens which are 2. A 10 m1 specimen of heparinised blood, 2 m1
most likely to yield useful information. When the of fluoridated blood and 10 ml of blood, without
poisoning is thought to be associated with the in- preservative or anticoagulant, taken on admission.
gestion of some specific material, any residual solid The use of swabs containing alcohols and of
or fluid found alongside the patient should be heparin containing phenolic preservatives should be
submitted for analysis; such substances may have avoided.
played no part in the episode, but subsequently it 3. The first urine specimen voided after admission
 37

and preferably before administration of diuretic Index 0/substances

drugs which may interfere with the tests.
4. Further samples of urine and blood should be Common proprietary drug names are italicised;
taken if the first analysis has proved negative and many others are indexed in Clarke (1969) or in Todd
there remain sound clinical reasons to suspect (1967).
poisoning.
These recommendations perhaps represent a Page
counsel of perfection; clearly in some cases blood Acetone as, UI 40
or urine specimens may not be readily available. Alcohols BS 40
Nevertheless, if medical and nursing staff are aware Allegron (Nortriptyline) TLC(U,S) 45
of the needs of the laboratory and the complexity of Amitriptyline (Tryptizol) TLC(U,S) 45
the analytical problem, there is a much better chance Amphetamine TLCm,S) 45
of a useful outcome to the investigations. Antimony S6 42
Arsenic S6 42
ANALYTICAL SCHEME Arvynol (Ethchlorvynol) SI 42
Aspirin B4,U1,S1 ~2
The analytical scheme involves preliminary tests A ventyl (Nortriptyline) TLC(U,S) 45
followed by solvent extraction procedures. Thin- Barbiturates B3, TLC (B) UV (B) 40-46
layer chromatography ('fLC) and ultra-violet (UV) Bismuth S6 42
spectrophotometry are used for further identification Bromate S7 43
or measurement. Bromvaletone SBb 43
Camphor SI 42
Uses 0/ the scheme Carbon Monoxide B6 40
The order in which the tests are performed will Carbrital (Carbromal and
depend to some extent on the information provided Pentobarbitone) B3,SBb 40,43
by the clinician. It should not be forgotten that Carbromal SBb 43
aspirin and barbiturates are still the commonest Chloral Hydrate US,SS 41,42
poisons; test for these first, preferably in blood. In Chlorate S7 43
the difficult case the whole scheme should be followed Chlorodyne US,SS 41.42
systematically. With the exception of the barbi- Chloroform US,SS 41,42
turates, for which a TLC procedure is recommended, Chlorpromazine
the spot tests should be carried out initially; other (Largacti/) U3,S3, TLC(U,S) 41-45
TLC procedures are intended only as a further screen Codeine TLC(U,S) 45
for the presence of drugs and for use in cases where Cyanide SI 42
a request for the detection of a particular drug has Desipramine (Perto/ran) U4, S4, TLC (U, S) 41-45
been made. Valuable information may be derived Dichloralphenazone
from UV spectra (Clarke, 1969; Sunshine and Ger- (WeI/dorm) U5,SS 41,42
ber, 1963) but such findings should be interpreted Diquat U7 42
with discretion. Disipal (Orphenadrine) TLC(U, S) 45
In every case a known negative sample should be Doriden (Glutethimide) B3,TLC(B), UV(B)40-46
tested at the same time and, where indicated, the Epanutin (Phenytoin) B3, TLC (B) 40,44
reaction of the unknown should be compared with Equanil (Meprobamate) US, sSa 42,43
that of a positive prepared with the authentic Ethanol B5 40
compound. Ethchlorvynol (Arvynol) SI 42
Reference has been made in the appendix to Glucose HI, UI 39,40
well-established quantitative methods for some Glutethimide (Doriden) B3,TLC(B), UV (B)~6
common poisons. Gramoxone (paraquat) U7 42
There are, as yet, no simple specific tests for the Imipramine (To/rani/) U4, TLC(U, S) 41,45
detection of the benzodiazepins and related com- Insulin (overdose of) HI 39
pounds (Librium, Valium, Mogadon, Nobrium). Iron SI 42
Ketones UI 40
Flow sheet Largactil
(Chlorpromazine) U3, S3, TLC (U, S) 41-45
B, Sand U stand for blood, stomach aspirate and Lysol SI 42
urine respectively, and the figures refer to the num- Mandrax (Methaqualone
bered tests in the list of Preliminary Tests. and Diphenhydramine) TLC (B), UV (B) 45,46
38

Page Copper/oil
Melsedin o-Cresol
(Methaqualone HCI) TLC (B), UV (B) 45,46 Dissolve 1 g of o-cresol in 100 ml of water.
Meprobamate (Equanil) U8,SSa 42,43 Dichromate reagent
Mercury S6 42 Dissolve 0.5 g of potassium dichromate (K.Cr10,)
MetaIdehyde B5 40 in 5 ml of water and add to a previously prepared
Methadone (Physeptone) TLC(U,S) 45 solution (CARE!) of 60 ml of concentrated sul-
Methanol B5 40 phuric acid in 40 ml of water.
Methaqualone (Mandrax,
Melsedin) TLC (B), UV (B) 45,46 Diphenylamine reagent
Methylamphetamine TLC(U,S) 45 Dissolve 0.5 g of diphenylamine in 100 ml of con-
Methyl salicylate 84, U2, SI, S2 40,42 centrated sulphuric acid.
Morphine TLC(U,S) 45 Fluorescein reagent
Nardil (phenelzine) SI 42 A saturated solution of sodium fluorescein in
Nicotine TLCCU) 45 ethanol.
Nitrate S7 43 FPN reagent (Forrest and Forrest, 1960)
Nitrite S7 43 Add 36 ml of 72 % perchloric acid to 144 ml of
Nor/lex (Orphenadrine) TLC(U,S) 45 water; add 100 ml of concentrated nitric acid to
Nortriptyline (Aventyl, 100 ml of water; dissolve 1 g of ferric chloride in
Allegron) TLC(U,S) 45 100 ml of water. Mix all three solutions.
Orphenadrine (Nor/lex, Fur/ural reagent
Disipal) TLC(U,S) 45 Add 1 ml of furfural to 9 rnl of absolute ethanol;
Panadol (paracetamol) U6 41 this reagent must be freshly prepared.
Paracetamol (Panadol) U6 41,46 Hydrochloric acid
Paraldehyde B5,SI 40,42 Make the following volumes of 36 % hydrochloric
Paraquat (Gramoxone, acid up to 100 ml with water: 18 ml (2 molfI); 9 ml
Weedol) U7 42 (l molfI).
Per to/ran (Desipramine) U4, S4, TLC(U, S) 41-45
Hydrogen peroxide solution (100 volumes)
Phenacetin U6 41
Imipramine reagent (Forrest, Forrest and Mason,
Phenelzine (Nardil) SI 42
U3, S3, TLC (U, S) 41-45 1960)
Phenothiazines
Phenytoin (Epanutin) 83, TLC(B) 40,44 Dissolve 0.2 g of potassium dichromate in 100 ml
Physeptone (Methadone) TLC(U,S) 45 of water; cautiously add 30 ml of concentrated
84, U2,S2 40,42 sulphuric acid to 70 ml of water; add 20 ml of 72%
Salicylamide
84,U2,S2 40,42 perchloric acid to 80 ml of water; add SO ml of
Salicylate
Surgical Spirit B5 40 concentrated nitric acid to 50 ml of water. Mix all
Tofranil (Imipramine) U4, S4, TLC (U, S) 41,42 four solutions.
Trichloroethylene U5,S5 41,42 Iodoplatinate spray
Tryptizol (Amitriptyline) TLC(U, S) 45 Dissolve 0.25 g of platinic chloride in 100 rnl of
Urea B2 40 water and add 5 g of potassium iodide. Add 2 ml of
Weedol (Paraquat) U7 42 concentrated hydrochloric acid.
Welldorm Mercurous nitrate spray
(Dichloralphenazone) U5,S5 41,42 A saturated solution of mercurous nitrate
(Hg2(NOa)2.2H20) in water.
Index 0/ reagents
a-Naphthol reagent
Except where indicated, these reagents are stable Dissolve 100 mg of a-naphthol in 5 ml of 2 molll
at room temperature. sodium hydroxide solution. This reagent must be
Acetic acid (glacial) freshly prepared.
Ammonium chloride solution Pal~wmchwridesomtwn
Dissolve 16 g of ammonium chloride in 100 rnl Dissolve 10 mg of palladous chloride fPdCI I ) in
of water. 100 rnl of water.
Ammonium hydroxide Phosphate buffer (pH 5.7)
Make the following volumes of '880' ammonia Dissolve 6.2 g of sodium dihydrogen phosphate
up to 100 ml with water: 85 ml (12 mol/I); 14 ml (NaH.PO,.2HI0) in 200 ml of water (solution A);
(2 mol/l), dissolve 7.1 g of disodium hydrogen phosphate
 39

(Na.HPO,.12H.O) in 100 ml of water (solution B). Trinder's reagent (Trinder, 1954)

Mix 47 ml of A with 4.0 ml of B and make up to Dissolve 10 g of mercuric chloride (HgCI.) in
100ml with water. The buffer is best freshly prepared 200 m1 of water; add 12 m1 of 1 mol/I hydrochloric
from the component solutions. acid and 4 g of ferric nitrate (Fe(NO.).. 9H.O).
Potassium carbonate solution Make up to 250 ml with water.
A saturated solution of potassium carbonate in
water. Suppliers ofspecial materials or equipment
Pyridine (analytical reagent grade)
Cavett Flask (Quickfit Scientific Supplies Co.
Salicylate standard (salicylic acid, 50 mg/IOO ml) and Quartz, assembly Ltd., Scientific House,
Dissolve 58 mg of sodium salicylate in 100 ml of 2BC) Vine Hill, London, E.C.l
water. (rei. 01-837 7765)
Sodium dithionite solution
Dissolve 0.1 g of sodium dithionite (NazSzOJ in Ethanol Estimation Kit Boehringer Corporation
10 ml of I mol/l sodium hydroxide solution. This (London) Ltd., Bilton
solution must be freshly prepared. House, Uxbridge Road,
Sodium hydroxide solutions London, W.5. (Tel.
Cautiously dissolve the following quantities of 01-5674551)
sodium hydroxide in water and make up to 100 ml: Sigma (London) Chemical
2 g (0.5 mol/l); 4 g (l rnol/l); 8 g (2 molfl); 20 g Co. Ltd., 12Lettice Street,
(5mol/]). London, S.W.6. (Tel.
Sodium nitrite solution 01-736 5823)
Dissolve 0.1 g of sodium nitrite (NaNOJ in 10 ml
of water. This solution must be freshly prepared. Labstix (No. 2810) Ames Co., Stoke Poges,
Slough, Bucks. (Tel.
Sodium phosphate solution 01-3692151)
Dissolve 10 g of trisodium orthophosphate
(Na.PO, . 12H zO) in 10 ml of water. Cellulose thin-layer plates Research Chemicals
Sodium sulphate (anhydrous) with fluorescent indicator Sales, Kodak Ltd.,
(No. 6065); Eastman Kirkby, Liverpool.
Solvents (analytical reagent grades) Chromatogram (Tel. 051-546 2101)
Chloroform, dichloromethane, ethanol, i-propanol Developing Apparatus
n-amyl methyl ketone, petroleum spirit (b.p, 60-80°). (Model 104)
Sulphuric acid
Cautiously add the following volumes of concen- Ucon Lubricant Union Carbide U.K. Ltd.,
trated sulphuric acid to 50 ml of water: 50 ml 8 Grafton Street, London,
(l0 molfl); 5 m1 (2 mol/I). W.1. (reI. 01-629 8100)

PRELIMINARY TESTS

Test Technical Notes Remarks

BLOOD
Bl Blood glucose
Carry out a blood glucose determination Hypoglycaemia is a feature of
diabetes and of overdosage with
insulin or hypoglycaemic agents;
it may also occur early in hepatic
failure caused by severe iron,
chlorpromazine or paracetamol
poisoning.
40

Test Technical Notes Re1TlQrks

H2 Blood urea
Carry out a blood urea determination.
B3 Barbiturates and related compounds
Test the blood for barbiturates and Whole blood is best used as the Qualitative detection is often all
related compounds by one of the TLC barbiturates are found in both that is necessary except in the
methods described on pages 44-46. cells and plasma. severely poisoned patient. In this
circumstance identification and
quantitative assay may be of value
in management, particularly if
phenobarbitone is involved.
B4 Salicylates
To 1 ml of plasma add 5 ml of Trinder's A violet colour in the super- Measurement of the plasma level
reagent; shake well; centrifuge. If the natant indicates salicylate or is of great value in deciding on the
supernatant is slightly turbid (due to salicylamide. For measurement need for forced alkaline diuresis;
lipid material), it may be clarified by treat 1 ml of the salicylate stan- for adults this should, in general,
shaking with 1 ml of chloroform. dard in the same way and read at be instituted if the level exceeds
540 nm (Trinder, 1954). 60 mg/l00 ml. A blood-gas analysis
must be carried out since severe
salicylate poisoning is characterised
by respiratory alkalosis and meta-
bolic acidosis. The results of
plasma electrolyte determinations
before treatment are unlikely to be
meaningful because of severe de-
hydration. Unless central depres-
sant substances have been taken,
impairment ofconsciousness oceurs
only in very severe poisoning with
acidaemia.
BS Alcohol and related compounds
Place 1 ml of blood with 1 ml of satur- Extreme cleanliness of containers Ethanol may be taken with other
ated aqueous potassium carbonate and purity of reagents is essen- drugs and may potentiate their
solution in the base of a Cavett flask. In tial. A green colour developing toxic effects. Measurement of the
the hanging cup put 0.5 ml of dichromate after i to 1 hour indicates ethanol, blood ethanol level may be helpful
reagent. Mix the flask contents and seal methanol, met- or paraldehyde or in assessing its contribution to the
the flask with Ucon lubricant. Incubate possibly acetone. The presence of clinical features of drug overdosage.
at 60" for 1 hour. If strongly positive the ethanol can be confirmed by the
blood ethanol level should be measured quantitative enzymatic method
(see appendix). given in the appendix.

B6 Carbon monoxide
Place 1 ml of blood with 1 ml of sul- The liberated palladium can be This test should not be ignored
phuric acid, 2 mol/I, in the base of a seen as a black film within about even if the blood is of normal
Cavett flask. Put 0.5 ml of palladium i hour if carbon monoxide is colour. The severity of poisoning
chloride solution into the hanging cup. present. Two quantitative pro- cannot be assessed by quantitative
Mix the blood and the acid, seal the top cedures are referred to in the carboxyhaemoglobin determina-
and leave to stand for 30 minutes. appendix. tion if oxygen has been adminis-
tered before the blood specimen is
taken.

URINE
VI Glucose, pH andketones
Dip a 'Labstix' briefly in urine and read Correlate with the blood glucose
after 10-15seconds. results. A positive ketone test may
indicate acetone or isopropanol
intoxication. This test may also be
positive in starvation or diabetic
ketosis.
 41

Test Technical Notes Remarks

UZ Salicylates
To 1 mI of urine add 4-5 drops of A violet colour indicates sali- A positive reaction may be obtained
Trinder's reagent. cylate or salicylamide. after the ingestion of a therapeutic
dose of a preparation containing
aspirin or p-aminosalicyclic acid.
A better indication of poisoning is
provided by determination of the
plasma salicylate level.

U3 Phenothiazines
To 1 mI of urine add 1 ml of FPN Blue to violet colours may deve- A strongly positive reaction is a
reagent. lop depending on the dose and good indication of the presence of
the metabolites present. phenothiazines but not necessarily
of overdosage.

U4 Imipramine and desipramine

To 1 mI of urine add 1 mI of imipramine Green or blue colours indicate If inco-ordination or coma is
reagent. imipramine or desipramine. To accompanied by hypertension or
obtain a known positive, grind hyperpyrexia, the possibility of
one Tofranil tablet (25 mg interaction of a 'therapeutic dose'
imipramine hydrochloride) with of a tricyclic antidepressant with a
10 mI of water. Take 0.1 mI monoamine oxidase inhibitor
diluted to 1 mI with water for the should be considered although no
test. simple tests are available for the
latter group of drugs.

US Chlorotorm and related compounds

To 1 mI of urine add 1 mI of sodium A red colour in the pyridine Carbon tetrachloride is much more
hydroxide,5 mol/I, and 1 mI, of pyridine; layer indicates chloroform, toxic than chloroform or tri-
heat in a boiling water-bath for 1 chloral hydrate, dichloralphen- chlorethylene and is only meta-
minute. azone or trichloroethylene. bolised in part to trichloromethyl
ChIorodyne and other medicines compounds. This test, therefore,
containing chloroform-water may fail in carbon tetrachloride
should also be considered. To poisoning; if this is suspected,
obtain a known positive use a evidence of direct hepatoxicity
dilute solution of chloral hydrate. should be sought by appropriate
N.B. This test is very sensitive serum enzyme assays.
and a blank must be carried
through the procedure at the
same time. Avoid the use of chlo-
roform since a positive result
may be obtained due to acciden-
tal contamination of the test
tubes or sample by this solvent.

U6 Paracetamol and phenacetin

(a) To 1 mI of urine add 1 mI of hydro- A red colour indicates a positive A positive reaction may be ob-
chloric acid, 1 mol/l, and, 2-3 drops of reaction. Aqueous paramino- tained after the ingestion of a
freshly prepared sodium nitrite solution phenol (0.2 g/IOO mI) may be therapeutic dose of a preparation
followed by 2-3 drops of a-naphthol used as a known positive. containing paracetamol or phen-
reagent. If negative proceed to (b) acetin. In overdosage with para-
cetamol, evidence of direct hepato-
toxicity should be sought by appro-
priate serum enzyme determina-
tion for up to 2-3 days after
admission.Consciousness is notim-
paired until hepatic coma ensues.
42

Test Technical Notes Re1l/Q1'ks

(b) To 0.5 ml of urine add 0.5 ml of A blue colour is observed if Overdosage with phenacetin may
concentrated hydrochloric acid and paracetamol or phenacetin were cause methaemoglobinaemia.
place in a boiling water-bath for 1 hour. present. Due to the hydrolysis of
Dilute 0.1 ml of this mixture to 10 ml drug conjugates, which are not
with water and add 1 ml of o-cresol detected by the above test (U6a),
solution and 4 ml of 2 molfl ammonium this test is the more sensitive
hydroxide. and is useful in cases where
several days have elapsed follow-
ing ingestion.
U7 Paraquat and diquat
To 1 ml of urine add 1 ml of sodium A strong blue or green colour If this test is positive it is possible
dithionite solution. indicates the presence of para- that clinical complications (renal,
quat or diquat or both. Where the hepatic and/or pulmonary) will
natural colour of the urine masks occur.
the colour produced, the sen-
sitivity of the test can be improved
by shaking with aluminia before
adding the dithionite solution.
US Meprobamate
Take 10 ml of urine and proceed as for
the test on stomach aspirate (SSa)

STOMACH ASPIRATE
Sl Smell,colourandpH
Note the smell, colour and general Characteristic smells such as
appearance of the stomach aspirate. those due to lysol, camphor,
Test the pH. phenelzine, methyl salicylate,
cyanide, paraldehyde, ethanol or
ethchlorvynol should be noted.
Recognisable tablets or capsules
may be present and colours may
derive from these. Blue ferrous
phosphate following ingestion of
'iron pills' is usually obvious. A
high pH may indicate ingestion
of alkalies.
S2 Salicylates
This test is preferably done on blood or Aspirin itself will not react with
urine. If using stomach aspirate proceed Trinder's reagent, since prelim-
as for test U2. inary alkaline hydrolysis to
salicylate is required.

S3 Phenathiazines
Carry out the test described under U3.
S4 Imipramine and desipramine
Carry out the test described under U4.
S5 Chloroform and related compounds
Carry out the test described under US.
S6 Arsenic, antimony, bismuth and
mercury
Place a quarter of the gastric aspirate in Clean the copper foil with sand-
a conical flask with an equal volume of paper until shiny before carrying
hydrochloric acid, 2 molfl, and 0.5 em out the test. Observe the colour
square of copper foil. Boil for 5 minutes. of the foil; a film of metallic
mercury is obvious and arsenic,
antimony and bismuth give
black stains. Confirmatory tests
are essential.
 43

Test Technical Notes Remarks

S7 Bromate, chlorate, nitrate and

nitrite
Add 1 ml diphenylamine reagent to two A blue colour indicates the pre-
drops of aspirate. sence of one of these substances.

sa Carbromal and meprobamate

Acidify a portion of the gastric aspirate If the sample contains fatty
with hydrochloric acid, 2 molfl, and material, this must be removed
extract with four times its volume of by extracting with an equal
chloroform. Separate and evaporate the volume of petroleum spirit prior
organic solvent to dryness. Dissolve the to extracting with chloroform.
residue in 0.5 ml of ethanol.

(a) Meprobamate
Evaporate 0.1 ml of the ethanolic A purple-black colour develop-
extract on a filter paper. Superimpose ing in about 1 minute indicates a
0.01 ml of furfural reagent and allow to carbamate. A positive control
dry. Expose to the fumes of concen- may be obtained by extracting
trated hydrochloric acid. one tablet of Equanil (400 mg of
meprobamate) with 5 ml of
hydrochloric acid, 1 mol/l, and
5 ml of chloroform, and treat-
ing 0.1 ml of the chloroform
extract as for the unknown.
Positive and negative controls on
the same piece of filter-paper are
essential for purposes of com-
parison. If the aspirate is very
fatty or otherwise unsuitable,
10 ml of urine can be used.
(b) Carbromal
Heat 0.1 ml of the ethanol extract to A red colour, due to eosin, in-' Very little carbromal is excreted
dryness on a white porcelain dish with dicates carbromal or brom- unchanged in the urine and, there-
2 drops of sodium hydroxide, 2 mol/l, valetone which are the most fore, this is only easily detectable
using a microbumer. Cool, add 2 drops common bromoureides. To ob- in gastric contents. The acute
of fluorescein reagent, 4 drops of glacial tain a known positive, extract clinical effects of overdosage with
acetic acid and 4 drops of hydrogen one tablet of Carbrital (250 mg Carbrital (pentobarbitone and car-
peroxide solution. Evaporate to dryness of carbromal) with 5 ml of 1 bromal) are entirely those of bar-
on a boiling water bath. mol/I hydrochloric acid and 5 ml biturate poisoning.
of chloroform, and treat 0.1 ml
of the chloroform extract as for
the unknown.

SOLVENT EXTRACTION PROCEDURES Whatman No. 90 paper into a dry conical tube.
3. Evaporate the extract to dryness. Redissolve
Barbiturates and related compounds in blood the residue in 0.1 ml of chloroform.
Note: If barbiturates are to be measured by the
1. To 1 ml of blood in a glass-stoppered tube, spectrophotometric method, they may be back-
add 1 ml of phosphate buffer and 10 ml of chloro- extracted from the initial chloroform extract into
form. Shake for five minutes and centrifuge. 1 ml of 0.5 mol/l sodium hydroxide solution,
2. Remove the upper (aqueous) layer with a scanned in the spectrophotometer between 220 and
Pasteur pipette and filter the chloroform through 320 nm and re-extracted into 10 ml of chloroform
44

(after adding 2 mol/l sulphuric acid) prior to TLC. together with 5 10'1 aliquots of authentic solutions of
This procedure reduces the amount of sample barbiturates or related compounds (l mg/mI in
required for analysis. chloroform). (N.B. Do not attempt to use the
sodium salts of barbiturates since these are insoluble
Barbiturates and related compounds in urine in chloroform.)
3. Develop the plate in chloroform/acetone (9 :1)
Proceed as for blood but using 10 mI of urine. mixture (CARE: chloroform and acetone can form
Because of the presence of metabolites, interpretation an explosive mixture in the presence of alkali and
of TLC plates is difficult and these drugs are pre- should be disposed of immediately after use).
ferably sought in blood. 4. Air-dry the plate and detect the spots by
examination in UV light (254 om) and subsequently
Basic drugs in urine by spraying with mercurous nitrate reagent. In
either case, barbiturates and related compounds
1. To 10 mI of urine in a glass-stoppered tube, appear as dark spots on a white background.
add 1 mI of 1 mol/l sodium hydroxide and 10 mI Approximate Rfvalues are given in Table 1.
of dichloromethane-isopropanol (9:1) mixture.
Shake for 5 minutes and centrifuge.
2. Remove the upper (aqueous) layer with a
Pasteur pipette and filter the organic phase through Table 1. Rf'values of barbiturates and related compounds
Whatman No. 90 paper into a dry conical tube. ill the two TLC systems
3. Evaporate the extract to approximately 0.1 ml
(CARE: some basic drugs, e.g. amphetamines, are Approximate Rf
highly volatile and may be lost if the extract is taken
to dryness). Drug System T System II
Note: The solvent extraction procedures may be
applied to stomach aspirate, but samples may first Phenytoin 0.50 -
require centrifugation of particulate material and Barbitone 0.60 0.20
Phenobarbitone 0.65 0.30
removal of fat by extraction with petroleum spirit. Cyclobarbitone 0.70 0.40
Butobarbitone 0.75 0.60
Pentobarbitone 0.75 0.80
CHROMATOGRAPillC PROCEDURES Amylobarbitone 0.80 0.75
Quinalbarbitone 0.85 0.85
Thin-layer chromatography Glutethimide 0.95 -

It is essential to realise the limitations of TLC

for the detection of poisons. Rf values are rarely
reproducible and should only be estimated in
relation to those obtained from simultaneous System l/: Cellulose/acetone-water
chromatography of authentic samples. The technique
is highly susceptible to interference from extraneous 1. Cut the cellulose plates to 4 x 10 cm size, and
compounds (e.g. nicotine, from the urine of heavy dip in sodium phosphate solution. Oven-dry at
smokers) and from urinary drug metabolites. A l00 cC for 30 minutes. Plates prepared in this way
positive result in one solvent system and with one may be stored indefinitely.
spray reagent is not an infallible proof of the pre- 2. Apply 1010'1 of the chloroform concentrate to
sence of a specific drug. For further confirmation, the plate alongside 10 10'1 aliquots of authentic
using other solvent systems and spray reagents, barbiturate solutions (1 rng/ml in chloroform). Dip
reference should be made to Clarke (1969) or the plate in an acetone/water (3:1) mixture and
Sunshine (1969). allow the acetone to evaporate at room temperature
by holding in a vertical position for 20 to 40 seconds.
Barbiturates and related compounds (CARE: This step is critical since over-drying
destroys the resolution.) Develop the plate with
System I: Silica gel/chloroform-acetone a-amyl methyl ketone in a covered beaker (or
preferably in an Eastman Chromatogram Developing
DC
1. Activate silica gel plates by heating at l00 Apparatus). Air-dry the plate and detect the spots
for 30 minutes shortly before use. as before. Approximate Rf values are given in
2. Apply 20 10'1 of the concentrate to the plate Table 1.
 4S

Methaqualone method for obtaining the ultra-violet spectra of

three drugs which are fairly readily distinguished
Apply 10 ,.,.1 of the chloroform concentrate ob- by this technique:
tained from a blood sample to a silica gel plate Blood (l ml) or stomach aspirate (1 ml) is shaken
alongside 10 ,.,.1 of an authentic solution of meth- for 5 minutes with 10 ml of re-distilled chloroform.
aqualone (1 mg/ml in chloroform). Develop the After centrifugation, the chloroform layer is separ-
plate in methanol-12 mol/l ammonium hydroxide ated and shaken for 10 minutes with 2 ml of O.SM
(100:1.5), air-dry and spray with iodoplatinate sodium hydroxide. The alkaline extract now contains
reagent. Methaqualone appears as a blue spot with barbiturates, and the chloroform layer glutethimide
an approximate Rf of 0.8. (N.B. Unchanged meth- and methaqualone.
aqualone may also be found in urine, and may be
extracted as a 'basic drug'.)
I•• IAlBITDIE 12-511II11011111I1
Basic Drugs pH 10
1. Apply 30 ,.,.1 of the concentrate to a silica gel
plate together with 10 ,.,.1 aliquots of authentic
sample solutions (l mg/rnl in chloroform).
2. Develop the plate in methanol-12 mol/l
ammonium hydroxide (100:1.5).
3. Air-dry the plate and detect the spots by spray- D·]
ing with iodoplatinate reagent. Most basic drugs
give blue colours against a pink background. A list
of approximate Rf values and colour reactions is
given in Table 2.
no 211 2100 25D 26D 270 2BD 29lI DI
D·9 Ib. GLUTETHIMIDE
Table 2. TLC Rf values and iodoplatinate colours of
some basic drugs 11l11g/loolllll in D·5M laDH
_Before h,drol,lil
Drug Approximate Rf Colour ....... -..-.-,

Desipramine 0.25 Blue ",\

Nortriptyline 0.35 Blue
Codeine
Morphine
0.40
0.45
Purple
Deep Blue
0·] Alter ~"
h,drol,lis
"
Amphetamine 0.45 White
Imipramine 0.50 Blue
Methadone 0.50 Purple
Methylamphetamine 0.50 White no 230 240 250 2&0 27D 210 290 DI
Chlorpromazine 0.50 Blue
Amitriptyline 0.55 Blue ..u O·g Ie. METHAQUALONE 10~1IIlI11JDII(1
Orphenadrine
Methaqualone
0.60
0.90 I
Blue
Blue ...
~ in ethanol I--i
in 2M H2SO, 1-----1
~ 0·&
N.B. Nicotine from the urine of heavy smokers gives a
strong brown spot with an approximate Rf of 0.52.

0·]
ULTRA-VIOLET SPECTROPHOTOMETRIC
PROCEDURES
The ultra-violet absorption spectrum of a drug
extract can give a useful lead to its identity but it 220 23IJ 240 250 2&0 27D 2BD 290 DI
should be noted that interference may occur when Wavelength I nllli
more than one drug is present; interpretation may Fig. I-Ultra-violet absorption spectra of barbitone (Ia),
then be difficult. Below are details of a simple glutethimide (Ib) and methaqualone (Ie),
46

Barbiturates Glutethimide-Dauphinais and McComb (1965),

Scan the alkaline extract immediately from 220 spectrophotometric.
to 320 nm against a blank of chloroform-saturated Methaqualone-Lawson and Brown (1967), spectro-
0.5 molJ1 sodium hydroxide. Compare the spectrum photometric.
with that shown in Fig. 1a. Re-scan after leaving Paracetamol-Routh et al. (1968) spectrophoto-
for 10 minutes at room temperature and note a metric.
change in absorbance at 235 nm; if there is a distinct
REFERENCES AND BIBUOGRAPHY
fall, suspect glutethimide. Add 1 ml of ammonium
chloride solution to both the sample and the blank Broughton, P. M. G. A rapid ultraviolet spectrophoto-
metric method for the detection, estimation and iden-
solution and re-scan the spectrum at this lower pH tification of barbiturates in biological material.
of 10. The common barbiturates display a peak of Biochem. J. 63 (1956) 207.
240 nm. Finally, add a few drops of 10 mol/I sul- Clarke, E. G. C. (Ed.) Isolation and Identification 0/ Drugs
phuric acid to both cells to give pH 2 or less, when the (1969) Pharmaceutical Press, London, £14.00.
absorption at 240 nm is reduced almost to zero. Curry. A. S. Simple Tests to Detect Poisoning. (1966)
Association of Clinical Pathologists Broadsheet No.
Glutethimide 52,25p.
Curry, A. S. Poison Detection in Human Organs. 2nd ed.
This is not quantitatively extracted into alkali, (1969) Thomas, Springfield, £5.78.
and should be sought in the chloroform fraction. Dauphinais, R. M., McComb, R. A specific procedure
Dry the chloroform phase with 2 g of anhydrous for serum glutethimide (Doriden) determination. Amer.
sodium sulphate, evaporate to dryness and re- J. C/in. Path. 44 (1965) 440.
dissolve in 2 ml of ethanol. Add 1 ml of 0.5 mol/l Forrest, I. S., Forrest, F. M., Mason, A. S. A rapid urine
NaOH and scan immediately from 220 to 320 nm. colour test for imipramine (TofraniI, Geigy): supple-
A peak at 235 nm which is considerably reduced mentary report with colour chart. Amer. J. Psychiat.
116 (1960) 1021.
after 10 minutes (Fig. 1b) due to alkaline hydrolysis Forrest, I. S., Forrest, F. M. Urine colour test for the
is indicative of glutethimide. detection of phenothiazine compounds. C/in. Chem. 6
(1960) 11.
Methaqualone Garvey, K., Bowden, C. M. The colorimetric deter-
Dry the chloroform phase with 2 g of anhydrous mination of barbiturates. Proc. Assoc. din. Biochem. 4
(1966) 20.
sodium sulphate, evaporate to dryness and re- Lawson, A. A. H., Brown, S. S. Acute methaqualone
dissolve in 2 ml of absolute ethanol or 2 ml of 2 mol/I (Mandrax) poisoning. Scot. med. J-. 12 (1967) 63.
sulphuric acid. Scan from 220 to 320 nm. Meth- Matthew, H., Lawson, A. A. H. Treatment 0/ Common
aqualone has the characteristic absorption spectra Acute Poisonings. 2nd ed. (1970), Livingstone, Edin-
shown in Fig. Ic, burgh, £1.00.
Routh, J. I., Shane, N. A., Arredondo, E. G., Paul,
APPENDIX W. D. Determination of N-acetyl-p-aminophtnol in
plasma. C/in. Chem. 14 (1968) 882.
References to simple quantitative methods of assay Sunshine, I. Handbook 0/ Analytical Toxicology. (1969)
for some common poisons Chemical Rubber Co., Cleveland, £14'00.
Barbiturates-Garvey and Bowden (1966), colori- Sunshine, I., Gerber, S. R. Spectrophotometric Analysis
metric; Broughton (1956), and Curry (1969, p, 59), 0/ Drugs. Including Atlas of Spectra (1963) Thomas,
spectrophotometric. Springfield, £4.50.
Carbon Monoxidt---eurry (1969, p. 179); Whitehead Todd, R. G. (Ed.) Extra Pharmacopoeia: Martindale,
and Worthington (1961). 25th ed. (1967) Pharmaceutical Press, London, £7.50.
Trinder, P. Rapid determination of salicylate in biological
Ethanol-The Boehringer or Sigma enzymatic fluids. Biochem. J. 57 (1954) 301.
estimation kits; note that this method is unsuitable Whitehead, T. P., Worthington, S. The determination of
for medico-legal purposes. carboxyhaemoglobin. C/in. chim. Acta 6 (1961) 356.

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