Med. Sci. Law (1976) Vol. 16, No.

2 91

The Detection of Drugs in Biological Fluids*

ANN E. ROBINSON, BPharm, PhD, CChem, FRIC
The Department of Forensic Medicine, The London Hospital Medical College,
Turner Street, London

INTRODUCTION then the m a i n point of interest lies in whether

Many factors influence the success or failure a specific d r u g or alcohol has been taken. For
°f tests for the detection of drugs in body example, is this patient 'high' on a m p h e t a ­
fluids. It is the purpose of this paper to outline mine or is that patient 'stoned' on barbitu­
the more important of these factors in relation rates or methaqualone or u n d e r the influence
to laboratory tests as a n aid to the recognition of some other drug of the m o m e n t purchased
and investigation of the suspected non­ on the 'street' ? J u s t as the alcoholic will deny
medical use of drugs. I n its widest sense, the drinking alcohol, so the non-medical d r u g
non-medical use of drugs includes not only user will deny taking drugs. Laboratory
self-medication with any drug b u t the use of evidence should provide a more reliable
Prescribed drugs other t h a n in the m a n n e r guide for the clinician b u t the selection a n d
and for the purpose for which intended, collection of specimens, a n d the analytical
^ n t g s acting on the central nervous system as limitations, are important in relation to the
stimulants, depressants or as hallucinogens interpretation of results.
Predominate a n d provide the illustrative O n the other hand, quantitative d a t a m a y
examples for this discussion. disclose whether lack of response to d r u g
Why is there any need to detect drugs in treatment reflects a n inadequate dose so t h a t
hody fluids ? Does it matter if a person takes a n effective blood level of the drug is not
drugs on prescription or as self-medication? achieved. T h e lapse of response during treat­
Perhaps not until antisocial behaviour or the m e n t with nortriptyline of some psychiatric
a
P p e a r a n c e of toxic effects provides a patients has been correlated with low plasma
^tirnulus for legal or clinical intervention. levels of the d r u g : w h e n the dosage was
^Jiitc a p a r t from the various medicolegal adjusted for the individual patient the desired
"^plications of drug use or drug intoxication therapeutic effects were achieved (Kragh-
1X1
relation to criminal offences, a n objective Sorensen et al., 1974).
assessment of d r u g use is clearly relevant to Quantitative measurement of plasma (or
he clinical m a n a g e m e n t of a patient. T h e blood) levels is appropriate in patients show­
Moratory assessment of d r u g taking, based ing symptoms of intoxication, or adverse
n
° analysis of body fluids, m a y offer a more effects as well as in those more obviously
^tisfactory explanation of a patient's condi- overdosed. W h e r e drugs are prescribed on a
l
° n a n d symptoms t h a n the story he tells. chronic basis, as in epilepsy (or in rheumatic
Qualitative evidence of drug taking m a y disorders) regular monitoring of the blood
Office when there is a possibility that the levels of the drugs should ensure that d r u g
Patient is not taking prescribed medication dosage can be adjusted before intoxication
w
?* e l l as in those circumstances where there becomes obvious a n d distressing to the patien t.
^ s u s p i c i o n of the non-medical use of drugs;
*A pi " "
« y L presented during the 5th Inter- M E T H O D S OF A N A L Y S I S
e n a r e c t u r e
l0
» nal Conference of the International Association Accepting that there is a need for analysis, the
A c
j n« d e n t and Traffic Medicine and the 3rd following factors must be considered in
> ^national Conference on Drug Abuse held in designing a n d developing suitable methods:
n d
° n during September, 1975.
 92 Med. Sci. Law (1976) Vol. 16, No. 2

1. T h e nature of the d r u g a n d its meta­ undergoing a molecular rearrangement to

bolites; norpropoxyphene amide. T h e latter sub­
2. T h e distribution a n d elimination of the stance, r a t h e r t h a n the actual metabolite, will
drugs a n d its metabolites in man; be found in solvent extracts obtained from
3. T h e n a t u r e a n d a m o u n t of specimens alkaline conditions.
available for analysis a n d the practical I n a few instances, the very extensive
limitations of the analytical methods. metabolism of a d r u g m a y necessitate
It is well known that heroin administered development of a n analytical method for the
to m a n is rapidly de-acetylated in the blood detection of the major metabolite rather than
a n d that, unless very special precautions are the drug. A p a r t from the example of mor­
taken in the collection a n d analysis of blood phine as a metabolite of heroin, there is the
or urine specimens, only morphine a n d its detection of benzoyl ecgonine in urine as
conjugate with glucuronic acid will be found. evidence of cocaine administration. It seems
I n morphine-dependent patients the urinary likely also that evidence of methyl phenidate
excretion products include the ethereal administration is more readily obtained from
sulphate, normorphine a n d normorphine the detection of ritalinic acid in urine rather
glucuronide, albeit in minor proportion in t h a n the unchanged drug.
comparison with the total morphine (Yeh, T h e distribution a n d elimination of a drug
1975). Recognition of metabolites as well as must be taken into account in relation to tests
the unchanged drug in urine, for instance, for drugs or d r u g metabolites in body fluids.
provides additional a n d more convincing It is only comparatively recently that detailed
evidence of actual d r u g taking. T h e surrepti­ pharmacokinetic d a t a in m a n has become
tious addition of a drug to a urine specimen available whereas, in the past, analytical
that is to be sent for analysis is not u n k n o w n procedures were evaluated in practical terms.
as a ruse to obtain a prescription for a T h u s , a n ether extract of a 5 ml volume of
dependence-producing d r u g . urine adjusted to p H 10 carefully evaporated
Analytical methods are usually developed a n d subjected to gas chromatography allowed
to allow recovery of a n d characterization of detection of a m p h e t a m i n e for at least 48
the u n c h a n g e d d r u g even when urine testing hours following a 5 m g dose.
is contemplated a n d only a very small Some of the m a n y factors which influence
proportion of a n administered dose is excreted drug distribution a n d elimination m a y
unchanged. However, some metabolites m a y be
be co-extracted, sometimes quantitatively. illustrated by published work on diazepam.
For example, m e t h a d o n e is excreted un­ This drug is very widely used as a tran­
changed a n d partly as 2-ethylidene-l,5- quillizer a n d sedative a n d is of particular
dimethyl-3,3-diphenylpyrrolidine (the major interest since it m a y adversely influence
metabolite), 2-ethyl-5-methyl-3,3-diphenyl-l- driving ability a n d also m a y be accepted by
pyrroline, the ring hydroxylated analogues of those abusing drugs when their preferred
the two cyclic metabolites a n d lesser amounts drugs are in short supply.
of several other metabolites (Sullivan a n d Hillestad et al. (1974) measured the serum
Due, 1973). Solvent extraction from alkaline concentration of normal subjects given 20 tog
conditions allows recovery of methadone, the diazepam doses a n d showed that the route of
major metabolite a n d the pyrroline metabo­ administration affected the values; the clinical
lite which m a y then be detected on thin-layer effects were related to the serum levels. They
a n d gas chromatograms of the extracts. found also that the metabolite N-desmethyi*
Similar remarks apply to m a n y other drugs diazepam was not formed in significant
—for example, propoxyphene (Nash et al., amounts following a single dose of diazepam-
eS

1975). However, in this instance a n artefact Repeated administration of diazepam ( H i H '

is introduced during analysis: the mono- tad et al., 1974; Korttila et al., 1975;
desmethyl derivative which is excreted in Linnoila et al., 1975) m a y lead to elevated
urine is unstable under alkaline conditions, serum levels of N-desmethyldiazepam, though
it does not a p p e a r that this metabolite >*
 Robinson: Drugs in Biological Fluids 93

active pharmacologically. T h i s observation convenient specimen—usually urine or blood-

might indicate that diazepam is acting as a n If the time of dosage is known or intoxication
inducer of its own metabolism, but it seems is suspected then determination of blood (or
more likely t h a t the metabolite is retained i n serum) levels may be desirable especially
some tissues for long periods. Such findings when this m a y be correlated with the
may be important when considering the pharmacological effect, i.e. when the d r u g in
analytical criteria for the demonstration of the blood is in equilibrium with the drug at
significant d r u g use by a person involved in the site of its action.
an accident whether in the home, at work or in Limitations imposed by the restricted
traffic. volumes of specimens available are m u c h
Klotz et al. (1975) found that in normal greater when the identity of the d r u g or
individuals, the terminal plasma half-life of drugs is not known, a factor not always
diazepam [t^ (P)] varied with age from about appreciated by the physician who submits a
20 h at 20 years increasing linearly with age 10 ml urine sample or 1 ml of clotted blood
to about 90 h a t 80 years. However, the with the request 'analysis for drugs, please'.
plasma clearance of diazepam in normal T h e published literature abounds with
subjects was not age-dependent. Neither the methods a n d modifications of methods for
Plasma binding ( 9 7 4 per cent) nor the blood: the detection a n d quantitation of drugs in
Plasma concentration ratio (0-58) altered blood a n d urine—many a p p e a r to be acade­
With age. Pharmacokinetic d a t a for diazepam mic exercises. I n contrast, the recent demon­
modified also in those with liver disease: stration of methadone a n d its metabolites in
reduced clearance of the drug was observed sweat by Henderson a n d Wilson (1973)
°y Klotz a n d his colleagues (1975) w h o suggest t h a t this specimen might perhaps b e
advised caution in the chronic administration used for monitoring purposes. Saliva analysis
°f this drug in patients with acute or chronic has been used to monitor a n d adjust dose
Parenchymal liver disease. schedules of theophylline in children attend­
Plasma diazepam concentrations may be ing a n allergy clinic (Johnson et al., 1975).
niarkedly affected by chronic alcohol intake, T h e detection of A -tetrahydrocannabinol
9

d e d u c e d plasma concentrations of diazepam in the saliva of cannabis users (Just et al.,

W e
r e observed by Sellman a n d others 1974) has been achieved but requires the use
(Sellman, K a n t o et al., 1975; Sellman, of a mass spectrometer. M o r p h i n e has been
fekkarinen et al., 1975) following oral or detected in saliva at least for 3-4 hours after
intravenous doses of the d r u g administered the last dose of morphine during chronic
t° chronic alcoholic patients during a n administration on a 6-hourly basis (Goro-
alcohol-free period. T h e y concluded that d e t z k y a n d Kullberg, 1974).
Unpaired intestinal absorption accounted for A p a r t from limitations imposed by speci­
reduced plasma levels w h e n the d r u g was men volumes, each analyst is limited by the
iv
8 e n orally a n d also that the distribution was equipment a n d facilities available to him.
accelerated by circulatory changes after Depending upon case-load, routine analyses
Parenteral administration (tachycardia, vaso­ using published research methods, particu­
d i l a t i o n , rise in blood pressure). T h e r e was larly those involving mass fragmentography,
n
° evidence of increased metabolism of
m a y not be feasible. M o r e encouraging is the
t a z e p a m in alcoholics, as is often seen with
development of sensitive immunological re­
ner drugs (warfarin, tolbutamide, pheny-
0 l n
agents such as are now available for a variety
, m e p r o b a m a t e , etc.).
of drugs. Essentially, the test specimen is
mixed with antibody complexed to a labelled
E
& M A N D S O N THE ANALYST drug, thus disturbing the equilibrium.
*yiat d e m a n d s are m a d e of the analyst? Measurement of the disturbed equilibrium
t h o d s need to be rapid a n d to provide by radioactivity for a radioactive label,
e

"equivocal identification of a large n u m b e r electron spin resonance spectrometry for a

drugs in the m i n i m u m volume of a spin-label or enzyme activity for a n inhibited
94 Med. Sci. Law (1976) Vol. 16, No. 2

e n z y m e allows estimation of t h e a m o u n t of Hillestad L., Hansen T., Melsom H. and Drivenes A.

d r u g (and cross-reacting substances) in the (1974) Diazepam metabolism in normal man. Clin.
Pharmacol. Ther. 16, 479-484.
specimen. A simple immunological m e t h o d
Johnson G. F., Dechtiaruk W. A. and Solomon H. M-
allows assessment of t h e drug-antibody (1975) Gas-chromatographic determination of theo­
reaction by inhibition of the agglutination of phylline in human serum and saliva. Clin. Chem. 21»
specially t r e a t e d red blood cells. Such 144-147.
methods a r e suitable for small volume Just W. W., Filipovic N. and Werner G. (1974)
Detection of A*-tetrahydrocannabinol in saliva of
samples a n d m a y be very r a p i d t h o u g h
men by means of thin-layer chromatography and
expensive. All t h e reagents m a y cross-react mass spectrometry. J . Chromatogr. 96, 189-194.
w i t h closely related substances other t h a n the Klotz U., Avant G. R., Hoyumpa A., Schenker S. and
d r u g of interest. While this m a y be a d v a n t a ­ Wilkinson G. R. (1975) The effects of age and liver
geous for screening purposes, all positively disease on the disposition and elimination of
diazepam in adult man. J. Clin. Invest. 55, 347-359.
reacting specimens must be analysed by
Korttila K., Manila M. J. and Linnoila M. (1975)
alternative methods to establish t h e identity Saturation of tissues with N-desmethyldiazepam as
of the suspected d r u g beyond d o u b t . I t is a cause for elevated serum levels of this metabolite
encouraging to note t h a t detection of after repeated administration of diazepam. Acta
cannabis products in u r i n e by a r a d i o ­ Pharmacol. Toxicol. 36, 190-192.
immunoassay m e t h o d has been achieved by Kragh-Sarensen P., Hansen C. E., Larsen N.-E.>
Naestoft J. and Hvidberg E. F. (1974) Long-term
M a r k s et al. (1975); t h e m e t h o d a p p e a r s very treatment of endogenous depression with nor­
suitable for clinical a n d epidemiological triptyline with control of plasma levels. Psychol. Mid.
purposes b u t w i t h o u t additional confirmatory 4, 174-180.
evidence of t h e identity of the substances Linnoila M., Korttila K. and Mattila M. J. (1975)
present is not acceptable in a medicolegal Effect of food and repeated injections on serum
diazepam levels. Acta Pharmacol. Toxicol. 36, 181-186-
context. Marks V., Teale D. and Fry D. (1975) Detection of
cannabis products in urine by immunoassay. Br.
CONCLUSION Med. J. 2, 348-349.
Nash J. F., Bennett I. F., Bopp R. J., Brunson M. K-
Analytical evidence for the detection of and Sullivan H. R. (1975) Quantitation of pro­
drugs in body fluids facilitates monitoring of poxyphene and its major metabolites in heroin
patients w h o a r e not taking prescribed drugs addict plasma after large dose administration of
propoxythene napsylate. J. Pharm. Sci. 64,427-433-
or whose dosage is i n a d e q u a t e as well as
Sellman R., Kanto J., Raijola E. and Pekkarinen A.
identifying those suspected of the non­ (1975) Human and animal study on elimination
medical use of drugs. A n u n d e r s t a n d i n g of from plasma and metabolism of diazepam after
the various interrelating factors is needed if chronic alcohol intake. Acta Pharmacol. Toxicol. 3*>
33-38.
erroneous interpretations of the d a t a are to be
Sellman R., Pekkarinen A., Kaugas L. and Raijola E.
avoided. (1975) Reduced concentrations of plasma diazepam*
in chronic alcoholic patients following an oral
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