DRUG ANALYSIS

G M KellermanHunter Area Pathology Service

REASONS FOR ANALYSISILLEGAL DRUGS

• In many cases where there is a request for identification or measurement the question is of illicit drug use

• This comes from sporting events or illegal street drugs

• In either case there is big money involved somewhere, and in many countries penalties are very severe for street drugs, up to and including the death penalty

• So it may be enough to identify that a drug is present, no matter how little, or there may be an upper limit permitted (e.g. caffeine in athletes)

• On occasions there is an attempt to monitor abstinence from a long-lasting drug, so decreasing levels are sought (e.g. cannabinoids)

• Tests may therefore be qualitative or quantitative

• In view of the legal requirements, it is essential to document the “chain of custody” rigorously

• There are some specialist veterinary laboratories for race horses etc

REASONS FOR ANALYSISPRESCRIPTION DRUGS

• Requests for analysis of prescription drugs are made to investigate:• Compliance by the patient – is the level as expected for dose?• Adequacy of dose – is plasma level within an established

therapeutic range, high or low (e.g. thyroxine)? • Is there evidence of unusual drug metabolism, with excessively high

or low plasma levels (e.g. INH, azathioprine)?• Is the excretory capacity as expected, or low (e.g. aminoglycosides,

digoxin, where toxicity can develop)?• Do we need to assay a trough level, before the next dose, or a peak

level, or a whole time/concentration graph to measure AUC (e.g. immunosuppressives such as cyclosporine)?

• Overdoses, where knowledge of nature or level of drug has therapeutic implications (e.g. paracetamol)

ILLICIT DRUGS - 1

• There are many ways to identify and quantitate such drugs, either by direct measurement, measurement of metabolites or measurement of physiological effects (e.g. high Hb with EPO)

• Simple slide based immunological tests which contain a built-in positive control are used for many purposes (home pregnancy kits, LH spike detectors etc) and they have been adapted for use in emergency departments for many of the so-called “recreational” drugs such as opiates, cannabis, amphetamines, cocaine. However, they have limited range (e.g. opiates detect the morphine rings and do not react to pethidine {demerol} or methadone) and there is no single antibody good enough to find all the possible benzodiazepines at relevant concentrations. So “date rape” investigations are difficult

• Most of these slides are used to guide therapy in hospital and the chain of custody requirements are seldom if ever met

ILLICIT DRUGS - 2

• Quantitative methods involve separation – usually HPLC – and analysis by suitable detectors. Frequently extraction steps and/or chemical or enzymatic hydrolysis are needed

• Modern methodology is favouring the use of mass spectrometry for the final identification and quantitation, usually with internal standard deuterated compounds to control for incompleteness of the steps in the preliminary workup. This technology has much broader range of identification and more reliable quantitation, but it is expensive for normal requirements. When the big money or severe penalties are considered, it is cheap!

• Sports drugs are so important to that sector of the community that they are the province of specialist laboratories and normal clinical chemists never see them. We do receive quite a number of samples from surveys of employees for illegal drug use. Many users become experts at eluding tests – watering urine etc

PRESCRIPTION DRUGS - 1

• To interpret such results, it is necessary to have a firm basic knowledge of the pharmacokinetics of the drug (the rate of absorption, excretion or metabolism), the pharmacodynamics of the drug ( dose-response curves, toxicities) and the extensive set of drug interactions that seems to grow every year

• Hence the collaboration of the clinical chemist with the pharmacologist or at least the hospital pharmacist is essential, and computerised data bases are being developed and widely used

• In our laboratory we assay only those drugs for which there is a clear relationship between level and either activity or toxicity, especially if the pharmacokinetics differ widely among the population either for genetic or disease reasons. For the genetic problems we are tending to assay the affected enzymes or even the genes for preference to dose/effect measurements

PRESCRIPTION DRUGS - 2

• There is no substitute for knowledge of the health of the organs which do the metabolism or excretion – so liver disease and especially kidney disease (even mild) is of great importance.

• Some of the drugs we measure are:• Digoxin – excreted by kidney in proportion to GFR and plasma

creatinine is not a really adequate criterion – eGFR is better. The K level at the same time is needed due to interaction at the Na-K ATPase level. As we recognise toxic effects even at low levels, especially in women, this assay becomes more critical

• Aminoglycosides – excreted by kidney and hence levels are higher as GFR decreases. This is even more important as it is believed that a period without the drug in the day is beneficial to its action, so we need to ensure that the level drops adequately. Further, the drug is nephro- and oto-toxic and levels must not be too high for long

PRESCRIPTION DRUGS - 3

• Antiepileptics, especially phenytoin because of its saturation kinetics of metabolism. This means that when the mechanism is saturated, the plasma level rises rapidly for small increments in dose

• Carbamazepine and valproate are usually monitored, especially if in combination, as they and phenytoin and phenobarbitone all compete for albumin binding sites and allowances have to be made when more than one is administered. N.B. free fatty acids in starvation and a host of other drugs are also protein bound and compete

• Lithium is a very difficult drug to control and levels are often high or low. Patients of this type are often variably compliant and also sometimes suicidal, and there is an interaction with the Na content of the diet to make it harder. Monitoring is mandatory

• We often get requests for atypical antiepileptics and antidepressives Most of these do not have a clear dose/response relationship, but are tested for compliance. Clozapine is probably worth while

PRESCRIPTION DRUGS - 4

• One could write a whole book on the interactions with warfarin – suffice it to say that we give advice to the patient on diet and VITK intake, and monitor with INR

• Increasing knowledge of the specific cytochrome P450 enzymes responsible for drug metabolism and the characteristics of their induction/repression/competition is opening a new field of understanding for people who regularly prescribe drugs, but it means that the occasional prescriber will need to consult experts

• There are a number of other drugs that we occasionally measure – theophylline, lidocaine, salicylate, flecainide, perhexiline, amiodarone, itraconazole, some HIV drugs, but such requests are becoming less frequent as the popularity of drugs changes

PARACETAMOL (ACETAMINOPHEN)

• This mild analgesic causes more trouble that probably any other single drug because it is still a favourite means of attempted suicide particularly in young women

• Most appear fairly soon after administration and their plasma level can be compared to the published nomogram to establish the risk of severe toxicity. They can then be given N-acetylcysteamine and suffer no ill effects – but we had one who used to come in 3 times a week for months!

• Occasionally patients delay presentation until they begin to feel ill on day 2 or 3, and then they have little or no residual drug but the liver function tests show derangement – some transaminases are in the thousands and bilirubin is raised. It is usually possible to obtain the history of overdose. So far as I know we have had only 2 deaths over many years

POISONING

• The surfeit of toxic chemicals available for suicidal attempts, accidental intake or low level intake in food, water etc makes this a difficult field.

• We send most of the requests to the government office called “Workcover” which maintains methods for many such chemicals

• We can (and do) analyses for traces of toxic metals such as As, Cd, Zn, Cu, Mn, Hg etc by mass spectroscopy

• The most urgent tests we have are for cholinesterase in suspected (or known) organophosphate or carbamylphosphate poisoning, including monitoring of crop sprayers using these chemicals. The screen is for “pseudocholinesterase” (= butyrylcholinesterase) and if in doubt the true “acetylcholinesterase” can be measured in red cells as an index of severe toxicity. Mixing tests with normal plasma can detect residual toxin

• Remember, people will drink “anything that pours” even 90% phenol