CANNABINOID ASSAYS

IN HUMANS

Editor

ROBERT E. WILLETTE, PH.D.

Division of Research

National Institute on Drug Abuse

May 1976

NIDA Research Monograph 7

National Institute on Drug Abuse

11400 Rockville Pike

Rockville, Maryland 20852

U.S. Department of Health, Education, and Welfare

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration
The NIDA Research Monograph series is prepared by the

Research Division of the National Institute on Drug Abuse. Its

primary objective is to provide critical reviews of research problem

areas and techniques, the content of state-of-the-art conferences,

integrative research reviews and significant original research. Its

dual publication emphasis is rapid and targeted dissemination to

the scientific and professional comunity.

EDITORIAL ADVISORY BOARD

Avram Goldstein, M.D.

Jerome Jaffe, M.D.

Reese T Jones, M.D.

William McGlothlin, Ph.D.

Jack Mendelson, M.D.

Helen Nowlis, Ph.D.

Lee Robins, Ph.D.

Addiction Research Foundation

Palo Alto, California

College of Physicians and Surgeons

Columbia University, New York

Langley Porter Neuropsychiatric

Institute

University of California

San Francisco, California

Department of Psychology, UCLA

Los Angeles, California

Alcohol and Drug Abuse Research

Center

Harvard Medical School

McLean Hospital

Belmont, Massachusetts

Office of Drug Education, DHEW

Washington, D.C.

Washington University School of

Medicine

St. Louis, Missouri

NIDA RESEARCH MONOGRAPH series

Robert DuPont, M.D. DIRECTOR, NIDA

William Pollin, M.D. DIRECTOR, NIDA RESEARCH DIVISION

Robert C. Petersen, Ph.D. EDITOR-IN-CHIEF

Eunice L. Corfman, M.A. MANAGING EDITOR

Rockwall Building 11400 Rockville Pike Rockville, Maryland, 20852
CANNABINOID ASSAYS

IN HUMANS
Thanks are due the people of Macro Systems, Inc.,

who capably organized and smoothly coordinated

the conference, held February 24th and 25th, 1976,

under NIDA contract #271-75-1139, from which the

papers in this monograph are derived.

DHEW publication number (ADM) 76-339

Library of Congress catalog card number 76-15843

This document is for sale by National Technical Information Service

Springfield Va. 22161

Stock Order #PB 251 905; Papercopy: $6.00; Microfiche: $2.25

i v
FOREWORD

This monograph describes ways of determining

the levels of cannabinoids in the human body

after smoking marihuana. Investigations of a

number of serious social and health problems

have had to wait for the refinements of these

techniques.

Chief among these concerns is the effect of

marihuana smoking on driving. That investigation along with others can now be undertaken

with an expectation of far more precise findings than was formerly possible. We now have

the means not only of detecting cannabinoids,

but also of determining in what quantity they

are present. Thus, we can begin to establish

specific correlations between cannabinoid levels

and driving impairment. This information is

necessary in order to build marihuana into the

highway safety campaign now largely restricted

to alcohol.

These assay techniques will be useful tools

for a range of other problems including simple

screening procedures, epidemiological studies,

forensic toxicology, as well as for more fundamental pharmacokinetic and pharmacological research. The developments in method and instrumentation described here occur at a crucial

juncture for drug abuse research, as we strive

to assess the impact of marihuana on our culture.

Robert L. DuPont, M.D.

Director

National Institute on Drug Abuse

v

PREFACE

For the past several years, there has been an

increasing demand for qualitative and quantitative

assays for identifying and measuring the constituents of marihuana in the human body. This demand

is prompted by the need for such assays in several

research investigations that are attempting to improve our understanding of how this complex drug

affects the body. In addition to these more

fundamental issues, there is a growing feeling that

suitable analytical methods will be required for

determining the presence of cannabinoids in drivers

suspected of being under the influence of the drug.

We have now reached a stage in the search for and

development of such methods that many of them can

now be employed in a routine manner. As we gain in

experience and confidence with these methods, their

validity will become increasingly better established

and their applicability to critical decisions more

acceptable. It will be apparent upon reviewing the

procedures described herein that some were designed

for or are by their complexity only suited for research purposes or in validating other methods.

Others described are more amenable to routine

screening or survey applications.

The road to acceptable methods has been long and

arduous. Early attempts continued to suffer from

lack of adequate sensitivity. It was eventually

learned, as the studies on the composition of

marihuana and the metabolism of its constituents

progressed, that the problem of detecting any

specific cannabinoid in the body after use would

be an extremely difficult task. The primary active

constituent, delta-9-tetrahydrocannabinol (THC) is

rapidly distributed and metabolized in the body,

making its quantification a major challenge. It is

now very gratifying to be able to present a collection of manuscripts that delineate the tremendous

progress that has been made over the past few years.

vii
The methods described are grouped together under

three major headings. The first are based on the

increasingly used immunoassay techniques. In general, immunoassays offer speed and sensitivity and

are very amenable to the screening of large numbers

of samples. They often suffer from lack of specificity, but this is often acceptable if they cross

react only with metabolites of the target drug and

no other drug. The four methods described are at

various stages of development and refinement, and

some are being employed in various research studies.

Methods of the second group are based on the older

technology of chromatography, but are applied in

rigorous and innovative ways to provide the degree

of sensitivity required to measure the low levels

of drugs and metabolites. Two different approaches

were taken to reduce the background interference.

Using a conventional gas chromatograph, Dr. Garrett

employed a high pressure liquid chromatograph (HPLC)

to "clean-up" the sample. The dual-column instrument designed by Dr. Fenimore reduces the amount of

effort required for sample preparation. As the

technology in HPLC progresses, this method is becoming extremely popular, offering the ability to

separate difficult mixtures at low temperatures.

The effort described here is moving forward and

parallels similar efforts that are being carried

out by Dr. Valentine, in an outgrowth of his project's mass spectroscopic method.

The last group of methods all employ the mass

spectrometer as the detector for identifying and

quantifying the cannabinoids. Rapid advances in

the development of mass spectroscopy have made it

the method of choice in terms of sensitivity and

specificity. Because of its present size, cost,

and complexity, it is not ideal for routine application to routine screening. It is, however, being

used for the routine validation of other methods

and to confirm the presence of cannabinoids in

samples found positive by less specific screening

methods, like the immunoassays. The six papers

included represent some of the most outstanding

work done in the field of mass spectroscopic

analysis. Since Dr. Agurell first published his

method in 1973, tremendous strides have been made

by him and the others included in this monograph.

viii
We now feel very confident in our ability to get on

with many of the critical studies that have awaited

these methods.

This collection of papers does not signal the end

of the road in the development of suitable methods

for quantifying cannabinoids in the body. As our

understanding of the effects of marihuana progresses, so must the methods used in studying it.

We now have methods to study the effects of marihuana on driving, and if the evidence indicates

that it poses a significant hazard, then a simpleperhaps roadside- test may be required. Other

examples could be cited, but it is sufficient to

end with the recognition of a notable achievement

in this difficult area of research and a sense of

satisfaction that our perseverance is paying off.

Robert E. Willette, Ph.D.

Uivision of Research

National Institute on Drug Abuse

April 1976

i x

CONTENTS

FOREWORD

PREFACE

QUANTITATION OF CANNABINOIDS IN BIOLOGICAL FLUIDS BY RADIOIMMUNOASSAY 1

Arleen R. Chase, Paul R. Kelley, Alison Taunton-Rigby, Reese T. Jones, Theresa Harwood

SEPARATE RADIOIMMUNE MEASUREMENTS OF BODY FLUID Δ

9

-THC AND 11-NOR-9-CARBOXY-Δ

9

-THC

Stanley J. Gross, M.D. and James R. Soares, Ph.D.

10

RADIOIMMUNOASSAY OF Δ

9

-TETRAHYDROCANNABINOL

Clarence E. Cook, Ph.D., Mary L. Hawes, B.A., Ellen W. Amerson, B.A.,

Colin G. Pitt, Ph.D., and David Williams, B.A.

15

DETERMINATION OF THC AND ITS METABOLITES BY EMIT® HOMOGENEOUS ENZYME IOIMMUNOASSAY:

A SUMMARY REPORT

G.L. Rowley, Ph.D., T.A. Armstrong, C.P. CrowL, W.M. Eimstad, W.M. Hu, Ph.D.,

J.K. Kam, R. Rodgers, Ph.D., R.C. Ronald, Ph.D., K.E. Rubenstein, Ph.D.,

B.G. SkeLdon, and E.F. Ullman, Ph.D.

SEPARATION AND SENSITIVE ANALYSIS OF TETRAHYDROCANNABINOL IN BIOLOGICAL FLUIDS

BY HPLC AND GLC

Edward R. Garrett and C. Anthony Hurt

DETERMINATION OF Δ

9

-TETRAHYDROCANNABINOL IN HUMAN BLOOD SERUM BY ELECTRON CAPTURE

GAS CHROMATOGRAP'HY

David C. Fenimore, Ph.D., Chester M. Davis, Ph.D., and Alec H. Horn

DETECTION AND QUANTIFICATION OF TETRAHYDROCANNABINOL IN IN BLOOD PLASMA

Agneta Ohlsson, Jan-Erik Lindgren, Kurt Leander, Stig Agurell

A METHOD FOR THE IDENTIFICATION OF ACID METABOLITES OF TETRAHYDROCANNABINOL (THC)

BY MASS FRAGMENTOGRAPHY

Marianne Nordqvist, Jan-Erik Lindgren, Stig Agurell

QUANTITATION OF CANNABINOIDS IN BIOLOGICAL SPECIMENS USING PROBABILITY BASED MATCHING

GAS CHROMATOGRAP'HY/MASS SPECTROMlETRY

Donald E. Green, Ph.D.

QUANTITATION OF Δ

9

-TETRAHYDROCANNABINOL IN BODY FLUIDS BY GAS CHROMATOGRAPHY/

CHEMICAL IONIZATION-MASS SPECTROMETRY

Ruthanne Detrick and Rodger L. FoItz

HPLC-MS DETERMINATION OF Δ

9

-TETROHYDROCANNABINOL IN HUMAN BODY SAMPLES

Jimmie L. Valentine, Ph.D., Paul J. Bryant, Ph.D., Paul L. Gutshall, M.S.,

Owen H.M. Gan, B.S., Everett D. Thompson, B.S., Hsien Chi Niu, Ph.D.

ANALYTICAL METHODS FOR THE DETERMINATION OF CANNABINOIDS IN BIOLOGICAL MATERIALS

M.E. Wall, Ph.D., T.M. Harvey, Ph.D., J.T. Bursey, Ph.D., D.R. Brine, B.S.,

and D. Rosenthal, Ph.D.

28

33

42

48

64

70

88

96

107

LIST OF CONTRIBUTORS 118

LIST OF MONOGRAPHS x i 120

QUANTITATION OF CANNABINOIDS

IN BIOLOGICAL FLUIDS

BY RADIOIMMUNOASSAY

Arleen R. Chase, Paul R. Kelley, Alison Taunton-Rigby

Collaborative Research, Inc.

Waltham, Mass.

Reese T. Jones

Langley Porter Neuropsychiatric Institute

University of California, San Francisco

Theresa Harwood

Drug Enforcement Administration

Washington, D. C.

INTRODUCTION

There is an expanding need to assay tetrahydrocannabinol (

9THC) and its metabolites in biological fluids, particularly

in the area of medical research. Methods

used for the quantitation of cannabinoids

include gas chromatography coupled with

mass spectroscopy (GC/MS) , liquid chromatography and radioimmunoassay. All

methods are of value, especially when used

to confirm the results of another procedure, so that the method of choice depends on the particular application.

Radioimnunoassay is a common clinical

method which is highly specific, sensitive

and particularly well suited to the

routine, simultaneous analysis of multiple

samples, often without prior purification.

The equipment required is routinely used

in university, research, industrial, and

clinical laboratories and hospitals.

Results can be obtained rapidly and are

easy to interpret.

THE PRINCIPLES OF RADIOIMMMUNOASSAY

Radioimmmunoassay depends on the affinity

of a biological molecule, the antibody, for

the antigen in question, in this case

9THC. Sensitivity is achieved through the

use of a radioactive tracer molecule of

high specific activity, called the labelled

antigen. The extent to which the unlabelled antigen (

9THC) competes with the

radioactive antigen for a limited number of

receptor sites on the antibody serves as

the basis for quantitation in the radioimmunoassay. The assays are simple to

perform. Mixtures containing the labelled

antigen, the antibody and the sample are

incubated, free labelled antigen is

separated from antibody-bound labelled

antigen and the exent of binding is determined by counting the disintegrations per

minute of the radiolabel. Unknown samples

are quantitated accurately by comparison

with the binding levels achieved with known

samples.

Antibodies generally show a remarkable

ability to bind selectively the antigen

that stimulated their production. This

specificity is comparable to that of an

enzyme for its substrate. The ability of

an antibody to discriminate between the

1
antigen and the myriad of other compounds

of widely diverse structure, which are

found in biological fluids, is of fundamental importance in its use as an analytical tool. Macromolecules, such as proteins

nucleic acids and polysaccharides, usually

elicit an immune response when injected

directly into an animal. However, low

molecular weight compounds, such as 9THC,

cannot elicit an immune response unless

they are bound covalently to an antigenic

macromolecule such as a protein or polypeptide. The development of a radioimmunoassay for a molecule, such as 9THC, involves (i) the synthesis of suitable covalent conjugates for immunization, (ii) the

production of antisera, (iii) the preparation of a radioactive antigen, and (iv)

the establishment of the assay based on the

antigen-antibody reaction.

SYNTHESIS OF COVALENT CONJUGATES

FOR IMMUNIZATION

Molecules containing an amino or carboxy

function can be coupled directly to the

amino or carboxy groups of amino acid residues in proteins or polypeptides by formation of amide bonds. Since the specificity

of an antibody is usually directed toward

those structures on the hapten that are

distal to the linkage group, the hapten

should be coupled to the carrier so that

characteristic functional groups are exposed to the antibody synthesizing cells.

In the case of 9THC several derivatives

containing carboxy groups have been synthesized. and coupled to macromolecules. The

use of a hemisuccinate ester of 9THC has

been reported (Teale et al, 1975) as well

as an azobenzoic acid-derivative (Grant et

al 1972 and Gross et al, 1974) . We have

used O-carboxymethyl-THC prepared by reaction of 9THC with iodoethylacetate

followed by basic hydrolysis as shown in

Figure 1.

Figure 1

Synthesis of O-Carboxymethyl-THC

2
PRODUCTION OF ANTISERUM

O-carboxymethyl-THC was coupled to bovine

serum albumin (BSA) using two different

dehydrating agents. In the first preparation the water soluble carbodiimide,

1-ethyl-3-(3-dimethyl aminopropyl) -

carbodiimide (EDC), was used and in the

second preparation, coupling was effected

by Woodward’s reagent, N-ethyl-5-phenyl

isooxazolium-3’-sulphonate. These reactions

are surmmarized in Figure 2. Uncoupled

CBM-THC and other small molecules were removed from the two preparations of

O-carboxymethyl-THC-BSA (CBM-THC-BSA) by

dialysis and ethanol precipitation. The

number of THC residues incorporated was

estimated by spiking the preparations with

1 4C- 9THC.

The two preparations of the conjugate,

CBM-THC-BSA, were both used to immunize

rabbits. While the response varied from

animal to animal? antisera were generated

to both preparations. The presence of

antibody was demonstrated by the fact that

the antisera would bind radiolabelled

9THC and, moreover, this binding could

be inhibited by unlabelled 9THC.

The response of two of the rabbits is

summarized in Figure 3. These plots show

the titer of antiserum required to give

approximately 50% binding of 3H- 9THC.

It can be seen that antisera were generated

to both preparations. Most of the data

described in the rest of this paper were

obtained using bleeding H of rabbit 56.

Figure 2

Synthesis of O-Carboxymethyl-THC-Bovine Serum Albumin

3
Figure 3

Titer of Antiserum Giving Approxomately 50% Binding of

3 H - 9THC Following Immunization

Note: Rabbit 56 was immunized with CBM-THC-BSA prepared using EDC.

Rabbit 67 was injected with CBM-THC-BSA coupled using

Woodward's Reagent.

RADIOLABELLED ANTIGEN, BINDING

AND INHIBITION STUDIES

High specific activity, radiolabelled compounds are used to develop sensitive radioimmunoassays. We have used both 3H and

14C- 9THC in our work.1

 The 14C- 9THC

9THC, is shown in Figure 4. The 50%

inhibition point occurred at 330 nanograms

had a specific activity of 0.07 μCi/μg. At o f 9THC. As expected, this system was

a final titer of 1:3 the antibody gave a too insensitive to be used as a working

maximum binding of this 14C- 9THC of radioimmunoassay.

4

19.3%. A standard curve, prepared by

inhibiting this binding with unlabelled
Figure 4

Inhibition of the Binding of 14C- 9 THC

and Antibody of 9 THC

The insolubility of 9THC in aqueous systems is well established. Correspondingly,

to provide an internal check on each standard solution, 14C- 9THC is used as a

standard. The exact number of nanograms of

IHC in each standard solution is then

verified by counting an aliquot in a liquid

scintillation in the 14C channel. The 14C

label does not interfere with the counting

of the 3H label in the 3H-channel.

The assay conditions described above were

used to obtain the standard curve shown in

Figure 5. The system is capable of assaying as little as 0.25 nanograms of

9THC reproducibly.

CROSS REACTIVITY STUDIES

The cross reactivity of various cannabinoids, drugs and other compounds was established for the assay system. The data is

summarized in Table 1, and shows that the

antiserum apparently reacts exclusively with

cannabinoids. None of the non-cannabinoid

drugs, hormones or other compounds cross

Figure 5

Inhibition of the Binding of 3H- 9 THC

and Antibody by 9 THC

Practical sensitivity could be achieved,

however, using 3H-labelled 9THC with a

specific activity of 41 μCi/μg. Binding

levels of 30-50% were routinely achieved

at a final titer of 1:750. The binding was

inhibited by nanogram levels of unlabelled

9THC as shown in Figure 5. The 50%

inhibition point occurred at 0.7 nanograms

9THC. This system was used as the basis

of a working radioimmunoassay.

THE TRITIUM BASED RADIOMMUNOASSAY SYSTEM

A working radioimnunoassay has been developed based on the use of 3H- 9THC and antiserum generated to CBM-THC-BSA. The assay

system which is described below is available

to interested investigators. The assay is

carried out in a buffer system of 0.1M

phosphate pH 7.0, 0.1% Triton X-405, 0.2%

sheep gamma globulin, fraction II. Tubes

containing antibody, 3H- 9THC and either

the standard or unknown THC sample are

incubated for four hours at 4°C. Antibody

bound and free labelled THC are separated

by dextran coated charcoal. The percent of

the radiolabel bound by the antibody is

determined by counting samples of the

supernatant in a liquid scintillation

counter.

5
Table 1

Gross Reactivity of Certain Cannabinoids and Other Compounds With

-

9THC Antiserum

Cannabinoids

50% Inhibition

In Nanograms

9

-THC

8

-THC

11-OH 9-THC

11-OH 8-THC

OH 9-THC

11-Nor 9-THC-9-COOH

11-Nor CBN-9-COOH

Cannabinol (CBN)

Cannabidiol (CBD)

Cannabidiolic acid (CBD acid)

Cannabicyclol

Cannabichromene

0.7

0.7

0.4

0.7

2.0

0.25

1.5

3.5

100

40

8

4 0

Other Level of Detection

compounds In Nanograms

Caffeine

“Aldactone" or Spironolactone

LSD tartrate

Secobarbitol (Technam spiked urine)

Gibberellic acid

Nicotine

Morphine Sulfate

Cocaine (Technam spiked urine) Drugs of Abuse

Methadone HCl

Phenobarbitol, sodium

Tetracycline

D,L-Ephedrine

Salicylic Acid

Carvone

(+) Limonene terpenes

 - Ihujone

Aldosterone-21-hemisuccinate

Progesterone

Estradiol steroids

-1 cortisone

Testosterone

Cholesterol hydrogen succinate

* Highest level available

>100,000

>5,000

>32,000

>100*

>100,000

>1,000,000

>100,000

>60*

>100,000

>10,000

>100,000

>10,000

>100,000

>95,000

>80,000

>90,000

>10,000

>10,000

>10,000

>10,000

>10,000

>20,000

reacted to any significant extent. The

results obtained with the various cannabinThe specificity of the antiserum for cannabinoids without absolute specificity for

oids show that the antiserum reacts as

well with 9THC as with some of its metaboTHC is advantageous since native THC is not

excreted in the urine and an assay capable

lites. The antibody recognizes 9THC,

8THC and several hydroxy metabolites. It

of detecting THC only would be of little

value. The assay system described here can

can differ ntiate to a limited extent

between 9THC and cannabinol, cannabidiol,

detect 9THC and its hydroxy metabolites,

and therefore can be used to detect cannacannabidiolic acid and cannabichromene. bis use.

6
APPLICATION TO BIOLOGICAL SAMPLES known amounts of 9THC and assayed. The

results are shown in Table 2, and it can be

concluded that (i) urine can be assayed

directly with no pre-treatment required,

(ii) urine does not interfere with the

quantitation of 9THC, and (iii) 20

microliter samples can be quantitated

accurately.

Before applying the assay to the detection

of cannabinoids in urine samples, normal

urines were assayed to determine any nonspecific interference. It was found that

normal urine was negative in the assay systern. A pool of normal urine was spiked with

Table 2

Assay of a Spiked Normal Urine Pool

Nanograms 9THC

Added to Sample

2.2 2.0

2.0

5 0.8 0.7

1 11.2 11.2

2 5.6 5.2

3

4 1.6

Sample No.

Nanograms 9THC

By Radioimmunoassay

ASSAY OF BIOLOGICAL SAMPLES

Clinical studies were undertaken to establish the validity of the assay under experimental conditions. All samples were

assayed blind, with no knowledge of the

key to the sample number code.

The first study involved analysis of 24

hour urine speciments taken from heavy pot

users. Prior to receiving oral doses of

THC, the subjects were maintained free of

any drugs for nine days. They were then

maintained for 12 days on a dose of 120 mg/

day of hashish oil in ethanol. Urine

specimens were assayed for cannabinoids on

day 9 and day 21. From an examination of

the data, summarized in Table 3, it can

be concluded that the assay did detect the

presence of large amounts of cannabinoids

in the urine samples obtained on day 21.

Table 3

Cannabinoid Levels Found in Urine Samples From

Heavy Pot Smokers, Before and After Oral Administration

of Hashish Oil

No drug for 9 days Level Found

Patient A 30 ng/ml

Patient B 40 ng/ml

Hashish Oil for 12 days

Patient A 7000 ng/ml

Patient B 4000 ng/ml

Note: At the time of these assays, the minimum detectable

level of cannabinoids was 12.5 ng/ml.

7
A second series of 24 hour urine samples

from a similar experiment were also

assayed. The patients were maintained

for seven days without any drugs and were

then treated with oral doses of THC.

Subject 2 was a fairly heavy cannabis user

before admission to the hospital, and the

initial urine cannabinoid level was consistent with this fact. At 8:00 a.m. on

day 8 treatment was initiated on a schedule

of 10 mg of THC in sesame oil every 4

hours, This schedule was maintained until

8:00 a.m. on day 12 when the dose was increased to 30 mg every 4 hours. The treatOn days 13 and 22 each subject was also

given two cigarettes each of which contained

20 mg of THC.

sults.

As can be seen from the results in Table 4,

inhaled doses of THC can be detected in the

urine for 5-7 days after the last exposure

to the drug. The administration of oral

doses of THC results in increased urine

levels of cannabinoids and the level falls

following removal of the drug. The urine

levels also reflect an increase in the dosage level. These studies indicate that the

radioimmunoassay will give meaningful rement was discontinued on day 24 at 4:00 p.m.

Day Dose Schedule Patient 1 Patient 2

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

No drug

10 mg/4 hr.

170 ng/ml

76

47

38

No sample

17

30 mg/4 hr.

240

1718

1850

1582

2768

3725

4175

7375

3338

6992

9413

5600

6563

4938

10254

10722

5088

5513

3550

1600

1662

808

1498 ng/ml

168

106

81

69

28

75

992

3721

2994

4255

5425

7819

3675

6088

9950

6313

15767

10813

13275

9556

11181

1325

8775

No sample

1075

442

223

Table 4

Cannabinoid Levels in 24 Hour Urine Samples

No drug

*Two cigarettes each containing 20 mg. of 9THC smoked in

addition to regular dose.

8
SUMMARY

A tritium based radioimmunoassay for 9THC

and its metabolites has been developed for

the use of investigators studying the epidemiological, medical, clinical, and research aspects of cannabis use. The assay

is sufficiently sensitive to detect cannabinoids in the urine of marijuana smokers

for several days after their last exposure

to the drug. The results obtained from a

28 day study indicate that the assay reflects the administration and removal of

oral doses of THC. The specificity of the

antisera, as determined in cross reactivity

studies, allows not only the assay of

metabolites in biological samples without

interference from other drugs, but also the

evaluation of extracts of other kinds of

samples which may contain unmetabolized

9THC.

The technique of radioimmunoassay has many

advantages over other methods of analysis.

It is simple to perform and can be readily

applied to the rapid analysis of large

numbers of samples, It can be used in the

direct analysis of physiological fluids and

other biological samples which ordinarily

have to be processed before other techniques

ACKNOWLEDGMENTS

can be applied. The method is non-destructive and obviates the need to use radiolabelled drugs in man during metabolic and

other studies.

This radioimnunoassay has been designed with

particular emphasis on ease of use by other

investigators. We anticipate that it will

prove useful to investigators and scientists

for determining the absence, or presence and

amount, of THC metabolite in a biological

specimen, for epidemiologists in determining the full extent of cannabis use and to

the medical/clinical community for establishing the minimum effective dose of 9THC for

each patient. The widespread application

of a single method of analysis should also

remove a great deal of the controversy surrounding marihuana studies performed to date.

1 14C- 9THC=(-) 1

-Tetrahydro (3',5'-14C) cannabinol, 31 mCi/mmole, was purchased from Amersham Searle. The radiolabel purity was found

to be 97-98% by radioscan in two different TLC

systems capable of separating 8 and 9 THC.

3H THC= 1

(G-3H) tetrahydrocannabinol. 13 Ci/

mmole was purchased from Amersham Searle. The

radiolabel purity was found to be 98% by radioscan in three different TLC systems capable of

separating 8 and 9 THC.

REFERENCES

This work was carried out under the sponsorship of the Department of Justice, Drug

Enforcement Administration, under Contract

No. DEA-75-4, the Department of Justice,

Bureau of Narcotics and Dangerous Drugs

under Contract No. J-70-24 and independently by Collaborative Research, Inc.

Grant, J. D., Gross, S. J., Lomax, P. and

Wong, R., 1972, Nature New Biol., 236,

216.

Gross, S.J., Soares, J. R., Wong, R. and

Schuster, R. E., 1974, Nature, 252, 581.

Teale, J. D., Forman, E.J., King, L.J.,

Piall, E. M., and Hanks, V., 1975,

J. Pharm. Pharmac., 27, 465

9
SEPARATE RADIOIMMUNE MEASUREMENTS

OF BODY FLUID THC AND

11-NOR-9-CARBOXY THC

Stanley J. Gross, M.D., James R. Soares, Ph.D.

Department of Anatomy

School of Medicine

University of California

Los Angeles, California

INTRODUCTION

Pharmacologic and metabolic studies with cannabinoids have remained largely unquantitative due to lack of simple assay techniques.

Initial gas chromatographic and mass spectroscopic methods were tedious (Fenimore 1973;

Garrett 1973; Agurell 1973). Our immune

approach led to early development of a simple

radioimmune assay for 9-THC in plasma and

urine (Gross 1974). This has now been extended to measure separately in body fluids

11-nor-9-carboxy- 9-THC (C-THC) (Soares) a

major cannabinoid metabolite (Bernstein

1972).

ASSAY METHODS

Institute of Drug Abuse. The generation of

antisera for 9-THC and C-THC has been described (Gross and Soares 1974; Soares and

Gross 1976). Plasma 9

-THC and C-THC were

extracted, the extracts reconstituted in 50%

ethanol or phosphate buffer and assayed.

Urine specimens were assayed directly.

Plasma and urine samples were obtained from

3H- 9

-THC (50 Ci/mM) and C-THC were obtained

from Research Triangle Institute, North Carolina. 9

-THC was obtained from the National

subjects at various intervals after smoking

one to three marihuana cigarettes (Containing

19.8 mg 9-THC) and subsequently assayed for

both 9-THC and C-THC. Pooled plasma from

non-users of marihuana spiked with various

known amounts of 9

-THC and C-THC was used to

establish the standard inhibition curves.

The urine standard curves were similarly determined.

Plasma 9

-THC and C-THC: Plasma samples1

were obtained from five subjects after smoking a single cigarette and from three subjects after smoking three consecutive cigarettes. None of the first group and only one

of the second group had measurable preintoxication levels of 9

-THC. However, four of the

eight subjects had measurable preintoxication

levels of C-THC.

In five single smoke subjects definitive increments (10-130 ng/ml) of 9

-THC occurred 15

minutes after a single cigarette becoming almost undetectable by 2 hours (Table 1). C-THC

peaked at 30-60 minutes; significant but low

levels remained at least three hours after exposure.

10
Table 1

Plasma 9-THC and C-THC levels in occasional THC

users 15-240 minutes after smoking a single 900 mg THC cigarette

(2.2% 9-THC/cigarette)

180 240

Post intoxication (minutes)

5 15

31

144

130

---

9 0

---

10

---

30

3 1

224

92 38 0

116 24 59

60 120

21 11

116 71

9

7

7

7 6

7

9 2 5

0 0

12 75

14 16

5 9

0 60

36 34

0

8 23

0 0

0 40

0 0

0 0

T. S. 9

-THC 0 73 32 1 4 0 0

C-THC 0 278 228 216 108 115

50

0

0

0

0

0

19

S. W. 9

-THC

C-THC

Z. W. 9

-THC

C-THC

B. W. 9-THC

C-THC

P. G. 9

-THC

C-THC

Pre

intoxication

0

Subject

7 -

5 5

The rapid shifts of plasma 9

-THC and C-THC

15 minutes to 48 hours following repetitive

tiple consecutive cigarettes than a single

one, permitting detection of 9-THC 2-3 hours

exposure are shown in Figure 1 and summarized

9

-THC peaks

after completion of the last cigarette. Sigin Table 2. Expectedly plasma nificant amounts of this metabolite were mea-

(100-260 ng/ml) were much higher after mul- sured in plasma 48 hours later.

Table 2

Plasma 9-THC and C-THC equivalent levels in occasional marihuana

users 15 min - 2 days after consecutively smoking three 900 mg

marihuana cigarettes (2.2% 9-THC/cigarette)

Pre

0 15' 30' 60' 120' 180'

8 185 78 123 39 72

7 285 112 292 211 251

0 260 - - 37 15 7 0

40 68 - - 158 43 111 140

100 63 87 37 5

375 460

B. W. 9-THC

C-THC

T. T. 9

-THC

C-THC

W. Z. 9

-THC

C-THC

Post intoxication ng/ml

240' 24hrs 48hrs

Subject cation

intoxi12 41 10

296 97 75

7 5 8

428 60 24

21

94

9

71

0

46 401 413 363

11
Figure 1

Figure 1: Post-intoxication plasma 9

-THC and C-THC (ng/ml) in a representative subject.

=

9

-THC; = C-THC.

Figure 2

Figure 2: Post-intoxication urine C-THC (ng/ml) in chronic subjects. Arrows indicate an

additional smoke.

12
Table 3

No.

1

2

3

4

5

6

7

8

9

10

11

Urine C-THC levels in chronic THC users 1-12 hrs after

consecutive smoking 900 mg THC cigarettes

(2.2% 9-THC/cigarette)

Subject

119

123

124

126

127

120

121

121

132

128

128

C-THC level (ng/ml)

Postintoxication (hrs)

2 4 6

Preintoxication

72

28

79

61

40

123

13

19

17

17

9

1

176

55

134

90

14

4

31

11

144*

106*

107

57

125

21

18

28

17

12

101*

148*

105

126

93

325*

97*

91*

*

166* 205

10 8

10* 23

23

UD UD

UD UD

8

129

196

48

196*

78

215

5

23

31

U D

U D

*Repeat cigarettes on subject demand

UD = undetectable

Table 4

Urine C-THC levels in occasional THC users 1-48 hours after smoking

900 mg THC cigarettes (2.2% 9-THC/cigarette)

Subject

B. W.

K. G.

R. B.

W. Z.

L. G.

T. T.

Preintoxication

5

UD

5

UD

2

4

Urine C-THC level (ng/ml)

Postintoxication (hrs)

1 4

33

3

9

7

6

20

2

18

17

10

18

7

26

3

17

2

12

21

4

44

33

16

21

9

7

59

6

39

2

7

54

15

--

8

23

3

7

31

3

29

12

15

--

4

--

--

14

24 4 8

3 2

5 3

4 3

5 U D

7 5

-- 9

12

164

290

UD = undetectable

13
Urine 9

-THC: None of four subjects had measurable urinary 9

-THC. This is consistent

with previous work (Hollister 1974) describing urine 9

-THC levels to be far below

present immune assay sensitivity,

Urine C-THC: Assays of urine from 9 chronic

users 1, 2, 4, 6 and 8 hours after completion

of the first standard cigarette are summarized in Table 3 and Figure 2. Preintoxication levels varied from 28-123 ng/ml. Large

C-THC increases occurred (Table 3) peaking at

2-4 hours after initial exposure. Unfortunately the rigid clinical protocol permitted

additional cigarettes during the 8 hour study

period, C-THC continuing to rise after each

additional cigarette. Urine C-THC levels in

occasional smokers were considerably lower

(Table 4) than in chronic users (Table 3).

Though relatively few subjects were studied a

pattern of relative 9-THC and C-THC levels

did emerge. In occasional smoker subjects

there was a 15-30 minute plasma 9-THC peak

and a 30-60 minute C-THC peak after use of a

single standardized marihuana cigarette. Importantly, 9

-THC became almost undetectable

in plasma 1-2 hours after exposure while significant amounts of C-THC persisted in circulation for several hours. This critical divergence of 9

-THC and C-THC was even more

obvious in subjects who had smoked 3 consecutive marihuana cigarettes. 9

-THC was almost

undetectable in all plasma samples four hours

after the last cigarette had been consumed

REFERENCES

Agurell, S., Gustafsson, B., Holmstedt, B.,

Leander, K., Lindgreen, J. E., Nilsson, I,,

Sandberg, F. and Asberg, M., J. Pharm. Pharmac. 25, 554 (1973).

Bernstein, S., Rosenfeld, J. and Wittstruck,

T., Science 176, 422 (1972).

Fenimore, D. C., Freeman, R. R. and Loy, P.

R., Anal. Chem. 45, 2331 (1975).

Garrett, E. R. and Hunt, C. A., J. Pharm.

Sci. 62, 1211 (1973).

despite the prolonged (48 hrs) persistence of

significant levels of C-THC.

Urine from occasional marihuana subjects was

negative or marginal for 9-THC while C-THC

(60 ng/ml or less) was detected 1-48 hours

after one standard cigarette. The pre-smoke

values were zero (

9

-THC and C-THC) for occasional smokers. However chronic smokers had

significant pre-smoke levels (17-23 ng/ml) in

addition to a vastly greater rise of urine

C-THC 2-4 hours after consumption of a single

cigarette.

Clearly, a single THC metabolite level or use

of a significantly crossreacting antiserum

(Marks, 1975; Teale, 1975) cannot be used to

ascertain “post-intoxication” intervals.

Such a result could reflect a single exposure

just prior to an examination or multiple exposures several days earlier. The failure to

detect plasma 9

-THC indicates unambiguously

that marihuana was not smoked within the preceeding hour, whereas detection of plasma

C-THC in the absence of 9-THC indicates distant exposure. A large controlled experimental population is now essential to correlate 9

-THC and C-THC (ratios) with individual metabolic variants for behavior differences.

1Samples were provided by the Department of

Psychiatry, UCLA School of Medicine.

Gross, S. J. and Soares, J. R., Nature 252,

581 (1974).

Hollister, L. E., Kanter, S. L., Board, R. D.

and Green, D. E., Res. Comm. Chem. Pathol.

Pharmacol. 8, 579 (1974).

Marks, V. , Teale, D. and Fry, D., British

Med. J. 3, 348 (1975).

Soares, J. R. and Gross, S. J. (Submitted for

pub1ication).

Teale, J. D., Clough, J. M., Pialli, E. M.,

King, L. R. and Marks, V., Res. Comm. Chem.

Pathol. Pharmacol. 11, 339 (1975).

14
RADIOIMMUNOASSAY OF

9

-TETRAHYDROCANNABINOL

Clarence E. Cook, Ph.D., Mary L. Hawes, B. A., Ellen W. Amerson, B. A.,

Colin G. Pitt, Ph.D., David Williams, B.A.

Research Triangle Institute, North Carolina

INTRODUCTION

Since its introduction by Yalow and Berson

(see Berson and Yalow, 1971), radioimmunoassay (RIA) has played an ever-increasing

role in the quantitative analysis of drugs

and hormones. A number of review articles

and books are available (inter alia, Skelley

et al., 1973; Spector et al., 1973; Abraham,

1974). In particular a dramatic improvement

in assay methodology for many steroid hormones has resulted from the application of

radioimmunoassay to these substances.

Due to its extreme sensitivity, RIA can permit the quantitation of substances present

in concentrations as low as a few pg/ml of

a biological fluid. The inherent selectivity of antibodies introduces a further advantage since it often makes sample preparation requirements minimal and assay methodology simple and thus permits the analysis

of large numbers of samples.

These obvious assets of RIA have naturally

led to its consideration as a means for measuring blood levels of cannabinoid compounds.

A valid RIA procedure for 9-THC would be

useful in studying the pharmacokinetics of

the drug. Furthermore it is conceivable that

RIA methods could be used for the identification of THC and/or its metabolites, thus

facilitating forensic investigations. Therefore a number of investigators have been

interested in the development of RIA procedures for the cannabinoids.

We believe it is fair to state that development of useful and simple RIA procedures for

9

-THC has proven to be a relatively difficult research problem. The difficulties involved in THC RIA development may be summarized as follows:

(1) The highly lipophilic nature of the

molecule causes problems in working with the

aqueous systems in which RIA is carried out.

The compound adheres well to glass and plastic--generally in preference to dissolving in

an aqueous medium.

(2) In plasma the compound is tightly

bound, principally to a lipoprotein fraction

(Klausner, et al., 1975).

(3) It has proven difficult to obtain

antibodies highly selective for 9

-THC vs its

metabolites and various analogs.

15
(4) The plasma levels of interest (ca.

1-100 ng/ml, although not low by the usual

standards of RIA, are sufficiently low that

dilution of plasma samples and direct analysis, a technique which has proven highly

successful with certain other drugs (Spector

et al., 1973; Cook et al., 1973, 1975b;

Christensen et al.,1974) is not readily

feasible for the lower levels.

(5) Highly concentrated (i.e., high

titer) antisera have been difficult to obtain.

(6) High specific activity radioligand

for competitive binding studies has been difficult to obtain and often unstable.

It is the purpose of this paper to illustrate

these problems and to discuss progress made

in overcoming them.

MATERIALS AND METHODS

Definitions

Titer - The dilution of original antiserum

which must be added to the assay to obtain

X% binding. If one adds 0.1 ml of antiserum

which has been diluted 1:500 and obtains 50%

binding of radioligand, the 50% titer (initial dilution) is 1:500 If in the above

example the total assay volume was 0.5 ml the

50% titer (final dilution) would be 1:2500.

In this paper we will quote initial dilution

titers unless otherwise noted.

T, B, and N-tubes - Designation for different assay tubes. At end of assay, volumes

in all tubes are equal. T tubes measure

total radioactivity; N tubes measure nonspecific radioactivity (activity not adsorbed by charcoal) and B tubes measure the

amount of labeled drug bound to antibody in

the absence (Bo) or presence (Bi) of added

unlabeled compound (see Table 1).

Table 1

DEFINITION OF ASSAY TUBES

Tube

Designation Tube Contents

Unlabeled

Radioligand Compound Buffer Antiserum Charcoal

T +

- + ± -

N +

- +

- +

Bo

+ - + + +

Bi

+ + + + +

Table 1. Definition of abbreviations for assay tubes

Materials

Chemicals used in the work were reagent grade

obtained from commercial sources. Bovine

serum albumin was obtained from Sigma Chemical Co., St. Louis, MO (crystallized and lyophilized, No. A4378; or Fraction V, fatty

acid free, No. A6003). Norit A (C-176) was

obtained from Fisher Scientific, Pittsburgh,

PA. Triton X-100 was obtained from Palmetto

Chemical Co., Monroe, NC. Antiserum was obtained from Dr. James Soares of the UCLA

School of Medicine (Lot No. G2532; Gross et

al., 1974) and from Dr. V. Marks thru theNational Institute for Drug Abuse (Teale et

al., 1974, 1975; S133Y/30/9). Additional

antiserum was prepared at Research Triangle

Institute (see below).

RIA buffer (0.1 M, pH 6.8) contained 16.35 g

Na2HPO4•7H2O, 5.38 g NaH2PO4•H2O, 9.0 g NaCl,

1.0 g NaN3, 1.0 g bovine serum albumin (BSA),

and 1000 ml double distilled water adjusted

to pH 6.8 and was stored in a refrigerator

for no more than one month.

Fine particles were removed from charcoal by

several decantations from a suspension in

distilled water. The charcoal was dried at

200°C and 25 g was suspended in 1000 ml of

buffer.

Scintillation fluid contained 2 liters toluene, 1 liter Triton X-100 [purified by stirring for 30 min with 606 Tell-Tale Silica

Gel (25 g/ ) and filtering] and 18 grams

OmnifluorR (New England Nuclear).

Undiluted serum was stored in a freezer (-20°C).

Serum diluted in RIA buffer could be stored

in a refrigerator for several months.

The radioligand (

9

-THC-3H; Pitt et al.,

1975) stock solution was kept in benzene/10%

ethanol and refrigerated. Solutions for

assays were made in 50% ethanol:50% water

(double distilled). The radioligand was diluted for assays to 10,000 cpm/10 μl which

gives ca. 100 pg 8-THC/10 μl. Dilutions in

EtOH/H2O may be kept refrigerated for no more

than 7-10 days. Radioligand decomposition

becomes significant after that time. Stock

solutions were tested frequently for decomposition by thin layer chromatography. Silica

gel plates were developed in 100% benzene and

the purity of the radioligand determined by

radioscan.

9

-THC for standard curves and metabolites

for cross reaction studies were prepared at

RTI. For radioimmunoassay, 1 mg/ml stock

solutions (prepared from solutions used as

glc standards and obtained from K. H. Davis

of this laboratory) were stored in 100% ethanol and refrigerated. Dilutions were pre16
pared in 50% ethanol/50% water. These dilutions may be kept refrigerated for approximately one month. Periodic analyses by thin

layer chromatography are required to test

the purity and stability of the compounds.

RESULTS AND DISCUSSION

Antigen synthesis

In the first part of this century, LandSteiner (see Landsteiner, 1962) showed that

covalent bonding of a small molecule (haptenic component) to a protein gave an antigenic substance and that antibodies formed to

this antigen were capable of selectively

binding the small molecule. He further established that the antibody selectivity is

influenced primarily by those portions of the

hapten which are removed from the linkage to

the protein carrier. Thus to achieve an

antibody which will be selective for the

parent drug in the presence of its metabolites, proper synthetic design of the antigen

is essential. Those portions of the molecule

which are subject to metabolic alterations

must be left free to influence antibody selectivity.

The very fact that the hapten must be

attached to the protein via a covalent bond

then means that it is impossible to devise a

conjugate in which all portions of the hapten

will equally influence antibody selectivity.

No linkage can be considered completely inert.

In addition, in designing the synthesis of

the antigen one must consider the chemistry

involved and the ease or difficulty in relation to the expected benefit. Depending upon

their needs, then, it can be expected that

different investigators will synthesize antigens in different ways.

Most of the metabolic alterations of 9-THC

occur in the cyclohexene moiety with hydroxylation of the 9

 compound occurring at the

11-and 8-positions along with conversion of

the 11-carbon to a carboxyl group. More recently it has been reported that hydroxylation can also occur in the amyl side chain of

the phenolic ring (Wall, 1975).

A number of previously reported antigens are

summarized in Figure 1. Teale et al. (1974,

1975), formed a hemi-ester linkage with the

phenolic hydroxyl group and then coupled this

compound to bovine serum albumin to form an

antigen. Tsui et al. (1974), report formation of a hemisuccinate from the phenolic hydroxyl, and also formed an ether linkage at

this position to give a carboxymethyl derivative. The products were coupled to a variety

of proteins as shown in Figure 1. The phenolic group apparently undergoes no metabolic

Figure 1

a ) CO-NH-BSA (Teale, et al., 1974)

b) CO-CH2CH2CO-NH-BSA (Teale, et al., 1975)

C ) CO-CH2CH2CO-NH-PGG (HSA, SGG, or PLL) (Tsui, et al., 1974)

d) CH2CO-NH-HSA (or SGG) (Tsui, et al., 1974)

e ) N=N CO- NH-PGG (HSA or SGG) (Tsui, et al., 1974)

f ) N=N CO-NH-KLH (Gross, et al., 1974)

(+4–isomer)

g ) 10-I-9-NH-CO-NH-HSA (or PGG) (Tsui, et al., 1974)

Figure 1. Some positions through which 9-THC

has been linked to protein

alterations, but spatially the attachment to

protein is relatively close to the metabolically important cyclohexene ring. To avoid

this problem Tsui et al. also prepared a 2-

azophenylcarboxy derivative, a substitution

empioyeh by Gross et al. (1974) as well.

This substitution frees the hydroxyl group

for binding, although at the possible expense of introducing a relatively immunodominant azobenzoyl moiety. Finally, Tsui et

al. (1974) also prepared a 10-iodo-9-ureido

linked THC.

Our attention was drawn to the amyl side

chain of the aromatic ring as a potential

position for attachment to the protein.

Such a linkage would certainly fulfill the

requirement of distance from the metabolically reactive cyclohexene ring (although

not from sites of hydroxylation on the amyl

side chain). Also it was of interest to examine the effectiveness of the flexible hydrocarbon chain in exposing the tricyclic moiety

to influence antibody selectivity. Use of

such a long flexible chain had proved quite

successful for us in synthesis of antigens

for caffeine (Cook et al., 1974) and phenylbutazone (Cook et al., 1975a). For synthetic

reasons our initial attempts dealt with the 8

-THC analog as a substrate.

Synthesis of the 8-THC antigen is shown in

Figure 2. 5'-Carboxy- 8

-THC labeled with

17
Figure 2

SYNTHESIS OF – THC ANTIGEN

serum prepared from the amyl-linked antigen

is shown in Figure 3. The affinity constants

booster administrations. A third rabbit eventually achieved a titer (final dilution) of

1:2250 at the fourth bleeding. Titers generally declined at the fifth and sixth bleedings.

These titers compare quite favorably with

those reported by others (Teale et al., 1975;

Gross et al., 1974) and demonstrate that with

this antigen the rabbit is a reasonable animal to use for antibody production. A further comparison of the azobenzoyl-THC-derived

antiserum obtained from UCLA and the antiFigure 3

Figure 2. Synthetic route to antigen based on

5'-carboxy- 8-THC

ANTISERUM CHARACTERISTICS

UCLA RTl-RS-81

tracer amounts of carbon-14 in the carboxyl Affinity Constant 1.6 X 109

2.5 X 109

group (Pitt et al., 1975) was converted to its

N-oxysuccinimidoyl ester by reaction with Nhydrbxysuccinimihe and diclohexylcarbodiimide. This active ester readily coupled

with bovine serum albumin in a mixture of dioxane and water to yield the desired antigen.

Radioactivity measurements indicated the incorporation of ca. 33 residues of 8

-THC/

molecule of bovine serum albumin. We have

(for 8-THC)

Std. Curve

Characteristics

(Logit - log Plot)

found this mode of coupling to be a very

useful one in certain circumstances. Where 50% Intercept 0.5 ng 1.4ng

it is applicable, the number of molecules of

drug moiety incorporated into the bovine

serum albumin is relatively easy to control.

Formation of antisera

Rabbits were immunized with the conjugate

dissolved in sterile 0.9% NaCl and homogenized with an equal volume of Freund's complete adjuvant. Immunization with 200 μg of

antigen was carried out by intradermal technique of Vaitukaitus et al. (1971). This was

followed after two weeks by another intraderma1 immunization (100 μg) and then at four

week intervals subcutaneous booster injections (100 μg of antigen) were given. Rabbits were bled on day 52 after the initial

dose and every 28 days thereafter. After the

third bleeding the immunization program was

discontinued for a three-month period. Antigen booster injections were then resumed and

the fourth bleeding was taken 10 days after

immunization. Reasonable titers (50% binding

of ca. 125 pg of labeled 8-THC at an initial

dilution of 240-540 or a final dilution of

1200-2700) were achieved in two out of four

rabbits at the first bleeding, but no significant increases were observed on subsequent

Linear Range 0.3 - 4.1 ng 0.3-6 ng

Figure 3. Characteristics of two antisera

as determined in this laboratory: UCLA

(Gross, et al.,

paper).

1974); RTI-RS-81-2 (this

are quite close. The slightly higher affinity of the 8

-antiserum for 8-THC is understandable. Due to the heterologous nature of

the systems employed ( 8-radioligand, unlabeled 9

 and antiserum generated to either

8

- or

9

-), the standard curve characteristics

are not similar for the two antisera. When

standard curves are converted to the logitlog basis (Rodbard et al., 1969), the UCLA

antiserum exhibits a steeper slope with a

lower 50% intercept and a somewhat narrower

linear range than does the RTI antiserum.

The lower limit in Figure 3 is a conservative number. If one defines sensitivity as

the amount of 9-THC which will reduce

binding to a level two standard deviations

below the initial value, the limit for the

RTI antiserum is ca. 100 pg.

18
Antibody selectivity

The avidity of the antisera for various metabolites and analogs of 9

-THC was measured

by determining the relative amount of compound required for 50% displacement of the

initially bound radioligand. This is a relatively crude measure of cross-reactivity.

It is strictly valid only if the displacement curves for the two compounds in question are completely parallel and it does not

take into account the possibility of small

subpopulations of antibodies with high avidity for one substance in preference to the

other. Nevertheless, it offers a useful if

rough guide to antibody selectivity if its

limitations are realized. Figure 4 shows

Figure 4

Figure 4.

Structure-binding relationships of various

antisera for cannabinoids. Numbers in

parentheses are from the references cited:

A (Teale, et al., 1975); B (Gross, et al.,

1974)

the cross-reactivity of three types of antisera, all measured in our own laboratory.

Selectivity can be significantly influenced

by the assay conditions. Therefore the

values reported by the producer of the antiserum are also given in the figure in parentheses. The displacement ability of 9

-THC

is taken as a standard at 100%. For reasons

discussed later, the radioligand used was

8

-THC-3H.

A change which alters the tricyclic character

of the molecule (see cannabidiol, CBD) essentially destroys binding to all of the antibodies. However, much more subtle changes

can also have significant effects. In the

case of the antibody to 8-THC, shift of the

double bond from the 8- to the 9-position

results in a two and one-half fold decrease

in cross-reaction. A similar (two-fold) decrease is seen on comparison of the binding

of 9- and 8-THC to antibody obtained from

the other two antigens.

Reduction of the cyclohexene double bond has

no greater effect with the 8-antibody than

does the shift to the 9-position and actually somewhat increases the cross-reaction

with the 9

-antiserum prepared from the azobenzoyl antigen. Aromatization of the cyclohexene ring (to CBN) results in a four-fold

decrease in cross-reaction as compared with

9

-THC in the case of the azobenzoyl- and

amyl-linked antigens. It has little effect

on antibody to the O-succinoyl antigen.

Oxygenated C-11 metabolites cross-react relatively little with the antibody from the amyl

antigen. Cross-reaction of the 11-hydroxy

metabolite with the azobenzoyl antiserum is

rather significant (49%) but the cross-reaction drops markedly when the 11-nor-9-carboxy metabolite is considered (2%). Both of

these metabolites cross-react strongly with

antibody from the O-succinoyl antigen.

Thus it appears that good, but not outstanding, selectivity for 9

-THC vs a number of

metabolites and analogs can be achieved and

that modest but usable antibody titers can be

obtained in either the goat, sheep, or rabbit.

Let us now consider some of the other problems in the development of an assay.

Assay parameters

General procedure.--The general assay procedure finally adopted is shown in Figure 5.

Data leading to definition of the various

conditions are presented in suceeding paragraphs.

Radioligand.--The sensitivity of a radioimmunoassay is very much a function of the

specific activity of the bound radioligand.

Thus, in general, one would prefer the highest possible specific activity. Storage of

such high specific activity compounds can

often lead to radiation-induced decomposition. The general experience of our laboratory in the preparation and storage of such

materials is that unlabeled substances which

readily decompose through autoxidation, etc.

are often particularly unstable when prepared radiolabeled and with high specific

activity. This was found to be the case for

19
Figure 5 Figure 6

Figure 5. General procedure for RIA of 9-THC

9

-THC which was prepared at a specific activity of ca. 50 Ci/mmole (Pitt et al.,

1975) but had a very short shelfilife. On

the other hand, 8-THC-3H, prepared in similar specific activity, has proven to be a

quite stable entity. Its binding characteristics with the antisera raised to either

8

- or

9

-THC make it a useful radioligand.

Hexahydrocannabinol also exhibits good

cross-reactivity with the antisera but synthetic problems have discouraged our use of

tritiated HHC as a radioligand.

Buffer Protein.--The relatively intractable

nature of THC in aqueous systems is by now

presumably well-known to all interested in

this field of research. Because of the small

amounts of material involved, RIA procedures

for most compounds call for addition of some

type of protein to the buffer in order to

enhance solubility of the various components

and prevent binding to the incubation tubes.

Our experience has been that the nature of

this protein is of crucial import in work

with 9-THC. This is illustrated in Figure

6. When ca. 5 ng of tritiated 8

-THC in a

small amount of ethanol was added to a normal

RIA buffer (phosphate buffered saline) containing 0.1% bovine -globulin as the protein, very significant amounts of the THC

were bound to the glass. This was illusFigure 6. Illustration of the binding to glass

observed with THC

trated by decanting the buffer into scintillation liquid. The glass tube was then broken up, and the pieces were placed in a

scintillation vial and treated with a little

methanol. Scintillation fluid was then

added. This experiment indicated that only

57% of the THC remained in the buffer and 41%

adhered to the glass tube (percentages are

corrected for quenching). Even allowing for

incomplete removal of buffer from the glass

prior to crushing it, this is still a very

significant loss of the THC.

Experiments on the effect of silanizing glass

tubes or washing them with nitric acid gave

no improvement and in most cases the results

were worse. However we found that in our

laboratory, the use of bovine serum albumin

as the protein component of the buffer resulted in much diminished adherence to glass

and a better assay system. This is illustrated in Figure 7. Labeled THC was incubated

with buffer containing either bovine serum

albumin (BSA) or bovine -globulin (BGG) plus

varying dilutions of antiserum. The solutions were then decanted into scintillation

liquid and radioactivity determined. Results

were not corrected for quenching, which had

an approximately equal effect on all values.

At high concentrations of antiserum, there

is no difference between the two buffer proteins. In effect the antiserum itself is

keeping the THC in solution. However, as

the antiserum concentration is decreased

(dilution increased) less and less of the

label can be recovered from the tubes containing the BGG and eventually the recovery

20
Figure 7 Figure 8

Figure 7.

Variation of THC solubility as a function of

antiserum dilution and buffer protein. Antiserum of Gross, et al. (1974); BSA = bovine

serum albumin; BGG = bovine gamma globulin

falls to essentially the level found in the

previous experiment. The adsorption to

glass may be looked upon as competing with

the binding to antiserum. Although the problem may be overcome by higher concentrations of antiserum, it was to us preferable

to avoid this effect if possible, remove

another source of variability from the assay,

and increase the practical titer of the antiserum by using bovine serum albumin in the

buffer system. At the concentrations employed, this protein did not decrease the

amount of radiolabel which was adsorbed to

charcoal.

Not only was the type of protein important,

but the assay characteristics were also

greatly influenced by the type of bovine

serum albumin used and even by different lots

of the same type of BSA. This is illustrated

in Figure 8, where the 50% titers for three

different antisera (one obtained from UCLA

and two prepared at RTI) are compared in the

presence of the same concentration of protein

(0.1%) but varying the type or lot of bovine

serum albumin used. The first lot of crystallized and lyophilized BSA used gave the best

overall binding characteristics. Subsequent

lots gave significantly lower titers with one

in particular giving extremely poor results.

Somewhat more consistent results were obtained

with Sigma’s fraction V fatty acid-free BSA.

Because this material is less expensive than

the crystallized type, we have settled on it

in practice. A third type (fraction V powder)

Figure 8.

Influence of different types and lots of

bovine serum albumin on apparent antiserum

titer (initial dilution). Each vertical

line represents a separate lot of BSA:

A4378 (crystallized once and lyophilized);

A4503 (Fraction V powder); A7511 (crystallized and lyophilized; less than 0.005%

fatty acids); A6003 (Fraction V; less than

0.005% fatty acids)

gave very poor results and a BSA type prepared

by Sigma specifically to aid in the RIA of insulin proved worst of all.

We were unable to pinpoint the difference in

the various lots which might have influenced

the results. The presence of fatty acids as

a contaminant was suggested since batches

characterized as fatty acid-free gave generally better results. Treatment of one of the

less useful batches with charcoal to further

remove fatty acids (Chen, 1967) gave some improvement, but did not result in raising the

quality to that of some of the better batches.

We have, therefore, concluded that (a) each

batch of BSA used should be checked out before large amounts of antiserum are diluted

in the buffer, (b) large batches should preferably be checked and then used in the assay

to attain consistency, (c) the quality of an

antiserum in terms of titer may be strongly

influenced by the composition of the protein

in the buffer (which may account for inter21
Figure 9 Figure 10

Figure 9.

Net binding as a function of BSA concentration

and type: Sigma fraction V (<0.005%

fatty acids); different lot of the above

after second removal of fatty acids;

crystallized and lyophilized

Figure 10.

Effect of pH on binding and non-adsorbed

radioactivity. Antisera were obtained from

UCLA (Gross, et al., 1974) and as described

in this paper (RTI-RS-81-4)

laboratory differences in antiserum titers)

and (d) some antisera are more sensitive to

changes in the protein than are others. This

is particularly true of RTI RS-81-3.

As would be expected, the concentration of

protein had an effect on the net percent

binding and this concentration dependence was

also somewhat a function of the antiserum and

the type of bovine serum albumin used. This

is illustrated in Figure 9. At low concentrations (<0.05% BSA) the net percent binding

[100 (B-N)/T] was low, principally due to the

fact that the total binding was very low.

Non-specific radioactivity (N tubes, radioactivity not adsorbed by charcoal) was also

relatively low up to 0.2% concentration of

BSA. At concentrations of 0.5-1% it became

a significant factor and resulted in a considerable lowering of the net percent binding.

Best results were generally obtained at 0.1-

0.2% protein for the antisera and protein

samples examined. Therefore for reasons of

both economy and best binding, 0.1% was chosen

as the best concentration.

pH Effects.--As shown in Figure 10, pH had a

definite effect on both total binding and

radioactivity not adsorbed by charcoal. This

effect was again somewhat sensitive to the

type of antibody used. The UCLA antiserum

exhibited considerably better binding at pH

values of 8 or above, but the non-specific

radioactivity also increased. Although an increase in apparent binding also appeared to

occur at higher pH with the RTI antiserum, the

net binding showed no change. To minimize

nonspecific radioactivity pH 6.8 was accepted

as the best compromise. Since relatively

little THC would be ionized under the conditions used, it seems likely that the change

in pH acts principally by its effect upon the

conformation of the binding globulin and/or

by protonation effects in the binding region.

Incubation Time.--The effect of the time of

incubation at 4°C on the percent binding

achieved was also studied (Figure 11). In

the case of the RTI antiserum a perceptible

rise in percent binding occurred up to about

6 hr, although binding appeared to begin

leveling off after about 4 hr. Binding equi37
Figure 11 Figure 12

Figure 11.

Percent of radioligand bound as a function

of time of incubation at 4°C

librium was established more rapidly in the

case of the UCLA antiserum and was complete

in approximately 2 hr. Incubation times of

4 hr or more would be suitable for either

antiserum and there was no deleterious

effect if incubation continued for an overnight period.

Charcoal Treatment.--Since the charcoal adsorbant removes free radioligand, one might

expect that exposure to charcoal would eventually shift the binding equilibrium so as to

remove essentially all of the label from the

antibody. The rate at which this would happen would be dependent upon the rate of binding reversal, which in turn should be a function of temperature. A finite time is also

required to adsorb the free radiolabel and to

effect its removal from weakly binding substances such as protein. The best equilibration time then is often a compromise and must

be studied for each antiserum.

Figure 12 shows the effect of letting the

charcoal suspension stand with the incubation

mixture. Non-adsorbed radioactivity in the

absence of antiserum reached the minimum

value in approximately 15 min and did not

fluctuate for up to 60 min thereafter. In

the presence of antiserum, a somewhat similar

situation obtained. By 15 min the minimum

percent bound was essentially reached. Therefore, in order to reduce the non-specific

binding to its minimum, a 15 min time for

standing with the charcoal is necessary.

Figure 12.

Effect of time on adsorption of radioligand

by charcoal at 4°C. One ml of 2.5% charcoal

suspension was added to incubation tubes containing 0.52 ml. Contents were mixed for

10 sec on a vortex mixer and allowed to stand

Other experiments on the time of mixing of the

charcoal suspension with the incubation mixture showed that this also affected the amount

of radioactivity left in solution. It was

necessary to compromise in this regard and it

was found that a 10 sec mixing on a Vortex

mixer gave the best results within a reasonable period of time.

Variation of charcoal concentrations (1 ml of

suspension was added in all cases) from 1-10%

showed that a 2.5% suspension gave best results. Addition of dextran to the charcoal

did not prove useful.

Plasma analysis

Having established assay conditions, one

would now like to transfer these to the analysis of samples of biological importance.

Ideally, one would like to be able to analyze

a diluted sample of plasma directly without

carrying out any extraction procedures. Whether this is successful depends upon the

amount of plasma which can be added to each

assay tube and therefore upon the concentration of the drug in plasma and the sensitivity of the assay itself.

Plasma was added directly to T, B, and N

tubes containing both the RTI and UCLA antisera and its effect on each was studied. As

expected, T tubes showed no change in going

from 0 to 60 μl of plasma in a total volume

of 0.52 ml. Addition of 15 or more μl of

23
Figure 13

Figure 13.

Procedure used for extraction of 9-THC

from plasma

plasma resulted in an increase in the N-tubes

and a concomitant decrease in the percent

binding so that the net percent bound values

at 15 μl of plasma were only 60-70% that of

the tubes containing no plasma. Further increases in the amount of plasma resulted in

lower levels of net binding. However, if one

assumes an assay sensitivity of 0.3 ng and

ignores any problems caused by the presence

of metabolites, direct assay of plasma down

to a level of 20 ng/ml should be feasible.

This technique assumes that one has a sample

of blank plasma from the subject or is willing to ignore the possibility of intersubject

variation in effects on the assay. This latter point will be considered later.

For pharmacokinetic studies, assays at levels

below 20 ng/ml are required. Solvent extraction to remove protein and other interferences must apparently be used to analyze low

levels of 8-THC. Some care must be used in

the choice of solvents since small amounts of

certain solvents can have a significant influence on the assay. For example, the addition of only 1 μl of isoamyl alcohol (the

solvent commonly used in conjunction with

hexane to increase the extractability of THC

from plasma) reduces the binding in an assay

24

tube to ca. 60% of that obtained in the absence of the isoamyl alcohol. Therefore

traces of this relatively non-volatile substance remaining from an extraction process

may have a very significant effect upon assay

results.

Ethanol has somewhat less of an effect. Indeed we routinely add up to 10 μl of ethanol

to each assay tube in preparing the standard

curve. Addition of 20 μl more ethanol reduces binding to about 75% of the initial value.

Nevertheless, extraction of 9-THC from plasma

by precipitation of proteins with ethanol has

been reported to be a useful procedure in RIA

of THC (Teale, et al., 1975) and we have

therefore studied this particular extraction

system with plasma samples. The overall extraction process used is shown in Figure 13.

Plasma is treated with twice its volume of

ethanol and precipitated material is centrifuged out. Aliquots of the supernatant may

be taken for analysis at this point. We

tested the possibility of utilizing this supernatant as has been reported by Teale et

al. (1975).

Since the plasma/ethanol extract has a definite influence on the standard curve, it is

necessary to compensate for this effect by

running the standard curve in the presence

of the same volume of plasma extract as that

to be analyzed. This works reasonably well

when it is possible to obtain a blank plasma

from the same individual whose plasma THC

concentration is being studied. However

this would not be easy to do in the case of

chronic users of THC and would represent a

distinct limitation on the applicability of

the method. It was therefore of interest to

compare standard curves obtained from different plasmas.

Four human male plasma samples were spiked

with five concentrations of THC from 5-100

ng/ml. Each plasma series was then used to

generate a standard curve. Each standard

curve, in turn, was used as the basis for the

“analysis” of the spiked plasma samples. We

then looked at the percent of the samples

which were analvzed with less than 20% error.

Results are shown in Figure 14. As expected,

when plasma series A was analyzed by standard

curve A (generated from plasma A), all of the

samples fell within ±20% of the actual spiked

values. Under these-conditions it was possible to add sufficient of the ethanol extract

to get down to levels of 5 ng/ml with reasonable accuracy.

With cross plasma comparisons the results are

not so good. Plasma samples B and D gave

reasonable accuracy when measured against

standard curves A and B. However, the accuracy of analysis of samples from plasma C was
Figure 14 Figure 15

COMPARISON OF ASSAY RESULTS FROM

SPIKED PLASMA SAMPLES AFTER

EtOH PRECIPITATION OF PROTEIN

Percent of Analyses with

< 20% Error

Plasma

Series

Analyzed

Standard Curve Employed

A B C D

A

B

C

D

Figure 14.

Accuracy of analysis when extracts from one

plasma spiked with 9-THC are analyzed by

means of standard curves generated from

extracts of other plasma samples. Antiserum

from UCLA (Gross, et al., 1974)

STD. CURVES FROM SPIKED PLASMA SAMPLES

(EtOH PRECIPITATION: Et2O EXTRACTION;

RECONSTITUTED IN BUFFER)

INITIAL SLOPE 50%

BlNDlNG (LOGlT - LOG) INTERCEPT

Std Curve 35% -3.54 0.59 ng

(Buffer)

+ 50μl extract A 32% -2.88 0.81 ng

+ 50μl extract A 32% -2.84 0.77 ng

Std. Curve 45% 4.06 0.59 ng

+ 50μl extract B 35% -3.50 0.71 ng

+ 50μl extract C 38% -2.36 0.71 ng

+ 50μl extract D 39% -2.49 0.68 ng

Figure 15.

CORRELATION

COEFFICIENT

0.99

-0.98

-0.98

-0.99

-0.99

-0.96

-0.99

Comparison of standard curves generated in

buffer alone with those containing plasma

extracts. Results are from two separate

experiments. Duplicate extracts A should

be compared with the first standard curve;

B, C, and D with the second. Antiserum

from UCLA (Gross, et al., 1974)

quite poor when the other plasmas were used

as the basis of the standard curve. This is

particularly true in the case of plasma C

analyzed vs standard curve A and plasma A

analyzed vs standard curve C, where only one

out of five samples fell within the 20%

limits. If one requires accuracy within

+10%, results are much worse.

We believe this study leads to two conclusions: (1) if one sets modest goals for

accuracy, a fair number of plasma samples can

be analyzed down to levels of 5 ng/ml of pure

9

-THC. However, (2) the influence of plasma

extracts on the standard curve is not highly

reproducible from one plasma to another and

could, on occasion, lead to severe errors in

the analysis. This study also does not take

into account the potential problems caused by

cross-reaction of other cannabinoid materials

which may be present in plasma at higher concentrations than the 9

-THC itself.

To further purify the plasma material before

analysis, we then followed a procedure which

was described to us by Soares (private communication, 1975) and which consists of the

last part of Figure 13. The ethanol extract

is treated with water and ether. After

equilibration, ether is removed and evaporated. The residue may be taken up either in

50% aqueous ethanol or in RIA buffer. Since

use of the latter eliminates the effects of

ethanol on the assay and does not result in

lower recoveries, it is the procedure of

choice.

Figure 15 shows the results of generating

standard curves from plasma samples spiked

with 5-100 ng/ml of 9-THC and carried

through this procedure. Adding 50 μl (5% of

the total extract from one ml) of extract

from plasma A did result in a change in the

standard curve from one run in buffer alone.

The initial binding was lowered, the slope of

the logit-log plot was reduced, and the 50%

binding intercept was increased. However,

duplicate samples of the same plasma gave

rather reproducible standard curves. When

three different plasma extracts were compared

there were found to be some differences. This

implies that caution again should be used in

analyzing plasma samples from one subject

based upon a standard curve generated from

the plasma of a second subject. The use of a

pooled plasma for generation of standard

curves is probably the best compromise at

present.

25
CONCLUSIONS (3)

The work we have done leads us to believe that there are factors which can

be extracted from normal plasma under

the conditions described which will interfere with the assay. Since these

factors have not yet been identified and

may vary from subject to subject and

perhaps from extraction to extraction,

extreme caution should be used in evaluating the data obtained in such a manner. Care must also be taken in obtaining plasma samples and storing them

to avoid inadvertent addition of such

potentially interfering materials as

plasticizers, etc. The effect of freezing and storage on the stability of the

THC and on the formation of interfering

substances needs to be further evaluated. (4)

(1)

(2) The sensitivity of the assay to conditions such as buffer composition and

plasma materials makes interlaboratory

comparisons difficult. Standardized

techniques and materials are therefore

a pressing need.

Radioimmunoassay has not yet lived up

to its potential in the analysis of

cannabinoid substances. The sensitivity of the assay is not as great as

one would expect based on other compounds

such as the steroid hormones. Nor have

the high titer antisera of the type available for the steroid hormones yet been reported for 9

-THC. Since factors such

as titer and sensitivity can apparently be strongly influenced by the

actual assay conditions, this suggests

that further manipulation of conditions,

the synthesis of even higher specific

activity radioligand, and the development of antisera to a variety of antigens

and in a variety of animals are all valid

approaches to further work.

The reasonable results obtained by immunization of animals with a conjugate

prepared from 5'-carboxy- 8-THC have

encouraged us to undertake the synthesis of similar antigens based on 5'-

carboxy- 9-THC. Work along these lines

is in progress and can be reported at

future meetings.

REFERENCES

Abraham, G. E. Radioimmunoassay of steroids

in biological materials. Acta Endocr.,

1974, Suppl. 183, 1-42.

Berson, A. S. and Yalow, R. S. Current

knowledge of basic concepts in immunology and their clinical application. In

R. A. Good and D. W. Fisher (Eds.),

Immunobiology. Sinauer Associates, Stanford, CN, 1971: 287-293.

Chen, R. F. Removal of fatty acids from

serum albumin by charcoal treatment.

J. Biol. Chem., 1967, 242: 173-181.

Christensen, H. D., Amerson, E., Myers,

M. W., and Cook, C. E. Comparison of diphenylhydantoin and phenobarbital serum

and blood levels. Pharmacologist, 1974,

16 (2): Abstract 215.

Cook, C. E., Kepler, J. A. and Christensen,

H. D. Antiserum to diphenylhydantoin:

Preparation and characterization. Chem.

Path. Pharmacol., 1973, 5: 767-774.

Cook, C. E., Tallent, C. R., Kepler, J. A.,

Amerson, E. W., and Christensen, H. D.

Caffeine radioimmunoassay: Synthesis of

antigen and characterization of antibody.

26th Southeastern Regional Meeting of the

ACS, 1974, Abstract No. 84.

Cook, C. E., Tallent, C. R., Amerson, E.,

Christensen, H. D., Taylor, G. and Kepler,

J. A. Phenylbutazone antiserum. Fed.

Proc., 1975a, 34: Abstract 3059.

Cook, C. E., Amerson, E., Poole, W. K.,

Lesser, P., and O'Tuama, L. Phenytoin

and phenobarbital concentrations in saliva

and plasma of man measured by radioimmunoassay. Clin. Pharmacol. Therap., 1975b,

18: 742-747.

Gross, S. J., Soares, J. R., Wong, S. L., and

Schuster, R. E. Marihuana metabolites

measured by a radioimmnune technique.

Nature, 1974, 252: 581-582.

26
Klausner, H. A., Wilcox, H. G., and Dingell,

J. V. The use of zonal ultracentrifugation in the investigation of the binding

o f 9-tetrahydrocannabinol by plasma lypoproteins. Drug Metab. Disp., 1975, 3:

314-319.

Landsteiner, K. The specificity of serological reactions, Revised Edition. Dover

Press, Inc., New York, 1962.

Pitt, C. G., Hobbs, D. T., Schran, H., Twine,

C. E., Jr., and Williams, D. L. The synthesis of deuterium, carbon-14 and carrier-free tritium-labeled compounds. J.

Label. Comp., 1975, : - .

Rodbard, D., Bridson, W. and Rayford, P. L.

Rapid calculation of radioimmunoassay

results. J. Lab. Clin. Med., 1969, 74:

770-781.

Skelly, D. S., Brown, L. P. and Besch, P. K.

Radioimmunoassay. Clin. Chem., 1973, 19:

146-186.

Spector, S., Berkowitz, B. Glynn, E. J. and

Peskar, B. Antibodies to morphine, barbiturates, and serotonin. Pharmacol.

Revs., 1973, 25: 281-291.

Teale, J. D., Forman, E. J., King, L. J. and

Marks, V. Production of antibodies to

tetrahydrocannabinol as the basis for its

radioimmunoassay. Nature, 1974, 249:

154-155.

Teale, J. D., For-man, E. J., King, L. J.,

Piall, E. M. and Marks, V. The development of a radioimmunoassay for cannabinoids in blood and urine. J. Pharm.

Pharmacol., 1975, 27: 465-472.

Tsui, P. T., Kelly, K. A., Ponpipom, M. M.,

Strahilevitz, M. and Sehon, A. H. 9-

Tetrahydrocannabinol-protein conjugates.

Can. J. Biochem., 1974, 52: 252-253.

Vaitukaitus, J., Robbins, J. B., Meschlay, E.

and Ross, G. E. A method for producing

specific antisera with small doses of

imnunogen. J. Clin. Endocr., 1971, 33:

988-991.

Wall, M. E. Recent advances in the chemistry

and metabolism of the cannabinoids. In

V. C. Runeckles (Ed.), Recent Advances in

Phytochemistry, Vol. 9, Plenum, New York,

1975: pp. 29-59.

2 7
DETERMINATION OF THC AND ITS METABOLITES

BY EMIT® HOMOGENEOUS ENZYME IMMUNOASSAY:

A SUMMARY REPORT

G.L. Rowley, Ph.D., T. A. Armstrong , C.P. Crowl W. M. Eimstad, W.M. Hu, Ph.D.,

J.K. Kam, R Rogers, Ph.D., R. C. Ronald, Ph.D., K. E. Rubenstein, Ph. D.,

B.G. Sheldon, E. F. Ullman, Ph. O.

Syva Research Institute

Palo Alto, California

INTRODUCTION

We found that certain enzymes could

be inhibited by antihapten antibodies

when these enzymes were covalently

attached to the corresponding hapten.

In 1972 we introduced the first EMIT®

homogeneous enzyme immunoassay based

on this principle (Rubenstein,

Schneider, and Ullman, 1972). Since

then we have demonstrated the generality of the technique and have developed immunoassays based on three

enzymes, lysozyme (Schneider,

Linquist, Wong, Rubenstein, and

Ullman, 1973), malate dehydrogenase

(Ullman, Blakemore, Leute, Eimstad

and Jaklitsch, 1975), and glucose-6-

phosphate dehydrogenase (Chang, Crow1

and Schneider, 1975). The change

of enzyme activity of hapten-enzyme

conjugates produced upon binding

antihapten antibodies permits direct

measurement of the amount of antibody

bound to the conjugates. Enzyme

immunoassays based on this principle

are classified as "homogeneous"

immunochemical techniques since

separation of free from bound hapten

is not required. Separation is

required when the signal of the

labeled hapten is not changed by

antibody binding as is the case in

radioimmunoassay and enzyme-linked

immunosorbent assay (ELISA) which are

termed "heterogeneous" immunochemical

techniques. The observed change

of activity in homogeneous enzyme

immunoassays is reminescent of inhibition or activation produced when

certain antibodies to enzymes bind to

their respective enzymes (Arnon,

1973). The mode of action may be

similar. Either the antigenic determinant is an integral part of the

enzyme surface or it is attached

covalently to the surface.

Malate dehydrogenase (MDH) was chosen

for development of a THC assay because

of its high stability, its availability and its ease of detection by

a simple highly sensitive assay. As

little as 10-11 molar enzyme can be

detected in a one minute spectrophotometric measurement. The mechanism of

28
antibody inhibition of morphine conjugates of MDH had previously been

studied and an immunoassay for morphine was developed (Rowley,

Rubenstein, Huisjen, and Ullman, 1975)

which permitted detection of 2x10-9

molar morphine in the assay mixture.

SYNTHESIS OF THE THC HAPTEN

Synthesis of the THC hapten, III,

that was used for producing the components of the assay is illustrated

below:

1) O3,-78°

2) Zn/HOAc

9,11-THC, I, was treated with one

equivalent of ozone at low temperature

to yield an ozonide which was reduced

with zinc in acetic acid to yield

ketone II in 55% yield. Compound II

was treated with 1-C14-O-carboxymethylhydroxylamine in anhydrous

refluxing methanol to yield the

desired acid, III, in 61% yield.

PREPARATION OF THC ANTIBODY

Acid III was conjugated to bovine

globulin via its N-hydroxysuccinimide

ester, IV, prepared by condensation

with N-hydroxysuccinimide using the

condensing reagent, N-dimethylaminopropyl-N'-ethyl carbodiimide hydrochloride in anhydrous dimethylformamide.

Subsequently, the dimethylformamide

solution of "active ester" was added

to a buffered solution of the protein

containing 30% dimethylformamide cosolvent at pH 8.5. The protein was

exhaustively dialyzed to remove noncovalently bound THC residues. The

number of bound residues was determined by scintillation counting of the

radiolabeled products to yield conjugates containing 21 and 32 THC residues per protein molecule in two

separate preparations. Antibodies

were obtained from sheep by immunizing with the conjugates.

PREPARATION OF THC-MDH CONJUGATES

Acid III was conjugated to MDH via IV

under almost identical conditions. A

series of THC-MDH conjugates were thus

prepared by adding increasing amounts

of IV to constant amounts of MDH in

separate reaction vessels. Noncovalently bound THC residues were

removed either by exhaustive dialysis

or by gel chromatography on Sephadex

G-25. The number of bound radiolabeled THC residues and the residual

enzyme activity of each conjugate was

determined (Figure 1). Enzyme

activity of the conjugates decreased

sharply with increasing substitution up to about 5 residues

bound (20% activity). Substitution

beyond 5 residues provided a conjugate

(12.2 residues) with only a moderate

further loss of activity (8% activity).

Figure 1

Addition of excess THC antibody to

the conjugates reduced their enzymatic

activity. The maximum inhibition in

the presence of excess antibody was

only slightly affected by the animal

source. Maximal inhibition was,

however, directly dependent on the

29
number of bound THC residues (Figure

2). It increased sharply

with the number of THC groups on the

enzyme and reached a maximum when at

about 4.2 residues (51% inhibition).

Upon substituting the enzyme more

heavily (12.2 residues) the antibody

induced inhibition decreased to 35%.

Figure 2

IMMUNOASSAY FOR THC AND ll-NOR- 9-

THC-9-CARBOXYLIC ACID

THC-MDH conjugate, which was maximally

inhibited by THC antibodies (4.2 THC

residues), was chosen for the immunoassay. Stability of the conjugate was

found to be very sensitive to pH and

ionic strength. The conditions

selected to store working solutions of

enzyme, 0.50 M potassium phosphate,

pH 7.4 , provide room temperature

stability of greater than 34 days.

The conjugate readily adsorbed on

glass and plastic measuring devices

thus preventing quantitative and

reproducible transfer of dilute solutions. Such adsorption is reminiscent of reported adsorption of THC

itself to glass and plastics (Garrett

and Hunt, 1974). Triton X-405 was

found to prevent troublesome adsorption of THC in radioimmunoassays for

THC (Teale, Forman, Ring, Piall, and

Marks, 1975). We likewise found that

adsorption of THC-MDH conjugate was

prevented by incorporation of 0.10%

Triton X-405 in dilute enzyme solutions.

The effect of varying the ratio of

antibody to enzyme is given in Figure

3. Upon titration of the conjugate

Figure 3

30
with antibody, the enzyme activity at

first dropped off rapidly and assymptotically approached a limiting value.

An antibody to enzyme ratio was chosen

for a THC assay where the enzyme was

inhibited 39% (arrow, Figure 3). A

drug-response curve was obtained at

that ratio by adding standard ethanolic THC solutions to the assay

mixture (Figure 4). In the assay

procedure 1 μl ethanolic THC standard

solutions were combined with solutions

containing antibody and substrates

followed by addition of the THC-MDH

conjugate. Enzyme rate measurements

were made over 3 minute periods. The

lowest detectable level of THC in the

assay mixture was 0.5 ng/ml (1.6 x

10-9 M). The total useable range was

0.5 ng - 10 ng/ml.

Likewise a drug-response curve for

11-nor- 9

-THC-9-carboxylic acid, V

was obtained using standard ethanolic

solutions in an identical assay with

the same antibody (Figure 4).

The useable range of detection was

the same as that for THC, 0.5 ng -

10 ng/ml. However, response to V in

mid-range was about 1.5 times higher

than response to THC. The crossreactive behavior of our antibody

both to THC and to V is different

from that exhibited by antibodies

raised by others against O-carboxymethyl-THC (Van Vunakis and Levine,

1974), 2- or 4- (p-carboxyphenylazo)

THC, (Grant, Gross, Lomax, and Wong,

1972), and O-succinyl-THC (Teale,

Forman, King, Piall and Marks, 1975)

conjugates. All of these produced

antibodies that reacted only poorly

with V. Our antisera, therefore,

appear suited for general screening

Figure 4

3 1
for THC and its metabolites, and Enzyme immunoassays for THC and its

could also be used to assay either metabolites in urine are presently

THC or V if these compounds are first being developed and a serum assay is

separated by an extraction procedure. under investigation.

REFERENCES

Arnon, R. (1973) in The Antigens

(Sela, M., ed.) Vol. 1 pp. 87-159,

Academic, New York, N.Y.

Chang, J. J., Crowl, C. P., and

Schneider, R. S. (1975) Clin. Chem.

21, 967.

Garrett, E. R., and Hunt, C. A.

(1974) J. Pharm. Sci. 63, 1056.

Grant, J. D., Gross, S. J., Lomax, P.,

and Wong, R. (1972) Nature, New Biol.

236, 216.

Rowley, G. L. Rubenstein, K. E.,

Huisjen, J., and Ullman, E. F. (1975)

J. Biol. Chem., 250, 3759.

Schneider, R. S., Linquist, P., Wong,

E., Rubenstein, K. E. and Ullman,

E. F. (1973) Clin. Chem. 19, 821.

Teale, J. D., Forman, E. I., King,

L. J., Piall, E. M., and Marks, V.

(1975) J, Pharm. Pharmac. 27 465.

Ullman, E. F., Blakemore, J., Leute,

R. K., Eimstad, W., and Jaklitsch, A.

(1975) Clin. Chem. 21, 1011.

Van Vunakis, H., and Levine, L.

(1974) in Immunoassays for Drugs

Subject to Abuse (Mule, S. J.,

Sunshine, I., Brande, M., and

Willette, R. E., eds.) pp. 23-25,

CRC Press Inc., Cleveland, Ohio.

Rubenstein, K. E., Schneider, R. S.,

and Ullman, E. F. (1972) Biochem.

Biophys. Res. Comm. 47, 846.

32
SEPARATION AND SENSlTlVE ASSAY OF THC

IN BIOLOGICAL FLUIDS BY HPLC AND GLC

Edward R. Garrett and C. Anthon Hunt

College of Pharmacy, University of Florida

Gainesville, Florida

ABSTRACT

HPLC systems were developed to permit quantitative separation of 9

-tetrahydrocannabinol

from many of the heptane extractable lipoidal

and other endogenous substances in biological

fluids. These substances interfered with the

quantification by flame ionization GLC of unmodified compound and by electron capture GLC

of pentafluorobenzoylated compound. Reverse

phase HPLC elution, with 47% acetonitrile in

water, and normal phase HPLC with 25% chloroform in heptane, separated tetrahydrocannabinol

from 11-hydroxy- 9-tetrahydrocannabinol and

other monohydroxylated tetrahydrocannabinols.

These systems also purified stock solutions

of tetrahydrocannabinol from accompanying contaminants. The various monohydroxylated

tetrahydrocannabinols were resolved from each

other in normal phase, 80% chloroform in heptane. The 8

 and 9-tetrahydrocannabinols were

separable in normal phase with 5% tetrahydrofuran in hexane. The GLC analysis of pentafluorobenzoylated tetrahydrocannabinol had a

sensitivity of 1 ng/ml of plasma with an estimated 5% standard error of an assay with the

extraction and GLC procedures given herein.

Radiochemical analysis of the HPLC separated

fraction had a sensitivity of 0.2 ng/ml of

plasma with an estimated 2% standard error of

an assay. There was no significant difference

between the liquid scintillation and electron

capture GLC assays of the HPLC separated 9-

tetrahydrocannabinol obtained from the plasma

of dogs administered the drug. Radiolabelled

compounds can be added to pasma samples as

internal standards to determine the recovery

efficiencies of the several procedures in the

analysis of unlabelled tetrahydrocannabinol.

INTRODUCTION

A GLC analytical procedure for tetrahydrocannabinol in biological fluids was developed

previously (Garrett and Hunt 1973) and used

electron capture detection of the derived

pentafluorobenzoylated tetrahydrocannabinol.

It could readily detect 0.5 ng of tetrahydrocannabinol added to a 5.0 ml blood sample

from a fasting dog. This sensitivity was

only obtained when it was realized that

tetrahydrocannabinol bound extensively (15%-

40%) to glass (Garrett and Hunt 1973;

Garrett and Hunt 1974) and rubber stoppers

(Garrett and Hunt 1974) and that the timedependent degree of adsorption could be minimized by prior treatment of all glassware

with an organic solution of a silyl reagent.

In the case of organic solutions, the tetra33
hydrocannabinol could be reincorporated from

the glass into solution on vigorous shaking

prior to any sampling.

The method’s validity was demonstrated in

the fasting dog with a low fat diet. Plasma

levels down to 1 ng/ml of blood from 5 ml

blood samples were monitored for 12 hours

after the administration of 0.1 mg of pure

tetrahydrocannabinol per kg (Garrett and

Hunt 1973). However when the same method

was applied to non-fasting animals a significant increase in GLC background from interfacing plasma constituents was observed,

particularly within 4 hours of feeding a

previously fasted animal. The resultant

minimal detectable quantity unfortunately

increased to 5 - 10 ng/ml when a 5 ml blood

sample was taken.

Since phannacokinetic studies were contemplated in both dogs and humans over longer

periods of time so that fasting would be

impractical, such interferences were anticipated that would lower the analytical sensitivity. Thus it was necessary to devise

suitable separation and clean-up procedures

prior to analysis to improve the sensitivity.

High pressure liquid chromatography (HPLC)

provided a powerful method of separation of

drugs from their potential metabolites and

from endogenous substances in biological

fluids. While the classical on-line monitoring devices such as refractive index or

UV spectrophotometry were too insensitive for

direct assay at the plasma levels anticipated,

the separated collected pertinent fractions

were analyzed by analytical methods that provided the proper sensitivities. This paper

presents HPLC techniques to separate tetrahydrocannabinol from various cannabinoids

and endogenous materials of biological fluids

with subsequent analysis by various appropriate methods.

RESULTS AND DISCUSSION

Purification and Reproducibility of Collection

from Injected Ethanolic Solutions of 9-Tetrahydrocannabinol on HPLC

The 14C- 9 -tetrahydrocannabinol used as

supplied by NIDA was reported low in radiolabelled contaminants by TLC. However,

reverse phase HPLC (Fig. 1) revealed the

presence of a labelled contaminant. The

major peak, I, contained 9 -tetrahydrocannabinol ( > 96%) and 8

 -tetrahydrocannabino1 ( 3%), quantified by flame ionization

Figure 1

Figure 1. Reverse phase HPLC of stock 14C9

-tetrahydrocannabinol. The total radioactivity per eluate fraction is plotted vs

retention volume (curve A). Peak I was 14C9

-tetrahydrocannabinol and peak II was an unknown radioactive contaminent. Curve B is a

plot of the mean UV detector response (arbitrary units) vs retention volume and is displaced relative to A by the solvent volume between

the detector and the collection point. Collection between volumes a and b (peak retention

volume ± 30%) for curve B recovered 92.5% of

the radioactivity in this case. Column: Bondapack C18; eluent: 45% acetonitrile in water

at 1.5 ml/min. Each fraction was corrected

for background CPM.

GLC (Garrett and Hunt 1974; Garrett and Tsau

1974). A labelled contaminant was also

found (Fig. 2) with a normal phase column.

The contaminant eluted prior to tetrahydrocannabinol in both cases. Since the more

polar compounds elute first on reverse phase

HPLC whereas the least polar compounds elute

first on normal phase HPLC, it suggests that

the contaminants observed in the two systems

were not the same. The contaminants were

not analyzed further.

34
Figure 2 Figure 3

Figure 2. Normal phase HPLC of stock 14C- 9-

tetrahydrocannabinol. The total activity per

eluate fraction is plotted vs milliliters of

eluate. Peak I was 14C- 9- tetrahydrocannabinol and peak II was an unknown radioactive

contaminant. Collection between volumes a and

b recovered 97.5% of the radioactivity in this

case. Column: μ-Porsil; eluent: 20% chloroform in heptane at 2.5 ml/min. Each fraction

was corrected for background CPM.

When the tetrahydrocannabinol under peak I

(Figs. 1 and 2) was collected, dried, reconstituted in ethanol and re-analyzed on the

same HPLC system, only the single peak I was

observed. All the stock 9

-tetrahydrocannabinol used herein underwent this purification procedure. The percent of the total

injected radioactivity under peak I recovered

was 97.6 ± 0.7 (standard error of mean) %

(Table I) in the reverse phase system for the

ranges of volumes collected (Figs. 1 and 3)

and was 96.3 ± 1.0 (standard error of mean) %

(Table II) for 1 to 1000 ng injected in the

normal phase system at a retention volume of

9.7 ml within the collection range of 7.3 -

16.2 ml (Fig. 2).

Figure 3. The retention volumes for the peak

amounts of 9-tetrahydrocannabinol and

11-hydroxy- 9-tetrahydrocannabinol for

reverse phase HPLC are plotted vs soluent composition. Each point represents the mean peak

retention volume for two determinations. The

vertical bars represent the ranges of retention

volumes which contained approximately 98% of

the area under the plot of recovered radioactivity vs retention volume.

The fact that the retention volume of peak I

and the appropriate collection ranges for

98% recovery of injected labelled tetrahydrocannabinol were sensitive to solvent composition (Fig. 3) necessitated the prior establishment of an appropriate collection range

for each newly prepared batch of solvent by

radiochemical analysis of the collected HPLC

fractions of previously purified 14C- 9

-tetrabydrocannabinol. The retention volume

and the appropriate collection range increased

with decreasing solvent polarity (less %

acetonitrile) due to peak spreading (Fig. 3).

35
Table 1 Table 2

TABLE I - Recovery of Radioactivity of 14C- Terahydrocannabinola

from Reverse Phase HPLC

TABLE II - Recovery of Radioactivity of 14C- Terahydrocannabinola

from Normal Phase HPLCb

The quality of the water used in the eluting

solvent acetonitrile-water in reverse phase

HPLC was an important factor in maintaining

the reproducibility of the percent radioactivity recovered for a given collection

range. The UV detector clearly indicated

that adsorbed contaminants in impure water

could be eluted from a previously used column

by 100% acetonitrile, a less polar solvent

than the used mixed eluent. Additional evidence of these contaminants was demonstrated

when the GLC background significantly varied

when distilled water from various sources

was collected after reverse phase chromatography, extracted as if 9

-tetrahydrocannabinol were present, then treated by the

derivatization procedure and analyzed by

electron capture GLC.

HPLC Separation of 9-Tetrahydrocannabinol

from Selected Cannabinoids and Metabolites

Tetrahydrocannabinol, cannabinol and cannabidiol were resolved by the normal phase HPLC

system (Fig. 4). If 98% of the tetrahydrocannabinol were to be collected after normal

phase HPLC in this system, it is apparent

that the chosen range (Fig. 2) would also

collect cannabinol and cannabidiol. The

retention volumes of cannabinol and cannabidiol relative to tetrahydrocannabinol increased as the percent of chloroform in

heptane increased.

‘The monohydroxylated metabolites had large

retention volumes (> 15 ml) on the normal

phase column when 20 - 25% chloroform in

heptane was the solvent (Fig. 2) and could

be completely separated from tetrahydrocannabinol on this system. They were resolved

from each other with a more polar solvent,

80% chloroform in heptane (Fig. 5).

9

-Tetrahydrocannabinol and 11-hydroxy9

-tetrahydrocannabinol were quantitatively

separable on the reverse phase HPLC system

at 47% (or less) acetonitrile in water

(Fig. 3). The collection efficiencies in the

ranges given were 98% of the recoverable

radioactivities of 3H-11-hydroxy- 9-tetrahydrocannabinol and 14C- 9

-tetrahydrocannabinol.

8

 and 9

 -Tetrahydrocannabinols were not

readily resolvable in any of these systems.

However an HPLC system that readily resolved

and separated these two compounds was 5% THF

in hexane on the normal phase column at 0.5

ml/min with retention volumes of 8.15 and

8.45 ml, respectively. The tetrahydrocannabinols collected under peak I (Figs. 1 and

2) could be further separated by this system.

36
Figure 4 Figure 5

Figure 4. Normal phase HPLC separation of

cannabinoids. The UV detector response (arbitrary units) is plotted vs retention time after

injection (inj.) of a mixture of cannabidiol

(CBD), 9-tetrahydrocannabinol (THC) and cannabinol (CBN). The amounts of 9

-tetrahydrocannabinol and cannabidiol injected were approximately twice that of the cannabinol. Column:

μ-Porsil; eluent: 20% chloroform in heptane

at 1.5 ml/min.

Figure 5. Normal phase HPLC separation of 9

-

tetrahydrocannabinol metabolites. The UV detector response (arbitrary units) is plotted

vs retention time after injection (inj.) of a

mixture of cannabinol (CBN), 11-hydroxy- 9-

tetrahydrocannabinol (C), 8 - and 8 -hydroxy9

-tetrahydrocannabinol (B and A, respectively).

Column: μ-Porsil; eluent: 80% chloroform in

heptane at 1.5 ml/min.

Table 3

TABLE III Percent Recoveriesa of 14C- 9

-Tetrahydrocannabinol in the Heptane Extract of Plasma, in

the Collection of the Proper Fraction of the Heptane Extract Separated by HPLC and the

Overall Recovery after Both Extraction and HPLC Collection as Monitored by Both Electron

Capture-GLC and Scintillation Analysis

14C- 9

-Tetrahydrocannabinolb,

ng/2 ml plasma

2.25

Extracted by Heptane, %

Scintillation

90.2 ± 4.7

Extract HPLC Overall Recoveryd

, %

Collectedc, %

Scintillation GLC Scintillation GLC

93 92 84 ± 8 83 ± 32

22.5 90.8 ± 2.6 88 97 80 ± 6 88 ± 12

225 90.9 ± 8.5 97 89 8.8 ± 4 81± 7

Overall Average ± std. error 90.6 ± 0.7 92 93 84 + ± 2 84 ± 5

aGiven as the mean from 4 separate plasma samples ± standard deviation. The scintillation analysis of recovered radioactivity

was performed on a different set of four studies and on a different day than the electron capture GLC analysis of the derivatized

HPLC collection. b

142.47 CPM/ng. Two ml of plasma was extracted with 15 ml heptane and 14 ml was analyzed for total 14C.

cQuotient divided by the extraction efficiency (0.91). Additional studies were conducted for 26 ng 14C- 9

-tetrahydrocannabinol

per 2 ml plasma on two other day for 4 samples each and the % recovered on HPLC from the extract were 94 ± 4 and 92 ± 3%

(standard error) respectively. dQuotient of amount recovered corrected for volumes of extract used and amount added, which is

the product of the extraction efficiency and the collection efficiency.

37
Effect of HPLC Separation on GLC Analysis of

9 Tetrahydrocannabinol in Plasma

An equal amount of 9 tetrahydrocannabinol

and 11-hydroxy- 9-tetrahydrocannabinol in

2 ml of dog plasma was extracted and separated from a majority of the extracted components by reverse phase HPLC. The reduction in potential contaminants from plasma

observable on GLC was demonstrated by flame

ionization GLC analysis (Garrett and Hunt

1974; Garrett and Tsau 1974) both before and

after HPLC treatment (Fig. 6).

The normal phase HPLC with 20% chloroform in

heptane could separate 9-tetrahydrocannabinol from monohydroxylated metabolites and

from 11-hydroxy- 9-tetrahydrocannabinol.

However, a minor overlap could be avoided by

collecting the tetrahydrocannabinol in a

slightly narrower volume range. The prior

heptane extraction of alkalinized plasma

had separated these non-polar constituents

from any acidic metabolite. This separation

of plasma extracts and normal phase HPLC

collection of volumes in the appropriate

range resulted in a substantial, reduction in

GLC background from plasma components for

derivatized tetrahydrocannabinol analyzed

with electron capture detection as shown by

the comparison of curves A and B or A and C

in Fig. 7.

Figure 6

Figure 6. GLC chromatograms (flame ionization

detection) of an extract of 2 ml of plasma

containing 9-tetrahydrocannabinol, I (200

ng/ml), before (A) and after (B) reverse phase

HPLC separation of both cannabinoids over a

range of predetermined collection volumes

(Fig. 1). One μl of 18 μl of extract was injected into the GLC prior to HPLC (A). Ten

μl of 18 μl of the extract was injected into

the HPLC, the collected fraction was reconstituted in 10 μl of chloroform and 1 μl was injected into the GLC (B). Peak III is the solvent; peak IV is an unknown from plasma.

HPLC: column, Corasil C18; eluent, 52% acetonitrile in water at 1.5 ml/min. GLC: column,

5' x 2 mm 1.9% OV-225 at 245° with a N2 flow

of 24 ml/min. and an attenuation of 8 x 12-12

for both chromatograms. The initial base

lines and injection times for both chromatograms are superimposed for comparison.

Figure 7

Figure 7. GLC (electron capture) analysis of

derivatized samples. The chromatograms represent: A) the injection of 1 of 300 μl from a

derivatized extract of 2 ml of blank plasma

without HPLC purification: B) the injection

of 1 of 200 μl of the final solution of derivatized compound which was 70% of the total

amount from 2 ml of dog plasma containing 200

ng/ml 9tetrahydrocannibinol pentafluorobenzoate (THC) are appropriately labelled. Typical estimated base lines are shown. GLC conditions: 6' OV 17 column, 225°; detector,

280°; injector, 255°; N2 flow, 45 ml/min;

attenuations 4 x 10-9 (A and B) and 8 x 10-l0

(C).

38
Plasma samples obtained from dogs administered 9

-tetrahydrocannabinol solutions intravenously were analyzed by the electron capture GLC in accordance with the modified

procedures described herein that included

extraction, normal phase HPLC separation and

derivatization except that no internal

standard was added. These procedures would

have included any cannabinol or cannabidiol

in the HPLC collection volume range (Fig. 2

and 4) used. However, no peaks were seen at

the retention times of cannabinol or cannabidiol pentafluorbenzoate and no significant

amounts of cannabinol or cannabidiol could

be detected as metabolites of 9-tetrahydrocannabinol in the dog. Thus, either

compound, when purified, should serve as an

appropriate internal standard in pharmacokinetic studies. However, since cannabinol

has been reported as a minor metabolite

(McCallum 1973; McCallum et al. 1975;

Widman et al. 1974), cannabidiol pentaflurobenzoate was chosen as the internal standard.

It must be realized that cannabinol is known

to be a contaminent of degraded 9 tetrahydrocannabinol (Garrett and Tsau 1974).

The GLC methodology presented herein differed

from the prior studies (Garrett and Hunt

1973) in that the short 30 cm column of 3%

OV 225 was supplanted by a longer OV 17

packed column to be consistent with the data

in the literature accumulated for the resoI.ution of the cannabinols (Fetter-man and

Turner 1972; Turner and Hadley 1973; Turner

et al. 1974).

Efficiency, Reproducibility and Sensitivity

of the Steps in the Radiochemical and Electron

Capture Assay of l4C- 9-Tetrahydrocannabinol

in Dog Plasma

The heptane extraction efficiency from plasma

was highly reproducible (Table III) over a

wide range of plasma concentrations, 90.6 ±

0.7% standard error of mean.

The recovery of 9-tetrahydrocannabinol from

the heptane extract of dog plasma by the

normal phase HPLC was reproducible (Table

III) over the range of plasma concentrations

studied. Equivalent overall recoveries

(Table II) were obtained by both radiochemical analysis (83.7 ± 1.8% standard error)

and by electron capture GLC analysis (84.0 ±

4.9% standard error) of the derivatized

tetrahydrocannabinol (Fig. 7). Both methods

permitted estimation of a 92.5% recovery

of the amount in the heptane extract injected

on the normal phase HPLC and collected in the

chosen range. The normal HPLC collection

range was chosen to be slightly smaller than

in the studies on ethanolic solutions of

tetrahydrocannabinol (Fig. 2, Table II) since

the procedure had to be modified to separate

tetrahydrocannabinol from possible monohydroxylated or 11-hydroxy- 9

-tetrahydrocannabino1 metabolites in plasma. A slight overlap

of their HPLC peak areas with tetrahydrocannabinol would have occurred if the larger

collection ranges had been used.

A similar study of the reproducibility of

collection of 14C- 9-tetrahydrocannabinol in

plasma assayed by liquid scintillation after

extraction and reverse phase HPLC was also

conducted. The amounts recovered were proportional to the amounts injected (Fig. 8)

and the HPLC recovery efficiency of the drug

in the heptane extract in this case was 95.7%.

Figure 8

Figure 8. Recovery of 14C- 9

-tetrahydrocannabinol calculated as concentration in plasma

corrected for an extraction efficiency of 91%

against the experimentally prepared concentrations. The drug extracted from plasma and

collected over the proper volume range (Table

I and Fig. 1) after reverse phase HPLC with a

45% acetonitrile in water eluent. The collection was analyzed by liquid scintillation. The

slope, 0.957, is the HPLC collection efficiency

for 9-tetrahydrocannabinol and was typical of

the values obtained. The vertical bar is the

range for ± one standard deviation (n=4).

39
Equivalency of Radiochemical Analyses of 14C9

-Tetrahydrocannabinol and GLC Electron

Capture Detection of Derivatized Material

after Normal Phase HPLC in Dog Plasma during

Pharmacokinetic Studies

The plasma of a do intravenously administered

solutions of 14C- 9

 - tetrahydrocannabinol was

monitored with time after heptane extraction

by both radiochemical analysis and electron

capture GLC of the derivative of the appropriately collected eluate fraction from

normal phase HPLC. Typical plots of the

time course of the results from both methods

are given in Figs. 9 and 10.

Figure 9

Figure 9. Semilogarithmic plots of fraction

of the 9-tetrahydrocannabinol 0.1 mg/kg dose

per ml of plasma against time for dog A plotted

from the liquid scintillation analysis of the

total 14C collected as 9-tetrahydrocannabinol

on normal phase HPLC and from the electron

capture GLC of the derivatized HPLC collected

fraction . The values were corrected for

the fractions of extracts and total collection

range used.

Figure 10

Figure 10. Semilogarithmic plots of fraction

of the 9-tetrahydrocannabinol 2.0 mg/kg dose

per ml of plasma against time for dog A plotted

from the liquid scintillation analysis of the

total 14C collected as 9-tetrahydrocannabinol

on normal phase HPLC and from the elctron

capture GLC of the derivatized HPLC collected

fraction . The values were corrected for

the fractions of extracts and total collection

range used.

The procedures for GLC analysis reported

herein gave a lower limit for quantitative

analysis of tetrahydrocannabinol in plasma

of approximately 1 ng/ml from twice the

standard deviation (0.32 ng) obtained for

the amount of tetrahydrocannabinol recovered

from 2.25 ng in 2 ml plasma (Table II).

Similarly, the procedure for radiochemical

analysis reported herein gave a lower limit

of approximately 0.2 ng/ml from twice the

standard deviation (0.084 ng). A statistical analysis of the apparent differences

between the tetrahydrocannabinol assays at

a given time from both analytical methods

(Figs. 9 and 10) showed no significance.

40
This demonstrated that all of the recovered

radioactivity from the HPLC separation procedure could be assigned to the 9

-tetrahydrocannabinol assayed specifically by

electron capture GLC and thus no significant

amounts of radiolabelled metabolites were

in the collected HRLC fractions.

It can be concluded that 9 -tetrahydrocannabinol can be extracted from plasma and

other biological fluids, that it can be

separated on HPLC from the simultaneously

extracted biologically endogenous materials

and metabolites that would interfere with a

chosen highly sensitive analytical method

such as GLC. It is not necessary to collect

all of the material to be analyzed; assurance that a reproducible or known fraction

of the total material injected on HPLC is

REFERENCES

E. R. Garrett and C. A. Hunt, J. Pharm. Sci.,

1211 (1973).

E. R. Garrett and C. A. Hunt, J. Pharm. Sci.,

63, 1056 (1974).

E. R. Garrett and J. Tsau, J. Pharm. Sci.,

63, 1563 (1974).

N. K. McCallum, J. Chrom. Sci., 11, 509

(1973).

N. K. McCallum, B. Yagen, S. Levy and

R. Mechoulam, Experientia, 31, (5), 520

(1975).

M. Widman, M. Nordquist, S. Agurell,

J. E. Lindgren and F. Sandberg, Biochem.

Pharmacol., 23, 163 (1974).

R. S. Fetterman and C. E. Turner, J. Pharm.

Sci., 61, 1476 (1972).

C. E. Turner and K. Hadley, ibid, 62, 251,

1083 (1973).

C. E. Turner et al., ibid, 63, 1872 (1974).

recovered is all that is necessary since it

is directly proportional to the total drug

concentration. If unlabelled tetrahydrocannabinol in a solution of plasma were analyzed, the calculated recovery of known

amounts of labelled tetrahydrocannabinol

added either to plasma prior to extraction

or to heptane extract subsequent to extraction would permit calculation of the extraction and/or HPLC collection efficiencies for

that particular biological sample. These

known efficiencies would permit the calculation of the original plasma concentrations.

If a labelled 14C- 9

 -tetrahydrocannabinol

were used in pharmacokinetic studies,

extracted and separated on the HPLC, a tritium labelled 3H-tetrahydrocannabinol could be

used as the appropriate internal standard to

monitor the recovery efficiencies.

ACKNOWLEDGMENTS

Supported in part by Grant DA-00743

from the National Institute on Drug Abuse,

Rockville, MD 20852

The technical assistance of

Mrs. Kathleen Eberst and the contributions

of Hendryk Roseboom are gratefully acknowledged.

43
DETERMINATION OF ETRAHYDROCANNABINOL

IN HUMAN BLOOD SERUM BY

ELECTRON CAPTURE GAS CHROMATOGRAPHY

David C. Fenimore Ph.D. Chester M. Davis, Ph.D., Alec H. Horn

Texas Research Institute of Mental Sciences

Houston, Texas

The application of electron capture gas

chromatography (ECGC) to the determination

o f 9-tetrahydrocannabinol in blood samples

has been reported by Garrett and Hunt

(1973) and Fenimore, Freeman, and Loy

(1973). In these studies laboratory animals

were used as experimental subjects which,

as later experience demonstrated to the

present authors, was a fortunate choice.

When attempts were made to extend the

technique to samples of blood from human

subjects with their more varied diet and

environment, the presence of interfering

components proved to be a serious problem.

Had this level of difficulty occurred

during the initial development of the

methodology, other less sensitive but

more tractable alternatives would surely

have been pursued. The initial success with

animal subjects, however, encouraged modifications of the method which now permit

quantitation of 9-THC in human blood at

concentrations approaching that attainable

with the animal subjects.

Although the cannabinoids do not possess

high affinities for thermal electrons, it

is a relatively simple matter to form polyhalogenated derivatives of these compounds

in quantitative yield to which the electron

capture detector responds with excellent

sensitivity. The limit of detection for the

heptafluorobutyrate or pentafluoropropionate esters of 9

-THC is approximately one

picogram (Fenimore, 1973). Because the

levels of 9-THC in the blood are about

1000 times this amount per milliliter for

a considerable period after a nominally

active dose of cannabis preparation

(Lemberger 1971, Agurell 1973, Garrett 1973),

it is obvious that sensitivity of detection

is not the limiting factor. The problems

arise from the fact that the derivatizing

reactions are relatively non-specific. Not

only are there a large number of components

present in biological samples at parts per

billion concentrations capable of forming

derivatives of this type, but also many

similarly reactive species may be carried

through the initial sample preparation in

amounts orders of magnitude greater than

the compound or compounds of interest. It

is apparent, then, that selectivity becomes

all important if the sensitivity of the

determination is to be maintained, and if

this selectivity cannot be achieved at the

detector, it must be restored through the

chromatographic process, the sample preparation, or both.

4 2
One approach toward eliminating the interfering components is to achieve a relatively

pure isolate prior to gas chromatographic

analysis through sample preparation involving other chromatographic separations

or through extensive partitioning processes.

Experience has shown, however, that sample

recovery is not favored by manipulations

of this kind. Consequently we have relied

primarily on improved gas chromatographic

technique to alleviate the problem of

interference. A dual column-dual oven

instrument described previously (Fenimore,

1973) and discussed in some detail in the

following section has provided the necessary resolution to enable the determinations

o f 9

-THC in human blood serum.

DESCRIPTION OF THE GAS CHROMATOGRAPHIC

APPARATUS

A schematic diagram of the dual column-dual

oven gas chronatograph is shown in Figure 1.

Oven No 1, containing a conventional packed

column, is temperature programmed while

oven N

o

 2, equipped with a capillary

column, remains isothermal. A switching

valve and cold traps are arranged to permit

transfer of only that volume of effluent

containing the compound or compounds being

measured from the packed column to the

capillary column. The exact retention

volume necessary for accurate transfer is

determined previously by detection of

relatively large amounts of compound (approx.

Figure 1

1 μg) by flame ionization. The transferred

effluent is further separated by the

capillary column and detected by a microvolume electron capture detector (Fenimore,

1971).

This system provides the means to eliminate three major problems encountered

in high resolution ECGC. First, temperature programming, which is usually not

feasible with EC procedures because of

exaggerated baseline drift, is accomplished

by raising the temperature of the first

column while holding the second column at

a constant temperature. Second, the packed

column tolerates large injection volumes

which would rapidly degrade a small bore

capillary column. Third, the two columns

may contain stationary phases of widely

different polarity which increases the

probability of separation of compounds

having similar partition coefficients

on any single chromatographic column.

43
The problem of transferring the solute

from the high volume mobile phase eluate

of the packed column to the much smaller

volume of the capillary column is solved

by interposing two water-cooled traps

between the columns. The first trap consists of 1/8'' ID nickel tubing containing

a 3 cm plug of material identical to the

packing of the fore-column, and the second

is short length of 1/16'' ID nickel capillary

coated with the stationary phase used in

the second column. Water is allowed to

flow through an annular space surrounding

the traps immediately before and during

the transfer interval. When the temperature of the first trap is restored to

oven temperature by interrupting the flow

of coolant, the solute is released at the

reduced carrier gas flow rate of the

capillary column. The band broadening

introduced by the volume of the first

trap is reduced by re-trapping the solute

in the capillary tubing and then releasing

it to the capillary column in the second

oven. An eight-port switching valve (Valco

Instruments, Houston, Texas) is used to

transfer the carrier gas flow between

columns and traps. All lines connecting

the various elements of the system are

nickel capillary tubing.

The columns and column conditions used in

the present study are as follows:

Packed column: 6 ft x 3 mm ID coiled

glass containing 10% OV-101 on 100 to

120 mesh Gas Chrom Q (Applied Science Lab,

State College, Pennsylvania). Nitrogen

carrier gas flow rate is 40 cc per minute.

Column is temperature programmed from

180°C to 240°C at 4°C/min.

Capillary column: 100 ft x 0.02'' ID

nickel-200 tubing (Handy and Harmon

Tube Co., Norristown, Pennsylvania)

coated with Poly I-110 (Applied Science

Lab, State College, Pennsylvania).

Nitrogen carrier gas flow rate is 4 cc/

min. Column temperature is 190°C. Additional gas flow is added prior to the

electron capture detector to provide a

total flow of 10 cc/min.

The electron capture detector contains

a 1 Curie scandium tritide foil (U.S.

Radium Corp. , Panorama City, California)

and is operated at a 1000 μsec. collection

pulse. The signal from the detector is

converted by an analog linearizer (Antek

Instruments, Houston, Texas) prior to

the recorder.

SAMPLE PREPARATION

A flow diagram of the blood serum extraction and derivatization procedure is

shown in Figure 2. All solvents are

Figure 2

reagent quality or better and are redistilled and stored in glass before

use. All glassware is silylated using

vapor phase silylation. Derivatization

is a standard esterification of the

phenolic hydroxyl group of 9-THC using

pentafluoropropionic anhydride (PFPA) in

the presence of trimethylamine. The final

volume of sample prior to gas chromatographic analysis is 20 μl of which 5 μl

is taken for each sample injection.

The internal standard is hexahydrocannabinol (HHC) prepared by hydrogenation of

8

-THC and purified by preparative scale

thin layer chromatography. For most determinations 4 ng of the HHC are added in

solution to the blood serum sample before

proceeding with the extraction procedure.

RESULTS AND DISCUSSION

As stated previously the amounts of 9-

THC which would normally be determined

in a convenient volume of blood serum

are some three orders of magnitude

greater than the limit of detectability

attained by electron capture of the pure,

derivatized compound. The primary factor

which prevents the realization of this

detection limit is the presence of inter44
fering substances originating in the blood

serum or introduced to the sample during

preparation. This latter source of contamination can, at least, be controlled

by exercising the usual precautions generally recommended for electron capture determinations, i.e. use of glass-distilled

solvents, use of small total solvent volume where evaporative concentration is

required, re-distillation of derivatizing

reagents, scrupulous cleanliness of glassware, and avoidance of all plastic materials with the exception of polytetrafluorethylene type polymers.

Interference from endogenous compounds in

the sample can only be reduced to a

practical minimum through selective

solvent extraction of the cannabinoid

and utilization of appropriate chromatographic separation. The limit to which

the extractive isolation of the compound

can be pursued is very much dependent on

how much loss of that compound can be

tolerated. The multiplicative nature of

sample loss thus dictates against all

but the very minimal number of partitioning manipulations prior to gas chromatographic separation. On the other hand,

loss of sample during gas chromatography

can be all but be eliminated by careful

choice and preparation of the various

components of the instrument. In the

present study the individual elements

of the dual oven chromatograph were

examined for effect on peak area of the

detected 9-THC-PFP and were found to

have no significant contribution to

solute loss even at the limit of detectability. It is for these reasons

that reliance was placed on gas chromatographic separation rather than extractive techniques for reduction of

interference.

In even the most meticulous extraction

procedures involving recovery of compounds at part per billion concentration,

some loss must be anticipated. Inclusion

of an internal standard prior to the

extraction and derivatization procedures

is therefore necessary to monitor compound

recovery, and the chemical and physical

properties of the standard should approximate those of the compounds in question

as closely as possible. The deuterated

9

-THC employed by Agurell in mass fragmentographic assays (Agurell, 1973) certainly approaches the ideal, but, of

course. cannot be differentiated from

unlabeled 9-THC by electron capture

procedures. Hexahydrocannabinol was therefore selected as having similar properties

to 9-THC while retaining good chromatographic separation.

There are obstacles to relying almost

entirely on gas chromatography to isolate

a component of a mixture for measurement,

because complete separation may require

excessive analysis time, even with highly

efficient columns. Thus the columns used

and the operating conditions utilized are

usually a compromise between resolution

and speed of analysis. The variability of

blood constituents among human subjects

complicates the selection of operating

conditions. Therefore in this study

numerous blood samples from individuals

believed to be abstinant from cannabis

use were examined for the purpose of

varying the chromatographic conditions

until extraneous peaks were absent in

the region of the chromatogram where

the 9-THC and HHC internal standard

were known to appear at the appropriate

level of sensitivity. Once the conditions

had been achieved which produced a

reliable baseline, known amounts of 9-

THC were added to random sera samples,

and the ratio of detected peak heights.

o f 9-lHC to the internal standard were

computed. The regression was linear from

30 ng per ml to a limit of sensitivity

of 500 pg per ml without dilution of

the more concentrated trial samples.

Figures 3 and 4 show electron capture

chromatograms of human blood serum extracts

obtained using this procedure. The blood

samples were taken before and one hour

after a volunteer had smoked a single

marihuana cigarette estimated to have

contained 6 mg of 9-THC. Blood serum

levels determined over a four hour period

are shown in Figure 5, and are in close

agreement with those reported by other

investigators (Agurell, 1973).

The major disadvantage in applying ECGC

to determinations of this type is the

amount of instrument time involved in

performing a single analysis. For the

chromatograms shown in the figures the

total time from injection to the point

at which the capillary column is clear

for a subsequent determination is approximately forty-five minutes, and this time

period would undoubtedly be excessive in

studies where large numbers of samples

were processed daily. On the other hand,

this reported time is by no means an

irreducible minimum, and improvements

in column technology, operating parameters,

and sample preparation could well lower

this figure substantially. In addition,

gas chromatography lends itself readily

to instrument modification. A multiple

column system utilizing the principles

demonstrated successfully in the operation

of the dual-oven instrument could be

assembled capable of performing analyses

45
Figure 3 Figure 4

Figure 5

46
limited only by the rapidity with which

the primary temperature-programmed

column can be serially operated. With

automatic sampling and computer control

such an instrument could run continuously

with very little technician supervision.

REFERENCES

Agurell, S., Gustafsson, B., Leander,

K., Lindgren, J-E., Nilsson, J.,

Saudberg, F., and Ashberg, M., J.

Pharm. Pharmacol. 25: 554 (1973).

Fenimore, D.C., Freeman, R.R., and

Loy, P.R., Anal. Chem. 45:2331(1973).

Fenimore, D.C., Loy, P.R., and

Zlatkis, A., Anal. Chem.43:1972

(1971).

Garrett, E.R., and Hunt, C.A., J.

Pharm. Sci. 62: 1211 (1973).

Lemberger, L., Axelrod, J., Kopin,

I.J., Ann. N.Y. Acad. Sci.191:142

(1971).

McCallum, N.K., J. Chromatogr. Sci.11:

509 (1973).

Rosenfeld, J.J., Bowins, B., Roberts,

Perkins J., and Macpherson, A.S.,

Anal. Chem.46:2232 (1974).

47

Although this report concerns the determination of 9

-THC onlv. the techniques and instrumentation'should be

applicable with minor modification to

quantitation of other cannabinoids and

metabolites of these compounds in biological samples.
DETECTION AND QUANTIFICATION OF

TETRAHYDROCANNABINOL IN BLOOD PLASMA

Agneta Ohlsson1, Jan-Erik Lindgren2,3, Kurt Leander2, Stig Agursll1,3

1

Faculty of Pharmacy, University of Uppsala, Uppsala

2Department of Toxicology, Karolinska lnstitutet, Stockholm

3Astra Läkemedel AB, Södertälje, Sweden

INTRODUCTION

During the last few years much effort has

been spent on developing methods for the

identification of Cannabis users by analysing different body fluids. Also, techniques for the quantitative determination

o f 1-tetrahydrocannabinol (

1

-THC) and related compounds have long been desired.

Recent developments in this area have

been summarized in a review by Grli

(1974) including some eighty references.

This field of detection of cannabinoids

in biological fluids is in a state of rapid

expansion and for the positive identification

and quantification of cannabinoids in body

fluids several highly sensitive methods are

available.

It must be emphasized that the definition

of what a "satisfactory method" is will vary

with the goal of the users. For epidemiological studies of Cannabis use, simple

screening procedures - where a high

reliability may be traded for rapidity

and expense - will probably be satisfactory. In forensic toxicology, the

requirement for unequivocal identification hardly allows the use of procedures

yielding ambigious results. Finally,

in pharmacokinetic and pharmacological

studies it may be necessary to have a

specific as well as accurate quantitative procedure. These considerations

would suggest that eventually different

48
procedures will be used for the determination of cannabinoids in body fluids

(blood, urine) depending upon the aim

of the investigator.

What a "complex and sophisticated" technique

is will also depend upon the trends

in analytical chemistry in a specific

country. In Sweden, where over seventy

gas chromatography - mass spectrometry

(GC-MS) instruments are available in a

limited scientific community, a mass

spectrometric method is obviously less

"sophisticated" than in a country where

such instruments are scarce.

Radioimmuno assay procedures for cannabinoids in blood and urine have been extensively investigated (cf. Gross et al.,

1974; Teale et al., 1975). Both authors

utilized goat antiserum obtained after

immunization with conjugates of 1-THC.

After reduction of non-specific binding

a sensitivity of ca. 7.5 ng/ml THC in

plasma or 1 ng/ml in urine could be achieved

The antibodies were specific for the three

ringed cannabinoid nucleus in THC and

cross-reacted with e.g. 7-hydroxy- l-

THC and cannabinol (CBN) but not with

cannabidiol (CBD). As pointed out by

Teale et al. (1975) this cross-reactivity

has disadvantages but also advantages in

e.g. epidemiological studies. Another

immunological approach - free radical

immunoassay - has been tested by Cais

et al. (1975).

49

Gas chromatography in combination with

sensitive detection systems has also

been widely investigated. In general, the

problems have been to remove lipid materials

interfering with the gas chromatographic

separation of e.g. THC and increase the

sensitivity in the detection step. Fenimore et al. (1973) used a dual column

separation, utilizing a capillary column

for the final resolution, and the electron

capture (EC) sensitive heptafluorobutyrate

derivative. McCallum (1973) utilized a

flame photometric detection of phosphate

esters of l-THC and related compounds.

Garrett and Hunt (1973) described another

EC-based procedure using the pentafluorobenzoate ester. Later, the same authors

(Garrett and Hunt, 1976 a) have discussed

the use of high pressure liquid (HPLC) and

gas chromatography (GLC) for separation

and analysis of THC in biological fluids.

We have published methods for the quantitative determinations of l

-THC and 6-THC

in the blood of Cannabis smokers (Agurell

et al., 1973, 1974). These methods are

based upon the purification of THC extracted from blood plasma by liquid chromatography on Sephadex LH-20 followed by

mass fragmentographic assay using the

deuterated THC-analogue as internal

standard. Rosenfeld and co-workers (1974)

also used mass fragmentography for the detection of 1

-THC in humans after smoking.

The latter authors used trimethylaniliniur

hydroxide for on column methylation of the

phenolic group and relied on the trideuteromethyl ether as internal standard in

the last step.

Many other techniques have been tried as

reviewed by Grlic (1974) but a later

result utilizing HPLC of dansyl derivatives should be mentioned (Loeffler et al.,

1975). Wall and co-workers in this

volume present in detail a mass spectrometric procedure for the determination of

THC in humans after Cannabis smoking.
Of course, the crucial test for any method

is its performance (sensitivity, specificity, capability) when applied to the

actual problem in man - this is unfortunately often lacking in the papers published.

The purpose of the present paper is to

summarize previously published results

and to present additional information on

the mass fragmentographic procedures for

THC and other cannabinoids developed in

our laboratory.

CBD-d7. Olivetol-d 7 (160 mg) was condensed

with (+)-trans-p -mentha-2,8-dien-l-ol in

benzene using traces of oxalic acid as described by Petrzilka et al. (1969). After

final purification by TLC (Agurell et al.,

1974) on pre-washed silica gel G plates

with methylene chloride-methanol (95:5)

as solvent, 33 mg of 97% pure (GLC)

METHODS

CBD-d7 was obtained. Deuterium content

according to mass spectrometry (m/e

314-321): d0

 2%, d1 2%, d2 2%, d3 3%,

d4

 3%, d5 9%, d6 30%, d7 100%.

to mass spectrometry, m/e (180-187): do 2%,

d1 2%, d2 2%, d3 3%, d4 3%, d5 9%, d6 30%,

d7

 100%.

Cannabinoids 1

-THC-d7

. Olivetol-d7 was condensed with

(+)-trans-p-mentha-2,8-dien-l-ol using

Synthesis of olivetol-d7. The synthetic

procedure of Pitt et al. (1975) was used

with the following modifications in order

to increase the amount of deuterium incorporated into the molecule (Fig. 1). The

ylide was prepared from methyl 4-bromocro-.

tonoate and triphenylphosphine, was reacted

with 3,5-dimethoxybenzaldehyde to yield

methyl 5-(3,5-dimethoxyphenyl)penta-2,4-

dienoate (1) (Buchta & Andree, 1959, 1960).

1 was then reduced with LiAld4

 in ether at

p -toluensulfonic acid (Petrzilka et al,

1969), The preparative purification was

carried out on silica gel G plates eluted

three times with 5% ether in light

petroleum to separate the 6 and l-

isomers. The purified (94% by GLC, 37 mg)

1

-THC-d7 showed the following deuterium

content (m/e 314-321): d0 2%, d1 2%,

d2

 2%, d3 3%, d4 3%, d5 9%, d6 30%, d7

100%.

room temp. for 2 h. The complex was degraded with d20 to insert one more d in CBN-d7

. 

6

-THC-d 7

 was treated with two

the side chain. The isolated compound, with equivalents of chloranil and traces of

the hydrogen atom of the alcohol group ex- p -toluenesulfonic acid in refluxing dry

changed for deuterium, and the catalyst toluene for 5 h (Mechoulam et al., 1968).

tris(triphenylphosphine)rhodium chloride The solution was chromatographed on a siwas stirred in benzene under d2

 atmosphere lica gel column with toluene eluent and

for 16 h at room temp. The 5-(3,5-dimethoxy- further purification by TLC yielded 96%

phenyl)pentan-1,1,2,3,4,5- d6-l-ol was then pure CBN-d7

. The isotope distribution was

reacted with PBr3, LiAl d4

 and BBr3 according the same as in l

-THC- d7; this in contrast

to Pitt et al. (1975) to yield olivetol-d7 to dehydrogenation methods using sulphur

(over-all yield from 3,5-dimethoxybenzal- which causes extensive deuterium scrambdehyde 24%). Deuterium content according ling.

5 0
Figure 1

Fig. 1. Scheme for synthesis of olivetol-d7

. Formulas of cannahinoids

and deuterium containing internal stundards.

51
Olivetol-d 3

 was prepared as described by CBD- d2

, 

l

-THC-d2 CBN-d2

. CBD-d2 was

Pitt et al. (1975). Compound 1 (see synthesized as described for the d7-

olivetol-d7

) was hydrogenated in the pre- analogue and crystallized from hexane.

sence of Pd and the product reduced with The CBD-d2 was 97% pure by GLC and showed

LiAld4

. Subsequent reactions with PBr3

,

LiAld4 and BBr3 giving olivetol-d3 were

a deuterium content (m/e 314-316): d0 8%,

d1 12%, d2 100%.

as described for olivetol-d7.

1

-THC-d2

 was prepared as l

-THC-d3

and more than 95% pure by GLC: Deuterium

l

-THC-d3 and CBN-d3 were synthesized as content (m/e 314-316): do

 10%, d1 20%,

described for the d7-analogues. Since larger d2

 100%. CBN-d2 was prepared from 6-

amounts were prepared l-THC-d3

 was separa- THC-d2 by dehydrogenation with sulphur

ted from 6-isomer on a column of Florisil at 260°C (Petrzilka et al., 1969). The

using 2% ether in light petroleum as sol- deuterium content (m/e 310-312): d0

 12%,

vent. 1

-THC-d3 was 95% pure according d1

 46%, d2 100%.

to GLC and the deuterium content (m/e

314-317: d0

 2%, dl 6%, d2 12%, d3 100%.

CBN-d3 eluted with 2-10% ether in light

petroleum from the Florisil column was

6

-THC-d4. The synthesis of this compound

(98.5% pure) was described previously

(Agurell et al., 1974); deuterium content (m/e 314, 318): d0

 1.5%, d4 100%.

95% pure according to GLC with a deucerium

content (m/e 310-313): d0 2%, d1 5%, d2

15%, d3 100%.

Olivetol-d2. The methyl ester of 3,5-

dimethoxybenzoic acid was reduced with

LiAld4

 in tetrahydrofuran and the resulting alcohol was reacted with PBr3

.

(See olivetol-d7). The bromide crystallized from ether-light petroleum was

mixed with Cu(I)I in ether and butyl

lithium was added drop-wise to the

mixture (Petrzilka et al., 1969). The

obtained 0,0-dimethylolivetol-1',1'-d2

was distilled at 110° C, 0.15 mm Hg and

Non-labelled cannabinoids. (

1

-THC,

6

-THC, CBN, CBD) were synthesized according to standard procedures and carefully purified and dried before use. Stock

solutions were maintained in ethanol

(1-5 mg/ml, 4° C, stored in the dark).

GLC was carried out on a Varian 2100

FID gas chromatograph using 2 mm (i.d.)

x 180 cm glass columns with 2% SE-30

ultraphase on Gas Chrom Q (100-120 mesh)

at 250°C.

Analysis of THC in blood plasma

showed the following deuterium content The following procedure was used for 6

-

(m/e 180-182): d0 0.3%, d1 4%, d2 100%. THC (Agurell et al., 1974). Analogous

After demethylation with hydriodic acid procedures utilizing the proper deutera-

(cf. Agurell et al., 1973) olivetol-d2 ted internal standard and mass numbers

was purified on a silica column with are applicable for 1

-THC, CBN, and CBD.

methanol-chloroform of increasing pola- The procedure for l

-THC has been pubrity as eluent (from 3% methanol). lished (Agurell et al., 1973).

52
Blood samples. Blood samples (10 ml) were

collected as desired in heparinized tubes

after smoking of the 6-THC sample.

Plasma is obtained by centrifugation and

stored in silanized glass tubes at -20°C

until analysed.

Extraction. To a 5.0 ml plasma sample is

added 100 ng (THC levels above 5 ng/ml)

or 20 ng (below 5 ng/ml) of deuterated

internal standard (

6

-THC-d4

) dissolved

in 50 μl ethanol. The plasma sample is

extracted three times with an equal volume

of light petroleum containing 1.5% isopentanol in a glass stoppered centrifuge

tube. After centrifugation the light

petroleum is drawn off and the combined

organic extracts evaporated under nitrogen at 50°C almost to dryness. This extract is quantitatively transferred to

the Sephadex LH-20 column using three

0.2 ml portions of the elution solvent.

Liquid chromatographic purification

Mantled silanized glass columns (1 x 40 cm,

void vol. ca. 15 ml) operated at 12°C

(const. temp. water cooling system) containing Sephadex LH-20 and eluted with

light petroleum-chloroform-ethanol

(10:10:1) were used for purification of

plasma extracts. Each column was provided

with a 200 ml solvent reservoir and after

each purification the column was washed

with 40 ml solvent, If not in use, the

columns were washed twice weekly with

fresh solvent to maintain constant elution

volumes.

The elution volume for 6-THC was determined by calibration with ng amounts of

6

-THC-3H (Fig. 2). The calibration can

also be carried out by GLC analysis of

Figure 2

Fig. 2. Elution pattern for 6

-THC-3H, l-THC,

CBD and CBN from a Sephadex: LH-20 column.

the concentrated fractions after application of 10 μg quantities to the column.

The column was run at 0.2 ml/min and the

pertinent 7-8 ml fraction was collected

and evaporated under nitrogen. The

residue was dissolved in ethanol and

transferred to a 50 μl conic vial

(Reactivial, Pierce) dried, and finally

dissolved in 10-15 μl of ethanol and

stored at 4°C in the dark until analysis.

This solution was subjected to mass

fragmentography.

Mass fragmentography was carried out

using an LKB 9000 GC-MS instrument. The

column was a 1.4 m x 2 mm i.d. silanized

glass column containing 3% OV-17 on Gas

Chrom CLP 100/120 mesh. Temperatures

were in the column 180-210°, flash heater

250°, and source 290°. Helium was carrier

gas (25 ml/min) and typical retention

times are: CBD 3.4 min, 6-THC 4.0 min,

1

-THC 4.5 min, CBN 6.1 min. For mass

fragmentography a multiple ion detector

was added (Elkin et al., 1973) and used

53
at 50 eV. For 6

-THC, the mass spectrometer was set to continously record the

intensities of m/e 314 (molecular ion

of non-labelled 6

-THC) and m/e 318

(internal standard, 6-THC-d4

) as well as

m/e 299, 303 as shown in Fig. 3.

Figure 3

Fig.3. Mass fragmentation of 6

-THC-d4(

8

-THC-d4)

as internal standard (m/e 318, 303) and 6-THC

(m/e 314, 299) from purified plasma extract.

Standard curves were prepared by adding

known amounts of 6-THC (0.5-100 ng/ml)

to blank plasma samples and carrying out

the described procedure (Fig. 4). The

correctness of the standard curves were

checked during the day.

Figure 4

Fig. 4. Standard curves for 6-THC (

8

-THC) in

plasma. 0-1- ng/ml (left), 10-100 ng/ml (right).

RESULTS AND DISCUSSION

Principles

The described method for the determination

o f 6-THC (cf. Agurell et al., 1974) in

blood is basically identical to the one

published for l-THC (Agurell et al.,

1973). The method is based upon the addition of the proper deuterated internal

standard ( 6-THC-d4) due to the blood plasma

sample. After extraction with light petroleum, the 6

-THC containing fraction is

purified by liquid chromatography on a

Sephadex LH-20 column. The pertinent

fraction containing 6-THC (absorbed by

smoking a spiked Cannabis sample) and

6

-THC-d4 (added as internal standard)

is collected. The relative amounts of the

two compounds are determined by mass fragmentography monitoring the molecular peaks

(m/e 314 and 318, respectively; Fig. 3.).

54
As subsequently discussed, this method can

also be used for the analysis of other cannabinoids, such as THC:s of different molecular weight, CBD, and CBN. However, at

present less information is available with

regards to these latter compounds.

1"- and 2"-positions of the pentyl side

chain (Fig. 3).

There are generally only ng-amounts of

cannabinoids present in blood plasma and

although the deuterated internal standards

serve as carriers, the method requires

scrupulously clean, silanized glass ware

and redistilled solvents.

Other deuterated cannabinoids. A synthesis

o f 1-THC-d4 was reported earlier

(Agurell et al., 1973). In the present

paper the syntheses of olivetol-d7,

olivetol-d3, and olivetol-d2

 - mainly

based upon the procedures of Pitt et al.

(1975) - are outlined. From these intermediates, correspondingly labelled 1

-THC,

CBD, and CBN can be prepared as exemplified

in Fig. 1.

Reference cannabinoids

In all methods pure cannabinoids are necessary for identification and calibration purposes. Since cannabinoids are

often rather unstable and, with few exceptions, not crystalline - to obtain

and maintain the purity of cannabinoids

is not an entirely easy task. However,

purification on pre-washed TLC plates as

described previously (Agurell et al.,

1974) followed by careful drying and

storage in ethanol solution at 4° in the

dark provides cannabinoid standard solutions of satisfactory stability.

The syntheses of the deuherium labelled

compounds were accomplished by condensation

of suitable deuterated olivetols and (+)-

trans-p -mentha-2,8-dien-1-ol according to

standard methods (Mechoulam et al., 1976)

- Fig. 1.

6

-THC-d4

. The synthesis of this internal The mass spectra of 1

-THC-d7 and nonstandard was described previously (Agurell labelled 1

-THC are shown in Fig. 5

et al., 1974). The label is located in the together with 1

-THC-d 3.

The sensitivity in the final mass fragmentographic assay may be limited by the

amount of non-labelled (d0) compound

present in the deuterated internal standard.

This interferes with the non-labelled cannabinoid present in the plasma. Thus, we have

tried to minimize this interference by preventing exchange reactions, increasing the

number of hydrogens substituted with deuterium, and by limiting the amount of internal standard in samples containing low

amounts of cannabinoids (Fig. 5). Thus,

the present limit of sensitivity (ca. 0.3

ng THC/ml plasma) is partly due to the

amount of THC-d0

in the internal standard

and not due to chromatographic or mass

spectrometric problems per se . As expected,

d7

-containing 1

-THC, CBD, and CBN showed,

together with 1-THC-d3, the least contamination with d0

-analogues (2%). This is

in the same range as found for 6-THC-d4

(1.5%; Agurell et al., 1974).

55
Figure 5

Fig. 5. Mass spectra of 1

-THC, 1-THC- d3, and 1THC-d7.

56
Extraction and purification bolites before mass fragmentography. The

elution volume for e.g. 6-THC is stable

for months if the column is operated at

low const. temp. (12°C) and, when not in

use, washed with fresh solvent regularly.

Our present set-up contains five columns

which can be handled by a technician

(10 samples per day). However, such systems can be automated (Sippell et al.,

1975). Over 90% of the 6-THC peak is

eluted in a 5 ml volume (Fig. 2) but a

7-8 ml fraction is collected. As a precaution ca. 3-4 ml on each side of the

peak is collected and stored.

6

-THC and its 1-THC isomer appear to be

stable in human plasma for months if

stored at -20°C i silanized glass tubes.

The extraction and purification procedures

for 1-THC, CBD, or CBN are analogous.

The extraction procedure for 6-THC, as

revealed by experiments with 6-THC-3H,

is quite efficient and the recovery after

both extraction and liquid chromatography

is usually over 80%. Also. early studies

on

1

-THC showed recoveries of 70 ± 6%

(s.d.) after the column purification

(Agurell et al., 1973).

The Sephadex LH-20 separation is essential

in removing interfering lipids and metaWe have investigated the elution patterns

of different cannabinoids and their metabolites on the Sephadex LH-20 column

(Fonseka et al., 1976). This is shown in

Fig. 6.

Figure 6

Fig. 6. Elution patterns of l

-THC, 6-THC, CBN, and CBD and their mono-oxygenated

derivatives on Sephadex LH-20. Abbreviations as exemplified by the l

-series 6-0 =

6-oxo- l

-THC, 3"-OH= 3"-hydroxy- l-THC, 6 , 7-dihydroxy- l- THC, etc.

57
The complexity of cannabinoid metabolism in

man and animal is indeed great and the requirements for specificity when assaying a

certain cannabinoid are consequently considerable. To give an impression of the

complexity, we have in a review (Agurell

et al., 1976 b) listed 35 metabolites of

1

-THC, 6-THC, CBN, and CBD, which have

been isolated in our and other laboratories

and are substituted in the pentyl side

chain. To make a complete list an equal

number of metabolites oxygenated in the

terpene nucleus (cf. Mechoulam et al.,

1976) should be added.

Mass fragmentography

The sensitivity achieved in the quantification of 6

-THC and other cannabinoids is

partly due to the liquid chromatography

clean-up of blood plasma extract. Mainly,

however, the sensitivity and specificity is

dependent upon the mass fragmentographic

analysis.

We have used the deuterium labelled analogue

6

-THC-d4 for the analysis of 6-THC in blood

plasma from four male, casual Cannabis smokers

who had smoked 8 mg of 6-THC. Such a mass

fragmentogram is shown in Fig. 3.

Two standard curves were prepared (0-10 and

10-100 ng/ml) by adding known amounts of

6

-THC to blank plasma samples and carrying

out the described procedure. Peak heights

o f 6

-THC-d4

 (m/e 318) were plotted against

known amounts of 6-THC (Fig. 4). Since

the same ratios were obtained simply by

mixing known amounts of 6-THC and

6

-THC-d4, usually such standard curves

were used. The correctness of the standard curves should be checked occasionally

during the day of analysis.

There are advantages as well as disadvantages in the use of deuterium labelled

analogues as internal standards. They are

carriers for the minute amounts of cannabinoids present in biological samples

and, being almost identical to the analyzed compound they can be added to the

original plasma sample and will then compensate for variations in extraction and

purification recoveries. A disadvantage

is that deuterium labelled standards

have to be synthesized. Hopefully these

might be provided by NIDA.

The present method for 6

-THC can be used

down to 0.3 ng/ml. As pointed out (see

discussion on "Reference cannabinoids")

the sensitivity is partly limited by the

small amount of 6-THC-d0 present in the

deuterated internal standard. Thus, we

have tried to eliminate do-contamination

in the internal standards for 6-THC,

l

-THC, CBD, and CBN - with best results

in the d7-analogues.

The d3-compounds can also be used as

standards but were mainly synthesized to

study single dose pharmacokinetics of

1

-THC, CBD, and CBN in heavy hashish

users. These users presumably have a high

"steady-state" blood level of cannabinoids

and their metabolites. By the use of a

d3-labelled single dose of e.g. 1

-THC,

the kinetics of a particular 1-THC dose

can be followed in spite of high background

levels of non-labelled 1-THC (Fig. 7a). The

d7

-labelled 1-THC is then used as internal

standard (Fig. 7b).

l

-THC and CBD can both be determined in the

same mass fragmentogram using the same

channels - m/e 314 and e.g. 321 for internal standard - whereas CBN is simultaneously

assayed using m/e 310 and 313 (Fig. 7c).

58
Figure 7a Figure 7b

Fig. 7 a. Mass fragmentation of purified

plasma extract containing 1-THC (0.9 ng/ml

plasma, m/e 314) and 1-THC-d3 as internal

standard (m/e 317). Column temp. 210°C.

Fig. 7 b. Mass fragmentogram of l

-THC-d3, and

l

-THC,

1

-THC-d7 (ca. 5 ng/ml plasma

of each). Column temp. 210°C.

So far we have not developed assays for

any of the possibly important l

-THC metabolites, e.g. 7-hydroxy- l

-THC. However,

it is likely that if quantitative metabolic

Studies in man warrant, this metabolite

could also be quantitated after elution and

derivatization using mass fragmentography.

It is also possible that cannabinoids monohydroxylated in the side chain can be assayed

as their trimethylsilyl ethers using specific fragments (Binder et al., 1974). Side

chain hydroxylation seems to be a general

metabolic route and of importance in at

least some species (Agurell et al., 1976 a,

Harvey & Paton, 1976). Hydroxylation in

the 3"- and 4"-positions are favored -

reactions which also seem to confer high

psychotomimetic activity in the rhesus

monkey (Agurell et al., 1976 a).

59
Figure 7c

Fig. 7 c. Mass fragmentograms of CBD (m/e

314) and internal standard CBD-d2 (m/e 316,

retention time 3.0); l-THC (m/e 314) and

internal standard l

-THC-d3(m/e 317, retention time 4.0); and CBN (m/e 310) and

internal standard CBN-d3 (m/e 313, retention

time 5.0). Column temp. 220° C.

Plasma levels

The plasma levels of 6-THC in man after

smoking 8 mg 6-THC - of which about half

is absorbed in the lungs - are shown in Fig. 8.

Immediately after smoking high values

(>100 ng/ml) of 6-THC are recorded but

drop rapidly to 10-20 ng/ml at 0.5 hour

and are about 1 ng/ml at 4 hours. The

present sensitivity of 0.3 ng/ml, thus

allows THC levels to be followed for

12-24 hours. This is more than enough to

study any correlations of 6-THC levels

with physiological and psychological

effects (Agurell et al., 1974) but not

enough to establish a true elimination

phase. Lemberger and co-workers (1971)

have estimated elimination phase halflifes in man of 1-2 days.

Garrett and Hunt (1976 b) have shown

that the terminal half-life of l-THC

in the dog is reached only slowly. They

also found that the return of l-THC from

the tissues is the rate determining step

of the drug elimination process after the

initial distribution and metabolism.

Similar plasma levels of l-THC as for

6

-THC (Fig.8 ) have earlier been found

in man by us (Agurell et al., 1973) and

Rosenfeld et al. (1974) using mass fragmentography, and by e.g. Galanter et al.

(1972) using 1

-THC-14C. Wall and coworkers (1974) have published plasma

levels of both l-THC, CBD, and CBN and

certain metabolites as well as subjective

psychological effects after i.v. administration of the labelled drug.

Hence, the sensitivity requirements for

the determination of l-THC are indicated

in Fig. 8. Plasma levels will obviously

be modified by the amount of THC absorbed

and by the rate of absorption but if

levels are to be measured later than 4

hours after administration a sensitivity

of 1 ng/ml is required. Such a sensitivity with sufficient specificity is perhaps

limited to the mass fragmentographic tech60
Figure 8

Fig. 8. Plasma levels of 6-THC after smoking 8.3 mg 6-THC.

niques. We have so far encountered little Radioimmuno assay procedures, where also

interference in the mass fragmentographic certain metabolites cross-react, would

determination of l-THC provided redistilled clearly be applicable to the qualitative

solvents, particularly ethanol, and all identification of Cannabis users. However,

silanized glass ware are used. the potential cross-reactivity to e.g.

61
steroids and other drugs also has to be

ascertained.

purification. With the non-automated system

now in use, a technician can process about

ten plasma samples per day. This might be

The capacity of the mass fragmentographic improved by automation, by high pressure

technique is more limited than the radio- liquid chromatography or by using the double

immune assay procedure, the main time re- extraction technique of Rosenfeld et al.

quiring step being the liquid chromatography (1974).

ACKNOWLEDGMENTS

The support of the Swedish Medical Research

Council is appreciated. The mass spectrometric

work was supported by grants to the Department

of Toxicology.

REFERENCES

Agurell, S., Gustafsson, B., Holmstedt, B.,

Leander, K., Lindgren, J.-E., Nilsson, I.,

Sandberg, F. & Åsberg, M. (1973). J. Pharm.

Pharmac. 25, 554-558.

Agurell, S., Levander, S., Binder, M.,

Bader-Bartfai, A., Gustafsson, B.,

Leander, K., Lindgren, J.-E., Ohlsson,

A. & Tobisson B. (1974). Pharmacokinetics

o f 8-THC in man after smoking - relations

to physiological and psychological effects.

Presented at Conference on the pharmacology

of Cannabis, Savannah, Dec. 3-6, 1974. In

press in Pharmacology of Cannabis, Eds.

S. Szara & M. Braude, Raven Press, New

York, USA.

Agurell, S., Binder, M., Fonseka, K.,

Gustafsson, B., Lindgren, J.-E., Leander,

K., Martin, B., Nilsson, I.M., Nordqvist,

M., Ohlsson, A. & Widman, M. (1976 a).

Cannabinoids: Metabolites hydroxylated in

the side chain. In press.

Agurell, S., Martin, B. & Widman, M.

(1976 b). To be published.

Binder, M., Agurell, S., Leander, K.

& Lindgren, J.-E. (1974). Helv. Chim. Acta

57, 1626-1641.

Buchta, E. & Andree, F. (1959). Ber. 92,

3111.

62
Buchta, E. & Andree, F. (1960). Ber. 93,

1349.

Cais, M., Dani, S., Josephy, Y., Modiano,

A., Gershon, H. & Mechoulam, R. (1975)

FBBS 55, 257-260.

Elkin, K., Pierrou, L., Ahlborg, U.G.,

Holmstedt, B. & Lindgren, J.-E. (1973)

J. Chromatogr. 81', 47-55.

Fenimore, D.C., Freeman, R.R. & Loy,

P.R. (1973). Anal. Chem. 46, 2232-2234.

Fonseka, K., Widman, M.& Agurell, S.

(1976). J. Chromatogr. In press.

Galanter, M., Wyatt, R.J., Lemberger, L.,

Weingartner, H., Waughan, T.B. & Roth,

W.T. (1972). Science 176, 934-935.

Garrett, E.R. & Hunt, C.A. (1973). J.

Pharm. Sci. 62, 1211-1214.

Garrett, E.R. & Hunt, C.A. (1976 a).

Separation and sensitive analysis of

tetrahydrocannabinol in biological fluids

by HPLC and GLC. To be published.

Garrett, E.R. & Hunt, C.A. (1976 b).

Pharmacokinetics of 9-THC in the dog.

To be published.

Grli , L. (1974). Acta Pharm. Jugoslav.

24, 63-72.

Gross, S.J., Soares, J.R., Wong, S.L.R.

& Schuster, R.E. (1974). Nature 252,

581-582.

Harvey, D.J. & Paton, W.D.M (1976). Examination of the metabolites of 1

-tetrahydrocannabinol in mouse liver, heart and lung

by combined gas chromatography and mass

spectrometry. In press.

Lemberger, L., Axelrod, J. & Kopin, I.J.

(1971). N.Y. Acad. Sci. 191, 142-152.

Loeffler, K.O., Green, D.E., Chao, F.C.

& Forrest, I.S. (1975). Proc. West.

Pharmacol. Soc. 18, 363-368.

McCallum, N.K. (1973). J. Chromatogr.

11, 509-511.

Mechoulam, R., Yagnitinsky, B. & Gaoni, Y.

(1968). J. Am. Chem. Soc. 90, 2418.

Mechoulam, R., McCallum, N.K. & Burstein,

S. (1976). Chem. Rev. In press.

Petrzilka, T., Haefliger, W. & Sikemeier, C.

(1969). Helv. Chim. Acta 52, 1102-1118.

Pitt, C.G., Hobbs, D.T., Schran, H., Twine

Jr., C.E. & Williams, D.L. (1975). J. Label.

Comp. 11, 551-575

Rosenfeld, J.J., Bowins, B., Roberts, J.,

Perkins, J. & McPhersson, A.S. (1974).

Anal. Chem. 46, 2232-2234.

Sippell, W.G., Lehman, P. & Hellman, G. (1975)

J. Chromatogr. 108, 305-312.

Teale, J.D., Forman, E.J., King, L.J., Piall,

E.M. & Marks, V. (1975). J. Pharm. Pharmac.

27, 465-472.

Wall, M.E., Brine, D.E. & Perez-Reyes, M.

(1974). Metabolism of cannabinoids in man.

Presented at Conference on the pharmacology of Cannabis, Savannah, Dec. 3-6,

1974. In press in Pharmacology of Cannabis, Eds. S. Szara & M. Braude, Raven

Press, New York, USA.

63
A METHOD FOR THE IDENTIFICATION OF ACID

METABOLITES OF TETRAHYDROCANNABINOL (THC)

BY MASS FRAGMENTOGRAPHY

Marianne Nordqvist1, Jan-Erik Lindgren2,3, Stig Agurell1,3

1

Faculty of Pharmacy, University of Uppsala, Uppsala

2Department of Toxicology, Karolinska Institutet, Stockholm

3Astra Läkemedel AB, Södertälje, Sweden

INTRODUCTION

Plans to identify Cannabis users by the

detection of 1-THC in urine had to be

abandoned after the reports by Lemberger

et al. (1970, 1971). Their experiments

with humans given 14C- 1-THC i.v. and

later work by Wall et al. (1974) showed

that the drug is almost completely metabolized and the metabolites excreted

mainly via faeces. Most of the urinary

radioactivity derived from acidic metabolites. The contents of unchanged l

-THC

were less than 0.02% of the administered

dose. Only small amounts of 7-hydroxy- l-

THC, the major metabolite of 1-THC in

vitro, were indicated in the urine by TLC

(Lemberger et al., 1970). In contrast,

the faecal contents of 7-hydroxy- 1-THC

are high, about 20% (Lemberger et al.,

1971; Wall et al., 1974). The low amounts

o f l-THC and also 7-hydroxy- l-THC excreted in the urine seem to be a result

of further metabolism yielding more polar

compounds with the 7-methyl group being

further oxidized to a carboxyl group via

the aldehyde (Ben-Zvi & Burstein, 1974)

with or without introduction of additional

hydroxyl group(s). The l

-THC-7-oic acid

has been identified as a major metabolite

in urine, faeces, and plasma from humans

given l

-THC or 7-hydroxy- l-THC i.v.

(Wall et al., 1974).

64
A method for detection of l

-THC or 7-

hydroxy- l-THC in urine or in plasma

samples taken more than 12 h after the

inhalation of a normal occasional dose of

l

-THC would have to be extremely sensitive. The plasma level of unchanged drug

is then less than 0.3 ng/ml, which is

the limit of our method for detection of

THC. 7-Hydroxy- l-THC, 6 -hydroxy- 1-THC,

-hydroxy- l

-THC and 6,7-dihydroxy- 1

-

THC are all minor metabolites in plasma

with concentrations much lower than that

of the parent compound at all times, according to Wall et al. (1974). On the other

hand, the amount of l

-THC-7-oic acid in

plasma is almost equal to that of 1-THC

within 50 min and then remains slightly

higher for more than 24 h.

The possible occurrence of other major acid

metabolites must not be overlooked, as

only l-THC-7-oic acid is identified in

man so far. The major ones isolated from

rabbit urine - 1"- and 2"-hydroxy- l-THC7-oic acid (Burstein et al., 1972) and

4",5"-bisnor- 1-THC-7,3"-dioic acid

Nordqvist et al., 1974) could also be expected to be formed in man by further

hydroxylation and oxidation of 7-hydroxyl

-THC. The unidentified part of metabolites

in human plasma, urine, and faeces classified

as more polar acids by Wall et al. (1974)

are probably similar to the hydroxylated

THC-oic acids identified by Harvey and

Paton (1976) in mice.

Our proposed method for the identification

of Cannabis intoxication is based upon

the finding that l-THC-7-oic acid is a

major metabolite. For forensic purposes,

when the identification but not accurate

quantitation is required, it should be a

practical method, if gas chromatography -

mass spectrometry (GC-MS) equipment is

available. It is related to our method for

detection of THC in blood plasma (Agurell

et al., 1973) and can as described here be

used for qualitative identification and

semiquantitative assay of a major acidic

metabolite in urine and plasma from

Cannabis users. This method is probably

with slight modifications applicable also

to related acid metabolites.

METHODS

Analysis of THC-7-oic acid in blood plasma

The procedure used was a modification of

that described for 1-THC (Agurell et al.,

1973). All glass ware was carefully washed

and silanized before use. The solvents were

of analytical grade and those constituting

the eluent mixture were distilled twice.

Extraction. 50 ng 6

-THC-7-oic acid

(Mechoulam et al., 1973) dissolved in 25

μl ethanol was pipetted into a glass

stoppered centrifuge tube containing 2.5

ml human plasma. It was equilibrated and

2.5 ml 1 M citrate-HCl buffer pH 4.1 was

added 5 min before extraction with 10 ml

diethyl ether. After centrifugation for

10 min at 4000 rpm the ether layer was

drawn off, transferred into a conic tube

and evaporated to dryness under a stream

of nitrogen at 40°.

Methyl ester formation. The residue was

dissolved in 100 μl methanol, an etheral

solution of diazomethane was added in

excess and the solution allowed to react

for 10 min before evaporation of reagent

and solvent under nitrogen.

65
C!olumn chromatography. The resulting

residue was transferred to a jacketed

Sephadex LH-20 column (1 x 55 cm, V0 =

17 ml) using three consecutive 200 μl

portions of the elution solvent light

petroleum-chloroform-ethanol (10:10:1).

At the elution rate 0.2 ml/min and the

temperature 12° the methyl ester of 6

-

THC-7-oic acid was eluted between 37 and

47 ml. This fraction was collected in a

10 ml conic tube. The solvent was evaporated under nitrogen, the residue dissolved

in ethanol and transferred to a' 1.0 ml

conic vial, where it was stored at 4' in

the dark until analysis.

Silylation. On the day of analysis the

methylated extract was dried under nitrogen,

dissolved in 25 μl dry acetonitrile, mixed

with 10 μl silylating agent [N,0-bis-(trimethylsilyl)acetamide] and kept at 50-60°

for 10 min. It was then dried again under

nitrogen and redissolved in 25 μl acetonitrile.

Mass fragmentography. The silyl ether of

6

-THC-7-oic acid methyl ester was subjected

to mass fragmentography (3% SE-30 Gas Chrom

Q. 100/120 mesh, 220°). The mass spectrometer

(LKB Model 9000) was adjusted to record the

intensity of m/e 430 (M+), 415, and 374 at

70 eV on three different channels. For

further details, see previous paper in this

volume (Fig. 1).

Analysis of THC-7-oic acid in urine

The procedure described above for analysis

of human plasma can also be applied to

urine. In a preliminary study we equilibrated

100 ng 3H- 6-THC-7-oic acid for 20 min with

5.0 ml human urine and added 2.5 ml 1 M

citrate-HCl buffer to maintain pH 4.1 during

the extraction with equal volume of diethyl

ether (7.5 ml x 1).

Figure 1

Fig. 1. Mass fragmentograms of the silyl

ethers of the isomeric THC-7-oic acid methyl

esters from purified plasma extracts.

RESULTS AND DISCUSSION

The partition of 3H- 6-THC-7-oic acid (prepared by acid catalyzed exchange) between

buffer solutions of different pH and an

organic phase (diethyl ether, light petroleum with 1.5% isopentanol, and toluene,

respectively) was studied to determine the

combination of pH and extraction solvent

giving the best recovery. Diethyl ether

provedtobe the most efficient solvent

(log D = 2.2, pH 4.1). The recovery from

66
plasma of added 3H- 6

-THC-7-oic acid (in

the range of 50-200 ng) was over 85%

after extraction with a double volume of

ether. The recovery from urine was over

90% using equal volumes. The major in

vivo metabolite l-THC-7-oic acid is expected to behave almost identically.

The diazomethane was effective in esterifying the 6

-THC-7-oic acid but the

corresponding acid metabolite of l-THC

was more difficult to esterify. However,

the proceduredescribed using a 10 min

incubation with excess of reagent was

applicable to both acids. The elution

volume on Sephadex LH-20 of the methyl

ester of 6-THC-7-oic acid was determined

by calibration with ng amounts of tritiated compound. From earlier experiments

l

-THC is known to have an elution volume

slightly higher than that of 1-THC. In

our study the delayed elution of the

ester of l-THC-7-oic acid, in comparison

to its 6

-isomer, had to be compensated

for by a wide fraction of collection.

Total recovery of 3H- 6-THC-7-oic acid

in plasma after extraction, methylation,

and column purification was around 70%.

The silylated methyl ester of 6-THC7-oic acid is not fully separable from the

l

-isomer on the conventional GLC columns

tested (SE-30, SE-52, OV-17, JXR, XE-60).

When not silylated, the methyl esters

were resolved but that of the 1-isomer

partly decomposed on the column. Thus,

the silylated methyl ester of 6-THC-7-oic

acid was used as an external standard for

the l-isomer since both compounds have

very similar retention times but different

intensities in the mass fragmentograms

(Fig. 2, 3). The 6-isomer can also serve

as a reference for semiquantitative estimaFigure 2

Fig. 2. Mass spectra of the silyl ethers of 6

- (upper panel) and l-THC-7-oic acid

methyl ester (lower panel).

67
Figure 3a Figure 3b

Fig. 3. a. The silyl ether of l

-THC-7-oic

acid methyl ester (m/e 430, 374, 415).

tions of l-THC-7-oic acid in plasma or urine.

The possible concomitant occurrence of 6-

THC-7-oic acid in plasma or urine samples

from smokers should be of little significance

since 6-THC at most occurs in 2% of the

amount of 1

-THC in Cannabis (Ohlsson et al.,

1977). There is only a limited need for

an accurate quantitation method for l-

THC-7-oic acid since pharmacological test

with Rhesus monkeys (Edery et al., 1971)

have shown that the isomeric 6-THC-7-oic

Fig. 3. b. The silyl ether of 6

-THC-7-oic

acid methyl ester.

acid is inactive in doses up to 10 mg/kg

i.v. If necessary, the deuterium labelled

l

-THC-7-oic acid can be synthesized as

an internal standard according to methods

described by Pitt and Wall (1974) and Pitt

et al., (1975).

There are no studies on the urinary excretion

rates of l-THC-7-oic acid in humans after

Cannabis smoking. However, the studies on

l

-THC metabolism in humans by Wall et al.

(1974) suggest that identification of this

acid may be a practical method to identify

Cannabis users. The sensitivity of the

method has not been thoroughly tested yet

but is at least in the range of a few ng/ml.

Further studies are in progress.

68
ACKNOWLEDGMENTS

The support of the Medical Research

Council was appreciated. Mass spectrometry was supported by grants to the

Department of Toxicology. Reference

acids were kindly provided by Drs. R.

Mechoulam and M. Wall.

Lemberger, L., Silberstein, S.D., Axelrod,

J. & Kopin, I.J. (1970). Science, 170,

1320-1322.

Lemberger, L., Axelrod, J. & Kopin, I.J.

(1971). N.Y. Acad. Sci., 191, 142-152.

Mechoulam, R., Ben-Zvi, Z., Agurell, S.,

Nilsson, I.M., Nilsson, J.L.G., Edery, H.

& Grunfeld, Y. (1973). Experientia, 29,

1193-1195.

Nordqvist, M., Agurell, S., Binder, M.

& Nilsson, I.M. (1974). J. Pharm. Pharmac.

6, 471-473.

REFERENCES

Agurell, S., Gustafsson, B., Holmstedt, B.,

Leander, K., Lindgren, J.-E., Nilsson, I.,

Sandberg, F. & Åsberg, M. (1973). J. Pharm.

Pharmac. 25, 554-558.

Ben-Zvi, Z. & Burstein, S. (1974). Res.

Commun. Chem. Pathol. & Pharmacol.

8, 223-229.

Burstein, S., Rosenfeld, J. & Wittstruck, T.

(1972). Science N.Y. 176, 422-423.

Edery, H., Grunfeld, Y., Ben-Zvi, Z. &

Mechoulam, R. (1971). Ann. N.Y. Acad. Sci.

191, 40-53.

Harvey, D.J. & Paton, W.D.M. (1976).

Examination of the metabolites of l-

tetrahydrocannabinol in mouse liver,

heart and lung by combined gas chromatography and mass spectrometry. In press.

Ohlsson, A., Abou-Chaar, C.I., Agurell,

S., Nilsson, I.M., Olofsson, K. &

Sandberg, F. (1971). Bulletin on Narcotics, 23, 29-32.

Pitt, C.G., Fowler, M.S., Sathe, S.,

Srivastava, S.C. & Williams, D.L. (1975)

J. Am. Chem. Soc., 97, 3798-3802.

Pitt, C.G.& Wall, M.E. (1974). Synthesis

of radiolabeled cannabinoids and metabolites. Annual report from Research

Triangle Institute, North Carolina, USA

to National Institute on Drug Abuse.

Wall, M.E., Brine, D.E. & Perez-Reyes,

M. (1974). Metabolism of cannabinoids

in man. Presented at Conference on the

pharmacology of Cannabis, Savannah, Dec.

3-6, 1974. In press in Pharmacology of

Cannabis, Eds. S. Szara & M. Braude,

Raven Press, New York, USA.

69
QUANTITATION OF CANNABINOIDS IN BIOLOGICAL

SPECIMENS USING PROBABILITY BASED

MATCHING GC/MS

Donald E. Green, Ph.D.

Veterans Administration Hospital

Palo Alto, California

INTRODUCTION

The major effort in our laboratory in the

development of THC analytical methodology

has been on evaluating the efficacy of extraction procedures, on the TLC fractionation of

individual in vivo metabolites, and on the

development of an automated GC/MS quantitative procedure. For the first two aspects,

we have relied heavily upon 14C-studies. In

California it is almost impossible to administer 14C-labeled drugs to humans for research

purposes, so our model animals were usually

the higher primates, Rhesus and baboon (PPA).

We found that the excretion rates of total

radioactivity in both urine and feces for

Rhesus monkey and for man (Lemberger et al,

1971) were very similar (Figures 1 and 2), so

most of our early development efforts employed Rhesus specimens.

EXPERIMENTAL

Baboon feces

Extractions: Fecal specimens, although

of no direct practical importance, were useful for orientation and for methods developFigure 1

Figure 1. Comparative urinary excretion of

9

-THC and its metabolites in man and three

primates.

70
Figure 2 Table 1

Figure 2. Comparative cumulative fecal

excretion of parent drug and metabolites

after oral administration of 9-THC in man

and three primates. (Human data from

Lemberger et al, 1971)

ment because they contained copious quantities of the parent drug and its less extensively metabolized forms. (In this latter

sense, they can serve as a general model for

THC in blood.) In order to minimize the un

pleasantness of working with feces, a general

technique, based upon continuous extractions,

was developed. In typical experiments in

which 14C- 9-THC was administered to chronically THC-dosed baboons, 24-hour fecal collections were homogenized in methanol using

a Waring Blender and then transferred into a

soxhlet thimble where overnight extraction

with methanol removed more than 99% of the

total counts.

Methanolic extracts from individual specimens

were evaporated to dryness and the residues

were suspended in distilled water and extracted for 48 hours with petroleum ether

in a liquid/liquid extractor (Fraction I).

The aqueous phases were adjusted to pH 10 and

extracted with diethyl ether for 18 hours

(Fraction II). After adjusting the aqueous

phases to pH 2.5, final ether extractions were

continued for another 18 hours (Fraction III).

These three extractions removed nearly 90% of

the original counts as shown in Table 1 (Green

et al, 1975).

Fraction I, which contained 4/5 of the total

radioactivity, was separated by TLC into three

main groups of compounds: A non-polar band

Continuous Liquid/Liquid Extractions

of Soxhlet-Extracted Residues

Petroleum Ether (neutral pII) 80.2%

Diethyl Ether (pII 10) 3.2

Diethyl Ether (pII 2.5) 6.4

Total Recovery 89.8%

Figure 3

Figure 3. Thin layer radiochromatogram of

petroleum ether extract of baboon feces.

(Silica gel, petroleum ether/diethyl ether

1:1, 3 passes.)

(42%) near the Rf of 9-THC; a moderately

polar diffuse region (9%) in the approximate

position of mono-hydroxy- 9-THC's; and some

highly polar compounds near the origin (16%)

(Figure 3). A corresponding non-radioactive

petroleum ether extract - obtained from feces

collected on the day prior to radio-label dosing - was separated by preparative TLC (on

silica gel with 3 passes in chloroform) into

5 Fast Blue B-reacting bands: Band I-A, Rf

0.88; Band I-B, Rf 0.68; Band I-C, Rf 0.14;

Band I-D, Rf 0.12; and Band I-E, Rf 0.08.

High pressure liquid chromatography: An

aliquot of Band I-A was dansylated and a small

portion was analyzed using a gradient, high

pressure liquid chromatograph (Varian AeroGraph Model 8500) equipped with a 25 cm NH2

-

bonded 10 μ silica column and an experimental

filter fluorometer detector (Figure 4)

71
Figure 4 Figure 5

Figure 4. High pressure liquid chromatography of Dansylated Fraction I-A from petroleum ether extract of baboon feces (top) and

mixture of Dansylated standards (bottom).

(Loeffler et al, 1975). From this chromatogram, it can be seen that I-A had a major component with the same retention time as 9

-THC.

Minor components at the approximate retention

times of CBN and CBD were also present.

Gas chromatography/mass spectrometry: A

similar aliquot of Band I-A was silylated with

BSTFA and analyzed by GC/MS (Figure 5). The

automated GC/MS report (the inset in Figure 5)

shows that 9-THC and CBN were abundantly

present (2.02 μg and 544 ng, respectively),

but that little, if any, of the common monoand di-hydroxylated or carboxy metabolites

were present in this TLC band.

On our instrument, the automated GC/MS analyses can also be generated and displayed in a

more complete and accurate mode, if desired.

The actual mass fragmentographic data from

which the analytical results are derived can

be printed for any single individual compound

at that point on the GC peak at which the best

mass spectral match between the experimental

sample and the library spectrum occurred.

ThiS type of data for the two compounds which

were judged to be present in Band I-A are

shown in Figures 6 and 7 which show that

Figure 5. FID Chromatogram of silylated

Fraction I-A of petroleum ether extract of

baboon feces. (Column: 6' X 2 mm ID pyrex

with 1.5% OV-17 on 100/120 mesh HP Chromasorb

G; 30 ml nitrogen/min.; 275°C.) Inset: Automated GC/MS analytical data. Shaded areas

indicate retention time intervals during which

positively identified components were scanned.

eight characteristic ions from the spectrum

o f 9-THC were present in the proper intensity ratios (and at the correct retention

time) and that nine such ions were recorded

for CBN. As an illustration, the five highest mass ions from the 9

-THC contracted spectrum were monitored as a function of time

(Figure 8) and it can be clearly seen that

the GC peak is entirely homogeneous with respect to 9

-THC. The ion currents reveal that

the typical pattern was already recognizable

at about 10 ng.

The quantities of 9-THC and CBN in these individual compound assays represent a total

daily output of 1.48 mg and 390 μg, respectively. The first of these figures indicates

that about 8.5% of the total 9

-THC which was

administered was excreted as unmetabolized

drug at "steady state."

The value obtained from CBN helps to settle

the question as to whether CBN, whose presence

has been reported in plasma (Wilman et al,

1975; McCallum et al, 1975), is truly a meta72
Figure 6 Figure 7

OLFAX/GC ANALYSIS

TS-207 TI-295

>HEXANE EXTRACT OF PPA #30 FECES: 0.23% OF FRACTION I-A.

DELTA-9-THC +POS QTY 3.08E+ 3 CONF 102 RT 81

MASS INTENSITY

6 7 C 2.22E- 7

9 3 C 6.16E- 8

181 5.68E- 9

196 3.52E- 9

219 8.38E- 9

231 6.32E- 9

246 1.20E- 8

330 1.49E- 8

343 2.78E- 8

371 8.03E- 8

PLOT Y OR N?Y

# OF DECADES=1

FS=2.22E- 7

0 1 2 3 4 5 6 1 8 9 1 0

+----+----+- ---+----+----+----+----+ ----+----+----+

+

+

67C+>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

93C+>>>>>>>>>>>>>>

181 +>

196 +>>

219 +>>

231 +>

246 +>>>

331 +>>>

343 +>>>>>>

371 +>>>>>>>>>>>>>>>>>>

+

+

+

OLFAX/GC ANALYSIS

TS=209 TI=298

>HEXANE EXTRACT OF PPA #30 FECES: 0.23% OF FRACTION I-A.

CANNABlNOL +POS QTY 8.08E- 1 CONF 136 RT 109

MASS INTENSITY

165 C 7.68E- 9

178 6.84E- 9

238 9.36E- 9

295 1.34E- 3

310 2.05E- 3

323 7.68E- 9

367 2.27E- 7

368 8.20E- 8

369 2.24E: 3

382 1.04E- 6

PLOT Y OR N?Y

# OF DECADES=1

FS=2.27E- 7

0 1 2 3 4 5 6 7 8 9 10

+----+----+----+----+----+----+----+----+----+----+

+

+

165C+>>

178 +>>

233 +>>

295 +>>>

313 +>>>>>

333 +>>

367 +>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

368 +>>>>>>>>>>>>>>>>>>

369 +>>>>

382 +>>

+

+

+

Figure 6. Single compound GC/MS analysis of Figure 7. Single compound GC/MS analysis

Fraction I-A of petroleum ether extract of (Confirmation mode) of Fraction I-A of petrobaboon feces indicating 3.08 ng of 9

-THC with leum ether extract of baboon feces indicating

a Confidence Index of 102 at a retention time 0.808 μg of CBN with a Confidence Index of

of 81 secs. Note that only 2 ions (m/e 67 136 at a retention time of 109 secs. (only

and 93) out of 10 are contaminated. m/e 165 is not present in the required relative abundance for pure CBN.)

bolic product or arises as the excretion of

a CBN impurity in the administered THC. Our

work with standards indicates that there is

usually a maximum of about 3% CBN in the

9

-THC obtained from NIDA. If this were also

true of the lot which was used to dose this

baboon, a maximum quantity of about 0.5 mg

could have been administered. The finding of

nearly 0.4 mg in this specimen indicates a

recovery of nearly 80% of the maximum amount

of impurity possibly present. Leo Hollister's

group, with which we have collaborated closely

for several years in their intensive studies

of the urinary excretion of various metabolites of 9

-THC, CBN, CBD, and 11-HO- 9

-THC,

has shown that orally administered CBN is

very extensively metabolized (Kanter, 1975).

Thus it seems unlikely that more than half of

the "administered" CBN should be recoverable

from the feces in a single fraction especially after ail the precessing this sample

received - and therefore it would appear that

CBN is a genuine metabolite of 9-THC. The

remaining four bands derived from the petroleum ether extraction contained none of the

eight compounds in our test panel.

The continuous ether extraction at pH 10

(Fraction II) contained 54 μg 11-HO- 9

-THC

(total daily output) and 210 μg cannabinol-11-

oic acid (providing further evidence for the

metabolic origin of CBN), but the presence of

9

-THC-11-oic acid could not be unequivocally

demonstrated. The ether extract obtained at

73
Figure 8

Figure 8. Top: Single ion mass chromatogram est masses used in the automatic assay pro-

(m/e 386) for 1 μg 9-THC. Center: 5-Mass gram). Bottom: Same as center, but with

selected ion record for 1 μg 9-THC (5 high. 3-decade logarithmic presentation.

pH 2.5 (Fraction III) contained 93 μg of 8 ,11-

dihydroxy- 9-THC, but no other metabolite in

our panel could be conclusively identified.

The foregoing GC/MS results were obtained using

a computer-managed Olfax II spectrometer system (Universal Monitor Corp., Pasadena, CA)

which utilizes Processor McLafferty's "Probability Based Matching" (PBM) algorithm

(McLafferty et al, 1974). In the PBM procedure, reverse sear& logic (Abramson, 1975)

is used to compare a many-line mass spectral

pattern derived from an experimental sample

with a corresponding spectrum stored in a

limited library. In the present study, the

library was specially tailored to the Problem

of differentiating between various cannabinoids

and their metabolites.

Probability based matching

McLafferty's PBM technique was specifically

designed to monitor individual compounds in

mixtures by "resolving" their merged mass spectra and therefrom to automatically generate two

kinds of information: 1) quantitation and

2) an estimate of the degree of specificity of

the determination. Both of these analytical

values are derived from an ability to automatically ascertain whether or not any particular

mass spectral line (fragment ionj is contaminated by the simultaneous presence of an impurity. Such judgments are possible because of

one particular characteristic physical property

of mass spectra; when two or more different

compounds contribute an identical molecular

weight fragment ion to a merged spectrum. the

74
combined ion currents are always additive.

This undirectional variation can be used by the

computer to detect an artificially enhanced

abundance of any individual ion in the mass

spectral pattern of a pure substance which

arises as result of a contribution from a

second substance.

Since Probability Based Matching concerns the

manner of specific ion recording and the processing of these data, a brief description of

the PBM algorithm may help in understanding

the type of results which it produces. In the

reverse search process, it is not necessary to

scan an entire mass spectrum, but only to measure a set of ions which are particularly

characteristic of the target compound. A principal advantage of this concept is that a complete search for a limited, or “contracted,”

spectrum can be accomplished in less than a

second and therefore the contracted spectra

of several different compounds can be accurately recorded repetitively during the

elution of even very sharp GC peaks.

In the Olfax II GC/MS system a microprocessor

directs the spectrometer to record the ion

currents for each of the pre-selected ions

in the contracted spectrum and subtracts the

instrument background from these values

(Hertel et al, 1975). It then automatically

computes the relative intensities and compares the resulting ratios with the stored

library spectrum in order to detect any contaminated ions; it simultaneously compares

the absolute intensities of each ion with the

intensities of the corresponding lines in the

background and disregards any ions which are

so weak that they are approaching background

levels. All contaminated lines and those

which are near background intensity are ignored in subsequent calculations.

Confidence Indices: From the remaining uncontaminated lines, a statistical probability

is automatically computed which reflects the

quality of match between the experimental

spectrum and the reference spectrum of the

pure compound. McLafferty calls this probability a “Confidence Index” (K-score). Its

magnitude depends mainly upon the absolute

number of uncontaminated lines, the relative

importance of each specific uncontaminated

line, and the “dilution” of the sample.

Of these parameters, the concept of relative

importance of a particular ion - “line

value” - is unique to the PBM algorithm. The

line value (or V-score) is a combination of

the uniqueness of that particular mass number

(frequency of occurrence in McLafferty’s

library of 18,800+ compounds) and the relative abundance of that particular ion in the

entire mass spectrum of that specific compound (Pesyna et al, 1975). Unequivocal

identification of a spectrum is obviously

easier when using unusual mass peaks than

when one is forced to rely on common peaks

and when using strong ions rather than weak

ones. Actually, it is often better to have

a weak unique ion than a strong common ion.

V-scores provide a convenient way to automatically make such judgments without being

experienced in the interpretation of mass

spectra.

Quantitation: All of these criteria (and

more, which time does not permit mentioning)

are incorporated into the K-score which is

calculated each time the contracted spectrum

is scanned (i.e., several times a second).

Simultaneously, the absolute intensity of

each uncontaminated ion is compared with the

absolute intensity of each corresponding

ion - resulting from a given quantity of the

pure material and stored as a part of the

computer library - to produce an independent

mass fragmentographic quantitation for each

uncontaminated ion. All of these scaled ion

currents are then averaged to provide an

estimate of the quantity, Q, of substance

present at that point in time at which the

contracted spectrum was scanned. A significant aspect of PBM quantitation is that

the computer automatically selects the uncontaminated ion which represents the lowest

quantity of the target compound (based upon

the required relative abundances) and therefore is a self-adaptive system which reports

the maximum quantity of that compound which

could possibly have been present in the

sample. Since the K-score reflects the mass

spectral specificity of the uncontaminated

ions upon which quantitation is based, its

magnitude is helpful in establishing the

confidence one can place in the quantitative

results.

A highly unusual feature of the Olfax system

is that it is entirely digital; the only

truly analog output it provides is a conventional FID record from the GC. The manner in

which data are recorded is shown in Figure 9.

Each repetitive set of 5 ions constitutes a

single cycle (i.e., one scan of the contracted spectrum) which is compared against

the library values and for each cycle a new

K and Q are calculated. The computer reports

only the very best fit observed during any

series of scans (highest K-score) and the

maximum ion currents generated (highest Q),

e.g., cycle 5 in this illustration.

To show how these digital data relate to conventional specific ion recording, a set of

data like those of Figure 9 have been plotted

in Figure 10. (In normal operation, at least

three to five cycles are scanned between each

75
Figure 9 Figure 10

2/ 8/76 OPERATOR # 23

PROGRAM D9-THC TYPE SCN2

RUN # 2 TS=197 TI=298

>0.75 UG DELTA-9-THC/TMS

MASS INTENSITY

246 3.76E-12

330 8.28E-12

343 2.26E-12

371 1.11E-11

386 1.81E-11

246 3.66E-11

333 3.52E-11

343 1.94E-10

371 3.84E-10

386 3.04E-10

246 5.44E-10

330 5.52E-10

343 1.58E- 9

371 3.84E- 9

386 2.40E- 9

246 2.43E- 9

330 3.40E- 9

343 7.00E- 9

371 1.65E- 8

386 7.88E- 9

246 3.28E- 9

330 4.26E- 9

343 9.92E- 9

371 2.34E- 3

386 1.09E- 8

Maximum

Cycle

246 2.72E- 9

330 3.48E- 9

343 6.24E- 9

371 1.78E- 8

Figure 9. Representative digital data

recorded during elution of 0.75 μg 9

-THC GC

peak. (Intensities are in amperes, e.g. m/e

371 in maximum cycle is 2.34 X 10-8A.)

of the points depicted in Figure 10 - and a

typical cycle contains 10 to 15 ions, rather

than 5 as shown here.) Figure 11 shows three

modes in which an Olfax contracted spectrum

may be displayed. At the top is a single ion

mass chromatogram; in the middle is a fairly

conventional specific ion record (note the

recurring “pattern” of relative ion currents);

and at the bottom is a logarithmic presentation of the same data as in the middle (note

how much more easily the relative abundances

can be compared from cycle to cycle and also

that the correct pattern can be readily discerned at a very low level of signal - i.e.,

at less than 10 ng).

Selection of specific fragment ions

The PEN reverse search technique is such a

powerful pattern matching procedure that

Figure 10. Plot of typical digital data

(like that of Fig. 9) for 0.9 μg 9-THC.

almost any set of ions selected from the

spectrum of a compound will give acceptable

qualitative and quantitative results. However, for sets of compounds having the similarity of the cannabinoids, especially where

high sensitivity is desired, selection of the

optimum mass lists requires careful consideration of many factors.

The main types of differential criteria which

can be incorporated into a PBM assay program

are: 1) positive attributes of the target

compound (namely, high V-scores); 2) negative

attributes of challenges (compounds which are

likely to be encountered at similar retention

times); and 3) absolute intensities of the

selected ions relative to their background

intensities (which affect the minimum detectable quantity at a given mass number).

Thus one must attempt to maximize the K-score

for the target compound while simultaneously

minimizing the K-score for challenges (by

deliberately selecting lines which result in

contaminated or weak ions for the challenges)

and all the while keep in mind the background

ion currents for each mass being considered.

76
Fugure 11

Figure 11. Top: Mass chromatogram for 9

-THC

in Fraction I-A of petroleum ether extract of

tion). Bottom: Same as center, but with

baboon feces. Center: 5-Ion selected ion

3-decade logarithmic presentation. (5-Highest

record for the same sample (linear presentamasses used in automatic assay program.)

The closer the challenge comes to the retention time of the target, the more critical it

is to select contaminated or weak ions. Thus

for 11-hydroxy- and 8 , 11-dihydroxy- 9-THC/

TMS, the retention times differed by less than

1% and it became more important to screen

against the challenging compound than to

screen for the target. Because their spectra

are quite different this could be accomplished,

but only by sacrificing some sensitivity. The

pair 8 , 11-dihydroxy- and 8 , 11-dihydroxy9

-THC/TMS had similar spectra but differed

markedly in retention time so that they could

be easily differentiated by GC alone. Unfortunately, a different situation was encountered in the pair 8 -hydroxy- and 8 -hydroxy9

-THC/TMS which have nearly identical spectra

and nearly identical retention times; therefore even PBM was unable to produce unique

assays for the individual compounds. Accordingly, a single program was produced which

was labeled “8 -hydroxy- 9-THC,” but which

was in reality an 8 - and/or 8 -hydroxy- 9

-

THC assay.

Responses of the PBM algorithm

The performance of the PBM algorithm can best

be understood by looking at the manner in which

it responds to specific single compounds. If

the Confidence Index is monitored as a function of sample size, a decreasing value is

observed with decreasing quantity of sample

(Figure 12). This is due primarily to two

effects. At the upper end of the plot, it

decreases due to a “dilution” correction be77
Figure 12

Figure 12. Effect produced on PBM Confidence

Index by varying the quantity of pure 9-THC

injected.

cause it is assumed that low ion currents are

frequently the result of dilution with impurities and, if this is truly the case, such

readings are statistically less valid. At the

bottom end of the curve, in addition to the

dilution correction, the weaker lines approach background levels, and are dropped from

consideration because they are too weak to

measure reliably.

Numerically, K is a binary logarithmic representation of the probability of an accidental

(random) match of the observed quality between the unknown spectrum and the library

spectrum. A rough approximation of the significance of the K-scores is that 2

Kth spectra,

chosen at random, would have to be compared

with the library spectrum before one encountered a spectrum which would match as well as

does the experimental spectrum. Thus a K of

10 means roughly that 2

10 (approximately 1,000)

spectra would have to be examined in order to

find as good a fit.

Obviously, the numbers grow very rapidly -

K=20 is approximately one million - so that

only a qualitative meaning can be given to

very large K-scores. As the result of two

years of experience with the method, we can

say that a K of 30 to 40 is somewhat equivocal, but a Confidence Index of 60 to 70 is

virtual certainty of proper identification,

within the limits of low resolution mass

spectrometry (even without retention time

considerations). Differential K-scores are

of great significance as is easily seen in

Figure 12 where 8-THC, when used to challenge the pattern for the 9

-THC program,

gives a K-score of 11, more than 100 points

lower than does 9-THC (the target compound).

Differentials in K-score are likewise used to

establish the "thresholds for reliable identification" for each program. Somewhat arbitrarily, this threshold is set at 10 units

greater than the maximum K-score generated by

any known challenging compound or control specimen (i.e. a "safety" factor of 1,000). In

the case of 9-THC shown in Figure 12, the

Confidence Index threshold for reliable identification was set at 21, indicating that one

can be reasonably certain that it is 9

-THC

which is actually being monitored in any

analysis which results in a K of 22 or

greater.

The ability of these PBM assay programs (all

of which contained ten fragment ions) to distinguish between closely related compounds can

be seen in the matrix shown in Figure 13. The

Confidence Index (the upper number in each

box) for each target compound is compared with

that of the THC metabolites eluting immediately before it and after it. In most cases, the

difference (in K-scores) between the correct

compound and its challenge is greater than

100. Besides this Confidence Index screening,

many adjacent pairs can be differentiated by

their GC retention times (the lower number in

each box).

As one gains experience with these Confidence

Indices, the analytical significance of this

statistical parameter becomes "abundantly

clear". Thus, for each and every quantitation,

the likelihood that the data are valid is immediately apparent by inspection.

Along with the computation of the Confidence

Index, the PBM algorithm provides a quantitative response which is linear for well over

two decades (as may be seen in Figure 14).

Olfax II quantitation usually has less than a

10% error and its precision has typically a

2-5% coefficient of variation.

For the test panel used in the present study,

the 10 best lines which were found for each of

the compounds, along with some of the other essential parameters for the Olfax algorithm

78
Figure 13 Figure 14

Figure 13. Confidence Indices and retention

times for nine cannabinoids. Reported as

K for 1 μg samples. Column tempera- RT(sec)

ture 275°C.; nitrogen carrier flow rate

30 ml/min.)

Table 2

OLFAX II ASSAY PROGRAMS

Figure 14. Quantitation reported by the mass

spectrometer (PBM algorithm) as a function of

the quantity of 9-THC actually injected into

the GC.

arator temperatures (TS), and line-values (V)

Table 2. Confidence Index “thresholds for for the ions (m/e) used in the 8 cannabinoid

reliable identification” (KT), molecular sep- assay programs.

79
Figure 15 Figure 16

Figure 15. Extraction scheme used for

-glucuronidase / aryl sulfatase hydrolyzed

9

-THC urines indicating derivation of the

H, E-I and E-II Fractions.

(V-scores for the individual lines, Confidence

Index “thresholds”, and optimum temperatures

for the molecular separator), are listed in

Table 2.

Human urines

Although working with feces is, relatively

speaking, easier than with most other biological specimens, no great practical value can be

derived from their analysis. Therefore, we

have concentrated our efforts upon investigating urine, which would appear to be the most

practical biological specimen, provided that

some metabolite could be identified and monitored which follows the time course of physical impairment due to THC. (We have some preliminary evidence that suggests that we have

seen such a metabolite.)

Extractions: Four years ago, we developed a

rather elaborate procedure for fractionating

THC metabolites according to their polarities

and acidities using simple changes of solvents

and/or pH (Figure 15) (Forrest et al, 1972;

Melikian et al, 1973). This scheme separated

the total radioactivity in enzyme-hydrolyzed

Rhesus urine into six approximately equal fractions: Hexane extractable, H (non-polar neutrals); ether (pH 12) extractable, E-I (weakly

polar neutrals); ether (pH 2.5) extractable,

E-II and E-III (weakly polar acids); ethyl acetate (pH 2.5) extractable, EA (moderately polar

acids); and tetrahydrofuran (pH 2.5) extractable, THF (highly polar acids). Figure 16

shows the relationships between the sizes of

these fractions in hydrolyzed, as well as unhydrolyzed, urines. The actual values are

given in Table 3. Subsequently, this procedure

Figure 16. Distribution according to polarity

(extractability) of 9-THC metabolites in vivo

(hydrolyzed and unhydrolyzed Rhesus urine).

Non-polar neutrals = extractable with hexane

at neutral pH

Weakly polar neutrals = extractable with ether

at pH 12

Weakly polar acids = extractable with ether at

pH 2

Moderately polar acids = extractable with

ethyl acetate at pH 2

Highly polar acids = extractable with tetrahydrofuran at pH 2.

Table 3

Extraction Efficiencies of Various Solvents (Percent

of total counts extractable from Rhesus monkey urine)

Solvent and pII Unhydrolyzed Urine Hydrolyzed Urine

Hexane 0.1% 6.7%

(natural pII)

Ether 0.9% 11.0%

(pII 12)

Ether 47.8% 48.3%

(pII 2)

Ethyl acetate 26.8% 15.2%

(pII 2)

Tetrahydrofuran 23.9% 14.6%

(pII 2)

Residue 1.4% 1.3%

Total 100.9% 37.1%

80
was refined by resolving the weakly polar acids

(the largest single fraction) into strong and

weak acids according to their solubility in

NaHCO3(E-III and E-II, respectively)(Kanter

et al, 1974).

Most of our efforts have been concentrated on

the three least polar fractions, H, E-I, and

E-II, which contain the parent cannafioids

and the mono- and di-hydroxys, as well as the

“11-oic” acids. Standards do not distribute

cleanly between these categories; 9-THC and

11-hydroxy- 9-THC, for instance, distribute in

the ratio of about 4:1 between the hexane fraction and the ether at pH 12 fraction (E-I).

8,11-Dihydroxy- 9-THC, on the other hand, is

found predominantly in the E-I fraction (80%)

with only about 5% each in fractions H, E-II

and E-III.

Hollister’s group has applied this fractionation scheme very extensively to urines obtained from human volunteers after oral administration of pure standards of 9

-THC, CBN, CBD,

11-HO- 9

-THC, singly and in combinations

(Hollister, 1973; Kanter, 1975). They have

observed many interesting phenomena which can

best be summarized briefly as: 1) the existence of a multitude of drug-related metabolites; 2) very few matches with available

standards; and 3) the extreme persistance (10-

15 days) of highly polar metabolites after a

single administration of drug.

High dose specimens: As part of our own work

using these same procedures, our group was

very-fortunate to be able to obtain urine specimens from subjects used in some unusual

experiments last spring at the University of

California Langley Porter Clinic in San Francisco (Jones et al, 1975). These subjects

received the extremely high oral doses of 210

milligrams of 9-THC per day.

The urine was hydrolyzed with -glucuronidase/

aryl sulfatase and fractionated according to

the foregoing extraction scheme. Aliquots of

the dried extracts were derivatized with BSTFA

and screened by GC/MS. All the “positives”

which were detected, and any determinations

which showed high K-scores (although “negative”), were re-run in the more comprehensive

single compound mode.

The hexane fraction showed only CBN on initial

screening (Figure 17), but the confirmation

mode re-run showed a marginal positive for

9

-THC at 10 ng/ml (Figure 18). Since this

positive identification was achieved with only

two ions. a more conventional mass chromatogram was run (Figure 19). This showed clearly

that there indeed was a GC component with the

highly unique m/e 371 which eluted at the

retention time of 9

-THC.

In addition to the 9-THC, a confirmation mode

re-run showed a fairly clear-cut presence of

CBN (K=28) at a level of 12 ng/ml (Figure 20).

The weakly polar neutral fraction (E-I) showed

a very marginal positive for CBN at 0.8 ng/ml,

which probably was due to a slight carry-over

from the hexane fraction, and a fairly strong

positive for 8 ,11-dihydroxy- 9-THC at 24 ng/ml

in the screening run (Figure 21). When fraction E-I was re-run in the single compound

mode, it no longer showed a definite positive

although the 8 ,11-dihydroxy- 9-THC was a nearmiss with three weak ions in the correct ratios

and corresponding to a maximum concentration of

22 ng/ml. The screening run for the weakly

polar, weak acid fraction (E-II) indicated positives for 9

-THC (K=24), 9-THC-11-oic acid

(K=94) and CBN-11-oic acid (K=37) (Figure 22))

but only the THC acid could be unequivocally

confirmed (Figure 23). The 9

-THC-11-oic acid

concentration corresponded to 206 ng/ml in a

highly specific determination. That these

intensity ratios were in fact derived from a

single substance which eluted at the proper

retention time can be clearly seen in Figure 24

(top), as can the fact that the observed intensity ratios do, in fact, correspond to those of

authentic 9-THC-11-oic acid (bottom).

One aspect of PBM quantitation should be stressed in connection with the concentration of

9

-THC-11-oic acid which was measured. The PBM

algorithm provides an upper limit to the possible quantity present. From this we must conclude that a maximum quantity of about 0.4 mg

of this acid could have been excreted in the

24 hours during which the intake was 210 mg of

9

-THC. Since the urinary portion of the total

excretion is about 20% (Lemberger et al, 1971),

there would probably have been about 40 mg of

metabolites in all forms. Therefore the 9

-THC11-oic acid represents something of the order

of 0.5 to 1% of the total metabolite pool.

Allowing for dose-dependent variations of up to

10-fold, this particular acid could amount to

no more than 10%, at best, of the total drug

excreted in urine. This finding is entirely

consistent with the TLC data which have been

obtained in our laboratory and in Hollister’s

laboratory (Kanter, 1975), as well as in

Jones’ laboratory (Ellman, 1975). There are

copious quantities of many acidic metabolites,

but the 9-THC-11-oic acid per se is definitely a minor component.

Moderate dose specimen: An examination of a

slightly more typical urine specimen was carried out on some extracts provided to us by

Saul Kanter of Hollister’s group. These fractions, H, E-I, and E-II, were extracted from

the urines of an individual before and after

he received a single oral dose of 30 mg of

9

-THC; specimens were collected 12 hours prior

to drug intake and during the interval of 12 to

24 hours post-drug.

81
Figure 17 Figure 18

too high (c. f. pattern in Figs. 8, 9 and 11).

Figure 18. Single compound GC/MS analysis

(Confirmation mode) of Fraction H from hydrolyzed human urine (same as Fig. 17) indicating

45.4 ng of 9-THC in 5 ml urine with K=24 at a

retention time of 82 secs. Note that the comnon-polar neutrals (H) fraction of human urine

because it knew that they would be too close

(enzyme hydrolyzed).- GC conditions same as

to background levels and that the computer

Fig. 5. Inset: Automated GC/MS analytical

judged 3 of the remaining ions to be contaminated, i.e. their relative abundances were

Figure 17.

puter elected not to scan the lowest 5 masses

FID Chromatogram of silylated

data (Autoscan mode).

Figure 19

Figure 19. Single ion (m/e 371) mass chromatogram for same sample as Figs. 17 and 18.

Note that this ion becomes cleanly measurable

at about 5 ng.

82
Figure 20 Figure 22

Figure 20. Confirmation mode GC/MS analysis

of Fraction H from hydrolyzed human urine

(same as Fig. 17) indicating 52.0 ng of CBN in

5 ml urine with K=28 at a retention time of

110 secs. Nine out of the 10 masses were

scanned, but 7 were contaminated.

Figure 21

Figure 21. FID Chromatogram of silylated

weakly polar neutrals (E-I) fraction of human

urine (enzyme hydrolyzed). GC conditions

same as Fig. 5. Inset: Automated GC/MS analytical data (Autoscan mode).

Figure 22. Chromatogram of silylated weakly

polar, weak acids (E-II) fraction of human

urine (enzyme hydrolyzed). GC conditions same

as Fig. 5. Inset: Autoscan mode GC/MS data.

Figure 23

Figure 23. 9

-THC-11-oic acid/TMS analysis

of Fraction E-II (same as Fig. 22) showing

1.39 μg/5 ml hydrolyzed urine with K=52 at a

retention time of 227 secs. All 10 lines

were scanned, but 6 were contaminated.

83
Figure 24 Figure 25

Figure 24. Top: Selected ion record of 9-

THC-11-oic acid GC peak from Fraction E-II

(same sample and quantity as Figs. 22 and 23). Figure 25. Effect of pH on extractability of

Bottom: Same kind of selected ion record of 9

-THC in vivo metabolites from unhydrolyzed

0.75 μg of authentic 9-THC-11-oic acid. Rhesus urine.

We found a maximum quantity of about 50 ng of

9

-THC per 10 mg creatinine in the hexane

fraction, but the Confidence Index was too low

to identify the compound with certainy. The

CBN, however, was clearly evident (K=59) at a

level of about 100 ng/10 mg creatinine;

The E-I fraction (weakly polar neutrals) gave

a very marginal confirmation for 8 -HO- 9

-THC

at a level of about 60 ng/10 mg creatinine and

a suspiciously high reading of nearly 0.6 μg

of 8 , 11-dihydroxy- 9-THC per 10 mg creatinine with a K-score of 35. The E-II fraction

(weakly polar, weak acids) from this specimen,

surprisingly, failed to give a positive for

9

-THC-11-oic acid.

Unhydrolyzed urines: Four years ago, we reported that an appreciable proportion of the

total counts in Rhesus urine could be extracted

with ether, even without prior hydrolysis

(Forrest et al, 1972; Melikian et al, 1973).

This extraction was highly pH-dependent with

the curve's inflection point being near pH 4

(Figure 25) indicating that it was probably

carboxylic acids which were being removed by

the ether.

As one part of an experiment on urines obtained

from the Langley Porter Clinic Study, we extracted and another specimen with ether before and

after treatment with enzyme at pH 5.5. Unfortunately this pH was far from the optimum pH

for removal by ether, but the results are

interesting, nevertheless. A highly significant Confidence Index (K=68) for 9

-THC-11-oic

acid was obtained along with a quantitation of

36 ng/ml (Figure 26). Had the extraction been

made at pH 2.5, a quantity of about 90 ng/ml

would probably have been extracted, if the

compound follows a normal ionization curve for

carboxylic acids. (This level suggests that

about half of the 9-THC-11-oic acid which is

present in urine can be extracted directly,

without hydrolyzing the specimen.)

Single specific compound analysis confirmed

the screening results as did a selected ion

record, as shown at the bottom of Figure 27.

84
Figure 26 Figure 28

Figure 26. Silylated ether extract (pH 5.5)

of human urine prior to enzyme hydrolysis.

Inset: Autoscan analysis.

Figure 28. Silylated ether extract (pH 5.5)

of human urine after enzyme hydrolysis.

Inset: Autoscan analysis.

Figure 27

Figure 27. Top: Selected ion record of urine at RT of 9-THC-11-oic acid. Bottom:

0.75 μg of authentic 9-THC-11-oic acid.

Center: E-II Fraction of hydrolyzed human

Ether extract of unhydrolyzed human urine at

RT of 9-THC-11-oic acid.

85
Figure 29

Figure 29. 8α,11-Dihydroxy- 9

-THC/TMS

analysis of ether extract of human urine

after enzyme hydrolysis showing 1.20 μg/5 ml

with K=35 at a retention time of 150 secs.

Three ions are uncontaminated.

This selected ion record also shows that one

ion (m/e 355) becomes contaminated shortly

after the peak has eluted and is the type of

situation which would have been rejected by

the computer if it had occurred a few seconds

earlier. The center record was obtained from

the E-II (weak acids) fraction of the previously discussed enzyme hydrolyzed urine, and

the top record is authentic 9-THC-11-oic acid.

Figure 28 shows the results from the posthydrolysis ether extraction. Only 8 ,11-dihydroxy- 9

-THC appeared to be present, but its

identification was very marginal (K=32)

although it was slightly better in the confirmation mode (K=35) (Figure 29).

SUMMARY

The results from these urine studies and a few

other isolated extracts we have run may be

generalized as follows: The hexane fractions

showed low levels of 9-THC (ca. 10 ng/ml)

with marginal Confidence Indices (25-30), but

both the quantities and the K-scores were well

above the values seen in extracts of control

urines; the hexane fractions showed similar

levels of CBN (ca. 12-25 ng/ml) with significant K-scores (60-80); the ether-extractable

neutral fraction contained 8 ,11-dihydroxy9

-THC (25-150 ng/ml) with low confidence and

sometimes a suggestion of 8 -hydroxy- 9-THC

(15 ng/ml) with a marginal identification; and

the E-II fraction (ether-extractable, weak

acids usually contained a strong indication

(CONF 60-95) of 9-THC-11-oic acid (100-200

ng/ml).

CONCLUSIONS

Obviously, the limited scope of these data

which I have presented cannot be considered to

be much more than a feasibility study, but the

results are quite promising. Three metabolites of 9

-THC are consistently observed and

9

-THC per se is seen and quartitated with a

degree of certainty which requires only slight

improvement. Since the 9

-THC determinations

were not entirely satisfactory, suggestions

for future efforts aimed at improving this

particular assay might include partially shifting the burden of identification from the mass

spectrometer to the GC by improving the GC

separation using such means as operating at a

lower column temperature or increasing the

length of the column. The effectiveness of

the PBM assay program might be improved by

processing a larger sample to decrease the

effect of weak ions in the algorithm or by

improving the sensitivity of the mass spectrometer itself. Also, since 11-hydroxy- 9

-THC

was not encountered in any urine samples, the

8α,11-dihydroxy- 9

-THC program should be modified so as to make it better able to withstand

general challenges even though in the process

it becomes less specific with respect to

11-hydroxy- 9-THC.

In all of the work described here, a

standard test panel of TMS derivatives of

9

-THC and seven of its metabolites was

employed. Assay programs for additional metabolites, such as the side-chain hydroxylated

compounds described by Professor Agurell,

could easily be added to the panel if appropriate reference samples were made available

to us.

The Olfax method, in which GC retention time

screening is combined with the PBM mass spectrum matching technique, appears to be a very

86
apparatus which are available in any laboratory, the labor and analysis time have been

minimized, and the final assay uses a commercially available, completely automated

instrument which any skilled technician can

operate and which does not require prior

experience with mass spectrometers or an

ability to interpret mass spectra.

promising procedure for the highly specific

quantitative analysis of cannabinoids because it automates and makes practical the

best features of mass fragmentography. It

cannot do anything which would not be possible using conventional GC/MS systems, but

its degree of automation provides an advantage in speed and convenience while retaining

the inherent specificity of mass fragmentography. The extractions employ solvents and

ACKNOWLEDGMENTS

Parts of this work were supported by Contract

HSM-42-71-100 and by USPHS Grants DA 00424-01

and DA 00748-02.

The baboon specimens were very kindly supplied

by Eva E. K. Killam of the University of California (Davis) whose cooperation and interest

is greatly appreciated. Special thanks are

expressed to George L. Ellman of the University

of California (San Francisco) for providing the

unique urine specimens and also to Saul L.

Kanter of this Hospital for providing special

urine extracts. I wish to thank Richard

Schneider of Syva Corporation for the sample of

CBN-11-oic acid and Robert H. Hertel of Universal Monitor Corporation for valuable discussions during the course of this work. The very

able technical assistance of Fu-Chuan Chao and

Kay O. Loeffler is gratefully acknowledged.

Loeffler, K.O., Green, D.E., Chao, F-C., and

Forrest, I.S. (1975) Proc. West. Pharmacol.

Soc. 18, 363-368

McCallum, N.K., Yagen, B., Levy, S. and

Mechoulam, R. (1975) Experientia 31, 520-

521

McLafferty, F.W., Hertel, R.H. and Villwock,

R.D. (1974) Org. Mass Spectrom. 9, 690-702

Melikian, A.P., Green, D.E., Skinner, J.L.

and Forrest, I.S. (1973) Proc. West.

Pkarmacol. Soc. 16, 234-239

Pesyna, G.M., McLafferty, F.W., Venkataraghavan, R., and Dayringer, H.E. (1975)

Finally, most special thanks are due to Duane P. Anal. Chem. 47, 1161-1164

Littlejohn and the Universal Monitor Corporation for the loan of a prototype Olfax II Widman, M., Nordqvist, M., Dollery, C.T. and

GC/MS instrument system, without which this Briant, R.H. (1975) J. Pharm. Pharmacol.

work would not have been possible. 27, 842-848

REFERENCES

Abramson, F.P. (1975) Anal. Chem. 47, 45-49

Ellman, G.L. (1975) Personal communication

Forrest, I.S., Green, D.E., and Würsch, M.S.

(1972) Proc. Fifth Intern. Cong. on

Pharmacol.

Green, D.E., Killam E.E.K., Loeffler, K.O.,

Chao, F-C. and Forrest, I.S. (1975)

Pharmacologist 17, 268

Hertel, R.H., Green, D.E. and Strauss, P.A.

(1975) Proc. 26th Pittsburgh Conf. on Anal.

Ckem. and Appl. Spectr.

Hollister, L.E. (1973) Experimentia 29, 825-

826

Jones, R., Ellman, G.L. and Lysko, H. (1975)

Unpublished data

Kanter, S.L., Hollister, L.E., Moore, F. and

Green, D.E. (1974) Res. Comm. Chem. Path.

Pharmacol. 9, 205-213

Kanter, S.L. (1975) Personal communication

Lemberger, L., Tamarkin, N.R., Axelrod, J.

and Kopin, J. (1971) Science 173, 72-74

87
QUANTITATION OF -TETRAHYDROCANNABINOL

IN BODY FLUIDS BY GAS CHROMATOGRAPHY/

CHEMICAL IONIZATION -MASS SPECTROMETRY

Ruthanne Detrick and Roger Foltz

Battelle, Columbus Laboratories

Columbus, Ohio

As part of a program for the development of methodology for quantitation

of cannabinoids in body fluids, we

have validated an extraction and GC/

CI-MS analysis for the determination

o f 9-THC in plasma. The procedure

has proven suitable for the analysis

of relatively large numbers of samples, yet has sufficient sensitivity

and selectivity for analysis of

9

-THC concentrations as low as 0.5

ng/ml in plasma. Using this procedure, we are currently analyzing a

variety of body fluid samples from

different sources submitted by outside researchers.

Previous work at Battelle and other

laboratories indicated that substantial clean-up of the body fluid extract was necessary prior to GC/MS

analysis in order to achieve adequate

sensitivity. Although several cleanup procedures have been used successfully, many of these are cumbersome

and not well suited for analysis of

large numbers of samples. The procedure we are currently using is a

modification of the RTI solvent extraction procedure (Rosenthal, 1975).

The analysis scheme is essentially an

organic solvent extraction and cleanup, concentration and derivatization

of the 9-THC, followed by quantitation by GC/CI-MS analysis.

Specifically, the plasma sample is

transferred with a calibrated pipet

to a culture tube. To this is added

pH 7.0 buffer solution and a known

amount of 9-THC-d3 internal standard. The mixtures is vortexed to

assure complete mixing, then extracted twice with hexane. The combined

hexane extract is successively washed with 0.1 N NaOH and 0.1 N HCl.

The hexane extract is transferred to

an evaporation tube and evaporated

to dryness under a stream of nitrogen

using a tube heater maintained at below 50 c. The tube is rinsed with

88
pentane and the pentane solution

transferred quantitatively to a

Reacti-Vial(R). After removal of the

pentane, BSTFA [N,O-bis-(trimethylsilyl)-trifluoroacetamide] with 1%

TMCS is added, and the vial is heated for a minimum of 30 minutes at

75 C.

The concentration of 9-THC is determined using the technique of selected ion monitoring (SIM) with a GC-MS.

A 6 ft x 2-mm glass column packed

with 3% OV-17 on 100/120 mesh Gas

Chrom Q is temperature programmed

from 180 to 280 C at 10°/minute.

Methane is used as the carrier gas

while a small amount of ammonia is

bled into the ion scource through a

make-up gas inlet. The masses monitored are m/e 387, the protonated

molecule ion of the trimethylsilyl

ether (

9

-THC-TMS) and m/e 390, the

protonated molecule ion of the TMS

ether of the trideuterated internal

standard (

9

-THC-d3-TMS).

Previously we used methane as the

reagent gas for chemical ionization

of the GC effluent. We have now

found that addition of ammonia to the

ion source improved the signal

strength for a specific quantity of

9

-THC-TMS by a factor of about 3.

Furthermore, ammonia being a very

mild reagent gas is more selective

than methane so that there tends to

be less interference from other components of body fluid extracts. Also,

the relative intensity of the M-2 peak

is significantly lower when ammonia

is used rather than methane. Therefore, the 9

-THC-d3-TMS will make

less of a contribution to the MH+ ion

current of 9

-THC-d0-TMS. Figure 1

shows the methane and methane-ammonia

CI mass spectra of 9

-THC-TMS, while

Figure 1

Figure 1. Comparison of mass spectra of 9-THC-TMS

89
Table 1

Ionization

Method m/e Monitored

% of Sample Relative Response Per

Ion Current Unit Weight of 9

-THC

EI 386 (M+) 7 5 0

CI (CH4) 387 (MH+

) 2 4 2 0

CI (CH4+NH3)

CI (N2+NH3)

387 (MH+

)

387 (MH+

)

67

67

100

8 0

TABLE 1. RELATIVE RESPONSES FOR PROMINENT

IONS IN THE EI AND CI MASS

SPECTRA OF 9-THC-TMS

Table 1 compares the response per

unit weight of 9

-THC for different

modes of ionization.

A disadvantage of using ammonia as

the reagent gas is the lack of a

second prominent ion which can be

monitored as a check on possible contributions to the MH+

 ion currents by

other components of the body fluid

extracts. Chemical ionization with

methane gives several prominent fragment ions in addition to the MH+

.

However, methane is a less selective

reagent gas than ammonia, and in our

experience intefering peaks are more

of a problem with methane than with

ammonia. Of course, it is also important to carefully select the GC

column and operating conditions to

minimize interference from endogenous

components of the body fluids. We

have found it necessary to use temperature programming of the GC column.

Quantitation of the 9-THC is achieved

by monitoring ion masses associated

with the 9-THC-TMS and the deuterated internal standard, and measuring

the peak height ratios of the ion9

currents. The actual amount of 9-

THC is determined by comparison of

the ratio to a standard curve established at the time of each set of

analyses. The standard curve is prepared by simply mixing aliquots of

standard solutions of 9-THC and

9

-THC-d3, removal of the solvent by

evaporation, derivatization, and SIM

analysis. Figure 2 shows a typical

calibration curve prepared in the

above manner. The slope is 1.03 and

the zero intercept corresponds to a

peak height ratio of 0.0074 or a

9

-THC concentration of 0.3 ng. The

curve has a correlation coefficient

of 1.000. In almost all cases the

standard curve has been linear over

the concentrations measured, and the

90
Figure 2

Figure 2. Calibration curve for analysis of 9-THC-TMS

Table 2

9

-THC

Added

Sample Ht./Standard Ht. Relative

Experimental Standard

Theoretical (Average) Deviation

Number of

Determinations

96.8 ng 2.20 2.28 1.23 3

48.4 1.10 1.11 0.32 3

9.7 0.22 0.23 6.08 3

4.8 0.11 0.13 6.65 3

1.0 0.02 0.03 14.00 2

0.5 0.01 0.02 1.80 2

0.0 0.00 0.02 1

TABLE 2. RELATIVE STANDARD DEVIATIONS FOR POINTS ON CALIBRATION CURVE

(44.1 NG of 9-THC-d3 ADDED TO EACH)

relative standard deviation at any

given peak height ratio has typically

been less than 20, even for the lower

concentrations. The relative standard deviations for the points on

this particular calibration curve are

listed in Table 2. Changes in the

slope and intercept of the curve have

been observed, but the greatest

differences in the 9-THC values

assigned to a sample by using different calibration curves have been less

than 10%. To allow for changes due

to instrument settings or to a change

in the concentration of the internal

standard solution, we continue to

establish a calibration curve for each

set of samples.

91
Table 3 summarizes the precision and

accuracy data for a series of analyses.

Known quantities of 9-THC and 9-THCd3 were added to 1-ml portions of

plasma. The final volume of each

derivatized extract was about 25 μl.

Injections into the GC were between 2

and 4 μl. The actual quantity of

9

-THC injected on column is therefore approximately one-tenth of the

amount of 9

-THC added to each ml of

plasma. Figure 3 is a typical computer plot of an extract of 1 ml of

plasma containing 1 ng of 9-THC. In

this example the peak in the m/e 387

ion current curve corresponds to about

100 pg of 9

-THC-TMS injected on column. The corresponding peak in the

m/e 390 ion current curve corresponds

t o 9-THC-d3 concentration of 44.1 ng/

ml. The m/e 390 ion current signal is

attenuated by a factor of 10 relative

to the m/e 387 ion current signal.

Figure 3

Figure 3. Computer plot of ion currents for

m/e 387 and 390 corresponding to 1 NG/ML of

9

-THC and 44 NG/ML of 9-THC-d3. The m/e

390 ion current curve is attenuated by a

factor of 10.

Table 3

9-THC 9

-THC

Added Measured

(ng/ml) (Average)

Relative

Standard

Deviation

Number of

Determinations

Percent

Error

9.7 7.7 1.7% 10 21%

4.8 3.8 2.5% 10 21.%

1.0 0.65 6.4 9 35%

0.5 0.28 19.0% 10 44%

0 -0.13(a) 15.0% 8 -

(a) The average of the measured weights corresponded to a value below the

zero intercept of the calibration curve.

TABLE 3. PRECISION AND ACCURACY DATA FOR MEASUREMENT OF 9-THC ADDED

TO PLASMA (44 NG OF 9-THC-d3 ADDED PER ML OF PLASMA AS

INTERNAL STANDARD)

92
Figure 4

Figure 4. Plot of data points from SIM analysis of extracts of 1-ML

plasma samples containing 0 to 9.7 NG of 9

-THC and 44.1 NG of 9-THC-d3.

Finally, Figure 4 plots data points

corresponding to 50 separate SIM determinations on 2 sets of extracts from

plasmas to which known amounts of

9

-THC and 9

-THC-d3 were added. The 9

-THC concentrations ranged from 0 to

9.7 ng/ml of plasma.

Using this procedure, we are currently

analyzing a variety of plasma samples

submitted through NIDA by other researchers. These have included rat,

monkey, and human plasma samples with

volumes in the range of 0.25 to 2.0 ml.

Levels of 9-THC in the samples have

been typically in the range of 5-50 ng/

ml of plasma, although samples containing concentrations of 9

-THC as high

as 400 ng/ml of plasma have been analyzed. The actual procedure for analyzing the unknowns is as previously

described. To increase our confidence

in the integrity of the results for

any given set of samples, spiked plasma

93
samples containing a known amount of 9

-THC are analyzed at the same time.

These have been useful in pointing out

various problems with interferences,

and help to establish some confidence

limits for the measured values, particularly at the lower levels. In addition, a GC column resolution check and

a sensitivity check of the GC-MS are

made at the beginning of each day to

pinpoint any instrumental problems.

Although further modifications in the

analysis scheme are likely, the procedure presently being used has

proven suitable for the analysis of

9

-THC in plasma to concentrations as

low as 0.5 ng/ml and can be conveniently used for relatively large

numbers of samples.

Experimental Section

The plasma sample (usually 1 ml) is

transferred to a 16-ml culture tube

using a calibrated pipet and the volume is recorded. To this is added 1.0

ml of pH 7.0 buffer solution and 100

μl of 441 ng/ml solution of 9

-THC-d3

in ethanol. The mixture is vortexed

for 30 seconds. The plasma is then

extracted with 5 ml of Distilled-inGlass(R) hexane for 30 minutes on a

rotator. The mixture is centrifuged

and the hexane extract transferred to

a second tube. The hexane extraction

is repeated and the extracts combined.

The combined hexane extract is successively washed with 2.5 ml 0.1 N

NaOH, and 2.5 ml 0.1 N HCl. The -

hexane extract is transferred to an

evaporation tube and evaporated to

dryness under a stream of nitrogen

using a Kontes tube heater maintained

at below 50 C. The tube is rinsed

with 1 ml of Distilled-in-Glass(R)

pentane and the pentane solution

transferred quantitatively to a 1.0-

ml Reacti-Vial(R). The pentane is

removed under a stream of nitrogen at

room temperature. The sides of the

vial are washed down with 100 μl of

pentane, and the pentane is again

evaporated. To the vial is added 25

μl of BSTFA with 1% TMCS. The vial

is capped using a Teflon liner

and heated for 2 hours at 75 C.

The concentration of 9-THC is determined using the technique of selected

ion monitoring with a Finnigan 3200

GC-MS. A 6 ft x 2-mm glass column

packed with 3% OV-17 on 100/120 mesh

Gas Chrom Q is temperature programmed

from 180 to 280 C at 10o/minute.

Methane is used as the carrier gas

while a small amount of ammonia is

bled into the ion source through a

make-up gas inlet. The partial pressure of ammonia in the ion source is

100 μ. The flow rate of methane is

20 ml/min giving a partial pressure in

the ion source of about 700 μ. Other

mass spectrometer parameters are:

electron energy, 210 v; ion energy,

15 v; lens voltage, %30 v; electron

multiplier voltage, 1.4 to 1.6 KV;

and filament emission, 0.8 to 1.0 ma.

Cross-contamination from the syringe

used for injecting the samples into

the gas chromatography is avoided by

rinsing 10-20 times with pentane between injections.

For every 5 unknowns, a spiked sample

containing 9.7 mg 9-THC/ml of plasma

is prepared by adding 100 μl of a 97

ng/ml solution of 9-THC in ethanol

to 1 ml of human plasma. This is

extracted in the same manner and at

the same time as the unknowns.

A standard curve is established at

the time of each set of analyses by

adding 100 μl of the 441 ng/ml 9-THCd3 solution in ethanol and 100 μl of

one of various concentrations of 9-

THC in ethanol directly to a ReactiVial A series (R)

.

standards with d0/d3

of four such

ratios ranging

from 0-1 for 50 ng of 9-THC-d3 are

prepared. The ethanol is removed at

below 40 C, and the THC derivatized

in the same manner as the unknowns:

25 μl of BSTFA with 1% TMCS is added,

and the vial is heated at 75 C for 2

hours. These standard samples are

analyzed by monitoring the m/e 387 and

390 peaks of the mass spectrum. Since

there should be no interferences in

these standards, the gas chromatographic column is used isothermally at

205 C. The values for the ratio of

d0/d3 are used to prepare a standard

curve.

All reusable glassware used in the

analysis is rinsed with methanol,

cleaned with detergent and water,

rinsed with water, soaked in hot sulfuric and nitric acid solution, rinsed successively with tap water, distilled water and methanol, and finally

dried in an oven at >100 C.

Aliquots of standard 9

-THC and 9-

THC-d3 solutions are measured out

94
using a 100-μl Eppendorf pipet with

disposable tips. Syringes used for The concentration of the 9

-THC-d3

measuring out BSTFA or injecting stock solution in ethanol was detersamples into the GC-MS are cleaned in mined by comparison with a primary

methanol. The hexane and pentane used standard of 9

-THC in ethanol using

are high-purity, Distilled-in-Glass(R) SIM analysis.

solvents.

REFERENCE

D. Rosenthal, Second NIDA Technical Review on Mass Spectroscopy, Oak Brook, Illinois,

October 22, 1975.

95
HPLC-MS DETERMINATION OF

-TETRAHYDROCANNABINOL

IN HUMAN BODY SAMPLES

Jimmie L. Valentine, Ph.D., Paul J. Bryant, Ph. D., Paul L. Gutshall, M.S.,

Owen H.M. Gan, B.S., Everett O. Thompson, B.S., Hsien Chi Niu, Ph. D.

University of Missouri, Kansas City, Missouri

INTRODUCTION

Social and chronic abuse of cannabis

is believed to occur throughout the

United States. However, most information on the societal use of marijuana, in particular, comes from

question-response type surveys. Precise quantitative data obtainable via

body specimen analysis has been unavailable due mainly to the lack of

an accurate biological assay technology for the chemical constituents

found in cannabis.

The marijuana commonly smoked in this

country contains four principal constituents, viz., 9

-tetrahydrocannabinol (

9

-THC), cannabidiol (CBD),

cannabinol (CBN) , and cannabichromene

(CBC) (Doorenbos, et al., 1971). One

of these constituents, 9

-THC is believed to be responsible for the psychomimetic properties of marijuana

(Edery, et al., 1971). Likewise some

of the physiological responses in man

have been shown to change following

smoking of cigarettes containing 9-

THC and these correlate with plasma

levels of 9-THC and its metabolites

(Galanter, et al., 1972). However,

such studies were accomplishable only

by administering radiolabeled 9-THC

to the study subjects and following

its deposition in accessible body

fluids.

More recently there have been a number of methods proposed for determination of 9

-THC in blood plasma. A

glc procedure which utilized an electron capture detector was detailed

and reported to be capable of detectins 0.5 nq per 1 ml of blood plasma

(Fenimore; et al., 1973). This method which required precolumn derivatization and a dual oven apparatus

was not used to assay the blood plasma of actual cannabis smokers. Another reported method employed glcmass fragmentography with d2-

9

-THC

as the internal standard (Agurell, et

al., 1973). This later method was

employed to analyze 5 ml samples of

blood plasma from three volunteers

each of whom smoked a cigarette containing 10 mg of 9

-THC. A chromatographic elution of the blood plasma

96
on a Sephadex column was required

prior to glc-ms analysis. Blood samples were taken from the volunteers

at 0, 0.2, 0.5, 1 and 2 hours following smoking. Peak levels of 9

-THC

were found to be 19-26 ng/ml at 10

minutes following smoking. The levels declined to 5 ng/ml or less at 2

hours. Another method which has been

reported to be capable of detecting

9

-THC is based upon radioimmunoassay

procedures (Teal, et al., 1974 and

Gross, et al., 1974).

The method reported herein makes use

of a high pressure liquid chromatography-mass spectrometry (hplc-ms)

technique for determining ng/ml quantities of 9

-THC in human'body specimens. Problems associated with direct coupling of the hplc to the ms

have been circumvented by collecting

fractions of the mobile phase eminating from the hplc and subsequently

analyzing the fraction via the direct

insertion probe of the ms. Inherent

in this method is the use of d3-

9

-

THC for controlling extraction efficiency, as a marker for collection of

the hplc effluent and as a convenient internal standard for ms quantification. Thus by this method the

body specimen has enough d3-

9

-THC

added to allow its detection by a

u.v. spectrophotometer connected to

the output of the hplc. Once the

d3-

9

-THC is detected, fractions of

the mobile phase eluant are collected and introduced to the ms via the

direct insertion probe for quantification. This method was then validated in various human body samples

over the concentration range of 1-

100 ng/ml or ng/g depending upon the

specimen of interest.

The new assay technology was then

used to determine the blood plasma

levels of 9

-THC in eleven male volunteers during a 24 hour period following smoking of a marijuana cigarette. Also the new procedure has

been used to determine the presence

o f 9-THC in exhaled breath and saliva of volunteers following marijuana smoking. Similarly, a correlation has been made, using the method, between the blood plasma, bile

and brain levels of 9-THC in human

post-mortem specimens. The method

has also been used to analyze human

blood samples from a

9

-THC aerosol

inhalation study on a blind basis.

EXPERIMENTAL

High pressure liquid chromatography

All hplc analyses were conducted on

a Varian 8520 gradient elution liquid chromatograph utilizing a Varian

635M spectrophotometer set at 273.7

mμ as the detector. The column was

a 10 μ silica gel (Varian Si-10) , 25

cm X 2 mm (i.d.). A gradient elution program was developed using heptane and methylene chloride. For a

satisfactory separation of the cannabinoids as well as to assure accurate

ms quantitation, it was necessary to

routinely record the u.v. spectrum of

each lot of heptane and methylene

chloride prior to its use in the gradient program. Only those lots of

heptane and methylene chloride which

gave minimum absorbance in the‘region

of 260-280 mμ were used.

The gradient elution program, developed for this application, began at

95:5%, heptane:methylene chloride

and proceeded to 95:5%, methylene

chloride:heptane over 9 minutes.

The program was reversed, i.e., from

95:5% methylene chloride:heptane to

the initial 95:5% heptane:methylene

chloride mixture, thereby regenerating the column. A solvent flow rate

of 120 ml/hr was used for all determinations. By employing these conditions 9

-THC as well as d3-

9

-THC

were found to have a retention time

of 4.7 minutes, i.e., they appear

at a gradient elution mixture of 52:

48 methylene chloride:heptane. The

other major constituents of marijuana, CBD, CBN and CBC were found

to have retention times of 4.4, 4.6

and 5.6 minutes, respectively. The

amount of d3-

9

-THC added to the

body sample was sufficient to allow

u.v. detection of 9

-THC (labeled

plus unlabeled) as it eminated from

the column. A 10 cm "zero dead volume" stainless steel tube was attached to the flow cell of the spectrophotometer to facilitate collection of the effluent droplets almost

instantaneously after passing through

the flow cell.

Mass spectrometer quantification

All ms analyses were accomplished

using a Varian MAT SMI-B high resolution, double-focus mass spectrometer. A new ion-counting technique

97
was developed in conjunction with

the peak matching accessory which

provided for a rapid comparison between data from the internal standard (d3-

9

-THC, mass 317) and the

assayed compound (

9

-THC, mass 314).

Each sample from the hplc was introduced into the ms via the direct insertion probe. The instrument was

initially focused exactly on the 317

(d3-

9

-THC) mass signal, then through

the action of the peak matching unit,

and with the high resolution capability alternately focused to the 314

(

9

-THC) mass signal, cf., Figure 1.

Figure 1

Liquid Sample (5 mcl)

Direct Insertion Probe

Mass Spectrometer

Peak Matching Acces.

317 314

Ion Counter

Data Processor - Plotter

Standard Data

Register A Register B

As this alternation from one signal

to another occurs the exact number

of ion counts for each compound is

recorded and stored in two channels

of a Princeton Applied Research Model

SSR1110 dedicated computer. This

unit performs a summation of the number of ion events occurring in both

mass peaks (314 and 317) and stores

these in two registers. Thus a running total of ions detected from the

317 internal standard and the unknown amount of the 314 mass are

Figure 2

Ion Countinq Cycle

Counting Period Counting Period

t RA = 0.067 sec. t RB = 0.067 sec.

M/e = 317 AMU M/e = 314 AMU

Ten Data Points

Fifty Cycles

5 0 5 0

Ion Counts (317) Ion Counts (314)

t R A = 1 tRB = 1

T = 50 t R A + 50 t R B

=6.7 sec.

One Data Point

10 x T = 67 sec.

500 Counting Cycles

1,000 Counting Periods

stored separately by the counter as

shown schematically in Figure 2. The

peak matcher accessory is set to

dwell for 67 milliseconds on each

mass signal before it alternates to

the other mass signal. Repeated experimentation has shown that a total

counting time of 67 seconds combined

with a probe temperature of 65° gave

optimum results. Thus in 67 seconds

the alternating cycle is repeated

500 times yielding 1000 bits of data

for comparison and quantification.

With time the sample is depleted but

the ratio based on the internal standard remains linear and provides for

dependable quantification as shown

in Figure 3. The curve shown in this

figure was obtained from a HewlettPackard 9820 computer-plotter by

using the data from register B for

the ordinate and register A for the

abscissa and determining the least

square best straight line. The

slope of the data line gives the 314/

317 ratio. The amount of 9

-THC

(mass 314) is determined by multiplying this ratio times the known

amount of internal standard added.

In actual practice the d3-

9

-THC employed contained a small amount of

undeutrated 9-THC (mass 314). A ms

determination of this amount of mass

314 was made by analyzing 10 samples

of 1.6 μg d3-

9

-THC which had been

added to 1 ml of blood plasma by the

98
Figure 3

Table 1

Table 1. Precision and Accuracy in Recovery of

9

-THC Added to Human Plasma.

Added Found,a

ng/ml ng/ml

0 11.3 (9.35-13.6)b

1 1.2 (0.6-1.5)

5 4.5 (4.0-4.7)

10 10.5 (9.5-12.4)

20 20.0 (19.1-21.1)

40 41.2 (39.6-42.5)

60 62.1 (61.3-62.8)

80 82.8 (74.9-90.7)

100 103.7 (99.3-106.0)

n

10

3

3

10

3

3

3

3

3

Average percent recovered 103.8

±RSD RE

11.15 --

43.3 20.0

9.0 -11.12

10.0 5.0

5.14 0.0

3.58 3.0

1.26 3.5

9.55 3.5

3.65 3.7

b

 This value represents the amount of unlabeled

9

-THC in 1.6 μg of d3-

9

-THC. Other found

values have been corrected for this amount.

a

 Average (range) of n determinations.

method described above, cf. Table I.

This background value for 9-THC from the total amount of 9

-THC re-

(mass 314), inherently present in corded to give the actual found

the internal standard, is subtracted value.

99
Figure 4

Methods of blood plasma analysis

All glassware used was silinized using a reported method (Garrett and

Hunt, 1974). Whole blood was centrifuged at 2600 rpm for 20 minutes

to obtain blood plasma. To 1 ml of

blood plasma was added 1.6 μg of d3-

9

-THC followed by three repetitive

extractions with 2 ml of petroleum

ether for each extraction. The extracts were combined and evaporated

to dryness under nitrogen at room

temperature. The resultant residue

was reconstituted in 300 μl of heptane and the entire solution injected

into the hplc. A 100 μl wash of heptane was used on the vessel which

contained the extracts and it was also injected into the hplc and the

gradient elution program begun as

described above. When the peak for

9

-THC was noted on the recorder, the

eluant was collected in a silinized

screw cap vial. Collection of the

eluant was synchronized with the recorder tracing such that equal amounts

were obtained from either side of

the symmetrical peak. In general,

approximately 1 ml of eluant was collected for each 9

-THC peak. Samples

thus collected from the hplc were

stored at -5° until ms analysis.

Prior to ms analysis each sample was

evaporated under nitrogen at room

temperature to approximately a 10 μl

volume. A microsyringe was used to

transfer this solution in two portions to a 5 μl gold cup. The solution was allowed to air evaporate

and the gold cup was introduced into

the ms by attaching it to the direct

insertion probe. Quantification was

accomplished as described above.

Validation of the assay method in

blood plasma

Blood from 10 laboratory workers

known to be non-users of marijuana

was drawn and analyzed as detailed

above. These blood plasmas constituted the control samples. Each sample contained some background amount

o f 9-THC (mass 314) since the 1.6

μg of d3-

9

-THC, added to each plasma

sample, contained an average of 11.3

ng of 9-THC (mass 314), cf. Table

I. To demonstrate the reproducibility and accuracy of the developed method, pooled human plasma

samples were analyzed to which known

amounts of 9-THC had been added.

Table I is a summary of this study.

Figure 4 is a plot of the data given

in Table I. As outlined above, the

intercept in Figure 4 does not pass

through the origin or zero value

since some 9-THC (mass 314) is

present in the d3-

9

-THC used as

the internal standard.

100
Methods of breath analysis

For human breath collection a standard face mask (Welch model 7500-

30G) was modified by placing a 2.5

cm diameter port directly in front

of the mouth. On the interior of

the mask was placed a 4.5 cm (o.d.)

by 2.0 cm thick rubber ring of sufficent flexibility to firmly hold

a specially molded polyethylene filter. The polyethylene was formed

into a small wafer size, i.e., a

disc 3.0 cm in diameter and 0.25 cm

thick. This polyethylene wafer was

thus held directly in front of the

subject's mouth and approximately

1.5 cm away from the lips. Figure

5 gives a cross-sectional view of

this modified mask. For actual

breath collections each subject was

asked to breathe for one minute with

the mask positioned over the nose

and mouth using deep inhalations and

exhalations through the mouth. The

polyethylene wafers were placed in

and taken from the mask using disposable examination gloves. After

breath collection, each sample was

placed in 10 ml of methanol in a

silinized beaker and then ultrasonicated for 30 minutes. To each

extract was added 1.6 μg d3-

9

-THC

and the solution was evaporated to

dryness at room temperature, under

nitrogen. The residue was reconstituted in 300 μl of heptane and

analyzed via hplc-ms as described

above.

Figure 5

101
Method of saliva analysis

Saliva was collected from each volunteer in silinized collection tubes

and immediately frozen until analysis. To 100 μl of saliva was added

1.6 μg of d3-

9

-THC followed by extraction 3 times with 2 ml of petroleum ether for each extraction. The

extracts were combined and evaporated to dryness under nitrogen at

room temperature. The residue was

reconstituted in 300 μl of heptane

and analyzed by hplc-ms as discussed

above.

Method of bile analysis

Bile taken at post-mortem examination was frozen until analysis. To

100 μl of the bile was added 1.6 μg

of d3-

9

-THC and 1 ml of pH 7.0 buffer. The mixture was extracted 3

times with 2 ml of petroleum ether

for each extraction and the extracts

combined and evaporated to dryness

under nitrogen at room temperature.

The residue was reconstituted in 300

μl of heptane and analyzed by hplcms as discussed above.

Method of brain analysis

Brain samples from the cerebrum taken at post-mortem examination were

frozen until analysis. To 5 g of

brain sample was added 20 ml of pH

7.0 buffer and 1.6 μg of d3-

9

-THC.

The entire mixture was homogenized

then extracted 3 times with 10 ml of

petroleum ether each time and the

extracts combined and evaporated to

dryness under nitrogen at room temperature.

Marijuana smoking studies

Eleven healthy male volunteers between the ages of 21 and 26 were

used in the marijuana smoking studies.

Each subject was within 10% of ideal

body weight and received both medical and psychological exams prior to

admission. Values for the following

tests were determined prior to the

study and each subject was required

to be within the normal range:

electrocardiogram, chest x-ray,

creatinine, BUN, LDH, SGOT, alkaline phosphatase, blood sugar, calcium, phosphorus, bilirubin, total

protein, albumin, cholesterol, uric

acid, hematocrit, hemoglobin,

platelet count and prothrombin time.

All subjects were moderate marijuana smokers. Each was requested not

to smoke marijuana for 2 days prior

to reporting for the study. The

subjects were brought into the hospital ward 12 hrs. prior to smoking

and they were not allowed any food

or drink after 12:00 am of the

study day. At approximately 8:00

am of the test day, each subject

had a heparin-lock placed in a forearm vein and 5 ml of blood was withdrawn and placed in a silinized heparin vacutainer tube. Each blood

sample was handled so that the blood

did not come in contact with the

rubber stopper. All blood samples

were immediately centrifuged at 2600

rpm and the plasma removed and

placed in another silinized tube and

frozen for later analysis. This

initial 5 ml of blood drawn prior

to smoking constituted the 0 hr sample. Each subject was then allowed

to smoke one marijuana ci arette

which contained 10.8 mq 9

-THC.

2.16 mg CBN, 0.9 mg CBC and 0.63 mg

CBD. Upon completion of smoking,

timing was begun. Blood samples (5

ml) were withdrawn at 0.25, 0.5, 1,

2, 3, 4, 12 and 24 hours. Each

blood sample was handled as described above. At the same time intervals saliva and breath samples

were taken from each subject.

9

-THC aerosol and oral administration studies

Two normal male volunteers, who met

the same physical criteria listed

for the marijuana smoking volunteers,

were used. Each subject was administered an aerosol spray which contained 10 mg of 9-THC. Blood samples were taken from each subject at

0, 0.25, 0.5, 1, 1.5, 2.0, 3.0, 4.0,

5.0 and 6.0 hours. Samples were supplied to our laboratory with a code

number. In addition to these samples,

some additional blood plasma samples

from subjects in an oral cannabanoid

study were also coded and submitted

for analysis. All samples were analyzed as discussed above.

RESULTS AND DISCUSSION

Analysis of trace substances in a

biological system is often limited

by the sensitivity of an analytical

method. This was precisely the case

with the analysis of 9-THC since

prior to the present work there were

102
not many analytical methods which

offered the selectivity and sensitivity needed. The newly developed

method reported herein was shown to

be reproducible, accurate and linear

in the range of 1-100 ng/ml of human

blood plasma. Use of hplc rior to

ms quantification permits 9-THC to

be selectively separated from the

other cannabinoids present in marijuana preventing an erroneous analysis with the other mass 314 cannabinoids, viz., CBD and CBC. Also

when the ms analysis is performed

the high resolution capability of

the ms prevents any possible confusion of compounds, i.e., only 9

-THC

will have the correct mass number.

Use of the stable isotopic' form of

9

-THC in the developed method allows

for a control of extraction efficiency as well as a marker for peak collection and quantification. Thus the

d3 -

9

-THC was added to the plasma

prior to extraction and gave a control for any losses which might occur during extraction or chromatographic procedures. In the described

method the plasma was extracted three

times. Ordinarily three extractions

would be redundant when an internal

standard has been added. However,

the main reason for using the multiple extractions is to introduce to

the ms a sufficient amount of the

stable isotope for accurate quantification while not using excessive

amounts of the isotope for the internal standard. The other important

use of the d3-

9

-THC was to permit

the total amount of 9-THC (both labled and unlabled) to be detectable

by the u.v. spectrophotometer attached to the output of the hplc

since 9-THC has a low extinction

coefficient.

A human marijuana smoking study was

conducted and blood samples taken at

appropriate time intervals for 24

hours. Each sample was analyzed by

hplc-ms and the results are given

in Table II. As observed from the

mean values in this table, the peak

blood plasma concentration of 9-THC

occurs in all individuals, except

L.G., at 0.25 hours. Also worthy of

note in Table II is the fact that

most subjects showed a level of 9

-

THC in their control samples. This

is readily explainable since all subjects were prior users of marijuana

and most likely had smoked sometime

before reporting to the study.

The breath samples from each subject

in the controlled smoking study were

also analyzed by the new method.

Since the volume of exhaled air was

not measured, no meaningful quantification data could be obtained. That

is, the subjects were asked to

breathe through the breath apparatus

for a period of one minute and in

this time the rate of exhalation in

Table 2

Table 2. Amount of 9

-THC (ng/ml) found in human

blood plasma following marijuana smoking.

SUBJECTS 0 .25

23.0

39.0

34.1

66.7

38.4

57.9

39.7

43.7

15.3

34.6

20.6

.5 4

D.L. 3.6

B.N. 1.2

B.B. 1.6

D.J. 0

F.R. 4.2

B.W. 0.4

W.Z. 1.2

R.B. 5.7

L.G. 7.6

T.V. 11.3

K.C. 4.7

10.1

11.7

23.3

19.7

20.1

24.8

19.7

18.9

39.9

16.5

22.4

TIME (hrs.)

1

12.2

3.8

16.8

11.8

6.8

14.8

6.8

27.6

8.1

10.1

14.5

7.1

0.9

1.4

3.0

8.4

10.7

5.3

6.7

2.1

5.1

8.4

2

MEAN 3.8 37.5 20.7 12.1 5.2 3.9 3.6 2.5 1.9

3

0.9

0

1.1

3.8

2.8

2.4

4.9

4.4

11.1

4.2

7.7

3.5

0.7

0

4.3

6.3

6.2

2.0

2.5

7.5

2.3

4.7

12 24

2.7 0

0 1.7

2.0 0.9

0 3.1

0.6 0.9

5.8 2.5

0 0

0.1 3.4

9.7 2.5

1.6 2.8

5.0 2.5

103
Table 3

SUBJECT 1

SUBJECT 2

ORAL

CANNABANOID

SUBJECTSd

Table 3.

Blood Plasma Levels of 9-THC

Code No.a

Time (min)

Following 9-THC ng of 9-THC

11A 0 7.4

10A 5 30.3

7A 15 9.6

16A 30 4.3

22A 60 14.6

12A 90 15.2

13A 120 14.9

5A 180 lostb

15A 240 24.5

4A 300 20.3

2A 360 4.9

18A 0 2.2

19A 5 10.4

14A 15 13.3

20A 30 11.8

9A 60 no samplec

3A 90 13.6

17A 120 no samplec

8A 180 7.1

21A 240 7.6

1A 300 1.4

6A 360 lostb

3 0

e

0.1

4 300f

0.0

5 300g

lostb

6 300e

0.0

7 300e

0.0

8 O

f

0.4

9 0

g

0.0

10 O

f

4.0

4 days prior to receiving the oral formulation.

e

 Receiving oral CBN.

f

 Receiving oral CBD.

g

 Receiving oral 9-THC.

104

a

 Code number of sample as supplied by Dr. Tashkin.

b

 Laboratory accident resulting in sample loss.

c

 Sample broken in transit.

d

 All subjects were confined in the hospital ward for
each subject may have varied. More

important, however, was the fact that

a positive and statistically meaningful level of 9

-THC could be determined in the breath of these known

marijuana smokers during the initial

60 minutes following smoking.

Saliva samples from marijuana smokers

have to be quantitatively treated

very much like the breath samples.

That is, in the present study no attempt was made to accurately control

the amount of saliva exudated. Thus

any attempt to quantitatively assign

levels of 9-THC per unit volume

would be meaningless. However, it

was observed by analyzing the saliva

samples that a positive, statistically significant level of 9

-THC

could be detected in the saliva of

known marijuana smokers during the

initial 60 minutes following smoking.

Analysis of the blood samples which

came from a

9

-THC aerosol study

provided an opportunity to analyze

samples on a blind basis. The data

obtained from this study is shown

in Table III. Positive levels of

9

-THC for the control samples (0

hour) is readily explainable since

each subject was a marijuana user

and was not confined prior to the

study. In contrast, however, the

oral cannabanoid subjects were confined for 4 days prior to the collection of their control samples.

Also as shown in Table III, other

cannabanoids were being administered

which could have conceivably interfered with the 9

-THC determination.

However, the hplc program successfully separated the 9

-THC fraction

as was verified by correlation following the blind sample assays. For

both studies there was a good correlation between the physiological

states recorded following administration and the 9

-THC levels measured

later by means of the quantification

technology reported in this paper

(Tashkin, 1976).

CONCLUSIONS

A method was developed for quantitatively determining 9

-THC in human

body specimens. The method was shown

to be accurate and precise over a

concentration range of 1-100 ng/ml

in human blood plasma. This methodwas

used to assay blood plasma, breath and

saliva of human subjects following

smoking of a marijuana cigarette.

Results from these studies demonstrated that during the early time intervals following marijuana smoking the

levels of 9-THC measured are of Sufficient magnitude to be clearly discernable from the amount of 9

-THC

(i.e., 11.3 ng) added in the internal

standard.

The newly developed method has also

been used to study the relationship

between 9-THC levels in blood plasma, bile and brain of post-mortem

samples. Such data has revealed

that 9-THC levels are higher in

brain and bile than in blood plasma.

Data was also accumulated on blood

plasma samples which came from subjects receiving either an aerosol

formulation of 9

-THC or oral formulations of CBD, CBN and 9

-THC.

Analysis of these samples on a blind

basis provided further validation of

the new technology and gave results

which were consistant with the observed physiological responses.

105
ACKNOWLEDGMENTS

The authors wish to thank Mr. Robert

Nowlan, Ms. Phyllis Lessin and Dr.

Donald Tashkin of the University of

California - Los Angeles for their

cooperation in the human studies performed there.

This work was performed under contract DOT HS 4 00968 from the Department of Transportation, National

Highway Traffic Safety Administration.

The technical advise given by Dr.

Fred B. Benjamin of this agency and

Dr. Robert Willette of the National

Institute on Drug Abuse was greatly

appreciated.

REFERENCES

Agurell, S., Gustafsson, B., Holmstedt, B., Leander, K., Lindgren,

J., Nilsson, I., Sandberq, F., and

Asberg, M.: J. Pharm. Pharmacol.,

25,554 (1973).

Doorenbos, N.J., Fetterman, P.S.,

Quimby, M.W. and Turner, C.E.: N.Y.

Acad. Sci., 191,3 (1971).

Edery, H., Grunfeld, Y., Ben-Zvi, Z.,

and Mechoulam, R.: N.Y. Acad. Sc.,

191,40 (1971).

Fenimore, D.C., Freeman, R.R., and

Loy, P.R.: Anal. Chem., 45,2331

(1973).

Galanter, M., Wyatt, R.J., Lemberger,

L., Weingartner, H., Vaughan, T.B.

and Roth, W.T.: Sci., 176,934

(1972).

Garrett, E.R. and Hunt, C.A.: J.

Pharm. Sci., 63,1056 (1974).

Gross, S.J., Soares, J.R., Wong, S-L.

R. and Schuster, R.E.; Nature, 252,

581 (1974).

Tashkin, D.P., Private communication.

Teal, J.D., King, L. J., Forman, E.

J. and Marks, V.: Lancet, 1974, 553.

106
ANALYTICAL METHODS FOR THE DETERMINATION

OF CANNABINOIDS IN BIOLOGICAL MEDIA

M. E. Wall, Ph.D., T.M. Harvey, Ph.D., J.T. Bursey, Ph.D.,

D.R. Brine, B.S., D. Rosenthal, Ph.D.

Research Triangle Institute, North Carolina

ABSTRACT

A pharmacokinetic tudy of the blood plasma

levels in man of 9-tetrahydrocannabinol,

11-hydroxy- 9-tetrahydrocannabinol and cannabinol has been carried out by means of combined gas chromatographic-mass spectral

analysis. In some cases comparison of the

data was obtained on the same sample using

thin layer chromatography of radiolabeled

samples and electron capture gas-liquid chromatography. For the mass spectral studies

appropriately deuterium labeled analogs of

the previously named compounds were used

both as internal standards and as a carrier

for the relatively small amounts of nonlabeled drug present in plasma. Blood

samples were obtained at periodic intervals

up to 24 hours from volunteers receiving 4-5

m g

9

-THC intravenously. After extraction

and "clean-up" by Sephadex chromatography,

the extracts were concentrated and subjected

to glc-ms in the electron impact (ei) mode

or alternatively with a chemical ionization

(ci) source, in which case preliminary chromatography could be omitted. In all cases

calibration curves were obtained from replicate analyses of spiked plasma containing

the internal standard and various quantities

of the cannabinoid under analysis. A typical

biphasic elimination of the drug was observed

with rapid elimination of 9-THC from the

blood over a period of 40 min followed by a

much slower elimination up to 24 hours. The

experimental data show that 11-hydroxy- 9-

THC is found in the plasma in quantities

only of about one-twentieth to one-twentyfifth the values found for 9

-THC. Cannabinol

was not found in significant quantities. Good

agreement was obtained between the mass

spectral analyses and the thin layer chromatography or electron capture gas-liquid

chromatographic procedures.

INTRODUCTION

In recent years there has been a great increase in interest in the pharmacology,

metabolism and biodisposition of the cannabinoids [for recent reviews cf. Mechoulam, Paton

and Crown, Wall (1975) and Wall, et al.

(1975)]. Until recently quantitation of the

various cannabinoids in blood, urine, feces

and other biological tissues could be carried

107
out only by means of the use of appropriately

radiolabeled analogs of the cannabinoids under

study (Wall, et al. (1975) and Lemberger) .

Because of the widespread and increasing

opposition to the use of radiolabeled isotopes

in studies involving man and because many of

the studies currently being conducted with

various cannabinoids involve the use of large

scale experiments in which radiolabeled cannabinoids are not used, the need for the development of non-radiolabeled quantitative

methodology for certain key cannabinoids has

become increasingly apparent. In addition,

the use of radiolabeled thin layer chromatography techniques, while useful in initial

studies, lacks sufficient accuracy in the

sense that when-biological extracts are

studied, separation of 9-THC from cannabinol

and 11-hydroxy- 9-THC from other monohydroxylated analogs is poor. If such interfering

substances are present in considerable quantity, one would obtain erroneously high

values, This will increasingly be the case

when one is analyzing biological materials

obtained from marihuana smokers which contain

9

-THC, cannabinol, cannabidiol, and 11-

hydroxylated analogs of these compounds.

Quantitative gas liquid chromatography combined with mass spectrometry (glc-ms) has

been used with excellent results for the

quantitative analysis of drugs in biological

materials, combining as it does the separative powers of glc and the inherent sensitivity of ms detection. Pioneer studies by

Hammar, Holmstedt and Ryhage introduced the

concept of mass fragmentography [now also

called multiple ion detection (MID)] and

greatly increased the sensitivity of ms

methodology so that it could be applied to

the nanogram and picogram levels. The concept has been applied to many drugs and recently by Agurell, Holmstedt and co-workers

to the determination of 9-THC in blood

plasma. We wish to present in this paper

methods for the quantitative determination by

combined glc-ms of 9-tetrahydrocannabinol

(

9

-THC), 11-hydroxy- 9-THC and cannabinol

(CBN) in human plasma and results on the same

samples obtained by the tlc-radiolabeled or

electron capture glc procedures.

METHODS

Clinical protocol

Human, male volunteers who were experienced

marihuana users were administered 4.0-5.0 mg

of 9-THC by the intravenous infusion method

of Perez-Reyes et al. The volunteers were

kept under medical supervision for 24 hours

in the Clinical Research Unit of the University of North Carolina, School of Medicine.

Blood samples (10 ml approximately) were

collected at periodic intervals over a period

of 24 hours. Plasma was obtained by centrifugation, immediately frozen and stored in

frozen condition until analyzed.

Internal standards

A key feature of our quantitative procedures

was the use of appropriate deuterated analogs

of the cannabinoids under study as both carriers for the small quantity of cannabinoids

expected to be present in many cases and as

internal standards for quantitation by mass

spectrometry. The structures of the cannabinoids and their deuterated analogs used in

these studies are shown in Figure 1. All of

Figure 1

Figure 1.

Structure of Cannabinoids and Internal Standards

the compounds used were synthetic and were

made available by the National Institute on

Drug Abuse Synthesis Program.1 Synthetic

methods for the various deuterated cannabinoids utilized in these studies have been

presented by Pitt, et al.

Analytical procedures prior to glc-ms

General Precautions.--Close attention must be

paid to the precedural details presented below

in order to obtain reproducible and quantitative data. In general in working with

cannabinoids exposure of samples or extracts

108
to light or air should be minimized. All

solvent evaporations should be conducted in

vacuo or under nitrogen at low temperature.

Cannabinoids in nanogram levels are subject to

adsorption on the surface of glassware. In

order to minimize this problem all glassware,

including chromatography columns, were coated

by silylation using 5% DMCS in toluene.

Extraction and Purification Prior to Analysis

by GLC-MS in Ei Mode.--When the mass spectrometers were operated in this mode the molecular ions or charged fragments utilized for the

quantitative analysis of underivatized cannabinoids were in a range of m/e of 320 or

lower. Preliminary studies-with plasma

extracts indicated that interference from

endogenous plasma constituents would be

encountered. This could be avoided by carrying out a preliminary cleanup by Sephadex

LH-20 chromatography prior to the glc-ms

step. The methods which are pretsented are for

the combined determination of 9-THC (1a),

11-hydroxy- 9-THC (2a), and cannabinol (3a).

The methods, of course, are equally utilizable

for the determination of individual constituents. Deuterated internal standards (cf.,

Figure 1) were added to a sample of 3.0 ml

of cold (not frozen) plasma as follows: 1b,

150.0 ng; 2b, 15.0 ng; and 3b, 15.0 ng. Each

internal standard was added in 15-30 μl

ethanol. Following addition of each internal

standard the plasma sample was stirred for

3-5 seconds in a vortex agitator and then

subjected to sonication (Cole-Parmer ultrasonic cleaner) for the same time. The

plasma samples (contained in a screw capped

centrifuge tube) were then extracted 4 times

with 6.0 ml petroleum ether (bp 30-60°,

Nanogram Grade or Burdick and Jackson)

containing 1.5% isoamyl ether. The tubes

were agitated 15 minutes each time in a

vortex agitator and the layers separated by

centrifugation after each extraction. The

petroleum ether extracts were combined, evaporated in vacuo at room temperature and freeze

dried overnight to remove water and isoamyl

alcohol. The dried residue was dissolved in

a minimal volume of petroleum ether/chloroform/ethanol (10:10:1) and chromatographed

on 1 x 40 an water jacketed Sephadex LH-20

columns at 26°C in the same solvent mixture.

Twenty-seven ml of column eluant were collected and discarded. Seven ml of eluant

were then collected as the fraction containing 9

-THC. The next 8 ml of eluant were

collected as the CBN-containing fraction.

Thirty-eight ml of column effluent were then

collected and discarded. Finally, 17 ml of

eluant were collected as the fraction containing 11-hydroxy 9

-THC. The 9-THC and

CBN fractions were evaporated to dryness and

dissolved in 35 μl hexane. The 11-hydroxy9

-THC fraction was evaporated to dryness

under vacuum and heated with 75 μl of Regisil

(BSTFA + 1% TCMS) in a closed vial at 110°

for 3 hours. The reagent was removed in

vacuum and the residue dissolved in 20 μl

hexane.

Extraction Prior to Analysis by GC-MS in CI

Mode.--In this mode of operation 9-THC and

CBN were determined as their pentafluoropropionate ester derivatives, Fig. 1, 1c and

3c, respectively, using the PFP ester of

hexahydrocannahinol, 4b as the internal

standard for both analyses. It was found

that endogenous plasma constituents did not

interfere at the molecular ion positions utilized which were in the range of m/e 455-465.

Hence preliminary purificatin of the extracts

was not required. To a sample of 2.0-3.0

ml of plasma were added the internal standard

hexahydrocannabinol 4b, 15 ng/ml plasma,

under conditions described in the section

above. The samples were extracted four

times with hexane, 2.0 ml/ml plasma as

described in the above section. The combined

hexane extracts were washed sequentially

with 2.0 ml 0.1 N sodium hydroxide, 2.0 ml

0.1 N hydrochloric acid, and 4.0 ml distilled

water. The washed hexane extracts were

evaporated and dried overnight in vacuo. To

the dried extracts were added 0.2 ml hexane,

50 μl pyridine/benzene (5:95), and 50 μl

pentafluoropropionic acid anhydride. After

heating at 40°C for one hour, the reaction

mixture was washed with 0.2 ml of saturated

sodium bicarbonate solution, which was then

backwashed with an additional 0.2 ml hexane.

The hexane extracts were combined and evaporated under nitrogen. The derivatized

extracts were dissolved in 20 μl hexane.

Gas chromatography conditions

LKB-GLC-MS.--With this instrument we utilized

2% OV-17 on Chromosorb W-HP, 80-100 mesh in

3' or 6' x 1/4" glass columns, temp. 220°C,

the former being used for 11-hydroxy- 9-THCbis-TMS ether; the latter for both 9

-THC temp.

220°C; and CBN, temp. 240°C. Helium was used

as the gas phase at a rate of 35 ml/min.

Under the above stipulated conditions retention times of 4-6 minutes were observed for

each compound.

Finnigan-GLC-MS.--Only 9-THC and CBN were

analyzed on this instrument in the ei mode.

We used 6' glass columns containing 1% SE-30

on 100/120 mesh Chromatosorb W-HP, column

temperatures 200-230°; He flow 30-35 ml/min.

In the ci mode, which was used only with

pentafluoropropionate derivatives, the same

column was used at 180°.

109
Mass spectrometry

Only basic operating details are presented in

regard to thems-computer systems described

below. For a much more detailed report cf.

Rosenthal et al.

LKB 9000 GLC-MS .--This instrument is of the

magnetic sector type and was operated at

ev in the ei mode. The instrument was fitted

with a peak matching accessory, modified fran

an instrument described by Klein, which permits detection of very law levels of compounds

by the technique of acceleratig voltage

alternation (AVA) (Hammar, 1968 and Agurell

1973). For 9

-THC the mass spectrum was set

to focus alternately on the ions m/e 314 and

317 which correspond to the molecular ion of

the compound and its trideuterio analog. For

CBN the molecular ions were m/e 310 and 313.

For analysis of 11-hydroxy- 9

-THC as the bistms-ether, the strong M-103 ion (Wall, et

al., 1970, Wall, et al., 1976) at m/e 371

and 374 respectively was selected. The AVA

accessory used with the mass spectrometer

scans through each peak in turn; a hardware

counter determines the area under the peaks

each time they are scanned. The ratio of the

selected response (either area or peak

height) for the two ions in question is the

value used to determine the quantity of unknown

cannabinoid in the plasma. Appropriate calibration curves (Fig. 2, 3) are constructed by

adding variable amounts of cannabinoid to a

fixed quantity of corresponding trideuterio

analog in human plasma. Extraction, preliminary purifcation, and glc are carried out as

described above, Analysis by GLC-MS-EI.

Finnigan 3000-GLC-MS.--This instrument is a

quadrupole mass spectrometer and was operated

at 70 ev in the ei mode. Its scan is controlled

by a dedicated PDP-12 camputer which also

acquires data from the instrument. Although

the computer operates on a different principle,

the ratios of peak area or peak heights are

determined as described above using the same

molecular or fragment ions described in the

previous section.

In the ci mode isobutane was used as the carrier gas at a pressure of 400 μ; 9

-THC and

the internal standard HHC were determined as

the corresponding PFP ester as the MH+ ion

at m/e 461 and 463, respectively. Similarly,

for the case of CBN the MH+ ions were m/e 457

and 463, respectively. Plasma calibration

curves were obtained in the manner described

previously.

Figure 2 Figure 3

Figure 3.

Figure 2. Finnigan 3300-ei Plasma Calibration Curve

LKB 9000-ei Plasma Calibration Curve for 9

-THC for 9

-THC

110
TLC-radiolabel procedure

The volunteer subjects (described in Clinical

protocol) all received 100 μCi of tritium

labeled 9-THC, along with the standard 4.0-

5.0 mg IV dose. Two to three ml aliquots of

plasma were analyzed by the procedure described

by Wall, et al. (1975).

Electron capture-glc procedure

In this procedure 9

-THC was determined as the

pentafluropropionate ester derivative using

hexahydrocannabinol as internal standard.

Extraction and derivatization were carried

out as described under the procedure and

extraction for gc-ms in ci mode. The instrumentation utilized was constructed at RTI by

Dr. Edo Pellizzari as a modification of the

design reported by Fenimore, et al.

A packed precolumn is utilized for initial

cleanup of the sample followed by a packed

column or a capillary column to furnish the

requisite resolution. By interposing a

valving system and cold trap between the two

columns, a small portion of the effluent from

the first column can be introduced to the

second column with minimum loss of efficiency.

Furthermore, this instrumental design provides

the capability for temperature programming

which is seldom used in ec systems because of

excessive base-line drift due to the detector’s

sensitivity to column bleed. In the reported

dual column-dual oven system, the precolumn

can be temperature programmed while the high

efficiency column is maintained at isothermal

temperature. Thus, in effect, rapid and

efficient separations are achieved on the precolumn without disturbing the base-line response of the ec detector.

The instrument which we have assembled in our

laboratory consists of two separately controlled ovens (Varian Model 1440, Walnut Creek,

California). As shown in Figure 4 , one oven

houses the packed columns and possesses the

capability of linear temperature progamming;

the second oven contains the capillary column

or a second packed column and is operated

isothermally. The effluent from the first

packed column leads to an eight-port, high

temperature, low dead volume switching valve

(CF-8-HTa, Valco Instruments, Houston, Texas).

The gas flow from the first column when in the

normal operating position passes to the valve

and through a capillary-loop trap and finally

to a flame ionization detector. The trap consists of about 1 ft of 1/8 in stainless steel

tubing to which is silver soldered a 2 meter

length of 1/16 in O.D. x 0.20 i.d. Ni-200

capillary tubing. A water supply at 4°C is

provided through the 1/2 in tubing for cooling

purposes.

Figure 4

Figure 4. Dual Gas Chromatographic System

111
At the exact time interval (retention time) at

which trapping of the constituents in the

effluent from the packed column is to occur,

the valve is rotated to the trapping position,

and the coolant is passed through the 1/2 in

tubing to cool the capillary loop. In this

position, a second carrier gas flows to the

high resolution capillary or second packed

column; thus, the base-line is undisturbed.

The trapped constituents are swept into the

second oven and column by returning the valve

to the original position and shutting off the

ambient water supply. The heat in the first

oven is sufficient to rapidly raise the temperature on the capillary trap for sample

vaporation. A heated gold-plated Ni transfer

line connects the packed column, valve and

capillary or second column. The transfer line

is also coated with the same stationary phase

as in the final column.

Our system allows the utilization of flame

ionization, alkali flame, or electron capture

(ec) in the second oven for measuring drugs.

The second oven will also accept conventional

packed columns, metal (Ni) or glass capillaries. A linear ec amplifier (Hewlett Packard,

Avondale, Pennsylvania) and a miniature ecd

developed in our laboratories (Pellizzari,

1974) are employed in this dual gas chromatographed system.

Because of the low nanogram levels of cannabinoids found in human plasma, it was decided to

use the electron capture detector to allow

maximum sensitivity in the cannabinoid analysis. To make the analytical method more

widely applicable by other researchers, it was

decided to use packed columns in both ovens.

Glc conditions.--Plasma impurities were separated on a ten-foot column of 2% SE30 on Supelcoport (80/l00 mesh) at 205°C with a carrier

gas (N2) flow rate of 15 ml/min. Column

effluent was collected at the retention times

of HHC and 9-THC and transferred for quantitative analysis to a six-foot column of 2% OV225

on Supelcoport (100/120 mesh) at 178°C with

a carrier gas (5% methane - 95% argon) flow

rate of 9.5 ml/min.

Plasma calibration curves for 9-THC obtained

with the LKB and Finnigan mass spectrometers

in the ei mode are shown in Figures 2 and 3;

the calibration curve obtained with the

Finnigan instrument in the ci mode is shown

in Figure 5. In each case a linear calibration curve was obtained in range 1-100 nanograms

g

-THC/ml plasma. Detection of 9-THC

below the lower limit could be obtained (detection limits 0.1). 0.5 Ng/ml of plasma is

regarded as the minimal concentration at

Figure 5

Figure 5.

Finnigan 3300-ci Plasma Calibration Curve

for 9-THC

which reliable data could be obtained. The

standard error of estimation for LKB data

as obtained by linear regression analysis

was 0.114, and the correlation coefficient

was 0.9970. For the Finnigan in ei mode, the

comparable values were 0.034 and 0.9994, respectively. In the ci mode of the Finnigan

mass spectrometer the corresponding values

were 0.20 and 0.9977. Plasma calibration

curves for CBN are shown in Figures 6 and 7,

which respectively present the data obtained

on the LKB-ei and Finnigan-ei. Linear curves

on both instruments were obtained between

0.2-10.0 ng/ml plasma with detection limits

about 0.1 ng/ml. The standard error of estimation for the LKB and Finnigan instruments

was respectively 0.05 and 0.078; the correlation coefficients in each case were 0.998.

Preliminary data for CBN determined in the ci

mode as the PFP ester indicate that similar

results are to be expected. The plasma

calibration curve for 11-hydroxy- 9-THC-bistms-ethrer obtained with the LKB-mass spectrometer is shown in Figure 8. The curve was

linear in the range of 0.2-10.0 ng of 11-

hydroxy- 9-THC/ml plasma. As in previous

cases the accuracy of the determination decreases below concentrations of 1.0 ng/ml.

The standard error of the estimation was

0.027 and the correlation coefficient 0.9975.

RESULTS

112
Figure 6 Figure 8

Figure 6.

LKB 9000-ei Plasma Calibration Curve for CBN

Figure 7

Figure 7.

Finnigan 3300-ei Plasma Calibration Curve

for CBN

Figure 8.

LKB 9000-ei Plasma Calibration Curve for

11-Hydroxy- 9

-THC

Figure 9

Figure 9.

Plasma Levels of 9-THC, 11-Hydroxy- 9-THC

and CBN Found Over a 24-Hour Period in Human

Plasma from Volunteers Receiving 9-THC by

IV Administration

113
Figure 9 presents the average values with

standard error obtained for 9-THC, ll-hydroxy9

-THC and cannabinol from plasma of male

volunteers receiving 9-THC by intravenous

infusion. The measurements covered a 24 hour

period. 9

-THC values obtained with the LKB9000 ei source were in close agreement with

the data obtained on the Finnigan 3300-ci

source. Other data not presented here also

show close agrerment between the LKB and

Finnigan instruments when both were in the ei

mode. 9

-THC values increased rapidly during

the first 10-20 minutes, the peak values in the

range of 50-60 ng/ml coinciding With the maximal psychomimetric activity. A typical biphasic

elimination pattern was noted; the 9-THC

plasma levels decreased rapidly between 15-40

minutes and then fell at a much slower rate.

With a particular group of volunteers (3 subjects) levels after 24 hours were between 3-5

ng/ml. Spot checks at lower levels utilizing

the Finnigan MID program confirmed that the

substance being evaluated was indeed 9-THC

and not instrument "noise".

Figure l compares the results obtained from

the average of four subjects, analyzed by msei tlc radiolabel and electron capture glc.

Correlation coefficients are calculated in

Figure 11. The results are in reasonable

agreement, and in particular the glc-ms and

electron capture glc procedures gave good

agreement for most points over the whole

curve. In the case of 11-hydroxy- 9-THC

much lower levels were found; peak values

in the neighborhood of 2.0 ng/ml were noted

between 30-40 minutes. The maximal values

declined in a more gradual manner than was

the case for 9

-THC, falling to a level of

1.0 ng/ml in 60-90 minutes and 0.5 ng/ml

after 24 hours. The values for CBN are considered unreliable as obtained by the ei

technique since for the most part the levels

were well below 1.0 ng/ml and show no consistent pharmacokinetic pattern.

DISCUSSION

The basic objective of this investigation was

to establish sensitive methodology, which

would not depend on radiolabeling for the

quantitative estimation of 9

-THC, its primary

metabolite (Wall, 1970), 11-hydroxy- 9-THC and

cannabinol, which has been reported to be a

metabolite of 9

-THC in the rat (McCallum,

1975). This objective has been realized,

utilizing glc-ms with a variety of techniques

and instruments. In addition, a specialized

double glc procedure which utilizes one column

for clean up and one for final estimation has

been developed. Several aspects of our results

merit detailed discussion.

Figure 10

Figure 10.

9

-THC Found in the Plasma Following Intravenous Administration of 5 mg 9

-THC,

Average of 4 Subjects

Figure 11

Figure 11.

Least-Squares Best Lines Comparing All Data

Obtained by Each Two Methods of Analysis

114
Choice of instrument

Two completely different types of mass spectrometers coupled with different means for

quantitation of data were utilized. One was a

relatively old (1968) magnetic sector ms, the

LKB-9000 which was coupled with an alternation

voltage acceleration device (Rosenthal, in

press: Klein, 1972) which permitted measurement

of the ratio of the peak area of the unknown as

compared with that of the internal standard.

The other was a relatively new (1974) quadrupole ms, the Finnigan 3300 which was interfaced

to a PDP-12 computer. The Finnigan ms has both

ei and ci sources. As shown in Figures 2, 3, 6

and 7 and in the text of the Results section,

both instruments in the ei mode gave virtually

identical plasma calibration curves with identical linear range and quite similar stand

error of estimation. Figure 9 gives pharmacokinetic data in man obtained on the LKB in the

ei mode and the Finnigan in the ci mode. The

results are quite similar. It is thus evident

that a wide variety of mass spectrometers can

be used with comparable results provided appropriate internal carriers and standards are

added. Before concluding this discussion one

word of caution should be given: the nature of

the separators is most important; the LKB with

the Ryhage separator and the Finnigan with a

silylated glass jet separator gave appropriate

sensitivity. On the other hand, another mass

spectrometer which utilized a double glass coil

separator showed poor sensitivity and could not

be utilized for cannabinoid studies.

General analytical considerations

Internal standards.--As indicated previously,

the final mass spectrometric measurements can

be conducted with great accuracy. The key to

success in the various analytical studies was

the utilization of appropriate compounds which

could be used as both carriers and internal

standards. For this purpose deuterium labeled

cannabinoids identical to the parent compound

except for the label are ideal and were utilized for all of the ei studies. It is possible to use with equal success an internal

carrier which is not isotopically labeled as

long as its properties are very similar to that

of the cannabinoid being studied but permit

separation by glc. Hexahydrocannabinol was

excellent fo

studies of

this purpose and was used in ci

9

-THC and cannabinol.

Ei vs ci source.--In recent years quantitative

glc-ms using ci source has become more and

more popular for estimation of drugs in

biological materials. This is due to the

fact that in many cases greater sensitivity

can be obtained in the ci mode since there

is little molecular fragmentation as compared

to the situation when operating in the ei

mode. In our studies we have found no particular advantage in terms of sensitivity as

far as the two methods are concerned, the

useable lower limits in both cases being

about 1 ng/ml plasma and detection limits as

low as 0.2 ng/ml. However there would seem

to be a major advantage for utilizing the ci

source if a choice is available. As noted

earlier (cf., Methods section) a generalized

background interference is found in human

plasma in the region for determination of

underivatized 9-THC and cannabinol in the

region of m/e 310-320. This endogenous interference cannot be removed by glc techniques

alone but requires a preliminary "clean up",

utilizing Sephadex LH-20 chromatography. This

method which was described by Agurell et al.

for 9-THC and extended by us to mixtures

o f 9-THC, 11-hydroxy- 9

-THC and CBN, works

well for the former two compounds but is

time consuming. In addition, there is danger

of some conversion of 9

-THC to CBN because of

the longer exposure period. All of these

problems are avoided when the cannabinoids are

converted to the corresponding pentafluoropropionate esters. The molecular ions are then

in the region of m/e 455-465 and no interference was found after extraction of plasma with

hexane, derivatization and glc-ms determination. The method at present has been applied

t o 9-THC and CBN which can be extracted with

non-polar solvents. Whether more polar cannabinoids which require more polar solvents can

be successfully analyzed without prior purification by the ci technique is an open question

at this time.

Metabolic and pharmacokinetic data

The development of sensitive and accurate glcms methodology permitted a preliminary study

in man utilizing these techniques for the

precise determination of 9-THC, 11-hydroxy9

-THC and CBN in plasma. Previously we have

made an extensive stud

of the metabolism of

(Wall, et al., 1975)

9

-THC in man using

radiolabeled tracers and thin layer chromatography. The procedures utilized (in addition

to the undesirability of a radiolabeled tracer

in man) suffer from two potential sources of

error. The method utilized would not permit

separation of 9-THC from CBN, and in the

case of 11-hydroxy- 9

-THC, would not permit

separation from other monohydroxy-metabolites

which might be present (Wall, et al., 1973;

Wall, et al. 1975). The data in Figure 9

for 9-THC are quite comparable to pharmacokinetic data obtained in earlier studies

(Wall, et al., 1975). In both instances a

biphasic elimination curve is noted with a

sharp decline after the initial maximum level

followed by a much more gradual decrease.

Maximal values in the current studies were

50-60 ng/ml. After 24 hours, 3-5 ng/ml of

9

-THC were still found in the plasma. Our

115
results for 11-hydroxy- 9

-THC are probably

the most accurate data yet reported in man.

The concentration of this active metabolite

(cf. Figure 9) was only 2-3 ng/ml at peak

levels declining at a slower rate than 9

-

THC to 0.5 ng/ml after 24 hours. Although

9

-THC is readily converted to 11-hydroxy- 9-

THC in the liver (Wall, et al., 1975) only

small quantities find their way into the blood.

Our interest in CBN was aroused by reports

from McCallum (1975) and McCallum, et al.

(1975) which indicate that CBN might be a

transitory metabolite found at very early

time periods after administration of 9-THC.

As shown in Figure 9, the level of CBN was

below our reliability limits in the ei mode.

Other studies we have carried out by electron

capture glc or glc-ms in the ci mode indicate

the virtual absence of this substance at all

time periods. Since we have found that CBN

has the same general pharmacokinetic pattem

as

9

-THC in man (Wall, 1975), we must conACKNOWLEDGMENTS

These studies were conducted with the

support of the National Institute on Drug

Abuse under contracts HSM-41-71-95 and ADM45-74-109. We wish to thank Mario PerezReyes, M.D. for clinical material used in

some of these studies and express to Mrs.

Valerie H. Schindler and Mr. M. Taylor

our appreciation for their technical assistance.

clude that CBN can be disregarded in terms

of its importance as a metabolite in man.

Comparison of gc-ms with other procedures

As shown in Figures 10 and 11, the gc-ms procedures show reasonable agreement in the case

of 9-THC with data obtained by two independent procedures, involving respectively thin

layer chromatography of radiolabeled cannabinoids, and a double glc-electron capture

procedure. In preliminary studies on cannabinol levels of subjects who received 9-THC,

good agreement was found between the mass

spectrometric glc-ci method and electron capture glc. In each case no cannabinol could

be found. Studies on 11-hydroxy- 9-THC

levels by the glc-electron capture method

are in progress. Initial attempts to use

high pressure liquid chromatography as

mother method indicate that the sensitivity

by this procedure with current detectors is

of the order 10 ng/rnl. Hence at present

hplc techniques do not have requisite

sensitivity.

FOOTNOTE

1

Research Triangle Institute Contract HSM42-71-95. Appropriately qualified investigators may obtain a variety of labeled and

unlabeled cannabinoids by application to

Dr. Robert Willette, Acting Chief, The

Research Technology Branch, Division of

Research, National Institute on Drug Abuse,

Rockwall Building, 11400 Rockville Pike,

Rockville, Maryland 20852.

REFERENCES

Agurell, S., Gustafsson, B., Holmstedt, B., Klein, P. D., Hauman, J. R. and Eisler, W. J.

Leander, K., Lindgren, J.-E., Nilsson, I., Gas chromatograph-mass spectrometer-accelSandberg, F. and Asberg, M. Quantitation

o f 9-tetrahydrocannabinol in plasma from

erating voltage alternator system for the

measurement of stable isotope ratios in

cannabis smokers. J. Pharm. Pharmac., organic molecules. Anal. Chem., 1972, 44:

1973, 25: 554-558. 410-493.

Fenimore, D. C., Free R. R. and Loy, P. R. Lemberger, L. The metabolism of the tetraDetermination of 9

-tetrahydrocannabinol in hydrocannabinoids. In S. Garratini, F.

blood by electron capture gas chromatog- Hawking, A. Golden and I. Kopin (Eds.),

raphy. Anal. Chem., 1973, 45: 2331-2335. Advances in Pharmacology and Chemotherapy.

Academic Press, New York, NY. 1972: 221-

Hammar, C.-G. and Holmstedt, B. Mass fragmen- 251.

tography identification of chlorpromazine

and its metabolites in human blood by a new McCallum, N. K. The effect of cannabinol on

method. Anal. Biochem., 1968, 22: 532- 1

-tetrahydrocannabinol clearance from the

548. blood. Experimentia, 1975, 31: 957-958.

116
McCallum, N. K., Yagen, B., Levy, S. and

Mechoulam, R. Cannabinol: a rapidly

formed metabolite of 1- and 6-tetrahydrocannabinol. Experimentia, 1975, 31:

520-521.

Mechoulam, R. (Ed.), Marihuana. Academic

Press, New York, NY, 1973: 1-409.

Paton, W. D. and Crown, J. Cannabis and its

derivatives. Oxford University Press,

London, England. 1972: 1-198.

Pellizzari, E. D. High resolution electron

capture gas-liquid chromatography. J.

Chromatog., 1974, 92: 299-308.

Perez-Reyes, M., Timnons, M., Lipton, M.,

Davis, K. and Wall, M. Intravenous injection in man of 9

-tetrahydrocannabinol and

11-OH- 9-tetrahydrocannabinol. Science,

1972. 177: 633-635.

Pitt, C. G., Hobbs, D. T., Schran, H., Twine,

C. E. and Williams, D. L. The synthesis of

deuterium, carbon-14 and carrier-free

tritium-labeled cannabinoids. J. Label.

Comp., 1975, 11: 551-575.

Rosenthal, D., Harvey, M. T., Bursey, J. T.,

Brine, D. R. and Wall, M. E. Comparison Of

gas chromatography-mass spectrometry

methods for the determination of 9-tetrahydrocannabinol in plasma. Biomed. Mass

Spec., in preparation.

Wall, M. E. Recent advances in the chemistry

and metabolism of the cannabinoids. In

V. C. Runeckles (Ed.), Recent Advances in

Phychochemistry. Plenum Publishing Co., New

York, NY, 1975: 29-61.

Wall, M. E. and Brine, D. R. Application of

mass spectrometry to structure of metabolites of 9

-tetrahydrocannabinol. Summaries, International Symposium on Mass

Spectrometry in Biochemistry and Medicine.

Milan, Italy, 1973, p. 52.

Wall, M. E., Brine, D. R. and Perez-Reyes, M.

Identification of cannabinoids and metabolites in biological materials by combined

gas-liquid chromatography-mass spectrometry. In G. G. Nahas (Ed.), Marihuana:

Chemistry, Biochemistry, and Cellular

Effects. Springer-Verlag, New York, NY,

1976: 51-62.

Wall, M. E., Brine, D. R. and Perez-Reyes, M.

Metabolism of cannabinoids in man. In M.

Brade and S. Szara (Eds.), Pharmacology of

Marihuana. Raven Press, New York, NY,

1975.

Wall, M. E., Brine, D. R., Brine, G. A., Pitt,

C. G., Freudenthal, R. I. and Christensen,

H. D. Isolation, structure and biological

activity of several metabolites of 9-

tetrahydrocannabinol. J. Am. Chem. Soc.,

1970, 92; 3466-3468.

117
LIST OF CONTRIBUTORS

Quantitation of Cannabinoids in Biological Fluids by Radioimmunoassay

Arleen R. Chase, Paul R. Kelley, Alison Tunton-Rigby

Collaborative Research, Inc.

1365 Main Street

Waltham, Mass. 02154

Reese T. Jones

Langley Porter Neuropsychiatric Institute

University of California

San Francisco, Cal. 94143

Theresa Harwood

Drug Enforcement Administration

1405 Eye Street, N.W.

Washington, D.C. 20537

Separate Radioimmune Measurements of Body Fluid 9

-THC and 11-nor-9-Carboxy- 9

-THC.

Stanley J. Gross, M.D. and James R. Soares, Ph.D.

Department of Anatomy, School of Medicine

University of California, Los Angeles

Los Angeles, Cal. 90024

Radioimmunoassay of 9-Tetrahydrocannabinol

Clarence E. Cook, Ph.D., Mary L. Hawes, B.A., Ellen W. Amerson, B.A.,

Colin G. Pitt, Ph. D., and David Williams, B. A.

Research Triangle Institute

P.O. Box 12194

Research Triangle Park, North Carolina 27709

Determination of THC and its Metabolites by EMIT® Homogeneous Enzyme Immunoassay:

A Summary Report

G.L. Rowley, Ph.D., T.A. Armstrong, C.P. Crowl, W.M. Eimstad, W.M. Hu, Ph.D.,

J. K. Kam, R. Rodgers, Ph.D., R.C. Ronald, Ph.D., K.E. Rubenstein, Ph.D.,

B.G. Sheldon. and E.F. Ullman. Ph.D.

Syva Research Institute

3181 Porter Drive

Palo Alto, Cal. 94304

Separation and Sensitive Analysis of Tetrahydrocannabinol in Biological Fluids by HPLC and GLC

Edward R. Garrett and C. Anthony Hunt

College of Pharmacy, J. Hillis Miller Health Center

Box J-4

University of Florida

Gainesville, Fla. 32601

Determination of 9

-Tetrahydrocannabinol in Human Blood Serum by Electron Capture

Gas Chromatography

David C. Fenimore, Ph.D., Chester M. Davis, Ph.D., and Alec H. Horn

Instrumental Analysis Section

Texas Research Institute of Mental Sciences

1300 Moursund Street

Houston, Texas 77025

118
Detection and Quantification of Tetrahydrocannabinol in Blood Plasma

Agneta Ohlssonl, Jan-Erik Lindgren2 3, Kurt Leander2

, Stig Agurell1 3

1Faculty of Pharmacy, University of Uppsala

BMC, Box 579, S-751 23 Uppsala

2Department of Toxicology

Karolinska Institutet,

S-104 01 Stockholm 60

3Astra Läkemedel AB,

S-151-85

Södertälje, Sweden

A Method for the Identification of Acid Metabolites of Tetrahydrocannabinol (THC)

by Mass Fragmentography

Marianne Nordqvist1, Jan-Erik Lindgren2 3

,

 Stig Agurell1 3

Faculty of Pharmacy, University of Uppsala

BMC, Box 579, S-171 23 Uppsala

2Department of Toxicology

Karolinska Institutet,

S-104 01 Stockholm 60

3Astra Läkemedel AB,

S-151-85

Södertälje, Sweden

Quantitation of Cannabinoids in Biological Specimens Using Probability Based Matching

Gas Chromatography/Mass Spectrometry

Donald E. Green, Ph.D.

Biochemistry Research Lab 151-F

Veterans Administration Hospital

Palo Alto, Cal. 94304

Quantitation of 9-Tetrahydrocannabinol in Body Fluids by Gas Chromatography/Chemical IonizationMass Spectrometry

Ruthanne Detrick and Rodger L. Foltz

Battelle Memorial Institute

505 King Avenue

Columbus, Ohio 43201

HPLC-MS Determination of 9-Tetrahydrocannubinol in Human Body Samples

Jimmie L. Valentine, Ph.D., Paul J. Bryant, Ph.D., Paul L. Gutshall, M.S.,

Owen H. M. Gan, B.S., Everett D. Thompson, B.S., Hsien Chi Niu, Ph.D.

University of Missouri, Kansas City

5100 Rock Hill Road

Kansas City, Missouri 64110

Analytical Methods for the Determination of Cannabinoids in Biological Materials

M.E. Wall, Ph.D., T.M. Harvey, Ph.D., J.T. Bursey, Ph.D., D.R. Brine, B.S.,

and D. Rosenthal, Ph.D.

Research Triangle Institute

P.O. Box 12194

Research Triangle Park, North Carolina 27709

119
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