THE

ANALYSIS OF

IN BlOlOGlCAL

FLUIDS

CANNABlNOlDS

U. S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
The Analysis of Cannabinoids in

Biological Fluids

Editor:

Richard L. Hawks, Ph.D.

Research Technology Branch

Division of Research

National institute on Drug Abuse

NIDA Research Monograph 42

1982

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

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Temple University School of Medicine

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NB Associates, Health Sciences

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Cornell University Medical College

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Washington University School of Medicine

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Langley Porter Neuropsychiotric Institute

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The Analysis of

Cannabinoids in

Biological Fluids
ACKNOWLEDGMENT

This monograph is based on papers presented at a technical

review held in Rockville, Maryland, on January 17, 1980, under

the sponsorship of the Division of Research, National Institute

on Drug Abuse.

COPYRIGHT STATUS

The material in this volume is in the public domain and may

be used and reproduced without permission from the National

Institute on Drug Abuse or the authors Citation of the source

is appreciated. Reproduction from the chapter by Moffat, Williams, and King requires acknowledgment that the material is

under British Crown Copyright.

The United States Government does not endorse or favor any

specific commercial product or commodity. Trade names or sup

pliers’ names appearing in this publication are used only because they are considered essential in the context of the studies

reported herein.

Library of Congress catalog card number 82-600586

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Printed 1982 Reprinted 1985

NIDA Research Monographs are indexed in the Index Medicus.

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Statistics Index, Biosciences Information Service, Chemical Abstracts. Current Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

For Sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402
FOREWORD

As the use of marijuana has increased over the last several

years, the need for improved quantitative chemical assays with

which to study the impact of marijuana use on the health of the

individual has grown. More sensitive, reliable, and workable assays

for cannabinoids (the active principles of marijuana) in body fluids

are essential to increase our knowledge of how these chemical constituents and their metabolites accumulate and how they are eliminated from the body.

In addition to research requirements, there is a growing demand

for less expensive and more reliable tests for use by the armed

forces, civilian law enforcement agencies, and private industry

where concern exists for the impact of marijuana use on critical job

performance. Of particular concern are the effects of marijuana on

complex performance related to driving motor vehicles, piloting

aircraft, operating industrial equipment, monitoring radar screens,

etc.

Concern about the negative impact of marijuana use on health

has been elevated by recent research findings as well as the availability of more potent marijuana, the trend toward use among

younger age groups, and the growing numbers of individuals with

long-term exposure to marijuana. On the positive side of the health

issue, tetrahydrocannabinol (THC), the primary active component

of marijuana, is under intense investigation as a treatment for the

often intolerable side effects of chemotherapy when other antiemetics are ineffective. Potential uses of THC for treatment of the

symptoms of glaucoma, spasms related to muscular dystrophy, and

certain asthmatic conditions are also under investigation. Research

to assess both the clinical potential of THC and the effects of marijuana on health must rely in part on sensitive chemical analyses.

Differing methodologies for cannabinoid analysis are required to

accommodate these various needs. Accordingly, the National Institute on Drug Abuse (NIDA) has supported a variety of efforts to

develop sensitive cannabinoid assay methods by techniques of immunoassay, gas chromatography, gas chromatography/mass spectrometry, and high-performance liquid chromatography. The devel-
opment of these methods was the subject of a technical review held

by NIDA in February 1976 and described in NIDA Research Monograph 7. Since that time, research on techniques in the analysis of

cannabinoids in biological fluids has been refined and new methods

have been developed. Even more important, perhaps, has been the

application of many of these methods to a wide variety of research

problems.

In January 1980 NIDA held a second technical review to bring

together key scientists working on the development of cannabinoid

assays and to summarize recent research where cannabinoid analysis is being used. The present monograph presents this information

as a compendium of the state of the art in the rapidly developing

field of cannabinoid analysis research today.

It is our hope that this volume will be useful to basic researchers

in the fields of biomedical and forensic science, where the availability of reliable, accurate, and accessible quantitative assays is critical to a better understanding of issues in marijuana abuse.

Marvin Snyder, Ph.D.

Director

Division of Research

National Institute on Drug Abuse

vi
CONTENTS

Foreword

Marvin Snyder

Introduction and Overview

Richard L. Hawks

The Use of High-Pressure Liquid Chromatography with

Electrochemical Detection for the Assay of Nantradol and its

Application to -Tetrahydrocannabinol

Richard M. Shepard and George M. Milne

Radioimmunoassays for Cannabinoids

C. E. Cook, H. H. Seltzman, V. H. Schindler, C. R. Tallent,

K. M. Chin, and C. G. Pitt

Radioimmunoanalysis of Delta-9-THC in Blood by Means of an

125I Tracer

S. M. Owens, A. J. McBay, and H. M. Reisner

Significant Developments in Radioimmune Methods Applied

to -THC and its 9-Substituted Metabolites

J. R. Soares, J. D. Grant, and S. J. Gross

Combined High-Performance Liquid Chromatography and

Radioimmunoassay Method for the Analysis of

Tetrahydrocannabinol and its Metabolites in Plasma and

Urine

v

Anthony C. Moffat, Peter L. Williams, and Laurence J.

King

An EMIT Assay for Cannabinoid Metabolites in Urine

M. J. deLaurentis, K. McNeil, A. J. Mann, S. Clark, and

Helena M. Greenwood

Laboratory Evaluation of Immunoassay Kits for the Detection

of Cannabinoids in Biological Fluids

Michael A. Peat, Bryan S. Finkle, and Mary E. Deyman

1

7

19

33

44

56

69

85

vii
Quantitative Analysis for -THC, 11-Hydroxy- -THC, and 9-

Carboxy- -THC in Plasma Using GC/CI-MS

Rodger L. Foltz and Bruce J. Hidy 99

Stability of -Tetrahydrocannabinol in Stored Blood and

Serum

Anthony S. Wong, Michael W. Orbanosky, Victor C. Reeve,

and James D, Beede 119

The Constituents of Cannabis and the Disposition and

Metabolism of Cannabinoids

Richard L. Hawks 125

List of Monographs 138

viii
Analysis of Cannabinoids

Research Monograph 42, R. Hawks, Editor

National Institute on Drug Abuse, 1992

Introduction and Overview

Richard L. Hawks

The analysis of cannabinoids in biological fluids is a subject of

considerable interest in several areas of public concern and also a

subject of tremendous scientific challenge. Scientists involved in

basic research on marijuana and its components are demanding

more sensitive and accessible methode of quantitative analysis,

civil enforcement organizations are interested in more effective

ways of chemically determining intoxication or past use of marijuana, and professionals in the clinical field are becoming increasingly

interested in tests to determine the extent of an individual’s marijuana use habit in conjunction with treatment protocols.

In any area of assay development, and particularly in the drug

abuse area, the questione of method sensitivity, specificity, and reliability are paramount. Another related but often overlooked area

is that of drug pharmacokinetics, critical to the interpretation of a

drug-concentration analysis,

The sensitivity of an assay is a measure of the least amount of

material the assay will reliably detect. Because there are many

ways to arrive at a number which represents this limit, the sensitivity claims for published assays do not always coincide with their

practical working limits. When eeneitivity is determined it is important to consider the type of body fluid involved and the way the

sensitivity is determined. An assay which reliably detects one nanogram of a given drug in one milliliter of saline may not provide

reliable detection at even 100 times that concentration in an actual

serum or urine sample because of the interference of biological

background noise. When determining the sensitivity of an assay it

is important to prepare and analyze a significant number of blank

body-fluid samples containing the same amount of analyte near the

limit of sensitivity so that the precision of the assay can be statistically determined. The statistical variation of this “spiked” level

1
2 HAWKS

should be compared with the variation observed in the blank samples to determine the probability of overlapping values. This is particularly important in assays designed for use as detection assays

where there is concern for both false positive results and false negative results.

It is equally important to be able to distinguish the given drug of

interest from other drugs, metabolites, and endogenous compounds

that might appear in the sample matrix. The extent to which the

assay can make this distinction is the specificity of the assay. This

becomes particularly important in the case of forensic samples,

where the qualitative analysis of the drug is often more important

than the quantitative analysis. In the case of immunoassays, specificity is a particularly important factor. Historically, immunoassays have lacked high specificity because of the way antibody material was generated. As demonstrated in the papers presented by

Drs. Cook and Soares, high specificity of radioimmunoassay (RIA)

methods can be achieved by careful design and sophisticated techniques of synthesis in the preparation of the immunogens used to

generate the reagent antisera. In some cases, however, an assay

with high specificity for a specific parent drug or metabolite is not

as desirable as one with limited cross-reactivity for a class of molecules, as in the case of the Syva homogeneous enzyme immunoassay (EMIT) presented by Drs. Greenwood and deLaurentis. Here

cross-reactivity with a variety of -tetrahydrocannabinol (THC)

metabolites is desirable since the assay is used to determine past

use of marijuana rather than a specific level of a given cannabinoid. By cross-reaction with more than a single metabolite, additional sensitivity is achieved.

Another analytical approach which benefits from the use of

cross-reacting antisera is that which combines high-performance

liquid chromatography (HPLC) with an RIA. This technique exploits the specificity to be achieved by HPLC chromatographic separation and the sensitivity possible by using an RIA as the detector

for the system. A good example of this approach is described in the

paper by Dr. Moffat.

Major advances since the time of the previous NIDA monograph

on cannabinoid assays in 1976 have included the development of

sensitive gas chromatography/mass spectrometry (GC/MS) methods for the detection of 11-hydroxy-THC and 11-nor-THC-9-carboxylic acid (9-carboxy-THC) at low-nanogram sensitivity. New achievements in immunoassays include the development of specific RIA

methods for 9-carboxy-THC as well as the development of iodinated

tracers which allow sensitivity levels near one nanogram per ml to

be achieved. The demonstrated ability to use some of these assays
INTRODUCTION AND OVERVIEW 3

for direct analysis of whole blood is also an important achievement

for forensic research applications.

Detection of past use of marijuana could facilitate drug-treatment counseling and provide a broader base by which to assess the

impact of marijuana on health and safety. This is of particular concern with respect to those groups which might be considered to be

in relatively high-risk situations, either to themselves or others,

and includes the young, members of the military, automobile drivers, and operators of complex industrial equipment. Systems like

the Syva EMIT may provide some of this assessment in its role as a

screening tool for particular populations.

In the case of drivers, equipment operators, and others whose

level of sobriety is a key safety factor, the issue of an analytical

means of determining intoxication is critical and a matter that has

proven to be a much more difficult problem than mere detection.

There are many specific factors to be considered when cannabinoid

assays are used in the forensic area, including the convenience of

obtaining the biological sample to be analyzed and the usual concerns of sensitivity, specificity, and other technical aspects of the

assay. Interpretation of results in forensic cases becomes as important as the accuracy of the assay performed.

It appears from available data that only a blood sample may correlate physiologically with an actual state of intoxication. A urine

sample which tests positive indicates prior use, but such an analysis cannot be directly related to intoxication since cannabinoid metabolites can persist in the urine for several days after smoking a

single cigarette.

The use of saliva or breath for detection is very attractive because it involves an easily obtainable sample. Studies are in progress to further assess the potential of saliva or breath as a noninvasive alternative to blood collection in roadside surveys. Dr Soares’

paper provides preliminary evidence that cannabinoids detectable

by immunoassay techniques may be found in breath samples.

While experiments involving intravenous injection of radiolabeled

THC into human subjects with subsequent monitoring of radioactivity in saliva have indicated that negligible amounts of drug or

metabolites cross the blood/saliva barrier, other experiments have

shown that breath and blood samples collected after smoking marijuana do show detectable levels of THC which persist for several

hours. These THC levels are apparently due to material sequestered directly, in a nonspecific way, in the mouth and lungs during

smoking and then slowly released into breath or saliva. If simple

methods for the analysis of these levels could be developed, it is

conceivable that they could be used as a presumptive test of recent
4 HAWKS

use of marijuana. Such a method of analysis would not, however,

be quantitatively related to blood-level equivalents under these circumstances.

One of the most important issues in the area of marijuana and

driving is the interpretation of a given blood concentration of THC

to explain the presumptive intoxication of the individual from

whom the sample was taken. In an attempt to determine whether

it is feasible to establish a presumptive level of impairment for

THC, studies were initiated under NIDA contract at the Southern

California Research Institute to study the effects of various doses of

marijuana on behavioral tasks related to driving ability. Coincident

with the behavioral study, a pharmacokinetic analysis of each sub

ject was performed. These studies were designed to generate two

sets of time-course data-one of blood concentrations and one of behavioral performance. The sensitive behavioral tests were carried

out periodically to provide a performance time-course curve which

could be statistically compared to the pharmacokinetic blood-level

curve. Analysis of this data is not yet complete, but what is obvious

so far is that even though some consistency exists across individuals smoking a given dose of marijuana, in terms of expected blood

levels, the associated performance effects of these doses do not

show the same consistency. It is not yet clear whether a practical

presumptive concentration of THC can be related to measurable

impairment.

Research on the therapeutic use of THC is a rapidly developing

area. Large State and national programs have been started to investigate the therapeutic potential of THC as an antiemetic for

chemotherapy patients. Most of this research involves oral formulations of THC in sesame oil capsules. The chapter on metabolism

and disposition indicates that the oral route of administration produces a relatively large amount of 11-hydroxy-THC, which is a

pharmacologically active metabolite, and an assay suitable for this

type of clinical research must be designed to determine both THC

and 11-hydroxy-THC levels. At the present time only the CC/MS

method and methods which couple HPLC with RIA can be applied

to this type of analysis. It is expected however that a specific RIA

method being tested at the Research Triangle Institute will be

available soon for separately determining 11-hydroxy THC in the

presence of THC.

There has been increasing interest in the use of nonspecific

RIA’s in combination with the chromatographic separation properties of HPLC instruments. The paper by Dr. Moffat demonstrates

one particular use of this system in the area of cannabinoid analyses. The combination of these two methods constitutes a powerful
INTRODUCTION AND OVERVIEW 5

research tool in that it enables the investigator to determine quantitatively with a single radioimmunoassay the concentrations of a

large number of metabolites. The tradeoff is a somewhat longer

time of analysis, but in a research setting or with a small number

of samples, time is not often a major factor. HPLC has also been

used as an integral part of the sample preparation procedure for

GC/MS analyses, as noted by Dr. Foltz. Drs. Shepard and Milne

report a method of reasonable sensitivity with use of HPLC and

standard detectors.

Dr. Moffat, in forensic cases examined by his group, reports significant amounts of glucuronide-conjugated metabolites in urine

and blood. Dr. Peat reported that a large number of EMIT-positive

urine samples, when subsequently analyzed by GC/MS, showed

levels that were low or undetectable for free 9-carboxy-THC. In

view of Dr. Moffat’s comments, further analysis of some of these

samples (after the technical review) was carried out. In a number

of cases, samples previously low or negative for 9-carboxy-THC

showed significant levels after enzymatic hydrolysis. This finding

indicates that in some urine samples virtually all of the 9-carboxyTHC may be found in a conjugated form, an important factor when

a specific method such as GC/MS is used as a confirmation tool for

a less specific assay such as the EMIT, which probably cross-reacts

with the glucuronide metabolite. This hydrolysis technique has

been applied to the confirmation of a large number of EMIT-positive samples resulting from a Department of Defense screening

study. The Armed Forces Institute of Pathology used GC/MS to

confirm over 95 percent of 1100 positive urine samples resulting

from the study. Most of the questionable positive results which

made up the 5 percent unconfirmed cases were close to the cutoff

level of detection of the EMIT assay.

Dr. Owens reported that his highly specific and sensitive RIA

worked quite well with a methanol extract of whole blood. Previously, many investigators assumed that, for an immunoassay to

function properly, the medium in which the antigen-antibody interaction occurred would have to be quite similar to a normal physiological environment. It appears, however, that at least in the case

of the cannabinoid RIAs, this is not necessarily the case. In fact the

assay was shown to work better in the presence of alcohol than in

its absence. This also had been the experience of Drs. Moffat and

Cook, an important finding because it enables a blood sample to be

partially purified by alcohol precipitation, centrifugation, and subsequent analysis of the resulting alcoholic supernatant without an

evaporation step. The presence of alcohol may facilitate the assay

by reducing “sticking” of THC to nonspecific binding sites.
6 HAWKS

Of interest to the forensic community are the comments of Dr.

Wong concerning his studies on the stability of cannabinoids in

whole blood and plasma under different conditions. These studies

were carried out under contract to the Department of Justice,

State of California, as part of that State’s research efforts to provide a basis for future legislation concerning marijuana and driving impairment.

Interpretation of data from forensic samples was an issue of considerable discussion at this technical review. Because THC has a

relatively short half-life after smoking, assay method sensitivity of

1 ng per ml may not be sufficient to detect the compound more

than 6 hours after smoking. This has led some investigators to

argue that if any level of THC is detectable in blood at all, one can

assume that the sample was taken at a time very close to the incident of smoking and that, therefore, the subject has a high probability of being intoxicated. This argument makes fairly broad assumptions concerning the presumptive concentrations of THC that

can be related to specific types of intoxicated behavior. Quite obviously we are still a long way from determining exactly what this

presumptive level is. It was generally agreed, however, that for any

THC blood assay to be of use in the forensic area, it would have to

have a sensitivity of at least 3 nanograms per ml since blood levels

fall to this level within an hour or two after a smoking incident.

While little information is currently available on THC blood levels

in chronic smokers, the possibility of accumulated drug being at detectable levels long after the acute period of intoxication must be

considered.

While it is obvious that considerable progress has been made in

the last few years on cannabinoid assays, research in this area continues to develop at a rapid pace. From the presentations and discussions at this meeting, it seems that the immediate future holds

interesting breakthroughs in even more sensitive assays as well as

improvements in methods which will lead to cannabinoid assays

being used for the first time in a variety of new forensic and social

settings.

AUTHOR

Richard L. Hawks

Research Technology Branch

Division of Research

National Institute on Drug Abuse

Rockville, Maryland 20857
Analysis of Cannabinoids

Research Monograph 42, R. Hawks, Editor

National Institute on Drug Abuse, 1982

The Use of High-Pressure Liquid

Chromatography with

Electrochemical Detection for the

Assay of Nantradol and its

Application to

Tetrahydrocannabinol

Richard M. Shepard and George M. Milne

High-pressure liquid chromatography with electrochemical

detection permits sensitive, selective and quantitative analysis of biological and pharmaceutical compounds containing electroactive functional groups. In support of pharmacollogical and clinical studieswith nantradol, a potent, nonopioid analgetic, a specific HPLC/electrochemical assay for

nantradol and related compounds in plasma was recently

developed. In preliminary studies we have now extended

this technique to the detection of and 11-OH

tetrahydrocannabinol. Results are resented which indicate

that HPLC coupled to an electroc emical detector provides h

sensitivity for these compounds in plasma in the low ng/ml

range.

INTRODUCTION

High-performance liquid chromatography (HPLC) is finding increasing application to the trace analysis of drugs, drug metabolites and biologically important compounds in complicated matrices

such as body fluids and tissue samples. In the case of cannabinoids,

HPLC has been used as a means of purifying

tetrahydrocannabinol ( -THC) from related materials and interfering endogenous materials in blood plasma. However, at the concen7
8 SHEPARD AND MILNE

trations of -THC normally encountered in plasma, the direct

HPLC on-line monitoring methods such as refractive index or ultraviolet spectrophotometry detection are not sensitive enough for

use in plasma level monitoring. As a result, subsequent analysis by

gas chromatography (1), mass spectrometry (2), or radioimmunoassay (3) has been required. The coupling of electrochemical detection

to HPLC (4) is a recently developed technique that often provides

the sensitivity and selectivity necessary for practical analytical pro

cedures. The application of this technology to the determination in

plasma of nantradol, a potent analgetic, has recently been de

scribed (5). It is the aim of this paper to describe briefly the principles of electrochemical detection linked to HPLC, to examine the

application of this technique to the analysis of nantradol in

plasma, and then to discuss efforts to extend this method to the determination of -THC in plasma.

PRINCIPLES OF HPLC COUPLED WITH

ELECTROCHEMICAL DETECTION

In HPLC with electrochemical detection (figure 1), material

eluted from the chromatographic column reacts at an electrode

surface under controlled potential conditions, and the current

which results from the net exchange of electrons is monitored as a

function of time (6). Since the amount of material converted by the

electrochemical reaction is proportional to the instantaneous concentration, the current will be directly related to the amount of

compound eluted as a function of time. If chromatographic conditions are carefully controlled, then amperometric detection is quite

precise, with a wide linear range of typically 104, and quantitative

data can be obtained at the picomole level for many compounds.

Limits of detection down to 1 pg or less are attainable in some

cases.

In addition to being extremely sensitive, the electrochemical detector is selective in that only compounds containing an electrochemically oxidizable or reducible functional group can be detected

(6). Furthermore, these compounds must be electroactive at the

given potential to which the detector is set. While these features

represent both an advantage and disadvantage, they often combine

to make sample preparation a relatively simple matter.

With direct conversion of a chemical signal into an electrical

signal, the electronic instrumentation of the electrochemical detector is therefore inexpensive and reliable. The detector cell is well

suited for HPLC analysis because it has an extremely low dead

volume. Because of the nature of electrochemical detection, the
HPLC ASSAY FOR NANTRADOL

Picomole sensitivity

Selective

Low cost

Can be combined with HPLC

Used in analysis of:

phenols

aromatic amines

catecholamines

antioxidants

ascorbic acid

uric acid

natural products

pharmaceuticals

FIGURE 1. Features and applications of electrochemical detection.

chromatographic systems most promising for this type of detector

are ion-exchange and reversed-phase systems where aqueous or

aqueous-alcohol solvent mixtures containing some dissolved ions

can be used (6).

A number of endogenous compounds, drugs, and drug metabolites have been studied by HPLC with electrochemical detection.

Examples include phenols (7), aromatic amines (6,8), catecholamines (9,10,11), ascorbic acid (12) and uric acid (13). This methodology has found increasing application in the field of neurochemical

analysis, having been used for the simultaneous determination, in

rat and mouse brains, of nanogram levels of catecholaminergic and

serotonergic neurotransmitters such as norephinephrine, dopamine, tryptophan and serotonin (14,15).

APPLICATION OF HPLC AND ELECTROCHEMICAL

DETECTION TO NANTRADOL

Nantradol (figure 2) produces analgetic activity 2 to 7 times

greater than morphine across a range of analgetic tests traditionally responsive only to the narcotic agonist analgetics, but it does not

bind to the opiate receptor in vitro (16). While nantradol possesses

certain features of cannabinoid pharmacology, it has a number of

structural features which distinguish it from the cannabinoids-including the absence of a pyran oxygen, the presence of a weakly

basic nitrogen and the introduction of an oxygen-containing aide

chain. In vivo nantradol is rapidly deacetylated to the free phenol,

desacetylnantradol, which is the only form of the drug detectable
10 SHEPARD AND MILNE

in plasma (figure 2). The phenol function, which is shared with

THC, makes possible the electrochemical detection of desacetylnantradol.

FIGURE 2. Structure of nantradol (CP-44,001), a potent non-opioid analgetic, and its

deacetylation to desacetylnantradol (CP-44,041).

Fortification of 2 ml plasma sample with internal standard

(N-methyl analog)

Extraction with 2% isopropanol in diethyl ether

Purification of drug by adsorption to and elution from

Sephadex C-25 (cation exchanger)

Analysis by reversed-phase HPLC with electrochemical detection

FIGURE 3. Flow scheme outlining the procedure for the analysis of

desacetylnantradol in plasma.

The assay procedure (figure 3) begins with the fortification of a 2

ml plasma sample with 20 ng/ml of an internal standard, the Nmethyl analog of desacetylnantradol (5,17). Extraction of the drug

and internal standard is achieved with 2% isopropanol in diethyl

ether. The extract is added to 2 ml of methanol and concentrated

under nitrogen to a final volume of 1 ml. The extracted drug and

internal standard are then adsorbed onto cation exchanger Sephadex C-25 (added in 1 ml as a 30% suspension in water), collected by

centrifugation and the supernatant discarded. Following a water
HPLC ASSAY FOR NANTRADOL 11

wash, the desacetylnantradol and internal standard are eluted

from the Sephadex C-25 with 1 ml of acetonitrile:0.05 M KH2PO4

(1:1). Analysis is achieved by injection of 50 μl of the assay solution

onto a reversed-phase HPLC Waters μBondapak phenyl column

eluted with a solvent system of acetonitrile:0.05 M KH2PO4 (1:1) at a

flow rate of 2.0 ml/min. Two typical HPLC chromatograms of the

analysis of desacetylnantradol isolated from human plasma samples fortified with 20 ng/ml of internal standard and either 5 or 20

ng/ml of drug are shown in figure 4. Unknown drug levels are first

expressed as the ratio of the peak height corresponding to desacetylnantradol versus that for 20 mg/ml of the internal standard.

Concentrations are then determined from an appropriately constructed standard curve (figure 5), in which known amounts of drug

have been added to plasma. Good linearity is obtained and the precision of the assay is about 10% relative standard deviation based

on the mean normalized peak height ratios within an assay series.

The sensitivity of the assay permits the analysis of desacetylnantradol in plasma down to levels of about 5 ng/ml. As little as 200

pg of drug injected on-column can be detected.

APPLICATION OF HPLC/ELECTROCHEMICAL DETECTION

TO -THC

In light of the common presence of a C-1 phenol in desacetylnantradol and -THC, we have carried out some initial experiments

designed to investigate the feasibility of analyzing - THC using

HPLC with electrochemical detection. The HPLC system for the

analysis of nantradol was adapted to -THC by using the same reversed-phase column and detector potential and is probably not optimal. A less polar solvent of 55:45 acetonitrile:0.05 M KH2PO4

 at a

flow rate of 2.0 ml/min was used. In this system -THC and 11-

hydroxy -tetrahydrocannabinol (11-OH - THC), are readily separated from each other (figure 6), eluting at 16.6 ml and 8.2 ml, respectively. As little as 0.7 ng of pure -THC injected on-column

can be detected in this system. When various amounts of pure

THC, from 0.7 ng to 154 ng, are injected onto the column the resulting peak response curve is linear over the entire range with a

correlation coefficient of 0.999 and a precision of 6.3% relative

standard deviation based on the mean normalized peak heights

(figure 7).

In preliminary attempts to determine -THC levels in plasma,

dog plasma (2 ml) was extracted three times with hexane (3 ml).

The pooled hexane extracts were washed 3 times with 3% KOH in
12 SHEPARD AND MILNE

FIGURE 4. HPLC chromatograms of desacetylnantradol and internal standard

isolated from human plasma, fortified with 20 ng/ml of N-methyldesacetylnantradol and either a) 5 ng/ml or b) 20 ng/ml of desacetylnantradol.
HPLC ASSAY FOR NANTRADOL 13

FIGURE 5. Standard curve illustrating the changes in the ratio of the peak heights

of desacetylnantradol versus internal standard as a function of varying

concentrations of desacetylnantradol in fortified human plasma.
14 SHEPARD AND MILNE

FIGURE 6. HPLC chromatogram illustrating the detection and separation of

THC and 11-OH- -THC.
HPLC ASSAY FOR NANTRADOL 15

FIGURE 7. Demonstration of the linearity of peak height response of -THC versus

the amount injected onto the HPLC, using electrochemical detection.

20% aqueous methanol. The pooled alkali extracts were acidified

with concentrated HCl, diluted with water and back-extracted with

hexane. This hexane extract was then evaporated under nitrogen

and the residue was redissolved in 200 μ1 of HPLC solvent. 100 μ1

of the solution was then injected onto the column. The HPLC profiles obtained from blank dog plasma and dog plasma fortified with

20 ng/ml -THC are shown in figure 8. The average recovery of

-THC in three samples was 38% ± 2%. Based on these results,

the limit of detection for the plasma determination of -THC by

this procedure is approximately 5 ng/ml.
16 SHEPARD AND MILNE

FIGURE 8. HPLC chromatograms of -THC isolated from a) blank dog plasma and

b) dog plasma fortified with 20 ng/ml -THC.

CONCLUSION

HPLC with electrochemical detection can often provide the sensitivity and selectivity necessary for practical analytical procedures,

as in the case of nantradol. It has been demonstrated here that thie

methodology is capable of detecting low nanogram levels of -THC

in plasma while providing a separation of -THC from its major

metabolite 11-OH- -THC. Thus it appears that, while further de-
HPLC ASSAY FOR NANTRADOL 17

velopment of this technique is needed for a routine plasma assay

for -THC and its metabolites, HPLC with electrochemical detection is a feasible approach to the analysis of nanogram concentrations of -THC.

ACKNOWLEDGMENTS

Dr. Hugh M. McIlhenny and Mr. Frank R. Mosher made useful

suggestions in assay development for -THC and helped to develop

the assay for nantradol. Dr. M. Ross Johnson synthesized and provided nantradol, desacetylnantradol and N-methyl-desacetylnantradol, and Dr. Carl Hausmeyer made available an electrochemical detector in the early stages of nantradol assay development. The National Institute on Drug Abuse (NIDA) provided -THC and 11-

OH- -THC.

REFERENCES

1. E.R. Garrett and C.A. Hunt. Separation and analysis of -tetrahydrocannabinol

in biological fluids by high-pressure liquid chromatography and GLC. J. Pharmaceut. Sci., 66, 20-26 (1977).

2. J.L. Valentine, P.J. Bryant, P.L. Gutshall, O.H.M. Gan, P.D. Lovegreen, E.D.

Thompson, and H.C. Niu. High-pressure liquid chromatographic-mass spectrometric determination of -tetrahydrocannabinol in human plasma following

marijuana smoking. J. Pharmaceut. Sci, 66, 1263-1266 (1977).

3. P.L. Williams, A.C. Moffat, and L.J. King. Combined high-pressure liquid chromatography and radioimmunoassay method for the quantitation of

tetrahydrocannabinol and some of its metabolites in human plasma. J. Chromatog., 155, 273-283 (1978).

4. P.T. Kissinger, C. Refshauge, R. Dreiling, and R.N. Adams. An electrochemical

detector for liquid chromatography with picogram sensitivity. Anal. Lett., 6, 465-

477 (1973).

5. R.W. Mast, H.M. McIlhenny, G.M. Milne, and M.R. Johnson. Behavioral effects

and pharmacokinetics of nantradol hydrochloride, a potent, cannabinoid-related

analgesic. Pharmacologist, 21, 270 (1979).

6. P.T. Kissinger. Amperometric and coulometric detectors for high-performance

liquid chromatography. Anal. Chem., 49, 447A-466A (1977).

7. R.M. Riggin, L.D. Rau, R.L. Alcorn, and P.T. Kissinger. Determination of phenolic sympathetic stimulants in pharmaceuticals by liquid chromatography with

electrochemical detection. Anal. Lett., 7, 791-798 (1974).

8. L.A. Sternson and W.J. DeWitte. High-pressure liquid chromatographic analyeb

of isomeric aminophenols with electrochemical detection. J. Chromatog., 138,

229-231 (1977).

9. R.M. Riggin, R.L. Alcorn, and P.T. Kissinger. Liquid chromatographic method

for monitoring therapeutic concentrations of L-dopa and dopamine in serum.

Clin. Chem., 22, 782-784 (1976).
18 SHEPARD AND MILNE

10. P.T. Kissinger, R.M. Riggin, R.L. Alcorn, and L.D. Rau. Estimation of catecholamines in urine by high-performance liquid chromatography with electrochemical detection. Biochem. Med., 13, 299-306 (1975).

11. C. Refshauge, P.T. Kissinger, R. Dreiling, C.L. Blank, R. Freeman, and R.N.

Adams. New high-performance liquid chromatographic analysis of brain catecholamines. Life Sciences, 14, 311-322 (1974).

12. L.A. Pachla and P.T. Kissinger. Determination of ascorbic acid in foodstuffs,

pharmaceuticals, and body fluids by liquid chromatography with electrochemical detection. Anal. Chem., 48, 364-367 (1976).

13. L.A. Pachla and P.T. Kissinger. Estimation of serum uric acid by high-performance liquid chromatography with electrochemical detection. Clin. Chim. Acta,

59, 309-312 (1975).

14. S. Sasa and C.L. Blank. Simultaneous determination of norephinephrine, dopamine and serotonin in brain tissue by high-pressure liquid chromatography with

electrochemical detection. Anal. Chim. Acta, 104, 29-45 (1979).

15. I.V. Mefford and J.D. Barchas. Determination of tryptophan and metabolites in

rat brain and pineal tissue by reversed-phase high-performance liquid chromatography with electrochemical detection. J. Chromat., 181, 187-193 (1980).

16. G.M. Milne, A. Weissman, B.K. Koe, and M.R. Johnson. CP-44,001, A novel

benzo(c)quinoline analgesic. Pharmacologist, 20, 243 (1978).

17. H.M. McIlhenny, R.W. Mast, M.R. Johnson, and G.M. Milne. Nantradol hydrochloride: Pharmacokinetics and behavioral effects following acute and chronic

treatment. J. Pharmacol. Exp. Ther., 219 (2), 363-369 (1981).

AUTHORS

Richard M. Shepherd and George M. Milne

Pfiir Research Laboratories

Groton, Conn. 06340
Analysis of Cannabinoids

Research Monograph 42, R. Hawks. Editor

National Institute on Drug Abuse, 1982

Radioimmunoassays for

Cannabinoids

C. E. Cook, H. H. Seltzman, V. H. Schindler, C. R.

Tallent, K. M. Chin, and C. G. Pitt

The simplicity, sensitivity, and specificity of radioimmunoassay have made it an attractive procedure for the analysis of delta-9-tetrahydrocannabinol (THC) in biological

fluids or tissues. The presence of closely related compounds

such as metabolites may interfere with radioimmunoassay

results. Appriopriate design of immunogens may diminish

such interference. This work has been directed towards the

use of the amyl side chain for linking cannabinoid compounds to proteins to form immunogens. Although the amyl

side chain is metabolized to some extent, the metabolites

are not quantitatively significant in most cases. 5’-Carboxydelta-8-THC and 5’-carboxy-delta-9-THC were linked to

bovine serum albumin. Immunization of rabbits with the

resulting conjugates resulted in the formation of antisera

with high selectivity for delta-9-THC vs. its carboxylic acid

metabolite, 11-nor-9-carboxy-delta-9-THC. Delta-8-THC radioligands (4’,5’-tritium and 5’-iodine-125) could be used

with these antisera for analysis of delta-9-THC in plasma.

Sensitivity with tritium-labeled material is about 2.5 ng/

ml. 5’-Oxo-11-nor-9-carboxy-delta-8-THC was used to prepare

an immunogen which led to the genemtion of an antiserum

highly specific for 11-nor-9-carboxy-delta-9-THC. This antiserum and iodine-125-5’-iodo-11-nor-9-carboxy-delta-8-THC

were used to develop a highly specific assay for 11-nor-9-

carboxy-delta-9-THC in plasma.

INTRODUCTION

The basic premise of immunoassay techniques is that a labeled

antigen will interact with a specific antibody to form a labeled

antigen/antibody complex. In the presence of unlabeled antigen

19
20 COOK, SELTZMAN, SCHINDLER, TALLENT, CHIN, AND PITT

there will be a competition for binding sites to the antibody, thereby decreasing the amount of the complex which is labeled. In order

to achieve a useful assay, then, one must be able to obtain a labeled antigen, to generate a specific antibody which will bind the

compound of interest, and to develop some means of distinguishing

the labeled complex from the free labeled antigen.

In order to stimulate the formation of antibodies which will selectively bind a small molecule such as tetrahydrocannabinol, the

molecule or hapten must first be linked to a large molecule, generally a protein (figure 1). This concept was demonstrated by Landsteiner many years ago (1). Landsteiner also showed that the position of the linkage was of considerable importance in determining

the selectivity of the resulting antibody. The fit between antibody

and antigen is often quite good, particularly in areas which are removed from the site of attachment to the protein. However, introduction of the covalent link inevitably results in a change in the

structural characteristics of the molecule. The resulting antibody

often reflects this by being insensitive to changes around the position of linkage, as is illustrated by the hatched area in figure 1.

FIGURE 1. Generation of antibodies to a drug. The drug (hapten component) is

covalently linked to a large molecule such as a protein to form the immunogen.

Injection of this into animals stimulates formation of antibodies. The binding site of

the antibody accommodates the hapten, with some ambiguity around the area

which corresponds to the point of linkage in the immunogen.
RADIOIMMUNOASSAYS 21

Since in general a drug and its metabolites will have many similar structural features, the metabolites will usually be the principal interfering constituents in an RIA analysis. Thus, in preparing

a conjugate to generate specific antisera, one tries to avoid sites on

the drug which are metabolically reactive, to leave them free‘to influence the formation of selective antibodies. Also one tries to avoid

covering up structural features which may permit development of

strong affinity binding sites. In addition, one must also consider the

chemistry involved and its difficulty in relation to the expected

benefit.

It is now generally accepted that the principal metabolic alterations in the THC molecule occur in the 8- and 11-positions. Hydroxylation may occur in these positions, and oxidation to an 11-

nor-9-carboxy metabolite is also a principal pathway. Metabolites

involving the amyl side chain have also been reported, but it appears that in quantitative terms these are relatively minor-at least

in humans (2).

Nonselective antisera are also useful, but our interest has been

in the development of assays with specificity for one compound.

Specificity of an assay may be increased by techniques such as differential extraction of the desired compound or addition of a

second antibody to tie up the interfering substance (3). However,

these techniques work better if the antiserum has good selectivity

to begin with. We therefore chose to examine immunogens formed

by attaching cannabinoid moieties to proteins through the amyl

side chain, as this would fulfill our requirement of distance from

the principal sites of metabolism.

IMMUNOGEN SYNTHESIS AND ANTISERA TO DELTA-9-THC

For synthetic convenience our initial attempts dealt with the

delta-8 isomer of THC. 5’-Carboxy-delta-8-THC was treated with Nhydroxysuccinimide and dicyclohexylcarbodiimide to yield an ester

which readily reacted with bovine serum albumin in a mixture of

dioxane and sodium bicarbonate solution to yield the desired immunogen containing about 33 residues of delta-8-THC per molecule

of bovine serum albumin (4).

This immunogen was injected into rabbits by a modification of

the intradermal method of Vaitukaitus et al. (4). Reasonable antiserum titers were achieved by 7.5 weeks after the first immunization and, as we have reported previously, the resulting antisera

showed good selectivity for delta-9-tetrahydrocannabinol vs. its metabolites (4,5).
22 COOK, SELTZMAN, SCHINDLER, TALLENT, CHIN, AND PITT

Since the antisera produced from the delta-8-immunogen showed

about 2.5 times as great an affinity for delta-8-THC as for delta-9-

THC, it appeared that perhaps even better antisera could be

achieved starting with a delta-9-5’-carboxylic acid hapten. The desired immunogen was synthesized as in the case of the delta-8-

compound. Rabbits were immunized with the resulting conjugate

and antisera which were able to bind delta-9-THC were obtained

(5). As figure 2 shows, the antisera exhibited very low cross-reactions with the 11-nor-9-carboxy metabolite of delta-9-THC. A significant cross-reaction with the 11-hydroxy metabolite was observed,

but this compound is present in very low concentrations in blood

after smoking or iv administration (6), so the cross-reaction would

be of concern only after oral administration of delta-9-THC.

Analytical Protocol

Vortex mix 100 μ L plasma with 400 μ L methanol

Let stand, 5-10 min; centrifuge; aliquot

50 μ L aliquot + 400 μ L buffer with radioligand (15,000 cpm)

+ 100 μ L antiserum/buffer

Vortex mix; incubate 4-24 hr at 4°C

Add charcoal/buffer (500 μ L of 1.58% suspension)

Vortex mix; incubate 20 min at 4°C

Centrifuge 15 min at 4°C; decant; count.

FIGURE 2. Protocol for analysis of delta-9-THC or its carboxylic acid metabolite in

plasma.

RADIOLIGANDS

Now let us turn to consideration of the radioligand. Tritium-labeled radioligands have the advantage of generally better shelf-life.

They also have an affinity for the antibody essentially equal to

that of the unlabeled compound. On the other hand, iodine-125 has

certain advantages over tritium for use as the radioisotope. These

include the higher theoretical specific activity, resistance to

quenching and ease of measurement. The size of the iodine atom,
RADIOIMMUNOASSAYS 23

however, makes its introduction into a small molecule such as THC

a problem from the standpoint of binding affinity. Direct substitution of iodine into such a molecule often inhibits binding to the

antibody and reduces the affinity below that which can give a

useful assay. However, direct introduction of iodine into the 5’-position of THC--that is into the position which was linked to the protein in the immunogen--might allow us to take advantage of the insensitivity of antibody affinity to changes at the position of linkage

in the immunogen. Therefore, 5’-iodo-delta-8-THC was prepared and

submitted to an exchange reaction with iodine-125 sodium iodide.

This procedure led to iodinated THC of moderate specific activity,

ranging from about 8-40 Ci/mmole (4-7). More recently we have

found that displacement of a 5’-tosylate with iodine-125-iodide can

lead to even higher specific activities (7).

ANALYSIS OF DELTA-9-THC IN PLASMA

The above antisera were used to develop an assay for delta-gTHC in plasma. Initially tritium-labeled delta-8-THC of specific activity 60 Ci/mmole (8) was used as the radioligand. Antiserum to

delta-9-THC was used at an initial titer of 1:6000. Standard curves

showed a practical sensitivity of 25 pg. (We define practical sensitivity as the amount which reduces binding of radioligand by 10%.

This is a pragmatic definition, based on experience. Sensitivity

limits based on 2 or 3 standard deviations from blank tubes would

be much lower.)

For plasma analysis we used the procedure shown in figure 2.

Plasma is mixed with methanol (9) and an aliquot of the resulting

supernatant is incubated with the radioligand and antiserum, usually overnight, at 4°C. Separation is carried out by addition of a

suspension of charcoal in buffer and after centrifugation the supernatant is counted.

Under these conditions our practical sensitivity limit was 2.5 ng/

ml with either radioligand. When the tritium radioligand was used,

control samples at 8 ng/ml showed a within-assay CV of 5.9% and

a between-assay CV of 6.4%. For 30 ng/ml controls these values

were 6.0 and 5.5%, respectively. Variances (10) are based on 28

assays and 4 tubes were analyzed per sample. Similar results were

seen with the iodinated ligand, but with many fewer analyses.

We have now used both of these ligands in an assay for

tetrahydrocannabinol from plasma. Through the courtesy of Dr.

John Merritt, University of North Carolina School of Medicine, we

obtained some samples from subjects administered delta-9-THC and
24 COOK, SELTZMAN, SCHINDLER, TALLENT, CHIN, AND PITT

analyzed them using both ligands. The sensitivity in each case appeared to be 2.5 ng/ml. Figure 3 shows the data from a single subject analyzed using both ligands. Results appear comparable and

this conclusion is supported by figure 4 which shows results from

the analysis of all 62 samples in which either sample gave results

above 2.5 ng/ml. Using tritium as the ordinate, we found a regression equation with a slope of 1.06 and an intercept of 0.72 ng/ml.

The correlation coefficient was 0.9907.

FIGURE 3. Plasma curve from a subject administered delta-9-THC. The

methodology of figure 2 and either tritium or iodine-125-substituted radioligands

were used.
RADIOIMMUNOASSAYS 25

FIGURE 4. Comparison of results on analysis of 62 plasma samples for delta-9-THC

with either a tritium or iodine-125-labeled radioligand.

DEVELOPMENT OF SPECIFIC RIA FOR 11-NOR-9-CARBOXYDELTA-9-THC

Having demonstrated the utility of our approach to the analysis

of delta-9-THC, we then set out to apply the same principles to the

analysis of various metabolites of delta-9-THC. The l l-nor-9-carboxylic acid (or 11-oic acid) has been shown to be a major metabolite

present in plasma and was chosen as our next target for development of an immunoassay. We were reluctant to use a 5’-carboxy

analog of this compound as the hapten to prepare an immunogen,

since this presented the possibility of obtaining a mixture of 5- and

11-linked conjugates. Therefore it was necessary to find some

means of linking through the 5’-position which did not involve a
26 COOK, SELTZMAN, SCHINDLER, TALLENT, CHIN, AND PITT

carboxylic acid. An aldehyde moiety would appear to be useful in

this respect since it would react with the free amino groups of a

protein such as bovine serum albumin to form an imine and the

latter substance could be reduced with sodium cyanoborohydride to

form a stable alkylamino linkage (figure 5). The synthesis of this

compound, however, proved to be no trivial matter (11).

FIGURE 5. General route to an immunogen containing 11-nor-9-carboxy-delta-8-

THC. Reagents: a) bovine serum albumin (BSA); b) sodium cyanoborohydride.

Again, for reasons similar to those in the THC series, the delta-8-

compound was chosen for synthetic work. In the procedure finally

adopted (figure 6), 5’-bromo-delta-8-THC was treated with tetramethyl ammonium acetate to introduce a 5’-acetoxy group. The

phenolic group was protected as its t-butyldimethylsilyl ether, and

the acetoxy group was selectively removed by treatment with lithium aluminum hydride to give the 5’-alcohol which could be oxidized with pyridinium chlorochromate to the 5’-oxo compound. The

aldehyde group was then protected as the ethylene acetal. C-11 was

next converted to an aldehyde with selenium dioxide. Oxidation of

the aldehyde using sodium cyanide and manganese dioxide in

methanol led to the methyl ester of the 9-carboxylic acid. When

this ester was saponified with potassium hydroxide and ethanol,

both the ester and the silyl protecting groups were cleaved. It then

remained only to remove the ethylene acetal to generate the de

sired compound.
RADIOIMMUNOASSAYS 27

FIGURE 6. Synthesis of ethylene acetal of 5’-oxo-11-nor-9-carboxy-delta-8-THC.

Reagents: a) tetramethylammonium acetate; b) t-butyldimethylchlorosilane; c)

lithium aluminum hydride; d) pyridinium chlorochromate; e) ethylene glycol,

toluenesulfonic acid; f) selenium dioxide; g) sodium cyanide, acetic acid, methanol,

manganese dioxide; h) potassium hydroxide; i) toluenesulfonic acid, acetone.

Tests using a model system with a methyl group at C-9 showed

that under standard acidic conditions used to hydrolyze acetals, the

intermediate aldehyde which formed quickly cyclized by interaction with the reactive phenolic ring. It was therefore necessary to

deactivate the phenolic ring by formation of the phenolic acetate

(figure 7). This compound could then be hydrolyzed under acidic

conditions to give the 5’-oxo compound with the acetate group still

present. Mild basic hydrolytic conditions were found to cleave the
28 COOK, SELTZMAN, SCHINDLER, TALLENT, CHIN, AND PITT

acetate. However, under the conditions used for coupling to protein, it was shown that internal cyclization was competitive with

the coupling to protein. Furthermore, the hydrolyzed aldehyde was

unstable and cyclized either on standing or attempted purification

by preparative thin-layer chromatography.

FIGURE 7. Synthesis of immunogen containing 11-nor-9-carboxy-delta-8-THC.

Reagents: a) acetic anhydride, pyridine; b) toluenesulfonic acid, acetone; c) bovine

serum albumin, methanol; d) sodium cyanoborohydride.

The phenolic acetate therefore was allowed to react with bovine

serum albumin in pure anhydrous methanol in the presence of

sodium cyanoboro-hydride (12) and the resulting conjugate was deacetylated by stirring with aqueous methanol and sodium bicarbonate. Differential ultraviolet absorption measurements were then

used to estimate a minimum incorporation of 11 hapten residues

per molecule of protein. Rabbits were immunized with this conjugate. An excellent antiserum titer was obtained by 4 months after

initial immunization.

For the radioligand, the methyl ester of 5’-iodo-11-nor-9-carboxydelta-8-THC was converted to the free acid by treatment with trimethylsilyl iodide followed by hydrolysis with water. Exchange of

the resulting 5’-iodocarboxylic acid with iodine-125 sodium iodide

led to labeled material (figure 8) with a specific activity of about 7.5

Ci/mmole. Using this iodinated radioligand we then examined the

cross-reactions of the anti-serum, which was used at a titer of ap-
RADIOIMMUNOASSAYS 29

proximately 1:20,000 initial dilution. As shown in figure 9, 11-nor-9-

carboxy-delta-9-THC displaced this radioligand over the range of

20-5000 pg. The practical sensitivity was between 20 and 50 pg. The

carboxylic acid in the delta-9-THC series exhibited only slightly

greater affinity for the antibody than did the 5’-iodo-delta-8 derivative. 11-Hydroxy-delta-9-THC and delta-9-THC itself exhibited extremely low cross-reactions, with less than 0.1% the affinity of the

9-carboxy compound. It still remains to be demonstrated that the

antiserum will not cross-react strongly with the very polar acids

which are also formed as metabolites of THC, but based on results

to date it appears that this is an extremely selective antiserum.

FIGURE 8. Synthesis of radioligand for analysis of 11-nor-9-carboxy-delta-9-THC.

Reagents: a) trimethyliodosilane, water; b) iodine-125-NaI, acetone.

ANALYSIS OF 11-NOR-9-CARBOXY-DELTA-9-THC IN PLASMA

We have used this antiserum and radioligand in the analysis of a

series of plasma samples provided to us through the courtesy of

Mr. Owens and Dr. McBay. Conditions for analysis were the same

as for delta-9-THC. Figure 10 shows results for one subject who received THC by smoking. The results of this analysis are in excellent qualitative agreement with data obtained by Wall and coworkers (2). THC itself rises rapidly to a maximum after cessation of

smoking and then falls off rapidly. The concentration of the carboxylic acid metabolite rises fairly rapidly, reaching a maximum at

around 20-40 minutes and thereafter declining at a slower rate.

Almost identical results were observed in a second subject.
30 COOK, SELTZMAN, SCHINDLER, TALLENT, CHIN, AND PITT

FIGURE 9. Displacement curves for delta-9-THC, metabolites and iodo compounds.

Antiserum was generated to 11-nor-9-carboxy-delta-8-THC immunogen; radioligand

was iodine-125-5’-iodo-11-nor-9-carboxy-delta-8-THC.

CONCLUSIONS

These studies have demonstrated that linkage of cannabinoid

molecules to protein through the 5’-position of the amyl side chain

leads to the formation of selective antisera. Antisera developed in

this manner can be used for the analysis of plasma samples for

both delta-9-THC and 11-nor-9-carboxy-delta-9-THC, a principal

plasma metabolite. Furthermore, in conjunction with these antisera, the use of a 5’-iodo radioligand has been shown to be both

practical and convenient. As we have previously pointed out, the

general concept of introducing the iodine at the point of linkage appears to be one which should have widespread utility in radioimmunoassay work (5). Although the results with the 11-nor-9-carboxy-delta-9-THC analysis are at this point somewhat preliminary

and the method requires some further development, it is to our

knowledge the first reported example of a highly specific radioimmunoassay for this metabolite in plasma. We are now in the process of applying these general concepts to the preparation of an

antiserum for 11-hydroxy-delta-9-THC.
RADIOIMMUNOASSAYS 31

FIGURE 10. Analysis of delta-9-THC and 11-nor-9-carboxy-delta-9-THC in a subject

who smoked a single 9 mg THC cigarette.

ACKNOWLEDGMENTS

Dr. Robert Willette originally suggested the synthesis of the 5’-

iodo radioligand to us. We also acknowledge helpful discussions

with Mr. Mike Owens and Dr. Arthur McBay. Mr. Owens suggested

incorporating the methanol extraction procedure into our assay

and both Mr. Owens and Dr. McBay have encouraged our develop

ment of iodinated radioligands.
32 COOK, SELTZMAN, SCHINDLER, TALLENT, CHIN, AND PITT

REFERENCES

1. K. Landsteiner. The Specificity of Serological Reactions. Dover Press, Inc., New

York (1962).

2. M.E. Wall, D.R. Brine, J.T. Bursey, and D. Rosenthal. Detection and quantitation of tetrahydrocannabinol in physiological fluids. In J.A. Vinson (ed.), Cannabinoid Analysis in Physiological Fluids, ACS Symposium Series 98, Am. Chem.

Soc., Washington, D.C., 39-57 (1979).

3. J.J. Pratt, M.G. Woldring, R. Boonman, and W. Bosman. Specificity of assays.

III. Use of two antisera of differing specificities to improve the specificity of

steroid immunoassay. Eur. J. Nucl. Med. 4, 171-177 (1979).

4. C.E. Cook, M.L. Hawes, E.W. Amerson, C.G. Pitt, and D.L. Williams, Radioimmunoassays of Delta-9-Tetrahydrocannabinol. In R.E. Willette (ed.), Cannabinoid Assays in Humans, National Institute on Drug Abuse Research Monograph

Series 7, Supt. of Docs., U. S. Govt. Print. Off., Washington, D.C., 15-27 (1976).

6. C.E. Cook. Radioimmunoassay of cannabinoid compounds. In J.A. Vinson (ed.),

Cannabinoid Analysis in Physiological Fluids, ACS Symposium Series 98, Am.

Chem. See., Washington, D.C., 137-154 (1979).

6. C.E. Cook, M.L. Hawes, E.W. Amerson, C.G. Pitt, D.L. Williams and R.E. Willette. Tetrahydrocannabinol (THC) radioimmunoassay: Immunogen and novel

iodine-125-radioligand based on 5’-substituted-delta-8-THC. Pharmocologist 18, 291

(1976).

7. C.G. Pitt, H.H. Seltzman, S.R. Setzer, and D.L. Williams. The Preparation of 5’-

Iodo-Iodine-l25-Delta-8-THC; A Radioligand for the radioimmunoassay of cannabinoids. J. Label. Comp. Radiopharm., 17, 681 (1979).

8. C.G. Pitt, P.T. Hobbs, H. Schran, C.E. Twine, Jr., and D.L.M Williams. The synthesis of deuterium, carbon-14 and carrier-free tritium labeled cannabinoids. J.

Label. Comp., 11, 551-575 (1975).

9. J.D. Teale, J.M. Clough, L.J. King, V. Marks, P.L. Williams, and A.C. Moffat.

The incidence of cannabinoids in fatally injured drivers: An investigation by radioimmunoassay and high pressure liquid chromatography. J. Forens. Sci. Soc.,

17, 177-183 (1977).

10. D. Rodbard. Statistical quality control and routine date processing for radioimmunoassays and immunoradiometric assays. Clin. Chem., 20, 1255-1270 (1974).

11. H.H. Seltzman, C.G. Pitt, D.L. Williams, and C.E. Twine. Synthesis of a hapten

for the radioimmunoassay of 9-carboxy-11-nor-delta-9-THC. 31st Southeastern regional meeting of the American chemical society, Abstract No. 249 (1979).

12. Cf.R. Muller, A. Scheuer, H. Gerdes and K.-O Mosebach. Direkte kupplung von

steroiden an einweiss durch reduktive aminierung zur gewinnung spezifischer

antikorper. Fresenius Z. Anal. Chem., 290, 164 (1978).

AUTHORS

C. E. Cook, H. H. Seltzman, V. H. Schindler,

C. R. Tallent, K. M. Chin, and C. G. Pitt

Research Triangle Institute

Research Triangle Park, N. C. 27709
Analysis of Cannabinoids

Reaserch Monograph 42, R. Hawks, Editor

National Institute on Drug Abuse, 1982

Radioimmunoanalysis of Delta-9-

THC in Blood by Means of an 125I

Tracer

S. M. Owens, A. J. McBay, and H. M. Reisner

A radioimmunoassay or delta-9-THC in plasma, whole

blood, or hemolyzed blood specimens has been presented.

Samples and standards were diluted with methanol and

centrifuged. An aliquot of the supernatant fluid was incubated with RIA buffer, 125I-labeled delta-8-THC and rabbit

anti-THC serum. Solid phase goat anti-rabbit immunoglobulins were added to separate bound from free THC. After

centrifugation the supernatant fluid was aspirated and the

radioactivity of the precipitate was counted in a gamma

counter. The concentration of THC was calculated from a

standard curve using the logit-log transformation of the

average counts of duplicate tubes. The assay had several

advantages. Methanol dilution gave better results than

direct analysis. The 125I-labeled THC had high specific activity and could be counted in a gamma counter. The immunological separation of anti body-bound THC from free

THC was better than separation techniques using ammonium sulfate and activated charcoal. THC was determined in

0.1 ml of sample with a sensitivity of 1.5 ng/ml in plasma

and 3.0 ng/ml in hemolyzed blood.

INTRODUCTION

There is a need for accurate, sensitive and specific radioimmunoassays (RIA) of delta-9-THC (THC) for pharmacological and forensic studies of various biological fluids. The RIA of plasma samples

(1,2) and hemolyzed blood (3) have been reported.

Forensic blood samples are usually grossly hemolyzed. These re

quire a RIA that is not affected by varying degrees of hemolysis.

33
34 OWENS, McBAY, AND REISNER

An 125I-labeled THC radioligand is needed for this application (4).

The advantages of 125I-THC over 3H- or 14C-labeled THC are that it

is not affected by color quenching during the counting of radioactivity and it can be synthesized at a higher specific activity (5).

THC is a highly lipophilic molecule that adheres to glass and

plastics and binds to various blood products. We found that a critical step in the RIA of THC was the separation of antibody-bound

radioactivity from other radioactivity. We compared five common

RIA separation methods and found that a solid phase second antibody improved the separation.

PREPARATION OF ANTI-THC SERA AND RADIOLIGAND

Delta-8-THC was conjugated to bovine serum albumin by the

method of Cook et al (1). Delta-8-THC was used to make the antigen because delta-9-THC was not available. The concentration of

delta-8-THC in marijuana, if present, is very low compared to the

concentration of delta-9-THC. It therefore will not contribute to the

concentration of delta-9-THC in this analysis.

Four New Zealand white rabbits were each injected with 200 μg

of the antigen in physiological saline emulsified with Freund’s complete adjuvant. All injections were administered subcutaneously

near regional lymph centers. The next four boosts were given every

3 weeks with 100 μg of antigen plus Freund’s complete adjuvant.

Seven months from the start of immunization the rabbits were

boosted with 100 μg of antigen and Freund’s incomplete adjuvant.

The serum was tested for THC binding after the fourth boost and

on all subsequent boosts. The titer was expressed as that dilution of

serum which bound 50% of 85 pg of the radioligand. The assay conditions were those stated in the procedure except that ammonium

sulfate was used to separate bound from free radioactivity. The

serum from the rabbit with the highest titer was chosen for use in

the assay.

The radioligand 5’-iodo-125I-delta-8-THC was synthesized in our

laboratory by the method of Pitt et al (5). The specific activity was

determined to be 250 Ci/mmole by the self-displacement method of

Morris (6).

SPECIFICITY OF ANTISERUM

The cross-reactivity of the antiserum was measured by determining the concentration of compound that caused 50% displacement

of the radioligand. Since delta-9-THC was the analyte of interest,
RADIOIMMUNO ANALYSIS OF DELTA-9-THC IN BLOOD 35

its concentration at 50% displacement was considered as 100%

cross-reactivity. All other compounds were compared to it (table 1).

The assay conditions were those described in the procedure.

TABLE 1. Cross reactivity of various cannabinoids, drugs. and steroids with antidelta-8-THC serum. All percent cross reactivities preceded by the symbol for “less

than” gave no indication of crow reactivity at the concentration equivalent to that

percentage.

SAMPLE PREPARATION

THC in absolute ethanol was obtained from the Research Triangle Institute (RTI), Research Triangle Park, NC. Standards and

controls were prepared the day of the analysis by adding THC in

absolute ethanol to plasma or hemolyzed blood sample pools.

Standards were prepared from 1-100 ng/ml. Hemolyzed blood was

made by storing whole blood for at least one month at 4°C with 1%

sodium fluoride as a preservative. All standards, controls, and unknowns were treated the same throughout the procedure.
36 OWENS, McBAY, AND REISNER

Sensitivity was increased and interferences were decreased by diluting the samples with methanol rather than analyzing the samples directly. The use of methanol has been reported by Teale et al.

(7).

An aliquot (100 μl) of sample was pipetted into a silanized 10 ×

75 mm glass test tube. The tube was vortexed at low speed while

methanol (500 μl) was added. The tubes were immediately corked.

After 30 minutes the tubes were centrifuged at 1000 × g for 15

minutes. A 100 μl aliquot of the supernatant fluid was used in the

assay. When approximately 10 ng/ml of the radioligand was added

to plasma or hemolyzed blood (n = 10) the percent recovery was 99.3

(%CV= 1.4) and 93.3 (%CV = 4.7) respectively.

RADIOIMMUNOASSAY PROCEDURE

The buffer was tris-NaCl with bovine serum albumin (0.05 M

tris(hydroxymethyl)-aminomethane and 0.15 M NaCl with 0.1%

bovine serum albumin and 0.2% NaN3 adjusted to pH 7.6 with concentrated HCl). The radioligand in 50% ethanol was added to the

buffer in a ratio of 40 pg per 500 μl. A 500 μl aliquot of radioligand

in buffer was added to untreated 10 × 75 mm glass test tubes.

Each sample was analyzed in duplicate. An aliquot (100 μl) of the

methanol supernatant fluid was added to appropriate tubes and

vortexed. The anti-THC serum was diluted 1:35,000 with buffer (20-

35% antibody specific binding) and 100 μl was added to all tubes

except control tubes for nonspecific binding and total counts. To

those tubes 100 μl of buffer was added. All tubes were vortexed.

The rack of tubes was sealed in a plastic bag to prevent evaporation and was incubated overnight at 4°C. The next day, 100 μl of

solid phase goat anti-rabbit immunoglobulins (Immunobeads, prepared by Bio-Rad Laboratories, Richmond, CA 94804) were added.

The tubes were vortexed and then refrigerated at 4°C for 1 hour.

The tubes were allowed to warm to room temperature for 1 hour.

They were then centrifuged at 1000 × g for 8 minutes to precipitate antibody-bound radioligand. The supernatant was aspirated

without disturbing the pellet and 2 ml of buffer were added. The

tubes were vortexed and centrifuged as before. The supernatant

fluid was aspirated without disturbing the pellet. This washing reduced specific and nonspecific binding approximately 5%. The

tubes were counted in a gamma counter, having an 84.6% counting

efficiency with a 125I source. The concentration of THC in controls

and unknowns was obtained from a standard curve using the logitlog transformation of the average counts of duplicate tubes.
RADIOIMMUNOANALYSIS OF DELTA-9-THC IN BLOOD 37

COMPARISON OF SEPARATION METHODS

The methods commonly used to separate free from antibodybound radioactivity are based on chemical or immunological differences between free and antibody-bound radioactivity. If the equilibrium between the bound and free forms is disturbed a misclassification of counts could occur. An ideal method should not affect this

equilibrium. Five radioimmunoassay separation methods were compared. These used polyethylene glycol, Staphylococcus aureus with

protein A, activated charcoal, ammonium sulfate, or solid phase

goat anti-rabbit immunoglobulins (second antibody). The factors

considered in choosing a method were the accuracy of classification

of bound and free counts and the variability of binding with

plasma and hemolyzed blood. The Staphylococcus aureus with protein A and polyethylene glycol methods were not usable due to

high nonspecific binding and variability from sample to sample.

Ammonium sulfate performed well with plasma even though there

was high nonspecific binding. It could not be used with hemolyzed

blood. The charcoal separation performed well with plasma. With

hemolyzed blood it had variable nonspecific binding with different

samples. Also, a refrigerated centrifuge was needed. The solid

phase second antibody separation gave the most consistent results

with different specimens. The nonspecific binding for plasma was

consistently 0.5% ± 0.1% and for hemolyzed blood it was 1.5% ±

0.2%. Another important feature was that standard curves of

plasma and hemolyzed blood, when assayed in the same RIA, were

almost superimposable (see figure 1). Since plasma had 2.8% greater antibody-specific binding of radioligand than hemolyzed blood,

all samples had to be compared to their own zero dose standard,

i.e., plasma unknowns to plasma zero dose standards and hemolyzed blood unknowns to hemolyzed blood zero dose standards.

VALIDATION OF PROCEDURE

To obtain blood containing THC, a smoking study was conducted

under the direction of Dr. Mario Perez-Reyes of the University of

North Carolina Department of Psychiatry. Mexican marijuana

cigarettes containing 1% delta-9-THC (approximately 9 mg) and

other naturally occurring cannabinoids were provided by the National Institute on Drug Abuse. Each experienced male and female

volunteer smoked on two separate occasions either a marijuana

cigarette or a placebo. The placebos, which were obtained from

NIDA, were prepared by extracting cannabinoids from marijuana.
38 OWENS, McBAY, AND REISNER

FIGURE 1. Typical RIA logit-log curves for plasma and hemolyzed blood. When

assayed in the same RIA they were almost superimposable. The plasma and

hemolyzed blood curves had slopes of -1.56 and -1.55, y-intercepts of 1.63 and 1.70,

and nonspecific bindings of 0.5% and 1.5%, respectively. Both curves had a

r2=0.999. The percentage of total binding was 2.8% less for hemolyzed blood than

for plasma.

All subjects were asked not to use marijuana for 4 days prior to the

study. Blood samples were collected from an antecubital vein at 0,

5, 10, 15, 20, 30, 45, 60, 90, and 120 minutes after the start of smoking. A portion of the whole blood was preserved with 1% sodium

fluoride. The remaining blood was heparinized and the plasma separated. All samples were stored in silanized scintillation vials at

- 20 °C until analyzed.
RADIOIMMUNOANALYSIS OF DELTA-9-THC IN BLOOD 39

A total of 50 positive plasma samples from 5 subjects were analyzed in a double-blind fashion. The results were compared with

time after smoking. This was to determine if the expected concentration vs. time curve would be produced (see figure 2). The biphasic curve that was produced was similar to that of Wall et al (8).

FIGURE 2. Five subjects smoked Mexican marijuana cigarettes that contained

approximately 9 mg of THC. It took from 7.5 to 13 minutes to finish the cigarettes.

The concentration vs. time curves were all biphasic and similar for each subject.

As further validation, 31 positive and 21 negative plasma samples from the smoking study were analyzed in double-blind fashion

at RTI by the delta-9-THC RIA of Dr. C. E. Cook and by our procedure (see figure 3). The correlation coefficient was 0.971. All negative samples were correctly identified by both laboratories.

The usual means of determining sensitivity of a RIA is to evaluate statistically the counting variances of zero dose standards (9).

By this criterion, the sensitivity for THC in plasma and hemolyzed
40 OWENS, McBAY, AND REISNER

FIGURE 3. Comparison of 31 positive samples analyzed in a double-blind fashion at

RTI by a THC RIA and by our THC RIA. The correlation coefficient was 0.971.

blood specimens would be less than 1 ng/ml. The analysis of presumed negative samples proved to be a more rigid criterion for sensitivity estimation.

The sensitivity was determined by comparing the calculated concentrations of all negative samples with the zero dose standards.

Negative blood samples were obtained from subjects smoking placebos and from 10 nonusers of marijuana. Altogether 61 negative

plasmas and 16 negative hemolyzed bloods from 16 different sub

jects were analyzed. No plasma was calculated greater than 1.0 ng/

ml and no hemolyzed blood was calculated greater than 2.5 ng/ml.

To assure no false positives, the least detectable concentration was

arbitrarily set at 1.5 ng/ml in plasma and 3.0 ng/ml in hemolyzed

blood.
RADIOIMMUNOANALYSIS OF DELTA-9-THC IN BLOOD 41

We are presently trying to confirm if all samples from the placebo smoking study are THC-free. Some had significantly higher concentrations than the concentrations found in nonusers of marijuana. Since individuals in the smoking study were marijuana users,

the low concentrations of THC found in some samples may represent low residual concentrations of the drug.

The intra-assay reproducibility was determined using plasma or

hemolyzed blood spiked with THC (see table 2). The interassay precision for plasma samples was determined by using samples from

the smoking study (see table 3). The hemolyzed blood interassay

precision is being determined.

TABLE 2. Reproducibility of the assay of known quantities of THC. THC was added

to a plasma or hemolyzed blood sample pool. The THC concentration was calculated

from standard curves which were similar to figure 1.

TABLE 3. The interassay precision was determined using plasma samples from a

marijuana smoking study. The replicates were analyzed in spearate assays over a 3-

week period.

CONCLUSIONS

The concentration of THC after smoking can be accurately determined in various biological fluids by this RIA, using an 125I tracer

and a specific separation method. This assay will be of use in forensic and pharmacological studies.
42 OWENS, McBAY, AND REISNER

ACKNOWLEDGMENTS

Supported in part by North Carolina Governor’s Highway Safety

Program Project No. 80-08-03-C-308-2.

The helpful discussions with Dr. C.E. Cook and the technical

advice during preparation of the radioligand by Dr. C.G. Pitt, D. L.

Williams, and Dr. H. H. Seltzman of the Research Triangle Institute are acknowledged. Dr. Mario Perez-Reyes of the University of

North Carolina at Chapel Hill performed the human studies.

REFERENCES

1. C.E. Cook, M.L. Hawes, E.W. Amerson, C.G. Pitt, and D.W. Williams. Radioimmunoassays of delta-9-tetrahydrocannabinol. In R.E. Willette (ed.), Cannabinoid

Assays in Humans, National Institute on Drug Abuse Research Monograph 7,

Supt. of Docs., U.S. Govt. Print. Off., Washington, D.C., l5-27 (1976).

2. S.R. Gross and J.R. Soares. Validated direct blood delta-9-THC radioimmune

quantitation. J. Anal. Toxicol.. 2, 98-100 (1978).

3. S.R. Gross, J.R. Soares, and V.C. Reeve. (Personal communication).

4. C.E. Cook. Radioimmunoassay of cannabinoid compounds. In J.A. Vinson (ed.),

Cannabinoid Analysis in Physiological Fluids,. ACS Symposium Series 98, Am.

Chem. Soc., Washington, D.C., 137-154 (1979).

5. C.G. Pitt, H.H. Seltzman, S.R. Setzer, and D.L. Williams. The preparation of 5’-

iodo-125I-delta-8-THC; a radioligand for the radioimmunoassay of cannabinoids. J.

of Labeled Compd and Radiopharm. 17. 681-689 (1980).

6. B.J. Morris. Specific radioactivity of radioimmunoassay tracer determined by

self-displacement: A re-evaluation. Clin. Chim. Acta, 73, 213-216 (1976).

7. J.D. Teale, J.M. Clough, L.J. King, V. Marks, P.L. Williams, and A.C. Moffat.

The incidence of cannabinoids in fatally injured drivers: An investigation by radioimmunoassay and high pressure liquid chromatography. J. Forens. Sci. Soc.

17. 177-183 (1977).

8. M.E. Wall, T.M. Harvey, J.T. Bursey, D.R. Brine, and D Rosenthal. Analytical

methods for the determination of cannabinoids in biological media. In R.E. Willette (ed.), Cannabinoid Assays in Humans. National Institute on Drug Abuse

Research Monograph 7, Supt. of Docs., U.S. Govt. Print. Off., Washington, D.C.,

107-117 (1976).

9. D. Rodbard. Statistical estimation of the minimal detectable concentration

(“sensitivity”) for radioligand assays. Anal. Biochem., 90, 1-12 (1978).
RADIOIMMUNOANALYSIS OF DELTA-9-THC IN BLOOD 43

AUTHORS

S. M. Owens* and A. J. McBay

Office of the Chief Medical Examiner

and

Department of Pathology

University of North Carolina

Chapel Hill, N. C. 27514

and

H. M. Reisner

Department of Pathology

University of North Carolina

Chapel Hill, N. C. 27514

*present address:

Department of Pharmacology

University of Arizona

Tucson, Arizona
Analysis of Cannabinoids

Research Monograph 42, R. Hawks, Editor

National Institute on Drug Abuse. 1982

Significant Developments in

Radioimmune Methods Applied to

THC and its 9-Substituted

Metabolites

J. R. Soares, J. D. Grant, and S. J. Gross

Novel RIA’s have been developed for marijuana detection.

Modified simple techniques measure the “acute” metabolites in minute saliva, breath, and dried blood samples?

Solid purified antibody matches the sensitivity and yields

sensitivity values identical to liquid assays. The importance

of the polar 9-substituted metabolites (9SM) of THC in

acute users is shown by time course data that indicate their

levels remain high in the critical post-intoxication period

when -THC clears from the blood. Therefore a single-tube

assay using solid-phase reagents was constructed to quantitate simultaneously THC and 9SM. When -THC values

are low or questionable in subject samples, 9SM would

appear useful to confirm or refute acute marijuana use.

INTRODUCTION

The evolution of radioimmunoassay (RIA) techniques has confirmed our original concept (1) that desirable specificity depends on

sterically free chemical functions in the hapten and marker moieties.

These principles have allowed the development of

tetrahydrocannabinol (THC) and polar g-substituted THC metabolite (9SM) assays requiring a single incubation of 20 μl samples of

serum or blood (hemolyzed). These sensitive assays (5 ng/ml) have

routine clinical applicability. For example, THC dosage for antiemetic effects can effectively be monitored by serum concentration.

44
NEW RADIOIMMUNE METHODS FOR CANNABINOlDS 45

Furthermore, such determinations will add substantially to clinical

epidemiologic and behavioral assessments. Given a high avidity

antibody, the assay is flexible and can encompass a broad spectrum

of liquids and solids. Since there are obvious forensic applications,

the existing methodology has been extended to breath.

Conventional RIA procedures require select reagents and critical

steps mainly to separate free radioligand from that bound to antibody. This can be accomplished by “pour out” or “dipstick” techniques utilizing newly developed solid matrices containing covalently bound antibody or hapten. These materials can now be used

to measure two (or more) different entities simultaneously in a

single container.

MATERIAL AND METHODS

Subjects

Fifteen adult male volunteers were recruited. Their marijuana

habits ranged from light (<1/month) to heavy (>3/day). They

were asked to refrain from private use of cannabis for 24 hours

prior to testing and the baseline (pre-smoke) negativity of their

samples (blood and breath) verified by RIA. Volunteers were allowed 15 minutes to complete a single marijuana cigarette containing THC equivalent to approximately 200 μg THC per kg body

weight of subject. Blood and breath were collected at various intervals up to 6 hours after smoking. Serum was obtained by centrifugation of samples in red top vacutainer tubes. Five uninterrupted

breaths were passed over a borosilicate solid matrix. Cannabinoids

were recovered by elution with ethanol, reconstituted in buffer and

tested for -THC and 9SM content by liquid phase methods (described below).

Haptens and Antigens

The 2-(4’carboxyphenylazo) derivatives of THC (Azo-THC), 11-

hydroxy- -THC (Azo-11-OH) and cannabinol (Azo-CBN) were covalently coupled to polymers for immunization (2) and affinity chromatography (3).

Antisera

Texas bush goats were immunized with Keyhole Limpet Hemocyanin conjugates of Azo-THC and Azo-11-OH as previously described (2). Sera harvested by plasmapheresis were tested as follows.
46 SOARES, GRANT, AND GROSS

Titer was defined as the dilution at which 0.1 ml of antiserum

binds 40% of added 3H- -THC (40,000 cpm). Cross reactivities were

calculated from standard curves and expressed as -THC/cannabinoid mass ratio at 50% inhibition ( × 100%).

Diluted THC-specific antiserum was used directly in standard

liquid-phase assay. 9SM antibodies isolated by affinity chromatography were coupled to an insoluble matrix for solid-phase methods.

lmmunosorbant

The Azo-CBN carboxyl (50 μmoles) was converted to an active

ester by isobutyl chloroformate (60 μmoles) and added to a 10%

suspension of aminoalkyl agarose (Affi-Gel 102, BioRad, 35.1

μmoles of alkylamine) in dioxane. The immunosorbant (AZO-CBN-S)

was washed with dry dioxane (1L) to remove uncoupled hapten,

and stored in distilled water.

Affinity Chromatography

Saturated ammonium sulfate was added to Azo-11-OH antisera to

a final concentration of 33% and the precipitate (crude IgG) further purified by passage through QAE Sephadex (QAE IgG). Antibody dilutions were concentrated (10 ng/ml) and 10 ml aliquots

were incubated with immunosorbant to remove all antibody from

the supernate. Azo-CBN-S was washed with PBS (0.01 M phosphate

buffer, pH 6.9, 0.9% sodium chloride) until free of nonspecific protein (O.D. < .005). There was a final wash with 1 M lithium bromide

(100 ml). Clean immunosorbant was pipetted into glass columns (1.2

× 15 cm). Elution with 0.09 M hydrochloric acid (30 ml) removed

specific antibody. The eluate was dialyzed against distilled water

(12 hours at 4°C) and phosphate buffer (24 hours at 4°C).

Purified antibody was concentrated with sucrose and concentrations calculated spectrophotometrically (280 nm). Final titer was

compared with parent solutions to document recovery of activity.

Matrix Antibody Conjugates

Antibody fractions of varying purity were coupled to a polyfunctional acrylate. Active polymer fractions (33 mg) were reacted with

400 μg per ml antibody (3.0 ml). Uncoupled IgG was removed with

PBS. Binding efficiency of immunosorbant-purified antibody-matrix

conjugate (Ab-C) was compared with IgG-polymer controls (nonimmune IgG, crude IgG and QAE IgG fraction). Solid-phase reagents (30% suspension in PBS) were stored at 3°C.
NEW RADIOIMMUNE METHODS FOR CANNABINOIDS 47

Liquid-Phase Radioimmunoassays

The following cannabinoids were obtained from the National Institute on Drug Abuse (NIDA): -THC, - -THC, 11-hydroxy

THC, 11-nor- -THC-9-carboxylic acid, cannabinol, cannabidiol, and

3H- -THC (50.3 Ci/mM).

Standard inhibition curves were constructed from non-user samples containing known amounts of - THC. Serum (50 μl) was incubated (16 hours, 4°C) with 0.1 ml antiserum (diluted to bind 40% of

added marker in the absence of unlabeled THC) and 188 pg (40,000

cpm) of 3H- -THC. Free and antibody-bound radioligands were separated using 0.5 ml dextran-coated charcoal suspension (50 gm

Norit A charcoal and 5 gm dextran T-70 in 1.0 L PBS) chilled at

4°C by refrigeration. Antibody-bound 3H- -THC was counted in a

Beckman LS-250 Beta counter after decanting supernates into scintillation vials containing 10 ml of Aquasol (New England Nuclear).

Logit transformation (4) was employed to construct standard

curves and to calculate -THC concentrations of unknown samples

using a Wang 2200 computer.

Solid-Phase 9SM RIA

Serum standards were prepared from pooled marijuana negative

serum. Stock solutions of 11-nor- -THC-9-carboxylic acid in 50%

ethanol (0.01 mg/ml) were diluted 1/100 with serum to give a final

concentration of 100 ng/ml. This was serially diluted in serum for

standard curves which were included in each assay by transferring

aliquots (20 μl) from a sequence of cannabinoid concentrations into

10 × 75 mm borosilicate glass tubes in duplicate. Unknown samples (20 μl) were assayed identically.

Ab-C (10 μl) and 3H- -THC (30,000 cpm/0.6 ml PBS, 0.1% albumin) were added and the tubes incubated for 12 hours at 4°C with

intermittent shaking. PBS (1.0 ml) was added, and the tubes vortexed. After centrifugation (4,000 rpm, 10 minutes), supernates

were discarded, and the percentage of 3H- -THC bound to antibody

calculated by counting the matrix directly in scintillation fluid.

Log-logit standard curves were used to estimate unknown 9SM concentrations,

Dual-Metabolite RIA

The foregoing method was modified to assay -THC and 9SM simultaneously in a single tube. -THC and 9SM serum standards

were added to separate sets of 10 × 75 mm borosilicate glass tubes

in duplicate. 3H -THC (30,000 cpm/0.l ml), Ab-C (10 μl) and soluble -THC specific antibody (0.5 ml) were added to each set and
48 SOARES, GRANT, AND GROSS

the reagents incubated (12 hours, 4°C) with intermittent shaking.

The tubes were centrifuged and the percent of label bound to 9SM

antibody determined by measuring matrix counts. 3H- -THC

bound to -THC specific antibody was measured in the supernate.

Unknown samples (20 μl) were assayed identically, and -THC

and 9SM concentrations estimated from liquid ( -THC) and solid

(9SM) phase standard curves.

RESULTS

Antisera Specificities

Cross-reactivities are summarized in table 1. 11-Hydroxy -THC,

11-nor- THC-9-carboxylic acid and -THC are equally recognized

by the 9SM antibody. The non-homologous radioligand (

3H- -THC)

contributed to this spectrum. -THC antiserum has far greater

specificity, the cross-reactivities for 11-hydroxy- -THC and 11-nor-

-THC-9-carboxylic acid being 26% and 0.5% respectively.

TABLE 1. Percent Crossreactivities*

Compound 9SM

Antiserum

-THC 100.0 100.0

-THC 98.2 96.5

11-hydroxy- -THC 97.6 26.0

11-nor- -THC-9-carboxylic acid 96.5 0.5

Cannabinol 15.5 22.0

Cannabidiol 0.1 0.15

THC/cannabinoid ratio at 50% inhibition (x 100%).

Cannabinoid Blood Levels

Pre-intoxication levels of 9SM and -THC were negative in sera

from all 15 volunteers. Initial samples taken 15 minutes after completion of smoking showed -THC concentrations between 9 and

42 ng/ml. Thereafter, levels rapidly decreased, becoming undetectable at 2 l/4 to 3 l/4 hours. 9SM levels were vastly higher (20-198

ng/ml) than corresponding -THC values. The persistence of meta-
NEW RADIOIMMUNE METHODS FOR CANNABINOIDS 49

bolites was longer than -THC in all subjects but varied with their

individual habits. For example, in all three subjects who used marijuana heavily (>3 times/day) 9SM levels remained elevated (>20

ng/ml) up to 6 hours after marijuana inhalation (figure 1). 9SM returned to baseline (0) in two subjects whose sera were obtained at

24 hours. Alternatively, 9SM decreased to the level of assay sensitivity (5 ng/ml) in all moderate smokers (1-4 times/week) by 6 1/2

hours. Finally, two light smokers’ (<1 time/month) sera contained

no measurable 9SM 3 1/4 hours following acute exposure.

FIGURE 1. Serum levels of THC 9-polar metabolite in individuals who differ in

degree of marijuana use. Levels of 9SM were determined at various intervals after

subjects smoked one marijuana cigarette containing 200 μg THC/Kg body weight.

In all cases, 9SM cleared more slowly from the blood than

THC. Indeed, the mean 9SM/ THC ratio for these subjects increased from 2.9 at 15 minutes to 5.7 at 2 1/2 hours.

Breath

Cannabinoids were measured in trapped breath from ten of the

above volunteers after smoking a single marijuana cigarette. Both

-THC and 9SM were detected immediately upon completion of

smoking (0 time). Levels of THC between 10 and 56 ng/sample

were measured at 15 minutes after smoking and were detectable to
50 SOARES, GRANT, AND GROSS

1 hour. Higher concentrations of 9SM were noted (table 2) with

maximum values (50-123 ng/sample) observed at 15 minutes. Significant amounts of 9SM were detected up to 2 1/2 hours.

TABLE 2. THC Metabolites in Breath at Various Intervals after

Smoking a Single Marijuana Cigarette (200 μg THC/Kg

of body weight)

Time* 9SM levels in trapped breath**

Post-intoxication

(minutes) Subjects

RR EP JT DG

*Zero time signifies completion of smoke.

**Trapped breath was analyzed by liquid-phase methods using spiked-buffer

standards and 9SM level expressed as ng recovered per sample.

Solid-Phase 9SM Assay

Standard Curves

Ab-C and QAE IgG-C standard curves are compared in figure 2.

Six replicate assays were used to construct the Ab-C curve. Loglogit transformation of this data yielded a linear response to 9SM

in the 2-50 ng/ml range. The uniformly low coefficients of variation

for each standard documented precision throughout the response

curve. Assay sensitivity (2 ng/ml) was defined as the point on the

standard curve greater by 2 standard deviations than 0.0 ng/ml.
NEW RADIOIMMUNE METHODS FOR CANNABINOIDS 51

FIGURE 2. Solid-phase 9SM standard curves. THC-negative serum samples spiked

with 9SM were added to tubes containing antibody-matrix conjugate and 3H- THC.

Matrix was counted after incubation with (B) and without (B0) hapten. Log-logit

transformations of mean values from six replicate assays were used to construct

standard curves. The coefficient of variation for each point of the linear Ab-C

standard curve is in parenthesis. The flattening of the QAE IgG-C curve at high

9SM concentrations is indicative of high capacity nonspecific binding.

In contrast, QAE IgG-C standard curves were nonlinear. This occurred because less antibody IgG was available for covalent linkage

per unit volume of matrix in competition with other proteins in

crude antibody fractions. Thus, larger aliquots of QAE 1gG-C (100

μl) than Ab-C (10 μl) were required to bind sufficient 3H- THC

(figure 3). Resultant nonspecific binding caused extreme distortion

of the QAE IgG-C standard curve resulting in loss of specificity and

sensitivity.
52 SOARES, GRANT, AND GROSS

FIGURE 3. SH- -THC binding to polyacrylate antibody conjugates. Binding of 3H-

-THC to insolubilized antibody fractions of various purity. Small aliquots of

immunosorbant-purified material (20 μl/30% v/v suspension) binds 3H- THC well.

The excessive amounts of QAE Sephadex antibody conjugate (100 μ1/30% v/v

suspension) needed for equivalent binding of 3H- -THC results in 30% nonspecific

binding.

Precision

Intra-assay precision was measured by analysis of aliquots of

single samples in the same assay. A mean coefficient of variation of

14% was obtained with four volunteers’ sera assayed in duplicate.

Inter-assay precision was evaluated by analysis of six samples in

two separate assays. The coefficient of variation for these results

was 17%.
NEW RADIOIMMUNE METHODS FOR CANNABINOIDS 53

Solid-Phase Dual-Metabolite Assay

Simultaneous quantitation of both THC and 9SM in a single

assay tube was done by partitioning antibody into solid (9SM) and

liquid ( -THC) phases. Soluble -THC antibody measured -THC.

The 9SM were measured by homologous insolubilized antibody.

Accuracy

Accuracy was assessed by analysis of cannabinoid negative

serum to which varying amounts of -THC, 9SM, or both had been

added (table 3). Observed (O) and expected (E) values were compared to calculate the percent recovery from spiked serum (O/E ×

100%). 9SM recovery was excellent (92-120%) for concentrations

ranging from 0-50 ng/ml except when -THC levels were high

(> 10 ng/ml). In this situation excess - THC displaces significant

amounts of 3H- -THC from its homologous antibody. A portion of

this label then binds to 9SM-specific antibody, resulting in slight

underestimation of 9SM (70-84% recovery). Importantly, -THC

values were accurately measured irrespective of 9SM content of

serum (92-110%).

TABLE 3. Recovery of 9SM and - THC Added to Cannabinoid

Negative Serum Measured by Simultaneous RIA

Cannabinoids added Cannabinoids measured Recovery (%)

(ng/ml) (ng/ml)

Aliquots of serum were spiked with varying amounts of -THC, 9SM, or both.

These were assayed in a single test tube by solid-phase methods.
54 SOARES, GRANT, AND GROSS

Subject Samples

9SM and -THC were measured by this method in the sera of

volunteer smokers. Liquid-phase assay was used to corroborate accuracy. Results from six subjects illustrate several important features (table 4). 9SM levels were precisely quantitated and were 4-6

times higher than corresponding -THC values. Additionally, in

certain subjects (3 and 5) 9SM were useful in confirming a marginal THC. Furthermore, the negative status of one control subject

(4) with a reported THC of 2.3 ng/ml was affirmed by the absence of measurable 9SM.

TABLE 4. Sera Assayed for 9SM and -THC by Simultaneous RIA

Subject Standard RIA Results Single Tube Assay Results

(ng/ml) (ng/ml)

1 25 26 24 32

2 6 29 5 22

3 3 12.5 4.5 16

4 2.3 3.5 0

5 7.0 36.3 10 45

6 0 0 0 0

Marijuana metabolites were measured In subjects’ sera within three hours of

acute exposure. Standard liquid assay was used to assess the accuracy of simultaneous metabolite measurements.

DISCUSSION

The early liquid-phase RIA is a flexible technique which has

been effectively employed for -THC and metabolite measurements in minute volumes of body fluids using samples ranging

from clear serum to hemolyzed blood. However, expertise in pipetting small volumes containing high concentrations of troublesome

macromolecules (5) is required to quantitate low nanogram/ml concentrations. In addition, physical (charcoal, ammonium sulfate) or

immunologic (second antibody) separation steps require critical

timing and may introduce dilution, “stripping” and nonspecific

binding errors. These limitations of liquid-phase RIA’s have stimulated development of solid-phase techniques in many laboratories
NEW RADIOIMMUNE METHODS FOR CANNABINOIDS 55

here and abroad. An antibody-coupled solid matrix simplifies and

can even eliminate the steps necessary to separate free from bound

hapten. Finally, since the solid phase is counted, water and macromolecule quenching of scintillant is avoided.

Reactive solid polymers can selectively remove and concentrate

desired hapten from a dilute body fluid containing a great mixture

of ingredients. Thus, such a matrix permits the collection and sub

sequent measurement of cannabinoids from breath samples.

It should be noted in this context that studies in which tritiated

-THC was administered intravenously (Mario Perez-Reyes, personal communication), no radioactivity was detected in saliva. If

the present findings reflect metabolic processes at the lung surface,

cannabinoid measurement in breath has potential significance.

The newer methods employing solids available in geometric

forms ranging from regular tubes to amorphous beads lead to simultaneous serum THC and 9SM measurements in a single container. This has potential merit in gauging marijuana impairment,

particularly when -THC concentration is marginal. Long-term

stability evaluations of purified antibody and the other solid reagents are now required. Polyphase supports will eventually encompass a large variety of molecules currently assayed by qualified

technicians in hospital and reference laboratories. The new generation of methods will simplify their tasks and perhaps allow quantification in the low nanogram/ml range by less experienced individuals.

REFERENCES

1. S.J. Gross. Specificities of steroid antibodies, chapter 3, in Immunologic Methods

in Steroid Determination, Appleton-Century-Crofts, 41-54 (1970).

2. J.D. Grant, S.J. Gross, P. Lomax, and R. Wong. Antibody detection of marijuana. Nature New Biology, 236, 216-217 (1972).

3. J.D. Grant, J.R. Soares, and S.J. Gross. Separation of high affinity hapten specific and crossreacting IgG population. Immunochemistry, 12, 481-484 (1975).

4. D. Rodbard, W. Bridson, and P.L. Rayford. Rapid calculation of radioimmunoassay results J. Lab. Clin. Med., 74, 770-781 (1969).

5. R.S. Yalow and S.A. Berson. Problems of validation of radioimmunoassays,

chapter XV, in Principles of Competitive Protein Binding Assays, J.P. Lippincott,

374-400 (1970).

AUTHORS

J. R. Soares, J. D. Grant, and S. J. Gross

Receptor Research Institute

1707 Gardena Avenue

Glendale, CA 91204
Analysis of Cannabinoids

Research Monograph 42, R. Hawks, Editor

National lnstitute on Drug Abuse, 1982

Combined High-Performance Liquid

Chromatography and

Radioimmunoassay Method for the

Analysis of

Tetrahydrocannabinol and its

Metabolites in Plasma and Urine

Anthony C. Moffat, Peter L. Williams, and Laurence J.

King

A high-performance liquid chromatography-radioimmunoassay met od for the measurement of cannabinoids in h

plasma and urine is described. The experimental procedure

consists of chromatraphing a plasma extract or hydrolysed urine sample by high-performance liquid chromatography and quantifying the elutin cross-reacting cannabinoids with radioimmunoassay. The concentrations of

tetrahydrocannabinol (THC), cannabinol, mono-hydroxylated metabolites, di-hydroxylated metabolites, THC-11-oic

acid, and -THC-11-oic acid ester glucuronide may be

measured by this technique.

INTRODUCTION

Radioimmunoassays (RIA) for the analysis of cannabinoids in

human body fluids have been described (1-5) and have a number of

advantages compared with other analytical techniques. Biological

fluids may be analysed without pretreatment, the sensitivity of the

method is high, large numbers of samples may be analysed at the

same time and the selectivity of the antiserum for cannabinoids

gives the assay a high degree of specificity. However, each antiserum cross-reacts with different cannabinoids to different extents

56
COMBINED HPLC/RIA ANALYSIS OF CANNABINOIDS 57

and these cross-reactions may be further changed by altering the

assay conditions, radioligand, etc. (6). An antiserum for use in a

RIA can be produced such that it may be specific for

tetrahydrocannabinol (THC). Thus, THC levels in plasma may be

measured directly with the use of this antiserum, even though

other cannabinoids or THC metabolites may be present. However,

if the 11-nor- THC-9-carboxylic acid (THC-11-oic acid) is to be

measured in urine, then a separate antiserum, and hopefully one

specific to the acid metabolite and no other, must be prepared for

use in another RIA (7).

An alternative approach is to produce an antiserum which crossreacts with a number of cannabinoids including THC and some of

its metabolites. This antiserum may then be used to assay plasma

or urine, but the levels found can only be expressed as cross-reacting cannabinoids, since the analytical results are the sum of all the

cross-reacting cannabinoids present in the sample (8). Although

only one RIA is now needed for any biological fluid to be assayed,

the ease of analysis has been exchanged for lack of specificity.

Other methods for the assay of cannabinoids in biological fluids

have been developed, but nearly all require lengthy analysis times,

due partly to the extensive purification procedures required prior

to the instrumental analysis. The use of chromatographic methods

has aided analyses because of the separating power of the techniques, but detection problems have always been evident. Various

devices have been used to increase the selectivity of ultra-violet

(UV) detectors in high-performance liquid chromatographic (HPLC)

analyses, e.g., by the use of a Sephadex column clean-up prior to

HPLC (9) or by the use of simultaneous dual wavelength UV detection (10). However, the great separating power of HPLC has constantly been hindered by the lack of a specific detector. The use of

HPLC as a clean-up procedure before the use of another analytical

technique has obvious advantages, and methods involving mass

spectrometry (MS) (11), gas chromatography (GC) using electron

capture detection (12), and liquid scintillation counting for radiolabelled metabolites (13) have been used.

The combination of RIA with HPLC has obvious advantages. The

drawback of the lack of a sensitive and specific detector for cannabinoids in HPLC can be overcome by the use of a radioimmunoassay, and the lack of complete specificity of an antiserum can be

overcome by the prior use of HPLC. Thus, cross-reacting components can be separated by the HPLC system, and by collecting fractions at the end of the column the individual cannabinoids can be

quantified. This procedure has been used for the quantification of

THC and its metabolites in human plasma (14) and also for the
58 MOFFAT, WILLIAMS, AND KING

analysis of THC metabolites in human urine (15). Plasma levels of

THC obtained by the HPLC-RIA method have also been compared

with those obtained by a GGMS procedure. The HPLC-RIA methods are presented below, together with a discussion on the advantages of the combined technique for use in forensic toxicology.

COLLECTION OF SAMPLES

Volunteers smoked tobacco cigarettes impregnated with either 10

or 8 mg of THC over a lo-minute period. Blood samples (10 ml)

were taken at timed intervals after the subject had finished smoking. Anticoagulant (potassium EDTA, 10 mg) was added to the

blood, which was centrifuged to separate the plasma. Urine samples from the same subjects were collected in silanised glass containers during the 24-hour period after smoking. Control urine

samples were obtained from volunteers with no experience of cannabis use. All samples were stored at -20°C until analysis.

RADIOIMMUNOASSAY

Antiserum (133Y/22/5) for the assay was obtained from Dr. J. D.

Teale, Department of Biochemistry, University of Surrey, Great

Britain. -THC and other cannabinoid compounds were provided

by the National Institute on Drug Abuse, Rockville, Maryland,

U.S.A. (G-3H)-THC was purchased from the Radiochemical

Centre, Amersham, Great Britain, polyvinylpyrrolidone-40 and

charcoal (Norit A) from Sigma, St. Louis, Missouri, U.S.A., Dextran

T70 from Pharmacia, Uppsala, Sweden, and methanol (AnalaR)

and all other chemicals and solvents were obtained from BDH,

Poole, Great Britain. An Intertechnique SL30 was used for liquid

scintillation counting.

Antiserum (133Y/22/5) was stored as aliquots in buffer (0.1M

phosphate buffer pH 7.5, containing 0.2 percent

polyvinylpyrrolidone-40) at -20°C and diluted to 1:300 before use.

Solutions of THC used to calibrate the assay were made up in

aqueous methanol (50 percent v/v) at concentrations ranging from

500 pg/ml to 50 ng/ml and stored at -20°C. (

3H)-THC (12 Ci/

mmole) was also stored at -20°C at a concentration of 0.25 μCi/ml

in aqueous methanol (50 percent v/v, pH 8.6) ready for use.

The plasma sample to be assayed was mixed with three volumes

of methanol, vortexed and allowed to stand for 30 min. It was then

centrifuged and the supernatant added directly to the assay tubes.

Normal human plasma similarly treated was used in the total,
COMBINED HPLC/RIA ANALYSIS OF CANNABINOIDS 59

nonspecific binding and zero binding tubes and for assay dilutions.

The plasma extract (100 μl) was mixed with the radiolabel (50 μl),

50 percent v/v methanol (100 μl), and antiserum (100 μl). The final

assay volume was made up to 600 μl with buffer and methanol to

give a final methanol concentration of 25 percent v/v. After

mixing, the solutions were left to stand at room temperature for 1

hour. Dextran-coated charcoal (200 μl, pH 7.4) was added, the tubes

were centrifuged, and after 2 minutes contact time the supernatant

(500 μl) from each tube was counted. Urine samples were treated in

a similar manner. All analyses were performed in duplicate.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHYRADIOIMMUNOASSAY

A constant-flow pump (M-6000A Waters Associates, Milford, Massachusetts, U.S.A.) was used to deliver a methanol-water eluent to

a stainless-steel HPLC column (10 cm × 4.6 mm i.d.) slurry-packed

with Spherisorb-5-ODS (Phase Separations, Flintshire, Great Britain). Samples were introduced into the HPLC column with a sixport injection valve (Spectroscopy Accessory, Sidcup, Great Britain)

fitted with a 10 ml injection loop. Column eluate was either monitored with an ultraviolet (UV) detector at 280 nm (Cecil CE212) or

collected with a Struers Samplomat fraction collector (Camlab,

Cambridge, Great Britain). A freezedrier (Model SB4, Chemlaboratory Instruments, Ilford, Great Britain) was used to remove solvents.

Plasma (0.2-1 ml) was mixed with three volumes of methanol,

vortexed, left to stand for 30 min and centrifuged. The supernatant

was removed, the residue mixed with methanol and the sample

again centrifuged. Water was added to the combined supernatants

to give a methanol concentration of 50 percent (v/v). Urine (0.1-3

ml) was mixed with methanol and a methanol-water (50:50) buffer

(0.lM acetic acid adjusted to pH 5.5 with sodium hydroxide) to give

a final volume of 6 ml and a methanol concentration of 50 percent

v/v. Metabolite conjugates in urine were hydrolysed by adding an

equal volume of methanolic sodium hydroxide (1M) to the urine,

evacuating the vessel to remove oxygen and leaving at room temperature for 30 minutes. Sufficient acetic acid was then added to

give a pH of 5.6 and the volume was made up to 6 ml with methanol-water (50:50) buffer (pH 5.5).

Plasma extract and unhydrolysed or hydrolysed urine samples

treated as above were then injected onto the HPLC column with

the loop injector. A stepped solvent elution programme was used:
60 MOFFAT, WILLIAMS, AND KING

10 ml of methanol-water (50:50), 10 ml of methanol-water

(62.5:37.5), 20 ml of methanol-water (72.5:27.5) at a flow rate of 1

ml/min. Eluate fractions were taken every 30 seconds and were

lyophilised. The freezedrier was flushed with argon before and

after drying to prevent atmospheric oxidation of sensitive metabolites. A solution of (

3H)-THC (0.025 μCi in 500 μl of a mixture of

methanoldiluent buffer, 30:70) was added to each of the dried tubes

with a solution of antiserum in diluent buffer (100 μl of 1:300 antiserum solution). The nonspecific binding tubes received diluent

buffer in place of antiserum solution. The subsequent RIA procedure was the same as that described above.

Cannabinoid retention volumes were determined by monitoring

the eluate with either RIA for cross-reacting compounds, or UV absorption (280 nm) for non-cross-reacting compounds. UV detection

required μg quantities compared with the ng quantities needed for

RIA detection.

GAS CHROMATOGRAPHY-MASS SPECTROMETRY

The internal standard (5’,5’,5’-2H3- THC, 10 ng) in methanol was

added to the plasma sample (1 ml) and the mixture extracted with

methanol as above. The plasma extract was chromatographed

using the HPLC column above and an eluent of methanol-water

(67.5:32.5). The THC fraction (retention volume 20 ml) was collected, lyophilised and the residue dissolved in hexane. Injections were

made into a Pye 104 GC using a 3 percent OV-17 column (0.3 m ×

2 mm i.d., Gas Chrom Q, 80-100 mesh) at 190°C when THC had a

retention time of 4 min with a helium flow rate of 20 ml/min. The

VG Micromass 16F mass spectrometer was used in the multiple ion

detection mode tuned to ions of m/z 299 and 314 for THC and 302

and 317 for the tri-deuterated THC. Plasma concentrations of THC

were calculated from the ratio of ion intensities for THC and internal standard using a previously prepared calibration graph.

RESULTS AND DISCUSSION

The antiserum used in this study did not cross-react with 44 noncannabinoid drugs at concentrations of 400 μg/ml in plasma and is

therefore very selective for cannabinoids. It does, however, crossreact with a number of cannabinoids (table 1). Minor modifications

to the molecule at the 8, 9, or 11 positions do not alter the crossreactivity significantly, so the antiserum can be used to measure

THC, or any of the metabolites formed by changes at the 8, 9, or 11
COMBINED HPLC/RIA ANALYSIS OF CANNABINOIDS 61

positions, in either plasma or urine. The level of cross-reaction normally obtained for blank plasma is in the region of 4 ng/ml, with

only exceptional cases being as high as 9 ng/ml. The supernatant

serum from a post mortem specimen of blood which is grossly haemolysed gives similar values, so that a suitable cut-off level for a

positive result is about 5 ng/ml. Urine samples have a much lower

cut-off level (1 ng/ml) and only in exceptional circumstances, e.g., if

a large amount of an antibacterial inorganic preservative is added,

may the level be above this.

TABLE 1. Cross-Reactivity of THC Metabolites and Analogues

in the Radioimmunoassay

Cannabinoid Amount requird for 50%

depression of binding (ng)

The plasma levels of cannabinoids, determined by direct RIA, for

volunteers who smoked 10 mg THC were between 70 and 6 ng/ml

over the 2-hour period examined. Table 2 gives the results for one

smoker who was a cannabis user and had THC metabolites in his

plasma before the experiment started.
62 MOFFAT, WILLIAMS, AND KING

TABLE 2. Plasma Concentrations of -THC in a Subject

Who Had Smoked 10 mg -THC Measured by

RIA, HPLC-RIA and GC-MS

Time after Plasma concentration of -THC (ng/ml)

smoking

(min) Direct RIA* GC-MS HPLC-RIA

* These values included contributions from THC metabolites.

** The subject was a cannabis user and this level represents

the level of cannabinoids present n his plasma before the

experiment started.

The results obtained by the GC-MS method indicate that no THC

was present at the start of the experiment and a high level (55 ng/

ml) was obtained 2 minutes after stopping smoking. The level

gradually fell to 2 ng/ml over the 2-hour period. The direct RIA results, however, do not show the same fall, since at 2 hours the

plasma still had a level of 48 ng/ml cross-reacting cannabinoids.

Much of the THC had left the plasma by this time, but there was

still an appreciable level of its metabolites cross-reacting in the

RIA.

The HPLC-RIA method can separate and individually quantify

these cross-reacting metabolites, and figure 1 shows a HPLC-RIA

chromatogram of a plasma sample taken 13 minutes after the volunteer had finished smoking a cigarette containing THC. Although

there was still some THC present, there were also cross-reacting

compounds eluting at the retention volumes of the mono- and dihydroxylated metabolites, with the greatest contribution coming

from the compounds with the elution volume 10.0 ml, which correspond to the THC-ll-oic acid and its ester glucuronide.

The retention volumes of THC, its metabolites, and other cannabinoids are given in table 3. The quantity of any of the compounds

may be obtained by integrating the individual points on the peak
COMBINED HPLC/RIA ANALYSIS OF CANNABINOIDS 63

FIGURE 1. HPLC-RIA chromatogram of a plasma sample from a subject taken 13

minutes after smoking 10mg THC. The retention volumes of THC, CBN, the

mono-hydroxylated metabolites and di-hydroxylated metabolites are marked.

to give the total contribution for that compound. Thus, when the

HPLC-RIA method is used to quantify the THC in the plasma samples, very good agreement with the CC-MS results is obtained

(table 2), since both methods are now measuring the same compound (THC) without any other cannabinoids interfering in the

assay.

The detection of cannabis smoking by an individual who habitually uses cannabis is very much easier when urine is analysed by

RIA because the levels of cross-reacting cannabinoids are much

higher than in plasma. For example, a single smoked dose of 10 mg

THC still gave levels of 49 ng/ml cross-reacting cannabinoids in the

urine 24 hours after smoking (table 4), although it must be noted
64 MOFFAT, WILLIAMS, AND KING

TABLE 3. Retention Volumes of Cannabinoids by

Reversed-Phase HPLC

Cannabinoid Retention volume (ml)

TABLE 4. Urine Concentrations of THC Metabolites In a Subject

Who Had Smoked 10 mg THC Measured by Radiolmmunoassay

Time after RIA cross- Urine volume Total crosssmoking (hr) reaction (ng/ml) (ml) reacting material

(μg/hr)

*The subject was a cannabis user; this level represents the level of cannabinoids

present in his urine before the experiment started.

that this subject, a cannabis user, started the experiment with a

level of 36 ng/ml.

When an unhydrolysed urine sample is chromatographed under

neutral conditions and the eluate analysed by HPLC, only one peak

is seen, which has a retention volume of 10 ml. This corresponds to
COMBINED HPLC/RIA ANALYSIS OF CANNABINOIDS 65

THC-11-oic acid and to its ester glucuronide. When a sample is hy

drolysed under the conditions given above and analysed by HPLC

using an acidic eluent (pH 5.5) for HPLC, three cross-reacting cannabinoids are seen (figure 2).

FIGURE 2. HPLC-RIA chromatogram of a hydrolysed urine sample from an

individual suspected of having smoked cannabis.

The first peak (retention volume 13.5 ml) is the glucuronide ester

of THC-11-oic acid, the second peak (retention volume 16.6 ml) is

THC-11-oic acid, and the third peak (retention volume 30 ml) is the

methyl ester of THC-11-oic acid. The identities of all three peaks

have been established by GC-MS. The predominant THC metabolite

in urine, as detected by the antiserum used in this work, is therefore the glucuronide ester of THC-11-oic acid. Under the conditions

of hydrolysis used above, this glucuronide is transesterified to the

methyl ester of THC-11-oic acid, which is then hydrolysed to the
66 MOFFAT, WILLIAMS, AND KING

free acid. The three peaks may be used as confirmation of the presence of cannabinoids in the urine sample in the same way as derivatives are used for proof of identity in GC analyses. In some

cases a quicker analysis for urine may be required, in which case

the hydrolysis conditions may be made more vigorous (100°C for 15

min using an equal quantity of 1M sodium hydroxide in methanol)

when the glucuronide peak is completely hydrolysed or transesterified and the methyl ester is completely hydrolysed to yield a single

peak in the HPLC-RIA chromatogram for THC-11-oic acid (16).

Because the antiserum used in this study is very selective for

cannabinoids, the RIA may be used to detect cannabis misuse in forensic cases by screening serum, plasma, haemolysed blood or

urine for the presence of cannabinoids. Those which are negative

may be reported as such, whilst those which are positive may be

analysed by the HPLC-RIA procedure. Not only will this give additional proof of the presence of cannabinoids, but it will enable each

cannabinoid to be identified and quantified. Three years’ experience using this method has shown that levels of THC in blood samples submitted for forensic analyses are usually very low, whilst

levels of the glucuronide ester of THC-11-oic acid are much higher.

This is an added reason for the use of a relatively nonselective

antiserum to detect all the cannabinoids whilst using the HPLC

procedure to separate them. The application of this method to the

analysis of cannabinoids in fatally injured drivers has been reported (17), as has its use for the analysis of samples submitted for forensic examination (18).

ACKNOWLEDGMENTS

We thank Professor J. W. Thompson (University of Newcastle

upon Tyne) for supplying plasma and urine samples from THC

smokers; Dr. S. Agurell and Ms. A. Ohlsson (Astra Lakemedel,

Sodetalje, Sweden) for the samples of deuterated THC; Dr. J. D.

Teale (University of Surrey) for the antisera; and Dr. R. E. Willette

(National Institute on Drug Abuse) for samples of cannabinoids and

their metabolites.

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1. J.D. Teale, E.J. Forman, L.J. King, and V. Marks. Production of antibodies to

tetrahydrocannabinol as the basis for its radioimmunoassay. Nature (London),

249, 154-155 (1974).
COMBINED HPLC/RIA ANALYSIS OF CANNABINOIDS 67

2. J.D. Teale, E.J. Forman, L.J. King, E.M. Piall, and V. Marks. The development

of a radioimmunoassay for cannabinoids in blood and urine. J. Pharm. Pharmacol., 27, 465472 (1975).

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8. J.D. Teale, J.M. Clough, L.J. King, V. Marks, P.L. Williams, and A.C. Moffet.

Antisera raised against tetrahydrocannabinol in the radioimmunoassay of cannabinoids. In J.A. Vinson (ed.), Cannabinoid Analysis in Physiological Fluids,

American Chemical Society, Washington, D.C., 155-173 (1979).

9. J.L. Valentine, O.H.M. Gan, H.C. Nio, and E.D. Thompson. HPLC analyses of

tetrahydrocannabinol and 11-nor- tetrahydrocannabinol-9-carboxylic acid in

human plasma. In J.A. Vinson (ed.), Cannabinoid Analysis in Physiological

Fluids, American Chemical Society, Washington, D.C., 175-205 (1979).

10. S.R. Abbot, J.R. Borg, K.O. Loeffler, S. Kantar, L.E. Hollister. J.H. Abrams, H.L.

Baras, and R.T. Jones. HPLC analysis of tetrahydrocannabinol and metabolites in biological fluids. In J.A. Vinson (ed.), Cannabinoid Analysis in Physiological Fluids, American Chemical Society, Washington, D.C., 115-136 (1979).

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68 MOFFAT, WILLIAMS, AND KING

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(1979).

16. B. Law. Unpublished observations.

17. J.D. Teele, J.M. Clough, L.J. King, V. Marks, P.L. Williams, and A.C. Moffet.

The incidence of cannabinoids in fatally injured drivers: an investigation by radioimmunoassay and high pressure liquid chromatography. J Forens. Sci. Soc.,

17, 177-183 (1977).

18. B. Law, P.L. Williams, and A.C. Moffat. The detection and quantification of cannabinoids in blood and urine by RIA, HPLC/RIA and GC/MS. Vet. and Human

Toxicol., 21, 144-147 (1979).

AUTHORS

Anthony C. Moffat and Peter L. Williams

Home Office, Central Research Establishment, Aldermaston,

Reading, Berks RG7 4PN, Great Britain

Laurence J. King

Department of Biochemistry

University of Surrey, Guildford,

Surrey GU2 5XH, Great Britain

British Crown Copyright
Analysis of Cannabinoids

Research Monograph 42, R Hawks. Editor

National Institute on Drug Abuse, 1982

An EMIT Assay for Cannabinoid

Metabolites in Urine

M. J. DeLaurentis, K. McNeil, A. J. Mann, S. Clark,

and Helena M. Greenwood

INTRODUCTION

An assay to detect cannabinoid metabolites in urine has been developed for application as a screening test for the use of marijuana.

The technique employed was homogeneous enzyme immunoassay

(EMIT), a non-isotopic immunoassay method first described for the

measurement of morphine in urine (1). The immunoassay employs

an enzyme as label. When the antigen to be assayed is conjugated

to an enzyme, the enzyme activity may be modified by antibody

binding. This forms the basis of the assay, where enzyme activity is

directly proportional to the free antigen concentration. Four enzymes have been employed in the development of assays for drugs,

both in urine and serum, for endogenous haptens and more recently for serum proteins; these applications of the technique have

been reviewed elsewhere (2,3).

Further application of the technique, using MDH conjugated

with a derivative of tetrahydrocannabinol ( THC), has demonstrated that the procedure is equally useful in the determination of

cannabinoids in urine. Pig heart mitochondrial malate dehydrogenase (MDH) E.C. 1.1.1.37 was chosen for development of a THC assay

because of its availability and its ease of detection. This, together

with antibodies raised in sheep, enabled development of a rapid,

sensitive and specific assay where as little as 20 ng of THC together with its metabolites can be detected in a 1-minute spectrophotometric measurement.

69
7 0 DeLAURENTIS, MCNEIL, MANN, CLARK, AND GREENWOOD

ASSAY DEVELOPMENT

In order to develop an assay which would detect most commonly

occurring cannabinoid metabolites present in urine, antibodies

were raised against a molecule derivatized at a point common to

many such metabolites. The principal metabolites believed to be

present in urine are shown in figure 1. The desired acid (I) was

FIGURE 1. Structures of THC and metabolites believed to be present in urine.
ENZYME IMMUNOASSAYS FOR CANNABINOIDS IN URINE 71

produced by the method previously described (4) and was conjugated to the enzyme, pig heart malate dehydrogenase. The mechanism

of inhibition of hapten conjugates of MDH has been previously described in an immunoassay for morphine (5). Antibodies to the cannabinoid metabolites were obtained from sheep by immunization

with a bovine gamma-globulin conjugate of the acid (I).

When the THC acid derivative was conjugated to MDH and

mixed with an antibody excess, a 60% inhibition of enzyme activity

resulted. This inhibition could be reversed by addition of increasing

amounts of THC acid such that it was possible to develop an

assay in which drug concentration is directly proportional to activity of the MDH conjugate. Using a spectrophotometer, the enzyme

rate is followed at 340 nm by measuring the rate of conversion of

the coenzyme NAD into NADH.

Assay Protocol

Using the Syva Model 1500 Pipetter/Diluter, 50 μl of urine

sample is delivered to a sample cup with 250 μl of buffer which

contains the enzyme substrate, malic acid. By sequentially adding

1) 50 μl of antibody reagent, which contains antibodies to a THC

derivative and coenzyme nicotinamide adenine dinucleotide (NAD),

together with 250 μl of buffer and 2) 50 μl of enzyme reagent plus

250 μl of assay buffer, the final reaction mixture is produced. The

mixture is aspirated into the flowcell of a spectrophotometer,

where the enzyme rate is measured at 30°C for 30 seconds following a 15-second delay. Enzyme activity is proportionally reduced

when the labeled drug combines with antibody; the active enzyme

converts NAD to NADH, resulting in an absorbance change that is

measured spectrophotometrically at 340 nm.
72 DeLAURENTIS, McNEIL, MANN, CLARK, AND GREENWOOD

Calibration

Three EMIT calibrators are employed in the assay; the Negative

calibrator contains no drug (0.0 ng/ml) and the Low (20 ng/ml) and

Medium (75 ng/ml) calibrators contain the drug derivative, 11-norTHCScarboxylic acid. Since -THC acid is difficult both to isolate and to prepare, -THC was selected to act as calibrator for

the assay. -THC acid is more readily available than the -THC

acid, and it exhibits virtually identical immunochemical reactivity

in this system.

For a positive/negative interpretation, the Negative (0.0 ng/ml)

and Low (20 ng/ml) calibrators are used. The difference between

the rate observed for the Negative calibrator and the rate observed

for the Low calibrator is used as a reference which permits differentiation of urine samples containing no detectable drug from

those containing drug. The Low calibrator (cut-off) contains a predetermined quantity of drug derivative which ensures that at least

96% of all samples that contain levels of cannabinoids equal to or

in excess of the detection limit of the assay (50 ng/ml) yield a positive response, and at least 96% of samples containing no cannabinoids give a response less than that of the calibrator.

To test for the presence or absence of cannabinoids in urine, it

was necessary to select this specific minimum reading (cut-off)

above which samples can be identified as positive. For the EMIT

Cannabinoid Assay, the response of the Low calibrator (nominal

concentration 20 ng/ml) serves as the cut-off, since it was found to

give a rate which discriminates effectively between samples containing 60 ng/ml and samples containing no drug. This was confirmed by assaying 60 samples known to be negative for THC, both

before and after spiking each of them with 60 ng/ml THC acid.

The results, when plotted as a histogram, show the separation be

tween the two populations with no overlap between positive and

negative samples (figure 2).

Alternatively, a standard curve (figure 3) for the assay can be

prepared by plotting the assay reading ( of the Negative, Low,

and Medium calibrators against the concentration on semi-log

graph paper. A semiquantitative result can then be estimated from

the standard curve. Because of sample-to-sample variation and the

fact that the assay detects certain cannabinoid metabolites and analogs with different sensitivity from that of the calibrator drug, semiquantitative results for positive samples can only be estimated

in analyte equivalents.
FIGURE 2. Histogram showing assay response of 50 THC negative samples and the

same samples spiked with 50 ng/ml 11-nor- carboxylic acid.
FIGURE 3. Response curve for the EMIT Cannabinoid Assay.
ENZYME IMMUNOASSAYS FOR CANNABINOIDS IN URINE 75

Specificity/Cross-Reactivity

The cross-reactivity of the EMIT Cannabinoid Assay to a wide

range of cannabinoid compounds, particularly those thought to

occur in urine, was studied by adding each compound to a THCnegative urine pool. Employing a representative lot of reagents, the

urine samples were assayed using the protocol described. Cross-reactivity was defined as the concentration necessary to produce a response in the assay equivalent to the cut-off, 20 ng/ml THC acid.

The compounds that were tested are shown in figure 4.

Cross-reactivity of the common urinary metabolites in the assay

was found to be high. In comparison the cross-reactivity to the

parent drug, THC, which is not found in urine, was slightly less.

Cross-reactivity between the THC acid and THC acid used to

calibrate the assay was determined to be approximately 100%.

To examine the cross-reactivity of the assay to various hormones,

drugs and their metabolites, each compound in table 1 was studied

by the same method. None of the non-cannabinoid compounds

tested exhibited clinically significant cross-reactivity.

TABLE 1. Non-Cannabinoid Compounds Tested That Produce a

Response Less Than That of the Cutoff Calibrator

(< 20 ng/ml 11-nor- -THC-9-carboxylic acid)

Acetylsalicyiic acid

Amitrlptyline

Amphetamine

Benzoyl ecgonine

Carbamazepine

Chlordiazepoxide

Codeine

Cortisol

Demerol

Dextromethorphan

Diazepam

Diphenyihydantoin

Ephedrine

Giutethimide

lmlpramlne

Medazapan

Methadone

Methaqualone

Methyiphenidate

Naioxone

PEMA

Phencyclidine

Phenobarbital

Phenyipropanolamine

Primidone

Promethazine

Propoxyphene

Propranolol

Quinidine

Secobarbitai

Theophylline

Thorazine
76 DeLAURENTIS, MCNEIL, MANN, CLARK, AND GREENWOOD
ENZYME IMMUNOASSAYS FOR CANNABINOIDS IN URINE 77

FIGURE 4. Cross-reactivity of a representative reagent lot to various cannabinoid

compounds.
78 DeLAURENTIS, MCNEIL, MANN, CLARK, AND GREENWOOD

CLINICAL STUDIES

Smoking Study

In a controlled study using the EMIT Cannabinoid Assay, three

male and three female subjects, all regular users of marijuana, abstained from smoking for at least 6 days (10 days maximum). Each

subject then smoked two marijuana cigarettes, each containing 9

mg THC, within a 2-hour interval. Urinary cannabinoid levels were

monitored over the following 10-day period (figures 5 and 6) during

which the subjects abstained from further marijuana use.

As can be seen from figures 5 and 6, the data from the controlled

study indicate that urinary cannabinoid levels of regular users of

marijuana, male and female, may be in excess of 20 ng/ml for 4 to

10 days and longer after smoking. Although the cannabinoid levels

detected with the EMIT Cannabinoid Assay were highest at the beginning of the study, it is not possible to distinguish between recent

use (within 24 hours) and infrequent use (up to 10 days).

Specificity Study

To evaluate the assay for potential cross-reaction due to nonTHC drug metabolites, clinical investigators were asked to assay as

many samples as possible that were known to contain drugs or

drug metabolites other than THC (figure 7). The presence or absence of cannabinoids in these samples was not confirmed by a

non-immunochemical technique.

As can be seen from the figure, there appears to be no correlation between samples positive for cannabinoids and samples positive for other drugs, implying that no crossreactivity exists between these compounds. In the case of alcohol, sulfasalazine, and

meprobamate, only samples positive for both THC and drug were

observed. The number of samples was small, however, and no conclusion regarding potential cross-reactivity with these compounds

and/or their metabolites should be made. No confirmation of the

presence or absence of THC metabolites was performed on these

samples.

Compounds appearing to cause no interference with the EMIT

Cannabinoid Assay include methadone, cocaine, morphine, PCP,

barbiturates, amphetamines, opiates, and benzodiazepines.
FIGURE 5. Cannabinoid smoking study (Male Subjects); data points represent

response produced by urine samples when assayed by the EMIT Cannabinoid Assay.
FIGURE 6. Cannabinoid smoking study (Female Subjects); data points represent

response produced by urine samples when assayed by the EMIT Cannabinoid Assay.
ENZYME IMMUNOASSAYS FOR CANNABINOIDS IN URINE 81
82 DeLAURENTIS, MCNEIL, MANN, CLARK, AND GREENWOOD

Sample Population Screening

Accuracy proved difficult to assess due to the lack of an established and available reference method. Confirmation in this evaluation was available only through confirmed recent use and experimental smoking studies.

Table 2 summarizes the data generated in population screens

conducted as part of the clinical investigations performed using the

EMIT THC Assay. At the methadone maintenance clinic (III), over

75% of the individuals whose urine produced positive results (>50

ng/ml) admitted to smoking marijuana during the previous 3-6

days, and at prisons and parole centers (IV), 100% of the individuals with positive samples admitted to recent use.

TABLE 2. Incidence of Samples Assaying Positive for THC by

EMIT ASSAY

Number

Samples

Run

Number %

Positive Positives

I. Smoking Study 2 8 7* -

II. Drug Treatment Program 9 5 64 87

III. Methadone Maintenance Clinic 211 115 5 5

IV. Prison/Parole Centers 246 125* 51

V. Toxicology/Forensic Laboratory 78 3 6 49

VI. Methadone Treatment Clinic 48 12 25

VII. Forensic/Criminal Laboratory 184 112 61

VIII. Hospital Emergency Room Patients 96 24 2 5

*100% Admission to Recent Use

Within the various populations studied, the incidence of samples

positive for cannabinoids ranged from 25% to 67%. The cumulative

results for all populations screened identified 491 positive specimens from a total of 958 samples, an overall positive incidence of

51%. During the study, it was reported that the investigators generally found the frequency of positive results to be realistic for the

populations studied.
ENZYME IMMUNOASSAYS FOR CANNABINOIDS IN URINE 83

DISCUSSION

A rapid, simple assay for the detection of urinary cannabinoid

metabolites has been developed for use as a screening test. Further

studies are underway to establish the duration of time after smoking that one may detect cannabinoids in urine by this method and

the effects of intersubject variability in the rate of metabolism of

the drug. As the assay has been designed as a screening test, any

results should be confirmed by a nonimmunological technique. It

should be noted, however, that the reference method, gas chromatography-mass spectrometry, may not employ a hydrolysis step in

the sample extraction, and therefore may only detect the unconjugated forms of the urinary metabolites. The degree of cross-reactivity of the EMIT assay to the conjugated metabolites is currently

being investigated. These metabolites may constitute a large proportion of the cannabinoids in urine and the degree of conjugation

may vary from subject to subject.

It is felt that the availability of a rapid, simple screening test for

cannabinoids will greatly facilitate studies regarding the medical,

demographic, and social use of marijuana.

REFERENCES

1. K.E. Rubenstein, R.S. Schneider, and E.F. Ullman. Homogeneous enzyme immunoassay. New immunochemical technique. Biochemistry and Biophysics Research Communications, 47, 846-851 (1972).

2. H.M. Greenwood and R.S. Schneider. Current developments in the application of

enzyme immunoassay. In Clinical Immunochemistry, S. Natilson, ed. American

Assoc. Clinical Chemistry, Washington, D.C., 455-473 (1978).

3. D. Kabakoff and H.M. Greenwood. Recent developments in homogeneous

enzyme immunoassay. In Recent Advances in Clinical Biochemistry, Vol. 2,

K.G.M.M. Alberti and C.P. Price, eds., Churchill Livingstone, Edinburgh, Scotland, 1-30 (1981).

4. G.L. Rowley, T.A. Armstrong, C.P. Crowl, et al. Determination of THC and its

metabolites by EMIT homogeneous enzyme immunoassay: a summary report. In

Cannabinoid Assays in Humans, National Institute on Drug Abuse Research

Monograph 7, R.E. Willette, ed. DHEW Publication No. (ADM)78-339, formerly

DHEW Pub. No. (ADM)76-339, Supt. of Docs., U.S. Govt. Print. Off., Washington,

D.C., pp. 28-32 (1977-78).

5. G.L. Rowley, K.E. Rubenstein, J. Huisjen, and E.F. Ullman. Mechanism by

which antibodies inhibit hapten-malate dehydrogenase conjugates. J. Biol.

Chem., 250, 3759 (1975).
84 DeLAURENTIS, MCNEIL, MANN, CLARK, AND GREENWOOD

ACKNOWLEDGMENTS

The authors are indebted to R. Bastiani for collecting the clinical

data and to Wendy Thole for her technical assistance.

AUTHORS

M. J. DeLaurentis, K. McNeil, A. J. Mann,

S. Clark, and Helena M. Greenwood

Syva Research Institute

3181 Porter Drive

Palo Alto, Calif. 94304
Analysis of Cannabinoids

Research Monograph 42, R. Hawks, Editor

National Institute on Drug Abuse, 1982

Laboratory Evaluation of

Immunoassay Kits for the Detection

of Cannabinoids in Biological Fluids

Michael A. Peat, Bryan S. Finkle, and Mary E.

Deyman

The Center for Human Toxicology has, together with other

toxicology laboratories, been involved in the field evaluation of a radioimmunoassay procedure for the detection and

quantitation of THC in serum and an enzyme multiplied

immunoassay procedure for the detection of the nor-carboxylic acid metabolite in urine. Each laboratory was selected

on the basis of proven experience with commercially available immunoassay procedures. Initially, the study was designed to screen samples collected from accident casualties;

however, at some of the study sites the data base was extended to include other samples.

The radioimmunoassay procedure evaluated was developed

at the Receptor Research Institute, and involves the use of

a tritium-labeled -THC tracer. A total of eight laboratories participated in the evaluation of this method, which is

prepared in kit form by the manufacturer. Thirteen laboratories evaluated the EMIT procedure developed at Syva

Corporation.

The study protocol required that each site screen serum and

urine samples and forward all presumptive positive specimens to the Center for Human Toxicology. These positive

specimens were then shipped to either Battelle Laboratories

or Research Triangle Institute for confirmation analysis by

gas chromatography-mass spectrometry.

The results of these evaluations will be presented and will

include:

1. Correlations of positive RIA and EMIT analysis with

gas chromatography-mass spectrometry results;

2. The results of quality-control tests;

85
86 PEAT, FINKLE, AND DEYMAN

3. Criticisms and suggestions made by analysts at each site;

4. The results of evaluations performed at the Center for

Human Toxicology.

INTRODUCTION

The widespread use of marijuana among the population of the

United States has led to the need for intensive research into the

pharmacological and psychological effects of tetra-hydrocannabinol ( THC), the principal active ingredient in marijuana. Furthermore, THC is now being studied for possible therapeutic use.

Together with the need to analyze plasma samples from these basic

research programs there is an increasing demand from forensic

and clinical toxicologists for improved analytical procedures to

screen and quantitate THC and its metabolites in biological

fluids.

One analytical technique which appears to be particularly favorable because of its sensitivity and specificity is radioimmunoassay

(RIA). In fact, several groups (1,2,3,4,5) have developed such procedures. Those developed by Cook et al. (1,2) and Gross et al. (3) are

reported to be specific for THC. The procedure developed by

Gross et al. at the Receptor Research Institute (RRI) was evaluated

under service laboratory conditions.

A variety of other immunoassay techniques are in common use,

including the homogeneous enzyme immunoassay (EMIT) procedure. This analytical method has been applied to urine samples for

the analysis of several drugs of abuse and to the quantitation of a

variety of drugs and metabolites in serum. Rodgers et al. (6) developed a semiquantitative EMIT assay for cannabinoid metabolites in

urine and this procedure has since been further refined. It was also

evaluated during the course of the project.

The RIA kit from RRI was designed specifically for the assay of

serum samples. It is a direct procedure involving no prior extraction or chromatography and uses a tritium-labeled THC tracer.

The method uses 10 μl of serum which is incubated with 100 μl of

THC antibody and 500 μl of the [

3H] THC tracer. Separation

of the unbound THC is by dextrancoated charcoal, and the technique is reported to be sensitive to 5 ng/ml.

The EMIT assay was designed specifically for the detection of

urinary metabolites structurally similar to 11-nor- THC-9-carboxylic acid (THC-9-acid). 11-Nor THC-9-carboxylic acid is used to

calibrate the assay. 50 μl of urine are mixed with THC antibody

reagent (malate dehydrogenase covalently linked to THC) and nico-
EVALUATION OF IMMUNOASSAY KITS 87

tinamide adenine dinucleotide, a substrate for the enzyme. The enzymatic reaction rate is measured spectrophotometrically at 340

nm for a 30-second time period at 30°C. The procedure can detect

50 ng/ml of THC-9-acid in urine with greater than 95% confidence.

This project was designed to evaluate both the RIA and EMIT

procedures under service laboratory conditions. Each laboratory in

the project was primarily an analytical toxicology laboratory involved in forensic and/or clinical toxicology. The study was designed initially to screen samples collected from accident casualties

and, therefore, each laboratory was requested to assure the confidentiality of the victim before inclusion in the study. During the

course of the study it became increasingly apparent that the anticipated sample population would not be obtained. This was particularly so with the RIA procedure, mainly because of the number of

hemolyzed blood samples encountered. In order to increase the

sample population, a few laboratories were asked to use the analysis kits on other types of specimens; for example, routine drug-suspected emergency room admissions.

The study protocol required that each site screen serum and

urine samples and forward all presumptive positive specimens to

the Center. They were then shipped to either Battelle Laboratories

or Research Triangle Institute for quantitative analysis by gas

chromatography-mass spectrometry (GC-MS).

SELECTION OF SITES

Each laboratory in the study was selected on the basis of proven

experience with commercially available immunoassay procedures.

This was achieved by personal communication and by evaluation of

responses to a questionnaire.

Twenty laboratories were asked originally to participate in the

study. In addition, during the course of the study four further sites

asked to be included and a number of sites withdrew. Seven laboratories evaluated the RIA kits and twelve the EMIT procedure. In

addition to these sites, the laboratory at the Center for Human

Toxicology was involved in the evaluation of both procedures.

RECEIPT AND SHIPMENT OF KITS

Evaluation of the RIA procedure began in December 1978 and

continued until August 1979. Two hundred kits were received at

CHT from RRI, of which 98 were used in the study. The dates of

receipt and kit expiration are shown in table 1.
88 PEAT, FINKLE, AND DEYMAN

TABLE 1. Date of Receipt and Expiration of RIA kits

Number of

Kits Received Lot Number

Date of

Receipt

Date of

Expiration

10 X4A 10-10-78 1-15-79

40 X4A 11-29-78 1-15-79

50 X5A 12-20-78 2-15-79

5 0 X6A 4-04-79 5-15-79

50 X7A 6-26-79 8-15-79

Some laboratories, including CHT, encountered abnormally high

nonspecific binding with the kits (Lot no. X6A) received on 4-4-79.

Replacement antibody was forwarded to CHT on 6-26-79. All of

these kits were shipped to CHT from RRI by overnight air courier

service and were received in a satisfactory condition.

A total of 120 EMIT kits were received during the period May

through August 1979. Although these kits were not in their final

marketing form, i.e., the enzyme preparation was not lyophylized,

inhouse evaluation indicated that they were satisfactory. Towards

the end of the study period a small number of lyophylized kits

were received and forwarded to some sites. Unfortunately, this was

too late for evaluation of these particular kits to be completed.

Approximately 60 EMIT kits were shipped by overnight service

to the sites or used at CHT. It is impossible to give a more accurate

estimate because towards the end of the project,, calibrator solutions and buffer solutions were shipped separately.

COLLECTION OF SAMPLES

The exact protocol for collection of samples at each site was the

responsibility of the investigator at the particular site. It was

stressed to those sites evaluating the RIA procedure that the kit

was designed for the assay of plasma/serum samples and not for

hemolyzed blood specimens. A number of sites, however, did in fact

use the kit to screen hemolyzed blood. After collection of serum/

plasma samples it was recommended that they be stored frozen in

glass tubes and, if possible, protected from light. Similar recommendations were made for storage of urine samples.
EVALUATION OF IMMUNOASSAY KITS 89

It was intended at first to limit the sample population to serum/

plasma or urine collected from accident victims; however, analytical problems with hemolyzed blood samples and the late arrival of

the EMIT kits made it clear that the anticipated sample population

would not be met. To increase the number of specimens analyzed,

it was decided to allow certain sites, including CHT, to screen other

samples using the test kits. Although this increased the number of

samples, the total number analyzed by RIA over a period of 12

months was still below that anticipated originally. A more statistically useful number of samples were screened in 6 months, using

the EMIT procedure.

TASKS OF THE PARTICIPATING LABORATORIES

Each participant was responsible for:

1. Collection of samples

2. Analysis of samples by immunoassay

3. Shipment of presumptive positive samples to CHT by

overnight air courier service

Participants were also asked to complete a questionnaire designed to obtain information about the accident and possible drug

involvement, and to record the analytical results. The completed

questionnaires were returned to CHT together with the presump

tive positive samples.

Although these tasks appear to be relatively simple, it is important to realize that each of the participants was responsible for a

forensic or clinical toxicology laboratory with a heavy routine

workload. In the case of the RIA procedure, the entire assay could

employ a skilled technician for as much as 2 days at a time, and

this also contributed to the small number of samples screened by

this technique.

CONFIRMATION OF PRESUMPTIVE POSITIVES BY GAS

CHROMATOGRAPHY-MASS SPECTROMETRY

All the presumptive positives were shipped from each site to

CHT by overnight air courier service They were then sent to either

Dr. R.. L. Foltz, of Battelle Laboratories, or Dr. J. Bursey, of Research Triangle Institute, for GC-MS confirmation. Dr. Foltz was

responsible for analyzing approximately half of the plasma samples

and Dr. Bursey for the remainder of these samples and all of the

urine samples.
90 PEAT, FINKLE, AND DEYMAN

The analytical method used to assay the plasma samples was designed to detect -THC specifically, and that used for the urine

samples to detect the 11-nor- -carboxylic acid metabolite. A small

number of urine specimens were hydrolyzed before extraction.

QUALITY CONTROL SAMPLES

At regular intervals throughout the study, serum samples

“spiked” with THC were supplied on a blind basis to each site

evaluating the RIA procedure. These were prepared according to

the following protocol: A 203 mg/ml THC solution supplied by

NIDA was serially diluted to a 20.3 (A) and 2.03 ng/μl (B) concentration with ethanol. These solutions were then used to “spike”

serum; for example, 50 μl of B in 25 mls of serum resulted in a

THC concentration of 4.06 ng/ml. Each batch of samples was

shipped in dry ice by overnight air courier service to each laboratory.

RESULTS

RIA

The participants evaluating the RIA procedure analyzed a total

of 310 samples of which 43 (14 percent) were identified as presump

tive positives. Approximately one-third of this latter group were hemolyzed. The results of quantitation of samples analyzed by both

RIA and GC-MS are shown in table 2. The number of hemolyzed

samples included in this group is unknown.

Quality control specimens, prepared in serum, were sent to each

site participating in the RIA evaluation. Table 3 shows the results

of these analyses. Some of the regression coefficients were as follows:

GC-MS against RIA:

“Spiked concentration”

against:

Total minus sample 4

GC-MS (samples 1-22*)

CHT (samples 1-22*)

RRI (samples 1-12*)

*Excluding sample 4

(n=97) r=0.777

(n = 21) r = 0.959

(n = 29) r = 0.802

(n = 19) r = 0.950

In addition to analyzing the quality control specimens, a number

of plasma samples from the Emergency Room of the University of

Utah Medical Center were also analyzed at CHT; these were paired
EVALUATION OF IMMUNOASSAY KITS

TABLE 2. Results of RIA and GC-CIMS Analysis

91

RIA

(ng/ml)

GC-MS

Site (ng/ml) Site

RIA GC-MS

(ng/ml)

I 6.6 0

5.0 Not Analyzed

6.4 0

6.5 Less than 1

15 9.3

9.0 9.9

23 Interference

6.5 6.5

9.4 0

6.5 6.2

15 5.0

II 7.0 2.0

III 15 10.5

9.0 3.0

4.0 0

IV Positive 86

Positive 31

Positive 220

Positlve 155

Positive 7 2

Positive 69

Positive 11

Positive 3.0

Positive 21

Positive 0

Positive 10

Positive 0

Positive 5.2

Positive 4.6

Positive 3.1

Positive Interference

with urine samples collected from the same patient and screened

positive by EMIT. Table 4 shows the data obtained by RIA and GCMS.

EMIT

A total of 13 laboratories, including CHT, were involved in the

evaluation of this procedure during the period June to December

1979. The 12 sites screened a total of 596 samples from accident victims, of which 165 (28%) were identified as presumptive positives.

Some of these sites used the EMIT procedure to screen for positives and negatives only, whereas others determined the concentration of metabolites present. It must be remembered that initially

the GC-MS procedure quantitated only the free 11-nor-carboxylic

acid metabolite, and Williams and Moffatt (7), reported that up to

80% of the acid metabolites are conjugated in the urine. From

these initial data, 18 of the samples were screened positive by

EMIT but were negative for the carboxylic acid metabolite by GCMS. Subsequently several samples with low or negative GC-MS results were hydrolyzed enzymatically and then reanalyzed by GCMS for the 11-nor-9-carboxylic acid metabolite. Table 5 shows the

results of these analyses.
TABLE 3. Results of Quality-Control Specimens (All Concentrations ng/ml)
EVALUATION OF IMMUNOASSAY KITS 93

TABLE 4. Plasma Samples from Subjects Whose Urine was

Screened Positive by EMIT

Sample RIA GC/MS Sample RIA GC-MS

No. (ng/ml) (ng/ml) No. (ng/ml) (ng/ml)

1 7 0 6 0 1.3

2 0 0 7 0 0

3 <5 0 8 1 0 <1

4 <5 0 9 0 2.1

5 11 0 10 0 0

TABLE 5. Results of EMIT and GC-MS Analyses After

Enzymatic Hydrolysis

EMIT GC-MS of Free Acid GC-MS After Hydrolysis

(ng/ml) (ng/ml) (ng/ml)

Positive 4.6 4 2

Positive 0 4

Greater than 7 5 36 600

53 7.1 32

0 0 4

Greater than 75 31 7 5

10 6.5 7.9

11 0 2.7

75 6.2 58

0 24 Less than 2

0 0 4 7

Greater than 7 5 5 31

Greater than 7 5 2.1 53

Greater than 7 5 0 43

Greater than 75 2.1 77
94 PEAT, FINKLE, AND DEYMAN

CHT was closely involved in the evaluation of the EMIT procedure and examined a large number of samples. Of these, 106 were

analyzed by both EMIT and GC-MS. The results are tabulated in

table 6. A total of 37 samples screened positive by EMIT but were

unconfirmed by GC-MS for the free acid metabolite. Subsequently 3

of these were hydrolyzed enzymatically and found to be positive by

TABLE 6. Results of EMIT Analysis at CHT and GC-MS Analysis

for the Free Acid Metabolite at RTI

EMIT GC-MS EMIT GC-MS EMIT GC-MS

(ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml)

12 0

30 4.0

Greater than 75 21

14 8.1

Greater than 75 5 5

Greater than 75 9.1

34 26

3 7 3 5

15 0

21 0

0 0

18 17

0 0

5 5 2 4

15 4.4

2 0 2 0

1 6 0

11 2.2

0 0

62 2 9

0 0

66 3 8

15 5

0 2 0

0 0

14 2 6

1 2 0

14 14

0 0

Greater than 75 5

3 0 0

21 3.5

57 2.9

52 3.6

37 0

5 2 4 2

0 0 57 20

0 0 Greater than 75 50

12 11 15 7.5

Greater than 75 3 6 5 8 22

0 0 3 6 2.6

5 3 7.1 60 3 0

2 3 0 Greater than 75 26

Greater than 75 31 2 7 3.3

10 6.5 Greater than 75 41

11 0 Greater than 75 7

0 0 60 9

Greater than 75 6.2 Greater than 75 45

0 24 Greater than 75 11

0 2.3 Greater than 75 5

19 0 Greater than 75 2.1

10 17 5 4 0

35 0 Greater than 75 2.1

15 4 3 3 145

0 29 10 4

0 2 0 24 0

Greater than 75 10 Greater than 75 2.1

10 4 22 2.5

74 22 66 78

24 2 5 Greater than 75 3 7

50 2.5 Greater than 75 3 9

0 10.5 5 6 70

Greater than 75 3.4 3 7 18

0 0 Greater than 75 67

0 7.8 3 3 2.5

0 7 Greater than 75 2 7

13 5 2 5 26

72 3 2 Greater than 75 0

3 5 4 3 4 0

25 0 4 0 2.6

2 3 0 18 0
EVALUATION OF IMMUNOASSAY KITS 95

GC-MS. More importantly, perhaps, 8 samples from the CHT data

screened negative by EMIT but positive by GGMS, although 4 of

these had free carboxylic acid metabolite concentrations of less

than 15 ng/ml (i.e. less than the low calibrator for EMIT) by GCMS. It should be noted that the 12 participants did not forward any

“negatives” for GC-MS confirmation, so the true number of “false

negatives” may have been higher.

Plasma and urine samples from some patients were analyzed by

EMIT, RIA and GC-MS methods. Of those plasma samples in which

no THC was detectable by GC-MS, 12 of the corresponding

urines were positive by both EMIT and GC-MS, 5 plasmas positive

by GC-MS, 11 of the corresponding urines were positive by both

EMIT and GC-MS, whereas 3 were positive by EMIT only, although

in these cases the urines were not hydrolyzed.

DISCUSSION

Certainly the most disappointing aspect of the entire study was

the lack of response to the RIA evaluation. The time-consuming

procedure and problems with the kit itself were the most common

explanations given by the participants. In addition, the kit was designed to assay serum samples, whereas the real need among those

laboratories chosen for the study was for a kit to screen hemolyzed

blood samples and, in fact, the kit was inappropriately used at

some laboratories to do this.

The correlation between GC-MS and RIA on the case samples

analyzed by the participants was far from adequate. At the 6 sites

reporting data, six false positives (1.9% of the total, 13.9% of the

presumptive positives) were found, although it is possible that some

of these were hemolyzed. Of 10 plasma samples analyzed at CHT

from subjects with positive urines (by EMIT), 2 showed values by

RIA above the sensitivity limit of the assay which were subsequently shown to be zero by GC-MS.

Six of the laboratories and CHT were involved in the analysis of

the quality control samples. The data from these samples is shown

in table 3. It is interesting to note that the performance at CHT

improved throughout the study as the laboratory staff became

more familiar with the procedure. It is also worthwhile to note that

those sites with personnel experienced in RIA techniques performed better than those new to the use of tritiated tracers.

The regression coefficients (GC-MS vs. RIA) for participating

sites are greater than 0.93 for all but two sites. One of these
96 PEAT, FINKLE, AND DEYMAN

achieved only 0.46, but this is much improved if one obvious error

is eliminated. Since the kit is prepared in a form suitable for marketing, consideration must be given to the fact that laboratory experience with this type of kit is essential for adequate results.

At the end of the study a questionnaire was forwarded to each

site. One of the questions asked was at what lower concentration

limit the kit was unreliable when applied to serum. There was, of

course, variation in the responses, one laboratory suggesting 2 ng/

ml, three auggesting 6 ng/ml and two 10 ng/ml. Our own experience at CHT suggests a limit between 5 and 10 ng/ml when the

procedure is used by inexperienced personnel. Whether this is adequate for forensic case work is questionable in view of recent data

on the blood concentrations of THC to be expected in medicolegal cases (Williams and Moffatt, 1980). The kit instructions indicate an expected sensitivity of 5 ng/ml when applied to serum.

Each participant was also asked to describe briefly his or her

views concerning the kit and the instructions and to comment on

the usefulness of the procedure. Generally, all the participants

found the kit and instructions to be satisfactory. Most, however, reported that they had trouble with the standards at the lower end of

the curve (5 to 10 ng/ml), particularly with reproducibility from

assay to assay, and they suggested that samples falling in this

range be repeated. One participant reported:

“Overall, I feel that the kit should be developed and laced

into laboratory use, especially in cases involving traffic accidents, drunken driving, bizarre accidents and coroner’s

cases in general.”

This view was echoed by the other participants.

A total of 12 laboratories and CHT evaluated the EMIT procedure. Because the technique was simple and familiar, all sites

found the kit easy to use. The kit was compared to a GC-MS assay

developed by Dr. Joan Bursey, of Research Triangle Institute. The

assay was designed to quantitate the 11-nor- THCScarboxylic

acid metabolite, whereas the kit was designed to detect “cannabinoid metabolites” which resembled this acid metabolite in structure.

This fact alone could explain the poor correlation between the

EMIT values and those obtained by GC-MS, particularly those samples with considerably higher EMIT values. However, if the data in

table 4 are considered, it is much more likely that the discrepancy

is due to the EMIT responding to the glucuronide conjugate of this

metabolite. Unfortunately, in most cases the sample volume was

insufficient for reanalysis of the urine by GC-MS after hydrolysis.
EVALUATION OF IMMUNOASSAY KITS 97

It is much more difficult, however, to account for those samples

with a significantly lower EMIT value:

EMIT (ng/ml) GC-MS (ng/ml)

0 20

14 2 6

0 29

0 2 0

0 10.5

0 7.8

0 7

33 145

As with the RIA procedure, a questionnaire was sent to each participant, requesting views and comments. Generally, the participants had few problems. Some questions were raised concerning

the procedure for running blanks and whether the kit was satisfactory for postmortem urine because of the extreme turbidity sometimes seen in such samples. The introduction of the lyophilized reagents has been reported to alleviate these problems.

The interpretation of EMIT urine analyses from both a medicolegal and clinical viewpoint should be approached with caution. Of

34 cases in which both plasma and urine were analyzed, both samples were positive in only 14 cases, showing that a positive EMIT

reading does not necessarily correlate with the recent smoking of

marijuana, if the presence of THC in plasma is taken to indicate

recent use. In addition, a practical difficulty is that the confirmation of the urine EMIT readings is not a straightforward process

because current GC-MS procedures are designed to quantitate only

the free 11-nor- -THC-9-carboxylic acid metabolite. Hydrolysis of

the conjugated metabolites becomes important if such techniques

are to be used.

CONCLUSIONS

There is a demand for analytical screening procedures for

THC in serum and/or blood and for cannabinoid metabolites in

urine. Development of the EMIT procedure has come a long way

towards satisfying the latter demand, although some effort should

be directed towards developing a more complete confirmatory procedure.

The analysis of THC in serum and/or blood is more complex.

In this case the GC-MS confirmatory method is acceptable, although further refinements are needed so that it will be applicable
98 PEAT, FINKLE, AND DEYMAN

to all types of blood samples and so metabolites can be clearly separated and identified.

Screening serum and blood samples using a tritiated RIA kit is

still a very difficult and time-consuming procedure. The kit should

not be used by inexperienced personnel for quantitation of THC.

To help overcome some of the problems encountered with the

RIA procedure evaluated, we would recommend the development of

an iodinated I

125) tracer for THC and also for either the 11-hydroxy or carboxylic acid metabolite. In our opinion, the latter

would improve both the quantitation and the confidence of the

screening assay.

REFERENCES

1. C.E. Cook, M.L. Hawes, E.W. Amerson, et al. Pharmacologist, 18, 291 (1976).

2. C.E. Cook. ACS Symposium Series 98, Am. Chem. Soc., Washington, D.C., 137-

154 (1979).

3. S.J. Gross, J.R. Soares, S.L. Wong, et al. Nature, 252, 581 (1974).

4. J.D. Teale, E. Forman, L.J. King, et al. Nature, 249, 154 (1974).

3. A.R. Chase, P.R. Kelley, A. Taunton-Rigby, R.T. Jones, and T. Harwood. Quantitation of cannabinoids in biological fluids by radioimmunoassay. In R.E. Willette, ed. National Institute on Drug Abuse Research Monograph 7, Cannabinoid

Assays in Humans. DHEW Pub. No. (ADM)76-339. Supt. of Docs., US. Govt.

Print. Off., Washington, D.C., 1-9 (1976).

6. R. Rodgers et al. Clin. Chem. 24, 95 (1978).

7. P.L. Williams and A.C. Moffat. J. Pharm. Pharmac., 32, 445 (1980).

AUTHORS

Michael A. Peat, Bryan S. Finkle, and Mary E. Deyman

Center for Human Toxicology

University of Utah

Salt Lake City, Utah 84112
Analysis of Cannabinoids

Research Monograph 42, R. Hawks, Editor

National Institute on Drug Abuse, 1982

Quantitative Analysis for THC,

11-Hydroxy- -THC, and 9-Carboxy-

 THC in Plasma Using GC/CI-MS

Rodger L. Foltz and Bruce J. Hidy

A method for quantitative measurement of THC concentrations in plasma has been developed and applied to the

analysis of more than 2000 samples over the

The method includes addition of deuterium-labeled THC

to the plasma for use as the internal standard, a simple

and relatively rapid solvent extraction procedure, formation

of the trimethylsilyl derivative, and quantitation by selected ion monitoring using ammonia chemical ionization. A

similar procedure has also been developed for simultaneous

analysis of THC and two of its major metabolites in

plasma. The latter procedure requires use of a glass capillary column for assays of plasma samples in which the

THC concentration is below 10 ng/ml. Sensitivities for both

methods permit quantitation of THC and its metabolites at

concentrations as low as 0.2 ng in 1-ml plasma samples.

INTRODUCTION

Interest in the pharmacology of cannabinoids continues at a high

level, owing to the widespread use of marijuana and the discovery

of potentially beneficial medical applications for tetrahydrocannabinol. Cannabinoid research has been hampered by

the limited availability of analytical facilities capable of measuring

the typically low levels of cannabinoids in physiological fluids after

cannabinoid administration. For the past 4 years Battelle Columbus Laboratories has provided an analytical service to NIDA-authorized scientists engaged in cannabinoid research. The cannabinoid assays developed and used at Battelle are based on the combi99
100 FOLTZ AND HIDY

nation of gas chromatography and chemical ionization mass spectrometry (GC/CI-MS).

QUANTITATION OF THC

Measurement of THC in plasma by a GC/MS method was first

reported by Agurell et al. (1). Their procedure includes addition. of

a deuterium-labeled THC analog to the plasma, extraction, and

liquid chromatographic purification on a Sephadex LH-20 column,

followed by quantitative measurement using gas chromatography

and electron impact mass spectrometry (GC/EI-MS). The method

used at Battelle is similar except that it uses a relatively simple

extraction based on the procedure (figure 1) developed at the Research Triangle Institute (2), and employs ammonia chemical ionization rather than electron impact ionization (3). Using this procedure, a set of 36 plasma samples can be extracted and prepared for

analysis by one technician in a period of 8 hours. An additional 8

hours is required to perform the GC/MS analyses.

FIGURE 1. Flow diagram for analysis of THC in plasma.
GC/CI-MS ANALYSIS OF CANNABINOIDS 101

Most of the THC analyses done at Battelle during the past 2

years have been performed on a Finnigan 4000 GC/MS coupled to

an Incos date system. With this system operating normally, as

little as 20 pg of derivatized THC injected into the GC/MS gives a

response sufficient to permit measurement with good precision.

Usually, only 10 to 20 percent of a derivatized extract is injected at

one time. Consequently, for a 1-ml plasma sample, concentrations

of THC as low as 0.2 ng/ml can be measured.

EXTRACTION PROCEDURE

All glassware should be thoroughly cleaned and silylated (4). The

THC standard solutions should be stored at –10°C in the dark but

allowed to warm to room temperature just prior to use.

Transfer a measured quantity (normally 1 ml) of plasma to a 20-

ml culture tube equipped with a Teflon-lined screw cap. Add 100 μl

of an ethanol solution of the THC-2H3 internal standard (200 ng/

ml) followed by 1.0 ml of a pH 7.0 buffer solution (Fisher Scientific

Co.). Vortex the mixture for 10 sec. Add 6 ml of high purity hexane

(Distilled-in-Glass, Burdick and Jackson Co.), tightly cap the tube,

and gently mix the contents for 20 min using an automated tube

rocker or rotator. Centrifuge for 6 min and then carefully transfer

the hexane (top) layer to a second culture tube using a disposable

Pasteur pipette. The remaining plasma residue can be discarded.

The hexane solution is now washed successively with dilute

alkali and dilute acid to remove strongly acidic and basic compounds. Add 2.5 ml of 0.1 N NaOH solution to the hexane extract.

After capping the tube, rotate or rock for 20 min and then centrifuge. Using another disposable pipette, remove the aqueous

(bottom) layer and discard. Add 2.5 ml of 0.1 N HCl solution to the

hexane extract. Tap the tube gently to cause gas bubbles formed by

the acid-base reaction to escape. Again, tightly cap the tube, rock

or rotate it for 10 min, and centrifuge. Carefully transfer just the

hexane (top) layer to a 15-ml glass tube with a tapered or coneshaped bottom. Gently concentrate the extract to near dryness at a

temperature of 35-40°C using either a nitrogen stream or a rapid

evaporation device such as the Buchler Vortex-evaporator (Buchler

Instruments).
102 FOLTZ AND HIDY

The extract residue is now transferred to a l-ml glass vial having

a cone-shaped bottom and equipped with a Teflon-lined screw cap

in the following manner. Add 0.5 ml of high purity pentane to the

tube containing the residue and vortex the tube for 10 sec. Using a

disposable pipette, transfer the pentane solution to the 1-ml glass

vial. Repeat the last step using an additional 0.5 ml of pentane to

transfer any remaining extract residue to the 1-ml vial. Add 10 μl

of high purity dimethylformamide to the pentane solution to act as

a “keeper” to minimize evaporative loss of the THC during the

final evaporation and to serve as a solvent for the trimethylsilation. Slowly concentrate the pentane solution by heating the glass

vial with a heating block maintained at 40°C. When all of the pentane has evaporated (approximately 20 min), add 15 μl of bis-(trimethylsilyl) trifluoroacetamide containing 1 percent trimethylsilyl

chloride to the vial and cap tightly. Vortex the vial and then heat

in an oven at 90°C for at least 1 hour. The sample is now ready for

GC/MS analysis. However, the sample can be stored at –10°C indefinitely.

GC/MS ANALYSIS

Chemical ionization with ammonia as the reagent gas is used for

quantitation of the trimethylsilylated THC because it affords

higher sensitivity and specificity than either electron impact ionization or chemical ionization using methane or isobutane as reagent gases (5). The method was developed on a quadrupole system,

but any mass spectrometer equipped for chemical ionization and selected ion monitoring should be satisfactory, provided it has adequate sensitivity. For systems in which the gas chromatograph is

connected directly to the mass spectrometer, methane (Ultrahigh

Purity grade, Matheson Gas Products, East Rutherford, NJ) should

be used as the carrier gas. If the GC/MS employs a separator,

helium may be used as the carrier gas. In both cases the ammonia

(Ultrahigh Purity grade, Air Products and Chemicals, Tamaqua,

PA) should be introduced into ion source via a makeup gas inlet so

that the ammonia and carrier gases enter the ion chamber in concentric gas streams. The ammonia flow rate into the ion source is

adjusted while observing ion intensities at m/z 18 and 35. As the

ammonia flow rate is increased, the intensity of the m/z 18 peak

(NH4

+

) should at first increase and then reach a plateau, at which

point a peak at m/z 35 (NH3•NH4

+

) appears and begins to increase

in intensity. The optimum ammonia flow rate is the point at which

the m/z 18 peak reaches a maximum and the m/z 35 peak just
GC/CI-MS ANALYSIS OF CANNABINOIDS 103

begins to increase in height. The total reagent gas pressure in the

ion chamber should be between 0.3 and 1.0 torr. If a separator is

used, it may be necessary to bleed in methane along with the ammonia to achieve this total pressure.

Various gas chromatographic columns can be used, but the best

results have been achieved with a 1.8-m × 2-mm ID glass column

packed with 3 percent OV-17 on 100/120 mesh Gas Chrom Q (Applied Science Labs, State College, PA). The injector, column, and

transfer-line temperatures are maintained at 280°, 250°, and 320°C,

respectively. Under these conditions and a carrier gas flow rate of

approximately 15 ml/min, the THC-TMS should elute in ap

proximately 4 min. The resolution of the GC column can be evaluated by injecting a solution containing an equal mixture of THCTMS and THC-TMS 50 μg/ml in hexane). Under the stated

GC conditions a good quality OV-17 column will give a chromatogram showing at least a detectable valley between the peaks corresponding to the two cannabinoid derivatives.

For selected ion monitoring the mass spectrometer is set up to

monitor the ion currents at m/z 387 and 390, corresponding to the

protonated-molecule ions (MH+) for THC-TMS and THC-2H2

-

TMS, respectively. After the CC/MS run is completed, the THC

plasma concentration is determined by measuring the areas of the

THC-TMS and THG2H2-TMS peaks in the selected ion current profiles (“ion chromatograms”) and relating the peak areas

ratio to a previously established standard curve. A new standard

curve should be established whenever new standard solutions are

prepared or when analysis of plasma samples containing known

concentrations of THC shows unacceptable accuracies. For routine analysis of plasmas containing unknown concentrations of

THC, at least 1 out of every 6 samples should be a spiked standard.

Figure 2 shows a typical selected ion current profile with peaks

corresponding to 5 ng/ml of THG-TMS and 20 ng/ml of THC

2H2

-TMS, while figure 3 is a standard curve for the THC concentration range 1 to 100 ng/ml of plasma. Precision data for various concentrations of THC in plasma are listed in table 1.

EXTRACTION OF HEMOLYZED BLOOD

The described procedure has proven to be a reliable method for

the analysis of THC in plasma and normal whole blood samples.

However, attempts to analyze hemolyzed and badly decomposed

blood samples gave erratic results due to poor extraction efficiencies. Both the THC originally in the blood and the added deuterium-labeled THC apparently are tightly bound to something in the
104 FOLTZ AND HIDY

FIGURE 2. Selected ion current profiles for quantitation of -THC in plasma.
GC/CI-MS ANALYSIS OF CANNABINOIDS 105

FIGURE 3. Standard curve for measurement of THC in 1-ml plasma samples.

TABLE 1. Precision Data for Measurement of THC

Added to Plasma*

-THC Added Relative Standard

(ng/ml) Deviation (%)

* 4 samples analyzed twice at each concentration.
106 FOLTZ AND HIDY

hemolyzed blood, thereby preventing efficient extraction with

hexane. Rosenthal and Brine reported a procedure for extraction of

THC from cadaver blood (6). A simpler procedure was developed at

Battelle for extraction of THC from hemolyzed blood. The procedure gave acceptable quantitative results for concentrations of

THC down to 1 ng/ml for most batches of hemolyzed blood. However, occasionally large variations in extraction efficiency did occur,

indicating that the method deserves further attention.

Figure 4 is a flow diagram for the extraction procedure used for

hemolyxed blood. An ethanol solution of the deuterium-labeled internal standard is added to 1 ml of hemolyzed blood followed by 1

ml of 3 N H2SO4 and 1 ml of high purity acetonitrile. The mixture

is sonicated and the resulting protein precipitate removed after

centrifugation. The supernatant is then neutralized with 3 N

NaOH and 1 ml of pH 7.0 buffer added. The aqueous solution is

extracted twice with 6 ml of high purity hexane. The remaining

steps involving successive washing of the hexane extract with

dilute alkali and dilute acid followed by concentration and derivatization are identical to the procedure for extraction of THC from

normal plasma.

QUANTITATION OF 9-CARBOXY- THC AND 11-HYDROXYTHC IN PLASMA

The 11-hydroxy- THC metabolite has psychotomimetic activity

comparable to that of THC. Consequently, any study attempting

to relate behavior effects to THC blood concentrations should also

include analysis of the 11-hydroxy metabolite blood concentrations.

Although the 9-carboxy metabolite has no psychomimetic activity,

it is a major THC metabolite, and its concentration in both blood

and urine can be measured for a longer period of time than the

concentration of THC. Consequently, there has been strong interest in developing methods for quantitation of these two metabolites in blood and other body fluids. A method capable of simultaneously measuring plasma concentrations of THC and the two metabolites would be particularly attractive.

Rosenfeld has published a method for simultaneous quantitation

of THC and the 11-hydroxy- -THC in plasma involving extraction into toluene followed by back extraction of phenolic compounds into Claisen’s alkali and derivatization prior to GC/MS

analysis (7).

A method for determination of -THC, cannabinol, and 11-hydroxy- -THC has been developed at the Research Triangle Insti-
GC/CI-MS ANALYSIS OF CANNABINOIDS 107

FIGURE 4. Flow diagram for analysis of THC in hemolyzed blood.
108 FOLTZ AND HIDY

tute (8). After addition of deuterated internal standards to the

plasma, the cannabinoids are extracted into petroleum ether containing 1 percent isoamyl alcohol. Chromatography on Sephadex

LH-20 is used to partially purify the cannabinoids. The THC and

cannabinol are collected in one fraction and analyzed by GC/MS

without derivatization. The 11-hydroxy- -THC is collected in a

later eluting fraction and converted to its bis-(trimethylsilyl) derivative prior to GC/MS analysis. The same publication (8) describes a separate procedure for analysis of the 9-carboxy metabolite of THC in plasma or urine. After addition of the deuterated

analog as the internal standard, acetone is added to the body fluid

to precipitate proteins, The acetone is removed by evaporation and

the aqueous residue extracted with diethyl ether. Reverse phase

high performance liquid chromatography is used to purify the carboxy metabolite which is subsequently converted to its dimethyl derivative by treatment with dimethyl sulfate. GC/MS with electron

impact ionization is used for quantitative determination of the

ratio of metabolite to internal standard.

A procedure for measurement specifically for 9-carboxy- -THC

was reported by Nordquist et al. (9). The method uses the isomer

of the metabolite as the internal standard. After acidification of

the plasma (pH 4), it is extracted with diethyl ether and the concentrated extract treated with diazomethane to form the methyl

ester of the carboxy metabolite. The methylated extract is then

partially purified by chromatography on Sephadex LH-20. Finally,

trimethylsilylation of the phenolic OH is achieved just prior to

quantitation by GC/EI-MS.

Investigation of LC/MS Analysis of 9-Carboxy- THC

In May 1978 Battelle acquired a Finnigan LC/MS system which

utilizes a moving belt to transport the eluent from a liquid chromatographic column into the ion source of a Finnigan 4000 mass spectrometer. The system offers the potential for direct analysis of

polar metabolites such as the 9-carboxy THC without derivatization. Experiments were conducted to determine the sensitivity of

this system for measurement of the metabolite. Figure 5 shows the

ion current profile obtained by depositing on the moving belt, by

means of a syringe, a hexane solution containing 1 ng of the 9-carboxy- THC. Unfortunately, this sensitivity could not be approached when the metabolite was passed through a reverse phase

HPLC column and then onto the moving belt. The major causes for

dramatically reduced sensitivity appeared to be increased background due to the solvent system and a longer period of elution.

Because of the relatively poor sensitivity of the LC/MS for this
GC/CI-MS ANALYSIS OF CANNABINOIDS 109

compound, attention was shifted to development of a GC/MS

method.

FIGURE 5. Ion current profile for MH+ of 9-carboxy- THC introduced into the

mass spectrometer via the LC/MS interface.

GC/MS Method

A procedure was sought for simultaneously extracting THC

and the two metabolites of interest from plasma. This was achieved
110 FOLTZ AND HIDY

by modifications of the previously described procedure for extraction of THC from plasma. The major changes consisted of use of

a more polar solvent and elimination of the dilute alkali wash.

Also, the best GC/MS reproducibility was experienced when the extracted carboxy metabolite was first converted to its methyl ester

prior to trimethylsilylation of the phenolic group.

FIGURE 6. Flow diagram for simultaneous analysis of THC, 11-hydroxy- -THC,

and 9-carboxy- THC in plasma.
GC/CI-MS ANALYSIS OF CANNABINOIDS 111

Figure 6 is a flow diagram for the extraction of THC and its

two metabolites of interest from plasma. To 1 ml of plasma in a 20-

ml culture tube equipped with a Teflon-lined screw cap, add 1 ml of

a pH 2 buffer, followed by 100 μl of an ethanol solution containing

50 ng each of the deuterium-labeled internal standards ( THC

2H3

, 11-hydroxy- THC-2H3, and 9-carboxy- THC-2H3). Vortex the

tube for 10 sec and then add 4 ml of a hexane-ethyl acetate solution (7:l v/v). Screw the cap on tightly and check for a leak. Slowly

rotate or rock the tube for 15 min. Loosen the cap and then centrifuge. Using a disposable Pasteur pipette, transfer the organic layer

(top) to a second culture tube and discard the aqueous plasma

layer. Add 2 ml of 0.2 N H2SO4 to the organic extract. Screw on the

cap, tighten, and again check for a leak. Repeat the rocking (10

min) and centrifugation (10 min). Transfer the organic layer (top)

to a 15-ml centrifuge tube. Be careful not to transfer any of the

aqueous (lower) layer to the centrifuge tube. Discard the aqueous

layer. Gently concentrate the extract to near dryness at a temperature of 35-40°C using a nitrogen stream. Wash down the sides of

the centrifuge tube with 0.5 ml of high purity methanol. Vortex

the tube for 10 sec and again evaporate to near dryness. Wash

down the sides of the centrifuge tube with 0.2 ml of methanol.

Vortex the tube for 10 sec and then transfer the methanol solution

to a 1-ml glass vial having a cone-shaped bottom and equipped with

a Teflon-lined screw cap. High purity diethyl ether (0.5 ml) is used

for a final wash of the centrifuge tube and the ether combined with

the methanol in the 1-ml vial. Evaporate the combined solvents to

near dryness using a gentle stream of nitrogen while gently heating the vial ( 40°C). To form the methyl ester of the carboxy metabolite, add a solution of diazomethane in diethyl ether until the

yellow color persists. Cap the vial and heat at 50°C for 0.5 hr. Cool

the vial to room temperature, remove the cap, and evaporate the

solvent to near dryness under a gentle stream of nitrogen. Add 50

μl of bis-(trimethylsilyl) acetamide containing 1 percent trimethylsilyl chloride to the vial, cap tightly and heat at 90°C for 1 hr. The

sample is now ready for GC/MS analysis, or it may be stored in a

freezer (–10°C) until it is to be analyzed.

For GC/MS analysis a 1.8-m × 2-mm glass column packed with 3

percent OV-17 on 100/120 mesh Gas Chrom Q (Applied Science

Labs, State College, PA) can be used. However, at THC concentrations below about 10 ng/ml, interference by an endogenous, acidic

component of plasma prevents accurate measurement of the THC
112 FOLTZ AND HIDY

FIGURE 7. Selected ion current profiles for packed column GC/MS analysis of

THC.

peak height (see figure 7). The interfering peak can be resolved

from the THC peak by use of a capillary column.

Figure 8 shows the selected ion current profiles corresponding to

the protonated-molecule ions for THC-TMS (m/z 387) and

THC-2H3-TMS (m/z 390) from an analysis conducted on a 15-m glass

capillary (0.25 mm I.D.) coated with SE-52.

In this example, the THC was present at a concentration of

only 1 ng/ml, and it is well resolved from other peaks in the chromatogram, in contrast to the packed column selected ion current

profile (figure 7) where the peak, due to the THC-TMS, is completely obscured by the interfering peak. The capillary column

assay involves a splitless injection at a column temperature of

160°C of 2 μl of the silylated extract plus 1 μl of n-tetradecane

which gives enhanced GC resolution due to the so-called “solvent

effect” (10). After 1 min the column temperature is rapidly programmed to 250°C. Under these conditions all three of the cannabinoids are eluted within 6 min as shown in figure 9.
GC/CI-MS ANALYSIS OF CANNABINOIDS 113

FIGURE 8. Selected ion current profiles for capillary column GC/MS analysis of

THC.

In order to achieve maximum sensitivity, only two ion masses

are monitored at a given time during the GC/MS analysis. Thus,

the ion masses corresponding to the protonated-molecule ions for

THC-TMS and its deuterated analog are monitored during their

elution window. Then, under computer control, the mass analyzer

voltages are switched so that the ions corresponding to the protonated-molecule ions for the ll-hydroxy -THC-TMS and its deuterated analog are monitored. Finally, the mass analyzer voltages are

set to monitor the protonated-molecule ion currents for the methyl

ester of the 9-carboxy- -THC-TMS and its deuterated analog. A

computer printout of the resulting ion current profiles is shown in

figure 10.

Improved capillary column GC resolution can be achieved without lengthening the analysis time by using hydrogen as the carrier

gas rather than helium or methane (11). Furthermore, the combination of hydrogen and ammonia gives better quality CI mass spectra than the combination of helium and ammonia. The hydrogen/

ammonia CI mass spectra for each of the trimethylsilylated cannabinoids are shown in figure 11.
FIGURE 9. Total ion current chromatogram from capillary column GC/MS analysis

of standard mixture of the trimethylsilylated cannabinoids.
FIGURE 10. Computer printout of selected ion current profiles for GC/MS analysis

of THC and two of its metabolites.
116 FOLTZ AND HIDY

FIGURE 11. CI-MS (H2/NH3) of trimethylsilylated cannabinoids.

Despite the high resolving power of capillary column GC and the

specificity of ammonia CI-MS, it is evident from figure 10 that interfering peaks are still a potential problem. This is largely due to

the relatively unselective extraction method used. An additional

negative consequence of the extraction procedure is that the life-
GC/CI-MS ANALYSIS OF CANNABINOIDS 117

FIGURE 12. Blood concentrations of THC and two of its metabolites following

oral administration of 10 mg of the drug to a human subject.

time of the capillary column is shortened due to the buildup of intractable, coextracted materials at the front of the column. Therefore, there is need for further work to develop a more selective extraction and sample cleanup method which is still amenable to routine analysis of multiple samples.

The described method for quantitation of THC and the two

metabolites has been applied to measurement of the plasma concentrations of these compounds following oral administration of 10

mg of THC to human volunteers. Figure 12 shows the concentrations versus time curves for a representative case.

ACKNOWLEDGMENT

These studies were supported by National Institute on Drug

Abuse Contract HSM-42-72-183.
118 FOLTZ AND HIDY

REFERENCES

1. S. Agurell, B. Gustafsson, B. Holmstedt, K. Leander, J.-E. Lindgren, I. Nilsson,

F. Sandbery, and M. Asberg. Quantitation of tetrahydrocannabinol in plasma

from cannabis smokers. J. Pharm. Pharmacol., 25, 554 (1973).

2. D. Rosenthal, T.M. Harvey, J.T. Bursey, D.R. Brine, and M.E. Wall. Comparison

of gas chromatography mass spectrometry methods for the determination of

tetrahydrocannabinol in plasma. Biomed. Mass Spectr., 5, 312-316 (1978).

3. R.L. Foltz, P.A. Clarke, B.J. Hidy, D.C.K. Lin, A.P. Graffeo, and B.A. Petersen.

Quantitation of tetrahydrocannabinol and 11-nor- tetrahydrocannabinol-9-

carboxylic acid in body fluids by GC/CI-MS. In J.A. Vinson (ed.), Cannabinoid

Analysis in Physiological Fluids, ACS Symposium Series 98, American Chemical

Society, Washington, D.C., 59-71 (1979).

4. D.L. Fenimore, C.M. Davis, J.H. Whitford, and C.A. Harrington. Vapor-phase silylation of glassware. Anal. Chem., 48, 2289-2290 (1976).

5. R.L. Foltz. Quantitative analysis of abused drugs in physiological fluids by gas

chromatography/chemical ionization mass spectrometry. In A.P. de Leenheer,

R.R. Roncucci, and C. van Peteghem (eds.), Quantitative Mass Spectrometry in

Life Sciences II, Elsevier Scientific Publishing Co., Amsterdam, 39-62 (1978).

6. D. Rosenthal and D. Brine. Quantitative determination of

tetrahydrocannabinol in cadaver blood. J. Forensic Sci., 24, 282-290 (1979).

7. J. Rosenfeld. The simultaneous determination of tetrahydrocannabinol and

11-hydroxy- tetrahydrocannabinol in plasma. Anal. Letters, 10, 917-930 (1977).

8. M.E. Wall, D.R. Brine, J.T. Bursey, and D. Rosenthal. Detection and quantitation of tetrahydrocannabinol in physiological fluids. In J.A. Vinson (ed.), Cannabinoid Analysis in Physiological Fluids, ACS Symposium Series 98, American

Chemical Society, Washington, D.C., 39-57 (1979).

9. M. Nordquist, J.-E. Lindgren, and S. Agurell. A method for the identification of

acid metabolites of tetrahydrocannabinol by mass fragmentography. In R.E. Willette (ed.), Cannabinoid Assays in Humans, NIDA Research Monograph 7,

DHEW Pub. No. (ADM)78-339. Supt. of Docs., U.S. Govt. Print. Off., Washington,

D.C., 64-69 (1976).

10. K. Grob and K. Grob, Jr. Splitless injection and the solvent effect. J. High Resolution Chromatogr. and Chromatogr. Commun., 1, 57-64 (1978).

11. K. Grob and G. Grob. Practical capillary gas chromatography-a systematic approach. J. High Resolution Chromatogr. and Chromatogr. Commun., 2, 109-117

(1979).

AUTHORS

Rodger L. Foltz

Center for Human Toxicology

University of Utah

Salt Lake City, Utah 84112

Bruce J. Hidy

Battelle Memorial Institute

505 King Avenue

Columbus, Ohio 43201
Analysis of Cannabinoids

Research Monograph 42, R. Hawks, Editor

National Institute on Drug Abuse, 1982

Stability of Delta-9-

Tetrahydrocannabinol in Stored

Blood and Serum

Anthony S. Wong, Michael W. Orbanosky, Victor C.

Reeve, and James D. Beede

INTRODUCTION

The literature has described delta-9-tetrahydrocannabinol (

THC) as a degradable molecule, subject to dissipation when exposed

to oxidation (1), elevated temperatures, and active surfaces such as

glass and plastic which contribute (2-6) to the loss of THC from

aqueous media by extensive and sometimes irretrievable binding.

Since our work deals principally with the analysis of THC in

blood, concern developed regarding whether THC would exhibit

similar instability in blood samples stored over relatively long periods. This study was designed to evaluate long term storage when

THC is present in blood in high physiological concentrations. We

examined the dissipation rate of THC during storage, the effects

of elevated temperatures on blood-THC samples, the effects of repetitive freeze/thaw cycles on blood-THC samples, and the effects

of agitation and ambient temperature fluctuation during mailing.

EXPERIMENTAL DESIGN

All blood and serum samples in this study were spiked with

THC at a concentration of 50 ng/ml by adding 50 ng of THC dissolved in 5 μl of alcohol to each ml of blood or serum. All glassware containing the concentrated standards was silylated prior to

use. Glassware used to contain blood-THC samples was left untreated. The quantitative analytical procedure used in this study in119
120 WONG, ORBANOSKY, REEVE, AND BEEDE

volved the addition of 50 μl of C-11 deuterated THC to 1.0 ml of

blood or serum, acidification of the sample to pH 4-5 using 3 ml 0.1

M KH2PO4 followed by multiple 5 ml extractions with 1.5% isoamyl alcohol in pentane. The combined organic phases were evaporated just to dryness and the residue dissolved in 50 μl of 0.1 M

trimethylphenyl ammonium hydroxide, of which 5 μl were injected

into the GC/MS. The fundamental difference between this method

and that developed by Rosenfeld (5) is the absence of a prior

cleanup of the extract.

A Finnigan 3200 GC/MS interfaced with a two-channel programmable multiple ion monitor (PROMIM) was employed for the analysis. Gas chromatography was conducted on a 60 × 0.4 cm glass

column containing 1.5% SP-2250/1.95% SP-2401 on 100/120 Supelcoport at 210°C. Selected ions were monitored at m/e 328 and m/e

331, the molecular ions for methylated THC and its deuterated

analog, respectively. Figure 1 and figure 2 represent an ion chromatogram for THC and a typical standard curve for unextracted

THC versus 5 ng deuterated THC.

The precision and accuracy of the analytical method in hemo

lyzed blood and serum were determined by spiking replicates at

two concentration levels: 10 ppb (10 ng THC in 5 μl of ethanol to

1 ml of substrate) and 50 ppb (50 ng of THC in 5 μl of ethanol to

1 ml of substrate). All the replicates of the same substrate were

analyzed on the same day immediately after spiking.

1. Long Term Storage: 1.0 ml samples of spiked blood or

serum were prepared and stored in glass under nitrogen at 5°C, under ambient atmosphere at 5°C, -5°C,

and -20°C. Storage stability was evaluated on a biweekly basis.

2. Elevated Temperature: Triplicates of 1.0 ml samples of

blood and serum were stored and maintained at 20°,

30°, 40°, 50° and 60°C for 25 hours prior to analyses.

3. Repetitive Freeze/Thaw Cycles: Triplicate 1.0 ml samples of blood and serum were frozen at -20°C for 8 days

during which time the samples were allowed to thaw

for 4 hours each day and refrozen. Analyses were performed on the eighth day.

4. Mailing Effect: THC (50 μg in 5 μl of ethanol) was

spiked to 1.0 ml of hemolyzed blood and serum in 2 ml

glass bottles equipped with Teflon-lined screw caps.

The containers were then mailed to southern California, returned to Sacramento, and the THC analyzed.
STABILITY OF DELTA-9-THC IN STORED BLOOD AND SERUM 121

FIGURE 1. Ion chromatogram of hemolyzed blood sample spiked with 10 ppb of

delta-9-THC.
122

FIGURE 2.

WONG, ORBANOSKY, REEVE, AND BEEDE

Standing curve of THC co-injected with THC-D3. The curve covers

range of 0 to 50 ng of THC in the original blood sample.

the

RESULTS

Relative recoveries and assay reproducibility of THC in ten

replicate analyses of 1.0 ml blood and serum samples spiked at concentrations of 50 ng/ml and 10 ng/ml respectively were determined. Mean recoveries for blood at the 50 ng/ml and 10 ng/ml

blood levels were 100% (CV=4%) and 97% (CV= 5%), respectively.

1. THC appeared to remain stable in blood up to and

including week 17. The analyses conducted on the
STABILITY OF DELTA-9-THC IN STORED BLOOD AND SERUM 123

samples stored to weeks 19, 21, and 23 showed significantly lower concentrations, indicating that loss of

THC begins at approximately week 19. Complete inability to detect THC occurred at week 23. High recovery of THC from serum was achieved up to week

13. Starting from week 15, however, lower and irregular analyses were obtained. As in the case of blood,

definite losses of THC occurred after 19 weeks in

storage.

2. In both blood and serum samples, neither atmospheric

conditions nor storage temperatures contributed significantly to the recovery of THC. Delta 9-THC was

found to be stable in blood samples when stored at

60°C for 25 hours. In serum, THC could be recovered from samples stored at 50°C for 25 hours. At 60°C

the serum matrix gelled, which inhibited extraction.

3. Both blood and serum samples containing -THC can

be repeatedly frozen and thawed without showing significant losses of the drug.

4. There was no significant loss from the substrates with

agitation in mailing during normal handling with the

containers utilized.

DISCUSSION

The sudden losses of THC that occurred around the 17th week

in storage cannot be sufficiently explained. It is thought, nonetheless, that the losses are not due to actual degradation or sudden

surface adsorption of the -THC molecule. Rather, it is surmised

that the dissipation of THC may be due to the inability successfully to extract the drug, possibly owing to irretrievable binding of

the molecule to degrading proteins.

The recovery data from the replicate analyses of the spiked samples clearly indicate the suitability of the analytical method for detection of -THC in both hemolyzed blood and serum. Comparison

of the ion chromatogram of a typical hemolyzed blood sample

spiked with 10 ppb of -THC (figure 1) and the standard curve

(figure 2) shows that the peak of the molecular ion, 328, of methylated THC is about 50% of full scale with a noise level of less

than 5% of full scale. The calculated detection limit of -THC

should be between 1 and 2 ppb (1 to 2 ng/ml of blood). This detection limit is comparable to that reported by other researchers

(2,5,7). Since the physiological levels normally observed in marijua-
124 WONG, ORBANOSKY, REEVE, AND BEEDE

na users range from 1 ng/ml to 100 ng/ml in blood (1), the method

developed during the present study to evaluate the in vitro, stability of THC can also be used to determine the THC in the

blood of the smokers.

The analytical technique is time-consuming and the CC/MS instrumentation is expensive. Therefore, the technique is not a practical screening procedure. The use of deuterated THC as an internal standard is essential in obtaining precise and accurate results.

REFERENCES

1. R.K. Razdan, A.J. Putlick, B.A. Zitko, and G.R. Hendrich. Experientia, 28, 121

(1972).

2. D. Rosenthal, D. Brine, and M. Wall. Quantitation of Delta-9-

Tetrahydrocannibinol in Human Serum by Gas Chromatography-Mass Spectrometry. A special report presented to National Institute on Drug Abuse, October 1, 1976.

3. R. Mechoulam. Science, 168, 1159 (1970).

4. R.F. Turk, J.E. Manno, N.C. Jain, and R.B. Forney. J. Pharmac., 23, 190 (1971).

5. J. Rosenfeld, B. Bowins, J. Roberts, J. Perkins, and A.S. Macpherson. Analytical

Chemistry, 46, 2232 (1974).

6. E.R. Garrett and D.A. Hunt. Journal of Pharmaceutical Sciences, 63, 1056 (1974).

7. D. Rosenthal and D.J. Brine. Forens. Sci., 24, 282 (1979).

AUTHORS

Anthony S. Wong and Michael W. Orbanosky

California Analytical Laboratories, Inc.

401 N. 16th Street

Sacramento, Calif. 95814

and

Victor C. Reeve and James D. Beede

California Department of Justice

Bureau of Forensic Services

3301 C Street

Sacremento, Calif. 95816
Analysis of Cannabinoids

Research Monograph 42, R. Hawks, Editor

National Institute on Drug Abuse, 1982

The Constituents of Cannabis and

the Disposition and Metabolism of

Cannabinoids

Richard L. Hawks

INTRODUCTION

Humans consume cannabinoids primarily by smoking preparations of Cannabis sativa L. When cannabis plant material is prepared for purposes of intoxication, it is referred to as “marijuana.”

Cannabis that is used as an intoxicant comes from many different

geographical sources around the world and is referred to as “drug

type” cannabis because it contains significant quantities of

tetrahydrocannabinol (THC), the primary chemical ingredient of

the plant material responsible for the psychopharmacological

effect. Another type of cannabis equally prevalent worldwide is referred to as “hemp type” because originally, about four centuries

ago, it was cultivated for the manufacture of rope material. Cannabis is not in fact a plant indigenous to the New World; it was introduced to the Western Hemisphere by Spanish explorers, who used

large amounts of hemp rope in their shipping industry.

More than 60 cannabinoid compounds have been identified in the

“drug type” cannabis used illicitly today. Some of the more important ones are illustrated in figure 1. In addition, as in any plant

material, there is a large number of other chemical compounds

present, several hundred of which have been identified in cannabis

(1).

Though cannabis contains many cannabinoid components and

other chemical substances, THC is the most prominent psychoactive cannabinoid component, and its concentration determines the

“potency” of marijuana. Other cannabinoids may contribute to

THC’s activity through interactions or direct effects on certain specific pharmacologic measures, but THC is the compound on which

125
126 HAWKS

FIGURE 1. Cannabinoids of primary interest found in Cannabis Sativa.

most studies to date have concentrated. The psychopharmacologic

activities of either cannabinol or cannabidiol, for instance, are estimated to be less than 10% as great in man as that of THC itself (2).

The predominant factor affecting potency is the genetic stock of

the plant. Seeds derived from cannabis of relatively high THC content will produce plants of relatively high potency, other factors

being equal. It should also be noted that the relative amounts of

cannabinoids in the plant can vary significantly depending on how

and where the plant is grown and harvested. Soil, climate, cultivation, and age can have considerable effect on the final “product.”
METABOLISM AND DISPOSITION OF CANNABINOIDS 127

Samples of marijuana from illegal shipments, confiscated between 1979 and 1982 by the Drug Enforcement Administration and

analyzed by the NIDA potency monitoring program, averaged

above 3.0% THC by weight. Potency has increased significantly

since the program started in 1974, when confiscated material was

found to contain less than 0.5%. A cannabis preparation called

“sinsemilla” has become increasingly available over the last 4

years and is currently averaging about 6% THC. Occasional samples with potencies as high as 10% have been encountered. The potential for ingesting psychologically dysphoric amounts of THC is

thus much increased with the availability of these more potent

forms of cannabis.

The THC in marijuana is predominantly in the form of THC2-(and a small amount of 4-)-carboxylic acid (also called

“tetrahydrocannabinolic acids” or “THC-acids”) (1). These THCacids are changed rapidly into THC itself when they are subjected

to the heat of the burning cigarette. The burning process also

causes pyrolytic destruction of a portion of the THC and THC acid

in the cigarette. The actual amount of THC delivered in the smoke

has been estimated at 20% to 70% in experiments conducted with

smoking machines (3). The estimate of 70% is derived from a study

in which the cigarette was burned with a continuous draw, and

20% was found when a 5-second puff each minute was used. The

latter rate is the standard for tobacco smoking experiments. Marijuana smokers probably fall in between these estimates since they

usually inhale longer and more frequently than once a minute for

5 seconds. This assumption is supported by a study which determined that the bioavailability of THC from marijuana cigarettes as

measured in blood of human subjects ranged from 8% to 24%

(mean 18±6%) in 11 subjects (4). This “bioavailability” is the fraction of THC in the cigarette which reaches the bloodstream. If for

instance, 35% of the THC in the cigarette were delivered in the

smoke and 50% of this passed through the lung to the blood, a

bioavailability of 17.5% would result, which is within the reported

range.

For the past 10 years NIDA has supplied cannabis cigarettes for

use in animal and human research on marijuana toxicity, pharmacology, pharmacokinetics, and chemistry. The cigarettes are sup

plied in potencies ranging from 0.5% to 3% by weight. The plant

material is grown under controlled conditions, and the material is

chemically characterized by quantitative analysis of eight major

cannabinoids. The cigarettes are supplied in a form similar in size

to unfiltered tobacco cigarettes.
128 HAWKS

Other THC dosage forms developed by NIDA and made available

for research include oral doses of THC in sesame oil contained in

gelatine capsules, intravenous solutions of THC which are either

suspended in human serum albumen or dissolved in ethanol, and

ophthalmic preparations of THC in mineral oil. Each of these

dosage forms has its own unique time course of effect and metabolic profile. Even though these dosage forms were developed for use

in research directed at consequences of marijuana abuse, they have

also served as dosage forms for studies in several areas of therapeutic research. The oral form is under investigation as an antiemetic

drug for patients undergoing cancer chemotherapy, and the ophthalmic preparation is being investigated as a treatment to relieve

intraocular pressure resulting from glaucoma.

METABOLISM AND DISPOSITION

When cannabis is smoked or ingested orally, THC and the many

other cannabinoids present undergo extensive metabolism into

many oxygenated species. Most of the research on cannabinoid metabolism has been carried out on THC itself. Metabolites of THC

which have been characterized and isolated from man (5,6,7) are illustrated in figure 2.

The total number of metabolites resulting from human administration of THC is undoubtedly much greater. In vitro and in vivo

animal studies indicate that a large number of hydroxylated and

carboxylated metabolites result from the metabolism of THC as

well as from the other cannabinoids found in the plant (8-14). Investigators have also shown the presence of fatty acid conjugates of

THC (15,16), a variety of 0-glucuronides (17,18), and C-glucuronides

(19).

The formation of glucuronide conjugates of THC and its metabolites has been reported to occur on the phenolic position (17), on

the carboxylic acid position in 11-nor- -THC-9-carboxylic acid (9-

carboxy-THC) (18), and directly on the two and four positions of the

aromatic ring (19). It is unclear at this time how this conjugation

affects excretion, storage, and recirculation of material, but it is

clear that a major portion of the cannabinoid metabolites excreted

in urine are in the form of glucuronide conjugates (5,18,20). In the

case of 9-carboxy-THC, conjugation may occur at both the phenolic

and the carboxy position, thus leading to the possibility of three

different conjugated species. A recent study has provided the characterization of 12 additional acid metabolites of THC (21). An extensive and complex cannabinoid metabolic profile in man is indi-
METABOLISM AND DISPOSITION OF CANNABINOIDS 129
130 HAWKS

cated when all the possible cannabinoid metabolites and their respective conjugates are considered.

The metabolite concentrations in figure 3 are typical of the significant differences found in blood after oral administration (22) of

THC as compared with administration via smoking (23). The 11-hydroxy metabolite probably plays a minor role after smoking as the

concentration is very low (5), less than 10% of the THC concentration at any time. After oral administration, however, 11-OH-THC

may play a more important role as its concentration is relatively

higher in blood, often higher than that of THC. Significant

amounts of THC are not observed after oral administration owing

primarily to the relatively slow absorption of THC and subsequent

extensive metabolism via the “first pass” through the liver. The

blood profile of the oral dose indicates 9-carboxy-THC as the predominant metabolite.

FIGURE 3. Plasma concentration versus time curve for THC and its metabolites

after oral and smoked administrations.

Several of the hydroxylated metabolites have been shown to have

some activity (24), but 9-carboxy-THC has shown little or no activity when administrated to either man (24) or animal. The only

active metabolite known to occur in any significant concentration

is 11-hydroxy- THC, which has been shown to have activity equal

to or perhaps slightly greater than THC itself when administered

alone to man (25). The psychopharmacological activity in man of 8-
METABOLISM AND DISPOSITION OF CANNABINOIDS 131

beta-hydroxy-THC has been reported as about 4% of that of

THC and 8-alpha-hydroxy-THC less than 10% (24).

In human studies it has been shown that 80% to 90% of the total

dose of THC administered is excreted within 5 days, mostly (65%)

in feces (26). The urinary fraction, which makes up the remaining

18% to 23% of the dose excreted, consists primarily of acidic metabolites such as 9-carboxy-THC and other related acids (21). Only

about 5% of the total urinary fraction consists of neutral cannabinoids, and THC itself makes up less than 1 % of this. The endpoint of

the renal pathway is therefore multi-oxygenated acidic metabolites.

The feces fraction consists of approximately equal parts neutral

and acidic metabolites, 9-carboxy-THC making up about 29% of the

extracted metabolites, THC about 8%, and 11-hydroxy-THC about

21% (5).

When the time course of the disappearance of THC from plasma

is compared with the very slow excretion of metabolites in urine, it

becomes obvious that some mechanism is contributing to this retarded elimination of material. As mentioned above, 80% to 90% of

the THC dose administered IV is accounted for in 5 days, the remainder being eliminated slowly over a longer period of time. One

mechanism which probably contributes significantly to the long excretion of these metabolites involves enterohepatic recirculation,

since the major portion of IV doses in humans is eliminated in the

feces (26) and reabsorption of biliary excreta has been demonstrated in the dog (27). Other mechanisms which could contribute include renal reabsorption and deeptissue distribution. Because of

the lipid solubility (28) of THC, a characteristic shared by many of

its metabolites, some portion of the dose is probably trapped in

fatty-tissue pools for slow release. Preferential absorption of THC

into the fat tissue of rats has been reported, while other tissues

such as brain, lung, and muscle absorbed a much smaller proportion of the dose (29).

It is important to define both the mechanisms involved in this

slow excretion and what specific metabolites are involved. Even

though the acid metabolites are not likely to have psychoactivity,

other types of toxic effects cannot be excluded. The characterization of fatty-acid conjugates of THC has led to speculation that species of this type may contribute to storage of cannabinoids in body

tissue (15,161. Much research remains to be done to ascertain the

chronic toxicity of these or the many other THC metabolites.

The character of the metabolites and their mode of elimination is

important since the elimination process is measured in days, not

hours, and repeated administration via marijuana smoking likely
132 HAWKS

leads to accumulation of both THC and metabolites and the possibility of subsequent toxic consequences.

Much of the forensic interest in cannabinoid analysis of body

fluids has focused on the 9-carboxy-THC metabolite, the principal

acid metabolite of THC that has been characterized and synthesized for study. This metabolite is an important species in both

blood and urine. As illustrated in figure 3, THC levels which are

initially quite high drop very rapidly and cross the rising 9-carboxy-THC curve at approximately 20 minutes. Although this curve

is based on a single dose, it does give credence to the hypothesis

that a blood analysis which shows similar concentrations for both

THC and 9-carboxy-THC could be an indication of very recent administration of marijuana and perhaps of a high probability that

the subject is in an intoxicated state. This figure also illustrates

that, because of the rapid decrease in the concentration of THC in

blood, analytical methodology less sensitive than 1 or 2 nanograms

per milliliter is not likely to detect the presence of THC after several hours except perhaps in a heavy chronic smoker. The curve

for 9-carboxy-THC in this figure shows a considerably longer time

course and therefore provides a better indicator of previous smoking.

Urine metabolite assays which have recently become available

on the commercial market have made cannabinoid detection available to a much broader spectrum of laboratories including those associated with the armed forces, with private industry, and penal institutions. These methods include the SYVA Co. EMIT system and

the Roche Diagnostics ABUSCREEN RIA system. The analysis of

urine using these types of assays provides an indication of past use

of marijuana. Due to the relatively long detection period of cannabinoid metabolites in urine, this ‘past use’ cannot be more specifically pinpointed than between 1 hour and 1 week. This makes any

urine analysis, regardless of the levels detected, inadequate for prediction of intoxication or specific time of use.

As mentioned above, a blood analysis does have the potential for

providing information relevant to intoxication. Blood samples are,

however, difficult to obtain for many forensic purposes because of

their invasive nature. Saliva and breath are much easier samples

to acquire for drug detection and both have been investigated for

the presence of marijuana components after smoking. The use of

TIC methods for detection of THC in saliva has been suggested by

several authors (30-34).

A study in humans using radiolabeled THC administered by intravenous injection (35) failed to detect any radioactivity in saliva

samples. This suggests that neither THC or its metabolites pass
METABOLISM AND DISPOSITION OF CANNABINOIDS 133

into the saliva or lung from the blood and that cannabinoids de

tected in these biological matrices are sequestered during the

smoking process.

In a recent study, an RIA specific for THC was used (36) to analyze a set of saliva samples (37) from subjects who smoked marijuana cigarettes containing cannabis of approximately 2% potency.

Mixed saliva samples were taken from these subjects over a 24-

hour period. The concentration of sequestered THC in these samples, illustrated in figure 4, is subject to considerably more fluctuation over time than plasma levels due to the inherent variability

of mixed saliva concentration and to the difficulty in precisely aliquoting from a mixed saliva sample. It is clear, however, that significant levels of THC remain in saliva for several hours after

smoking marijuana. During the first hour, in the absence of food

intake, some levels exceeded 100 ng/ml and levels above 5 ng/ml

FIGURE 4. Human saliva levels after smoking a single marijuana cigarette

containing 18 mg THC. N=8 at 20 min., N=7 at 60 min., N=5 for others.

Variability expressed as standard deviation.
134 HAWKS

were still detectable in most samples beyond 8 hours even though

normal food and liquid intake was resumed after 3 hours.

These results give further indication that saliva could be a useful

fluid for presumptive detection of recent use. Even though no quantitative correlation can be made between saliva levels and intoxication since only sequested THC is being detected, the capacity to

reduce the “past use” time frame from weeks to hours could have

useful research and forensic applications.

The use of breath as a screening medium has been investigated

using solvent and cryogenic traps with subsequent analysis by

HPLC/MS (38) and also using solid matrix traps followed by conventional RIA (39). The levels in breath appear to be lower than

those found in saliva after smoking, and use of such a medium for

detection would consequently require relatively more sensitivity in

the assay method as well as a more complex means of sample collection.

PHARMACOKINETICS

Studies of the pharmacokinetics of THC have been receiving increasing emphasis in the last few years as analytical techniques for

the quantitative determination of cannabinoids in biological fluids

have improved. The pharmacokinetics of THC and its metabolites

is important to several research areas. A variety of studies have

suggested that long-term, chronic use of marijuana may lead to adverse health effects in the individual (40). Some of these effects

could be related to a toxic accumulation of either THC or metabolites of THC in the body. Pharmacokinetic studies of the mechanisms by which these cannabinoids are eliminated from the body

are important in understanding the potential extent and rate of

this accumulation.

Detailed pharmacokinetic studies of THC in humans under both

acute and chronic dosing schedules are beginning to provide the

basic data that will allow blood levels of active drug to be related

to psychotomimetic, behavioral, and physiological effects (4,23,26).

With the increasing availability of analytical methods for analysis

of cannabinoids in blood and urine, there is increasing interest in

providing analytical data for forensic purposes which can be related to the intoxication of individuals involved in accidents, as well

as individuals who are legally liable for drug-taking behavior in

the armed forces, on probation, or in industry. It is important to

understand the kinetic mechanisms involved with THC and other

cannabinoids in order to relate these forensic questions to the
METABOLISM AND DISPOSITION OF CANNABINOIDS 136

meaning of a given blood level, particularly in cases where intoxication is a factor. The availability of more sensitive and more accessible analytical technology for the quantitative analysis of marijuana components in body fluids will play a key role in these efforts.

REFERENCES

1. C.T. Turner, M.A. Elsohly, and E.G. Boeren. Constituents of Cannabis Sativa L.

XVII. A review of the natural constituents. J. Natural Products, 43, 169-234

(1980).

2. M. Perez-Reyes, M.C. Timmons, K.H. Davis, and M.E. Wall. A comparison of the

pharmacological activity in man of intravenously administered delta-9-THC,

cannabinol and cannabidiol. Experientia, 29, 1368-1369 (1973).

3. K.H. Davis, Research Triangle Institute, private communication.

4. A. Ohlsson, J-E. Lindgren, A. Wahlen, S. Agurell, L.E. Hollister, and H.K. Gillispie. Plasma delta-9-THC concentrations and clinical effects after oral and intravenous administration and smoking. Clin. Pharm Ther., 98, 409-1413 (1980).

5. M.E. Wall, D.R. Brine, and M. Perez-Reyes. Metabolism of cannabinoids in man.

In The Pharmacology of Marijuana, Vol. 1, M.C. Braude and S. Szara, eds.,

Raven Press, New York, 93-116 (1976).

6. M.E. Wall, D.R. Brine, C.G. Pitt, and M. Perez-Reyes. Identification of delta-9

THC and metabolites in man. J. Am. Chem. Soc., 94, 8579-8681 (1972).

7. L. Lemberger, S.D. Silberstein, J. Axelrod, and I.J. Kopin. Delta-9-THC in man.

Science, 170, 1320-1322 (1970).

8. S.H. Burstein, F. Menezes, E. Williamson, and R. Mechoulam. Metabolism of

delta-l(6)-THC, an active marihuana constituent. Nature, 225, 87-88 (1979).

9. I.M. Nilsson, S. Agurell, J.L.G. Nilsson, A. Ohlsson, F. Sandberg, and M. Wahlqvist. Delta-1-THC: structure of a major metabolite. Science, 168, 1228-1229 (1970).

10. M.E. Wall, D.R. Brine, G.A. Brine, C.G. Pitt, R.I. Freudenthal, and H.D. Christensen. Isolation, structure, and biological activity of several metabolites of

delta-9-THC. J. Am. Chem. Soc., 92, 3466-3468 (1970).

11. S. Agurell, I.M. Nilsson, M. Widman. Cannabinoids metabolites hydroxylated in

the pentyl side chain. In Marihuana Chemistry, Biochemistry and Cellular Effects, G. Nahas, ed., Springer-Verlag, New York, 141-158 (1976).

12. S. Burstein. A survey of the metabolic transformations of delta-1-THC. In Cannabinoid Analysis in Physiological Fluids. J.A. Vinson, ed., American Chemical

Society, Washington, D.C., 1-12 (1979).

13. M.E. Wall and D.R. Brine. Applications of mass spectrometry in cannabinoid research. In Marihuana: Biological Effects, G. Nahas and W.D.M. Paton, eds., Pergamon Press, London, 15-43 (1979).

14. D.J. Harvey, B.R. Martin, and W.D.M. Paton. Identification and measurement of

cannabinoids and their in vivo metabolites in liver by gas chromatography-mass

spectrometry. In Marihuana: Biological Effects, G.G. Nahas and W.D.M. Paton,

eds., Pergamon Press, London, 45-62 (1979).

15. E.G. Leighty, A.F. Fentiman, and R.E. Foltz. Long retained metabolites of delta9-THC identified as novel fatty acid conjugates. Res. Commun Chem. Pathol.

Pharmacol., 14, 13-23 (1976).
136 HAWKS

16. W. Yisak, S. Agurell, J-E. Lingren, and M. Widman. In vivo metabolites of cannabinol identified as fatty acid conjugates. J. Pharm. Pharmacol., 30, 462-463

(1978).

17. S. Pallante, M.A. Lyle, and C. Fenselau. Synthesis and characterization of glucuronides of 5’-hydroxy-delta-9-THC and 11-hydroxy-delta-9-THC. Drug Metab.

Dispos., 6, 389-395 (1978).

18. P.L. Williams and A.C. Moffat. Identification in human urine of delta-9-THC-11-

oic acid glucuronide: A THC metabolite. J Pharm. Pharmacol., 32, 445-448

(1980).

19. S. Levy, B. Yagen, and R. Mechoulam. Identification of a C-glucuronide of delta6-tetrahydrocannabinol as a mouse liver conjugate in vivo. Science, 200, 1391-

1392 (1978).

20. S.L. Kanter, L.E. Hollister, and J.U. Zamora. Marijuana metabolites in urine of

man XI. Detection of unconjugatad and conjugated delta-9-THC-11-oic acid by

thin layer chromatography. J. Chromatogr., 235, 507-512 (1982).

21. M.M. Halldin, S. Carlsson, S.L. Kanter, M. Widman, and S. Agurell. Urinary

metabolites of tetrahydrocannabinol in man. Arzneimittelforschung, in press.

22. H. Gross, M.H. Ebert, V.B. Faden, S.C. Goldberg, W.H. Kaye, E.D. Caine, R.L.

Hawks, and N. Zinberg, A double-blind trial of delta-9-tetrahydrocannabinol in

primary anorexia nervosa. Submitted for publication, 1982.

23. M. Perez-Reyes, S. DiGuiseppi, K.H. Davis, V.H. Schindler, and C.E. Cook. Comparison of effects of marihuana cigarettes of three different potencies. Clin.

Pharmacd Ther., 31, 617-624 (1982).

24. M. Perez-Reyes, M.D. Timmons, M.A. Lipton, H.D. Christensen, and K.H. Davis.

A comparison of the pharmacological activity of delta-9-THC and its monohy

droxylated metabolites in man. Experientia, 29, 1009-1010 (1973).

26. M. Perez-Reyes, M.C. Timmons, M.A. Lipton, K.H. Davis, and M.E. Wall. Intravenous injection in man of delta-9-THC and 11-OH-delta-9-THC, Science, 177;

633-635 (1972).

26. A.C. Hunt and R.T. Jones. Tolerance and disposition of THC in man. J. Pharm.

Exp.. Ther., 215, 35-44 (1980).

27. E.R. Garrett and C.A. Hunt. Pharmacokinetics of delta-9-THC in dogs. J. Pharm.

Sci., 66, 395-407 (1977).

28. E.R. Garrett and C.A. Hunt. Physicochemical properties, solubility, and protein

binding of delta-9-THC. J. Pharm. Sci, 63, 1056-1064 (1974).

29. D.S. Kreuz and J. Axelrod. Delta-9-tetrahydrocannabinol: localization in body

fat. Science, 179, 391-393 (1973).

30 I.S. Forrest, D.E. Green, S.E. Rose, G.C. Skinner, and D.M. Torres. Flourescentlabeled cannabinoids. Res. Commun. Chem. Pathol. Pharmacol, 2, 787-792

(1971).

31. A.P. Melikian and I.S. Forrest. Dansyl derivatives of delta-9- and delta-&

tetrahydrocannabinols. J. Pharm Sci., 62, 1025-1026 (1973).

32. W.W. Just, G. Werner, and M. Weichmann. Bestimmung von delta-l- and delta1(6) Tetrahydrocannabinol in Blut, Urin und Speichel von Haschisch-Rauchern.

Naturwiss., 59, 222-223 (1972).

33. F. Friedrich-Fiechtl, G. Spiteller, W.W. Just, G. Werner, and M. Weichmann. Au

Nachweis und Identifizierung von Tetrahydrocannabinol in Biologischen Flussighkeiten. Naturwiss., 60, 207-208 (1973).

34. W.W. Jut, N. Filipovic, and G. Werner. Detection of delta-9-THC in saliva of

men by means of thin-layer chromatography and mass-spectrometry. J. Chromatogr., 96, 189-194 (1974).

36. M. Perez-Reyes, University of North Carolina, personal communication.
METABOLISM AND DISPOSITION OF CANNABINOIDS 137

36. C.E. Cook, Research Triangle Institute, NC, performed under NIDA contract

271-80-3705.

37. H. Moskowitz, Southern California Research Institite, CA, samples collected

under NIDA contract 271-76-3316.

38. J.L. Valentine, P.J. Bryant, P.L. Gutshall, O.H.M. Gan, and H.C. Niu. Detection

of delta-9-THC in human breath following marijuana smoking. Anal. Letters, 12

(B8) 867-880 (1979).

39. J.R. Soares, J.D. Grant, and S.J. Gross. Significant developments in radioimmune methods applied to delta-9-THC and its 9-substituted metabolites This

volume.

40. Marijuana and Health. Report of a study by a Committee of the Institute of

Medicine, AS. Relman, chairman. National Academy Press, Washington, D.C.,

1982.

AUTHOR

Richard L. Hawks

Research Technology Branch

Division of Research

National Institute on Drug Abuse

Rockville, Maryland 20857
monograph series

While limited supplies last, single copies of the monographs may be

obtalned free of charge from the National Clearinghouse for Drug

Abuse Information (NCDAI). Please contact NCDAI also for

Information about availability of coming issues and other

publications of the National Institute on Drug Abuse relevant to

drug abuse research.

Additional copies may be purchased from the U.S. Government Printing

Office (GPO) and/or the National Technical Information Service

(NTIS) as lndlcated. NTIS prices are for paper copy. Microfiche

copies, at $4.50, are also available from NTIS. Prices from either

source are subject to change.

Addresses are:

NCDAI

National Clearinghouse for Drug Abuse Information

Room lOA-43

5600 Flshers Lane

Rockville, Maryland 20857

GPO NTIS

Superintendent of Documents National Technical Information

U.S. Government Prlntlng Office Service

Washington, D.C. 20402 U.S. Department of Commerce

Springfield. Virginia 22161

1 FINDINGS OF DRUG ABUSE RESEARCH. Not available from NCDAI.

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2 OPERATIONAL DEFINITIONS IN SOCIO-BEHAVIORAL DRUG USE RESEARCH

1975. Jack Ellnson, Ph.D., and Davld Nurco, Ph.D., eds. Not

available from NCDAI.

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3 AMINERGIC HYPOTHESES OF BEHAVIOR: REALITY OR CLICHE? Bruce J.

Bernard, Ph.D., ad. Not available from NCDAI.

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138
139

4 NARCOTIC ANTAGONISTS: THE SEARCH FOR LONG-ACTING PREPARATIONS.

Robert Wlllette. Ph.D., ed.

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5 YOUNG MEN AND DRUGS: A NATIONWIDE SURVEY. John A. O'Donnell,

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6 EFFECTS OF LABELING THE "DRUG ABUSER": AN INQUIRY. Jay R.

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7 CANNABINOID ASSAYS IN HUMANS. Robert Wlllette, Ph.D., ed.

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8 Rx: 3x/WEEK LAAM - ALTERNATIVE TO METHADONE. Jack Blaine, M.D.,

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9 NARCOTIC ANTAGONISTS: NALTREXONE PROGRESS REPORT. Demetrios

Julius, M.D., and Plerre Renault, M.D., eds. Not available from

NCDA I.

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11 DRUGS AND DRIVING. Robert Wlllette. Ph.D., ed. Not available

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12 PSYCHODYNAMICS OF DRUG DEPENDENCE. Jack D. Blaine, M.D., and

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13 COCAINE: 1977. Robert C. Petersen, Ph.D., and Richard C.

Stillman. M.D., eds. Not available from NCDAI.

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14 MARIHUANA RESEARCH FINDINGS: 1976. Robert C. Petersen, Ph.D.,

ed. Not available from NCDAI.

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15 REVIEW OF INHALANTS: EUPHORIA TO DYSFUNCTION. Charles Wm.

Sharp, Ph.D., and Mary Lee Brehm. Ph.D., eds.

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16 THE EPIDEMIOLOGY OF HEROIN AND OTHER NARCOTICS. Joan Dunne

Rittenhouse, Ph.D.. ed. Not available from NCDAI.

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140

17 RESEARCH ON SMOKING BEHAVIOR. Murray E. Jarvik, M.D., Ph.D.,

et al., eds. Includes epidermiology, etiology. consequences of

use, and approaches to behavioral change. From a NIDA-supported

UCLA conference.

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18 BEHAVIORAL TOLERANCE: RESEARCH AND TREATMENT IMPLICATIONS.

Norman A. Krasnegor, Ph.D., ed. Theoretical and empirical studies

of nonpharmacologic factors in development of drug tolerance.

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19 THE INTERNATIONAL CHALLENGE OF DRUG ABUSE. Robert C. Petersen,

Ph.D., ed. Papers from the VI World Congress of Psychiatry.

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20 SELF-ADMINISTRATION OF ABUSED SUBSTANCES: METHODS FOR STUDY.

Norman A. Krasnegor, Ph.D., ed. Techniques used to study basic

processes underlying abuse of drugs, ethanol, food, and tobacco.

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21 PHENCYCLIDINE (PCP) ABUSE: AN APPRAISAL. Robert C. Petersen,

Ph.D., and Richard C. Stillman. M.D., eds. For clinicians and

researchers, assessing the problem of PCP abuse.

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22 QUASAR: QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIPS OF

ANALGESICS, NARCOTIC ANTAGONISTS, AND HALLUCINOGENS. Gene

Barnett, Ph.D.; Milan Trsic, Ph.D.; and Robert Willette, Ph.D.;

eds. Not available from NCDAI.

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23 CIGARETTE SMOKING AS A DEPENDENCE PROCESS. Norman A.

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maintenance, and cessation of the cigarette smoking habit.

Includes an agenda for future research.

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24 SYNTHETIC ESTIMATES FOR SMALL AREAS: STATISTICAL WORKSHOP

PAPERS AND DISCUSSION. Jos. Steinberg, ed. Papers from a workshop on Statistical approaches that yield needed estimates of

data for States and local areas. Not available from NCDAI.

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25 BEHAVIORAL ANALYSIS AND TREATMENT OF SUBSTANCE ABUSE. Norman

A. Krasnegor, Ph.D., ed. Papers on commonalities and

implications for treatment of dependency on drugs, ethanol, food,

and tobacco.

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26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor, Ph.D.,

ed. Reprint of the behavioral section of the 1979 Report of the

Surgeon General on Smoking and Health; introduction by editor.

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141

27 PROBLEMS OF DRUG DEPENDENCE, 1979: PROCEEDINGS OF THE 41ST

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG DEPENDENCE, INC. L.S. Harris, Ph.D., ed. Not available from NCDAI.

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28 NARCOTIC ANTAGONISTS: NALTREXONE PHARMACOCHEMISTRY AND

SUSTAINED-RELEASE PREPARATIONS. Robert Willette, Ph.D., and

Gene Barnett, Ph.D., eds. Papers report research on sustainedrelease and long-acting devices for use with the narcotic antagonist naltrexone. Not available from NCDAI.

GPO Stock #017-024-01081-2 $7.00 NTIS PB #81-238875 $23.50

29 DRUG ABUSE DEATHS IN NINE CITIES: A SURVEY REPORT. Louis A.

Gottschalk, M.D., et al. Not available from NCDAI.

GPO Stock #017-024-00982-2 $6.50 NTIS PB #8O-178882 $17.50

30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY PERSPECTIVES.

Dan J. Lettleri. Ph.D.; Mollle Sayers; and Helen Wallenstein

Pearson, eds. Volume presents summaries of major contemporary

theories of drug abuse by each of 43 leading theorists.

GPO Stock #017-024-00997-1 $10.00 Not available from NTIS

31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen, Ph.D..

ed. The text of the 8th Marijuana and Health report to Congress

and the background scientific papers on which it was based.

GPO out of stock NTIS PB #8O-215171 $20.50

32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz,

Ph.D.; Allison F. Fentlman, Jr., Ph.D.: and Ruth B. Foltz. A

collection of methods for quantitatlve analysis of several

important drugs of abuse by gas chromatography- mass spectrometry.

GPO Stock #017-024-01015-4 $6.00 NTIS PB #81-133746 $19

33 BENZODIAZEPINES: A REVIEW OF RESEARCH RESULTS, 1980. Stephen

I. Szara. M.D., D.Sc.. and Jacqueline P. Ludford. M.S.. eds.

A RAUS (Research Analysis and Utilization System) Review Report

on the abuse liability of the benzodiazepine "tranquilizers."

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34 PROBLEMS OF DRUG DEPENDENCE, 1980: PROCEEDINGS OF THE 42ND

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. Not available from

NCDAI.

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35 DEMOGRAPHIC TRENDS AND DRUG ABUSE, 1980-1995. Louise G.

Richards, Ph.D., ed. Estimates of probable extent and nature of

nonmedical drug use, 1980-1995. based on age structure and other

characteristics of U.S. population.

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142

36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A REVIEW OF MULTIDISCIPLINARY PAIN CLINICS AND PAIN CENTERS. Lorenz K.Y. Ng, M.D.,

Discussions by active practitioners in the treatment of pain.

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37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE. Travis

Thompson, Ph.D., and Chris E. Johanson, Ph.D., eds. Presents a

growing body of data, systematically derived, on the behavioral

mechanisms involved in use and abuse of drugs.

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38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettieri,

Ph.D., and Jacqueline P. Ludford. M.S., eds. A RAUS Review

Report, emphasizing use of marijuana: epidemiology, sociodemographic and personality factors, family and peer influence,

delinquency, and biomedical consequences.

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39 YOUNG MEN AND DRUGS IN MANHATTAN: A CAUSAL ANALYSIS.

Richard R. Clayton, Ph.D., and Harwin L. Voss, Ph.D. Examines

the etiology and natural history of drug use, with special focus

on heroin. Includes a Lifetime Drug Use Index.

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40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES. Herbert

Hendin. M.D.. Ann Pollinger, Ph.D.. Richard Ulman, Ph.D., and

Arthur Carr, Ph.D. A psychodynamic study of adolescents

involved in heavy marijuana use, to determine what interaction between family and adolescent gives rise to drug abuse.

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41 PROBLEMS OF DRUG DEPENDENCE, 1981: PROCEEDINGS OF THE 43RD

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. Not available from

NCDAI.

Not available from GPO NTIS PB #82-190760 $41.50

42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS. Richard L.

Hawks, Ph.D., ed. Varied approaches to sensitive, reliable, and

accessible quantitative assays for the chemical constltutents of

marijuana. for researchers. Not available from NCDAI.

GPO Stock #017-024-01151-7 $5 NTIS PB #83-136044 $1643

43 PROBLEMS OF DRUG DEPENDENCE, 1982: PROCEEDINGS OF THE 44TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D.. ed. Not available from

NCDAI.

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a143

44 MARIJUANA EFFECTS ON THE ENDOCRINE AND REPRODUCTIVE SYSTEMS.

Monique C. Braude, Ph.D., and Jacqueline P. Ludford. M.S., eds.

A RAUS Review Report of animal studies and preclinical and

clinical studies of effects of cannabinoids on human endocrine

and reproductive functions.

GPO Stock #017-024-01202-5 $4. NTIS PB #85-150563/AS $14.50

45 CONTEMPORARY RESEARCH IN PAIN AND ANALGESIA, 1983. Roger M.

Brown, Ph.D.; Theodore M. Pinkert, M.D., J.D.; and Jacqueline P.

Ludford, M.S., eds. A RAUS Review Report on the anatomy,

physiology, and neurochemistry of pain and its management.

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46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE TREATMENT.

John Grabowski. Ph.D .; Maxine L. Stitzer, Ph.D., and Jack E.

Henningfield, Ph.D., eds. Reports on behavioral contingency

management procedures used in research/treatment environments.

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47 PREVENTING ADOLESCENT DRUG ABUSE: INTERVENTION STRATEGIES.

Thomas J. Glynn, Ph.D .; Carl G. Leukefeld, D.S.W.; and

Jacqueline P. Ludford, M.S., eds. A RAUS Review Report on a

variety of approaches to prevention of adolescent drug abuse, how

they can be applied, their chances for success, and needed future

research.

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48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF SMOKING

BEHAVIOR. John Grabowski, Ph.D., and Catherine S. Bell, M.S..

eds. Based upon a meeting cosponsored by NIDA and the National

Cancer Institute to delineate necessary and sufficient measures

for analysis of smoking behavior in research and treatment

ettings.

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49 PROBLEMS OF DRUG DEPENDENCE, 1983: PROCEEDINGS OF THE 45TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. A collection of

papers which together record a year's advances in drug abuse

research; also includes reports on tests of new compounds for

efficacy and dependence liability.

GPO Stock #017-024-01198-3 $12 NTIS PB 85-159663/AS $22.

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

Grabowski, Ph.D., ed. Content ranges from an introductory

overview through neuropharmacology, pharmacology, animal and

human behavioral pharmacology, patterns of use in the natural

environment of cocaine users, treatment, through commentary on

societal perceptions of use.

GPO Stock #017-020-01214-9 $4 NTIS PB 85-150381/AS $14.50
144

51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES, PROGRESS, AND

PROSPECTS. Frank M. Tims. Ph.D., ed. A state-of-the-art review

of drug abuse treatment evaluation, identifying research needs,

promising approaches, and emerging issues.

GPO Stock #017-020-01218-1 $4.50 NTIS PB 85-150365/AS $17.50

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND ABUSE

LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas, Ph.D.,

eds. Describes animal and human test procedures for assessing

dependence potential and abuse liability of opioids, stimulants,

depressants, hallucinogens, cannabinoids, and dissociative

anesthetics.

GPO Stock #017-024-0204-1 $4.25 NTIS PB 85-150373/AS $16

54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm. Sharp,

Ph.D., ed. Review of basic knowledge concerning the mechanism of

action of opiates and other drugs in producing tolerance and/or

dependence.

GPO Stock #017-024-01213-1 $8.50 NTIS PB No. to be assigned.

55 PROBLEMS OF DRUG DEPENDENCE, 1984. PROCEEDINGS OF THE 46TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

IN PRESS OR PREPARATION

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION.

John Grabowski, Ph.D., ed.

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR PREVENTION. Coryl

LaRue Jones, Ph.D., and Robert J. Battjes, D.S.W., eds.

57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING CURRENT

CHALLENGES TO VALIDITY. Beatrice A. Rouse, Ph.D., Nicholas J.

Kozel, M.S., and Louise G. Richards, Ph.D., eds.

58 PROGRESS IN THE DEVELOPMENT OF COST-EFFECTIVE TREATMENT FOR

DRUG ABUSERS. Rebecca S. Ashery, D.S.W.. ed.

59 CURRENT RESEARCH ON THE CONSEQUENCES OF MATERNAL DRUG ABUSE.

Theodore M. Pinkert. M.D., J.D., ed.

60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS. C. Nora

Chlang, Ph.D., and Charles C. Lee, Ph.D., eds.
DHHS publication number (ADM)85-1212

Printed 1982 Reprinted 1985