» Qnfik HEALTL: LIBRARY National Institute on Drug Abuse QafCh monograph series 7 CANNABINOID ASSAYS IN HUMANS AU8121976 umvmsnfafi m,m~" I W Wm M 379 / U.S. DEPARTMENT OF HEALTH. EDUCATION, AND WELFARE- PUBLIC HEALTH SERVICE ALCOHOL DRUG ABUSE AND flea #:évr \(‘1'1 X LCANNABINOID ASSAYS IN HUMAN§J Editor ROBERT E. WILLETTE, PH.D. Division of Research National Institute on Drug Abuse May 1976 NIDA Research Monograph 7 L National Institute on Drug Abuse “400 Rockville Pike Rockville, Maryland 20852 US. Department of Health, Education,and Welfare Public Health Service Alcohol, Drug Abuse, and Mental Health Administration lmmmmm EDITORIAL ADVISORY BOARD Avram Goldstein, MD. Jerome Jaffe, M.D. ReeseT. Jones, M.D. William McGlothlin, Ph.D. Jack Mendelson, M.D. Helen Nowlis, Ph.D. Lee Robins, Ph.D. 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 community. 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, DC. Washington University School of Medicine St. Louis, Missouri NIDA RESEARCH MONOGRAPH series Robert DuPont, M.D. William Pollin, MD. Robert C. Petersen, Ph.D. Eunice L. Corfman , M.A. DIRECTOR, NIDA DIRECTOR, NIDA RESEARCH DIVISION EDITOR-IN-CHIEF MANAGING EDITOR RockwaII Building 11400 Rockville Pike Rockville, Maryland, 20852 RHIZH’Z C17 WSW /?7e PUB; 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 (AIWD 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: 50-60; Microfiche: $3-25 iv 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 investi- gation along with others can now be undertaken with an expectation of far more precise find- ings 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 funda- mental pharmacokinetic and pharmacological re- search. The developments in method and instru- mentation 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 PREFACE For the past several years, there has been an increasing demand for qualitative and quantitative assays for identifying and measuring the consti- tuents of marihuana in the human body. This demand is prompted by the need for such assays in several research investigations that are attempting to im- prove 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 re- search 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 collec- tion 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 gen- eral; immunoassays offer speed and sensitivity and are very amenable to the screening of large numbers of samples. They often suffer from lack of speci- ficity, 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 instru- ment designed by Dr. Fenimore reduces the amount of effort required for sample preparation. As the technology in HPLC progresses, this method is be- coming 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 pro- ject'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 appli- cation 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 pro- gresses, so must the methods used in studying it. We now have methods to study the effects of mari- huana on driving, and if the evidence indicates that it poses a significant hazard, then a simple- perhaps 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. Division of Research National Institute on Drug Abuse April l976 ix CONTENTS FORE WORD PREFA CE QUANTITATION 0F CANNABINOIDS IN BIOLOGICAL FLUIDS BY RADIOIMVIUNOASSAY 1 Arleen R. Chase, Paul R. Kelley, Alison Taunton—Rigby, Reese T. Jones, Theresa Harwood SEPARATE RADIOIMVIUNE MEASUREMENTS OF BODY FLUID A9-THC AND 11—N0R-9-CARBOXY—A9-THC 10 Stanley J. Gross, MD. and James R. Soares, Ph.D. RADIOIWRJI‘DASSAY OF A9-TETRAHYDROCANNABINOL 15 Clarence E. Cook, Ph.D., Mary L. flames, B.A., Ellen W. Amerson, B.A., Colin G. Pitt, Ph.D., and David Williams, B.A. DETERMINATION OF THC AND ITS METABOLITES BY EMIT®HOIVDGENEOUS ENZYME IDIMUNOASSAY: A SUMMARY REPORT 28 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. SEPARATION AND SENSITIVE ANALYSIS OF TETRAHYDROCANNABINOL IN BIOLOGICAL FLUIDS BY HPLC AND GLC 33 Eduard R. Garrett and C. Anthony Hunt DETERMINATION OF A9-TETRAHYDROCANNABINOL IN HUMAN BLOOD SERUM BY ELECTRON CAPTURE GAS CHROMATOGRAPHY 42 David C. Fenimore, Ph.D., Chester M. Davis, Ph.D., and Alec H. Horn DETECTION AND QUANTIFICATION OF TETRAHYDROCANNABINOL IN BLOOD PLASMA 48 Agneta Ohlsson, Jan—Erik Lindgren, Kurt Leander, Stig Agurell A METIDD FOR THE IDENTIFICATION OF ACID METABOLITES OF TETRAHYDROCANNABINOL (THC) BY MASS FRAGVIENTOGRAPHY 64 Marianne Nordqvist, Jan-Erik Lindgren, Stig Agurell QUANTITATION OF CANNABINOIDS IN BIOLOGICAL SPECIMENS USING PROBABILITY BASED MATCHING GAS CHROMATOGRAPHY/MASS SPECTROMETRY 70 Donald E. Green, Ph.D. QUANTITATION OF Ag—T'ETRAHYDROCANNABINOL IN BODY FLUIDS BY GAS CHROMATOGRAPHY/ CHEMICAL IONIZATION-MASS SPECTRGVIETRY 88 Ruthanne Detrick and Rodger L. Foltz HPLC-MS DETERMINATION OF A9-TETRAHYDROCANNABINOL IN HUMAN BODY SAMPLES 96 Jimmie L. Valentine, Ph.D., Paul J. Bryant, Ph.D., Paul L. Gutshall, M.S., Owen H.M. Gan, B.S., Everett D. Thompson, 3.3., Hsien Chi Niu, Ph.D. ANALYTICAL METHODS FOR THE DETERMINATION OF CANNABINOIDS IN BIOLOGICAL MATERIALS 107 ME. Wall, Ph.D., T.M. Harvey, Ph.D., J.T. Bursey, Ph.D., D.R. Brine, 3.3., and D. Rosenthal, Ph.D. LIST OF CONTRIBUTORS 118 LIST OF MONOGRAPHS xi 120 QUANTITATION OF CANNABINOIDS IN BIOLOGICAL FLUIDS BY RADIOIMMUNOASSAY Arleen R. Chase, Paul R. Kelley, Alison Taunten-Rigby Collaborative Research, Inc. Waltham. Mass. Reese 1'. Jones Langley Porter Neuropsychiatric Institute University of, alifornia, San Francisco Theresa Herwoe Drug Enforcement Administration Washington, D. C. INTRODJCTION There is an expanding need to assay tetra— hydrocannabinol (A9'IHC) and its metabo- lites in biological fluids, particularly in the area of medical research. Methods used for the quantitation of cannabinoids include gas chromatography coupled with mass spectroscopy (CC/NE) , liquid chrom— atography and radioimmlmoassay. All methods are of value, especially when used to confirm the results of another pro- cedure, so that the method of choice de- pends on the particular application. Radioinnnmoassay is a conmon 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. 'IHE PRINCIPLES OF RADIOINMUNOASSAY Radioimmlmoassay depends on the affinity of a biological molecule, the antibody, for the antigen in question, in this case A9'IHC. Sensitivity is achieved through the use of a radioactive tracer molecule of high specific activity, called the labelled antigen. The extent to which the unla- belled antigen (A9'IHC) competes with the radioactive antigen for a limited number of receptor sites on the antibody serves as the basis for quantitation in the radio- innnmoassay. 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 deter— mined 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 antigen and the myriad of other compounds of widely diverse structure, which are found in biological fluids, is of funda- mental importance in its use as an analyti- cal tool. Macromolecules, such as proteins nucleic acids and polysaccharides, usually elicit an immune response when injected directly into an animal. However, low molecular weight compomds, such as A9'IHC, cannot elicit an immune response unless they are bound covalently to an antigenic macromolecule such as a protein or poly— peptide. The development of a radioinmuno— assay for a molecule, such as A9'IHC, in— volves (i) the synthesis of suitable cov- alent conjugates for inmmization, (ii) the production of antisera, (iii) the prepara- tion of a radioactive antigen, and (iv) the establishment of the assay based on the antigen-antibody reaction. SYNTHESIS OF COVALENT CONJUGATES FOR IMVIUNIZATION Molecules containing an amino or carboxy function can be coupled directly to the amino or carboxy groups of amino acid resi- dues in proteins or polypeptides by forma- tion 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 ex- posed to the antibody synthesizing cells. In the case of Ag'IHC several derivatives containing carboxy groups have been synthe- sized, and coupled to macromolecules. The use of a hemisuccinate ester of AngC has been reported (Teale et a1, 1975) as well as an azobenzoic acid-derivative (Grant et al, 1972 and Gross et al, 1974). We have u—s‘ed O-carboxymethy‘F’IHC prepared by re- action of A9'IHC with iodoethylacetate followed by basic hydrolysis as shown in Figure 1. Figure 1 Synthesis of 0—CarboxymethyZ-THC OH OCHZCOOC2H5 ICHZCOOCZHS K2003 O CsHu O CsHu APTHC KOH/ MeOH OCH ZCOOH O CsHu 0-Carboxymethyl-THC O—carboxymethyl—THC was coupled to bovine serum albumin (BSA) using two different dehydrating agents. In the first prepara— tion the water soluble carbodiimide, l-ethyl-S— (IS-dimethyl aminopropyl) - carbodiimide (EDC), was used and in the second preparation, coupling was effected by Woodward's reagent, N-ethyl—S-phenyl isooxazolium-S' -sulphonate . These reactions are summarized in Figure 2. Uncoupled CBM-THC and other small molecules were re— moved from the two preparations of O-carboxymethyl-THC—BSA (CBM-THC-BSAJ by dialysis and ethanol precipitation. The number of THC residues incorporated was estimated by spiking the preparations with 14c- A9ch. PRODUCTION OF ANTISERUM The two preparations of the conjugate, CBM-‘IHC—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 A9THC and, moreover, this binding could be inhibited by unlabelled A9llic. The response of two of the rabbits is summarized in Figure 3. These plots show the titer of antiserum requirgd to ive approximately 50% binding of —159 C. It can be seen that antisera were generated to both preparations. lbst of the data described in the rest of this paper were obtained using bleeding H of rabbit 56. Figure 2 synthesis of 0-CarboxymethyZ—THC—Bovine Serum Albumin NH -BSA EDC \ OCHZCOOH CsHu NHZ —BSA Woodward's Reagent l C) OCHZCMS—NH—BSA C5H“ O—Carboxymethyl-THC—BSA Figure 3 Titer of Antiserum Giving Approxomately 50% Binding of 3H-A9THC Following Immunization Titer Rabbit S6 Titer Rabbit 67 mooo - mooo - . I-I-I 1:800 - /\/ \ 1-.soo - I I I 1:600 - “600 _ I 1:400- 1:400- I-I I I I "2°“ ' I 1.200 — I I I / I/ a ’ / I/ ABCDEFGHIJKLMNOP ABCDEFGHI Bleeding No. Bleeding No. Note: Rabbit 56‘ was imrrmnized with CBM—THC—BSA prepared using EDC. Rabbit 67 was injected with CBM-THC—BSA coupled using Woodward ’s Reagent . RADIOLABELLED ANTIGEN, BINDING AND IN-IIBITION STUDIES High specific activity, radiolabelled com— pounds are used to develop sensit've radio— immmoassays. We have used both and 14C- AQ'IHC in our work.1 The 14C- AQ’IHC had a specific activity of 0.07 pCi/pg. At a final titer of 1:3 the antibody gave a maximmn binding of this 14c—A 9'IHC of 19.3%. A standard curve, prepared by inhibiting this binding with unlabelled A9mc, is shown in Figure 4. The 50% inhibition point occurred at 330 nanograms of A9'IHC. As expected, this system was too insensitive to be used as a working radioinmmoass ay . Figure 4 Inhibition of the Binding of 1"‘C‘-A‘3T1‘IC and Antibody of ASTHC 2° .96 Bound A \- 1s - I no - 5- ! lllu_.l_L A I llllll L I I I‘ll The insolubility of AngC in aqueous sys— tems is well established. Correspondingly, to provide an internal check on each stand— ard solution, 14C- A9'IHC is used as a standard. The exact number of nanograms of A91Hc in each standard solution is then verified by counting an aliquot in a liquid scintillation in the 4C channel. The 14C label does not interfere with the counting of the 5H label in the -channel. The assay conditions described above were used to obtain the standard curve shown in Figure 5. The system is capable of assay- ing as little as 0.25 nanograms of AQ'IHC reproducibly. CROSS REACTIVITY STUDIES The cross reactivity of various cannabin— oids, drugs and other compounds was estab- lished for the assay system. The data is summarized in Table l, 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—A9THC 0.1 1.0 Micrograms AQTHC Practical sensitivity could be achieved, however, using 3H-labelled [Mac with a specific activity of 41 pCi/pg. Binding levels of 30-50% were routinely achieved at a final titer of 1:750. The binding was inhibited by nanogram levels of unlabelled A9'IHC as shown in Figure 5. The 50% inhibition point occurred at 0.7 nanograms AglI-IC. This system was used as the basis of a working radioimnumoassay. ’IHE TRITIUM BASED RADIOIBMUNOASSAY SYSTEVI A working radioinnmoassay hag been develop- ed based on the use of 3H- A THC and anti- serum generated to CBM—lHC-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- A9'IHC and either the standard or unknown 'IHC sample are incubated for four hours at 4°C. Antibody bound and free labelled 'IHC 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. and Antibody by A9THC 50 t 96 Bound 4o - A so - I 20 - I I I 10 ' \- k I \I \ 0.1 L0 10 Nanograms A9THC Table. I Cross Reactivity of Certain Cannabinoids and Other Compounds With Ag-THC Antisen_1m_ 50% Inhibition Cannabinoids In Nanogams AZ-mc 0.7 A —'IHC 0.7 ll—OHA9-THC 0.4 11-OHA8—THC 0.7 SBOH Ag—THC 2 .0 11-NorA9-IHC-9-cooa 0.25 ll-Nor CBN—9—C0(H 1.5 Cannabinol (CBN) 3.5 Cannabidiol (CBD) 100 Cannabidiolic acid (CBD acid) 40 Cannabicyclol 8 Carmabichromene 40 Other ’LeVel ‘of Detection Compounds In Nanogam Caffeine >100 ,000 ”Aldactone" or Spironolactone >5,000 LSD tartrate ' >32 ,000 Secobarbitol (Technam spiked urine) . >100* Gibberellic acid >100,000 Nicotine >1 ,000 ,000 Morphine Sulfate >100,000 Cocaine (Technam spiked urine) Drugs of Abuse >60* Methadone HCl >100,000 Phenobarbitol, sodium >10,000 Tetracycline > 100 ,000 D,L-Ephedrine >10 ,000 Salicylic Acid >100 ,000 Carvone >95,000 (+) Limonene ] terpenes >80,000 uB-Thujone >90,000 Aldos terone-Zl—hemisuccinate >10 ,000 Progesterone >10 ,000 Estradiol steroids >10 ,000 A -1 cortisone >10,000 Testosterone >10 ,000 Cholesterol hydrogen succinate >20,000 * Highest level available reacted to any significant extent. The results obtained with the various cannabin- oids show that the antiserum reacts as well with Ag'IHC as with some of its metabo— lites. The antibody recognizes A9'IHC, ASTHC and several hydroxy metabolites. It can differsntiate to a limited extent between A 'IHC and cannabinol, cannabidiol, cannabidiolic acid and cannabichromene. The specificity of the antiserum for can- nabinoids without absolute specificity for 'IHC is advantageous since native THC is not excreted in the urine and an assay capable of detecting 'IHC only would be of little value. The assay system described here can detect Ag'IHC and its hydroxy metabolites, and therefore can be used to detect canna— bis use. APPLICATION TO BIOLOGICAL SAMPLES Before applying the assay to the detection of cannabinoids in urine samples, normal urines were assayed to determine any non- specific interference. It was found that normal urine was negative in the assay sys— tem. A pool of normal urine was spiked with known amounts of A9'1HC 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 A9'IHC, and (iii) 20 microliter samples can be quantitated accurately. Table 2 Assay of a Spiked Normal Urine Pool W Nano ams Ag'IHC Sainple No. e to arm e By—R'g'ifim—mnfis—saz 1 11.2 11.2 2 5.6 5.2 3 2.2 2.0 4 1.6 2.0 5 0.8 0.7 ASSAY OF BIOLOGICAL SAWLES Clinical studies were undertaken to estab- lish the validity of the assay under exper— imental 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, smnmarized 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 Patient A Patient B Hashish Oil for '12 days Patient A Patient B Level Found 30 ng/ml 40 ng/ml 7000 ng/ml 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 con- sistent 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 in— creased to 30 mg every 4 hours. The treat- ment was discontinued on day 24 at 4:00 p.m. On days 13 and 22 each subject was also given two cigarettes each of which contained 20 mg of THC. 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 dos- age level. These studies indicate that the radioimmunoassay will give neaningful re- sults. Table 4 Cannabinoid Levels in 24 Hour Urine Samples T No drug H N <—— N H A No drug 1 NNNNNNN NHr—II—Io-u-IHH n—u—I ES ooxlo‘mAmI: ovoooxncxmc-g: HD‘DmNO‘mhMNr—J Dose Schedule 10 mg/4 hr. 30 mg/4 hr. Patient 1 Patient 2 170 ng/ml 1498 ng/ml 76 168 47 106. 38 81 No sample 69 17 28 240 75 1718 992 1850 3721 1582 2994 2768 4255 3725 5425 4175 7819 7375 3675 3338 6088 6992 9950 9413 6313 5600 15767 6563 10813 4938 13275 10254 9556 10722 11181 5088 1325 5513 8775 3550 No sample 1600 1075 1662 442 808 223 *TWo cigarettes each containing 20 mg. of APTHC smoked in addition to regular dose. SUMMARY A tritium based radioimmunoassay for ZhgTHC and its netabolites has been developed for the use of investigators studying the epi— demiological, medical, clinical, and re- search aspects of cannabis use. The assay is sufficiently sensitive to detect can— nabinoids 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 re- flects 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 netabolites in biological samples without interference from other drugs, but also the evaluation of extracts of other kinds of les which may contain unmetabolized sigma The technique of radioimmunoassay has nany 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 This work was carried out under the sponsor- ship of the Department of Justice, Drug Enforcement Administration, under Contract No. DEA—7S-4, the Department of Justice, Bureau of Narcotics and Dangerous Drugs under Contract No. J-70-24 and independ- ently by Collaborative Research, Inc. 'can be applied. The method is non-destruct- .ive and obviates the need to use radio— labelled drugs in man during metabolic and other studies. This radioinmunoassay 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 specinen, for epidemiologists in determin- ing the full extent of cannabis use and to the medical/clinical community for establish— ing the minimum effective dose of Z§9THC for each patient. The widespread application of a single method of analysis should also remove a great deal of the controversy sur- rounding marihuana studies performed to date. 1 ll‘C—A9T1-1C=(-)Al—Tetrahydro (3',5'-1”C) cann— abinol, 31 mCi/mmole, was purchased from Amer- sham Searle. The radiolabel purity was found to be 97—98% by radioscan in two different TLC systems capable of separating A8 and A9 THC. aHATHC=A1(G—3H) tetrahydrocannabinol. 13 01/ mmole was purchased from Amersham Searle. The radiolabel purity was found to be 982 by radio- scan in three different TLC systems capable of separating A5 and A9 THC. REFERENCES Grant, J. D., Gross, S. J., Lomax, P. and WOng, R., 1972, Nature New BioZ., 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 SEPARATE RADIOIMMUNE MEASUREMENTS or BODY FLUID A9-1Hc AND n-NOR-9-CARBoxv-A9- THC Stanley J. Gross, M.D., James R. Scares, Ph.D. Department of Anatomy School of Medicine University of California los Angeles . California INTRODUCTION Pharmacologic and metabolic studies with can— nabinoids have remained largely unquantita— tive due to lack of simple assay techniques. Initial gas chromatographic and mass spectro- scopic methods were tedious (Fenimore 1973; Garrett 1973; Agurell 1973). Our immune approach led to early development of a simple radioimmune assay for A9-THC in plasma and urine (Gross 1974). This has now been ex— tended to measure separately in body fluids ll-nor-9-carboxy-A9-THC (C-THC) (Scares) a major cannabinoid metabolite (Bernstein 1972). ASSAY METHODS 3H-A9-THC (50 Ci/mM) and C-THC were obtained from Research Triangle Institute, North Caro- lina. A9-THC was obtained from the National Institute of Drug Abuse. The generation of antisera for A9-THC and C-THC has been de— scribed (Gross and Scares 1974; Soares and Gross 1976). Plasma A9-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 10 subjects at various intervals after smoking one to three marihuana cigarettes (Containing 19.8 mg A9-THC) and subsequently assayed for both A -THC and C-THC. Pooled plasma from non-users of marihuana spiked with various known amounts of A9-THC and C-THC was used to establish the standard inhibition curves. The urine standard curves were similarly de- tennined. Plasma A9-THC and C-THC: Plasma samples1 were obtained from flve subjects after smok- ing a single cigarette and from three sub- jects after smoking three consecutive cigar- ettes. None of the first group and only one of the second grou had measurable preintoxi- cation levels of A -THC. However, four of the eight subjects had measurable preintoxication levels of C-THC. In five single smoke subjects definitive in- crements (10-130 ng/ml) of A9-THC occurred 15 minutes after a single cigarette becoming al— most undetectable by 2 hours (Table 1). C-THC peaked at 30-60 minutes; significant but low levels remained at least three hours after ex- posure.’ Folale 1 Plasma A9—THC and C-THC levels in occasional THC users 15-240 minutes after smoking a single 900 mg THC cigarette (2.2% A9-THC/cigarette) Pre intoxi- Subject cation Post intoxication Oninutes) 0 5 15 ‘30 60 120 180 240 s. w. A9-THC 7 - 31 31 21 11 14 16 C-THC 50 144 224 116 71 5 9 z. w. A9-THC 0 130 92 38 0 0 6o C-THC o --- 116 24 59 36 34 B. w. A9-THC 0 90 9 7 5 5 0 C-THC 0 --- 7 9 25 8 23 P. G. Ag-THC o 10 7 0 o o o C-THC 19 -—- 76 12 75 o 40 T. s. A9-THC 0 73 32 14 0 0 0 0 C-THC o 278 228 216 108 115 0 0 The rapid shifts of plasma A9-THC and C-THC 15 minutes to 48 hours following repetitive exposure are shown in Figure l and summarized in Table 2. Expectedly plasma A9-THC peaks (100-260 ng/ml) were much higher after mul- tiple consecutive cigarettes than a single one, permitting detection of A9-THC 2-3 hours after completion of the last cigarette. Sig- nificant amounts of this metabolite were mea- sured in plasma 48 hours later. llalflea 2 Plasma A9—THC and C-THC equivalent levels in occasional marihuana users 15 min — 2 days after consecutively smoking three 900 mg marihuana cigarettes (2.2% Ag-THC/cigarette) Pre intoxi- Subject cation Post intoxication nv/ml o 15' 30' 60' 120' 180' 240' 24 hrs 48 hrs w. z A9-THC 8 185 78 123 39 72 12 41 10 C—THC 7 285 112 292 211 251 296 97 75 T. T. A9—THC 0 260 -- 37 15 7 0 21 9 C-THC 40 68 -- 158 43 111 140 94 71 B. w. Ag—THC o 100 63 87 37 5 7 5 8 C-THC 46 375 460 401 413 363 428 60 24 ~11 Figure I 300- 250- 200- ISO- A9-THC c-T°H'c IOO - (ng/ml.) 50~ O ‘r l l I)‘)I $‘v—Sfi—I V4 1/2 | 2 3 4 24 48 Hours (Post-Intoxication) ! Figure l: Post-intoxication plasma A9-THC and C-THC (ng/ml) in a representative SUbjeCt- = A9-THC; = c-mc. Figure 2 3004 250 - IZO 200 - I50- C‘THC (ng/ml) IOO- 50- Hours (Post-Intoxication) Figure 2: Post-intoxication urine C-THC (mg/ml) in chronic subjects. Arrows indicate an additional smoke. 12 Urine C—THC levels in chronic THC users 1-12 hrs after Table )3 consecutive smoking 900 mg THC cigarettes (2.2% A9-THC/cigarette) C-THC’level (ng/ml) Sub- Preintox- Post1ntox1cat10n (hrs) No. ject ication l 2 4 8 12 1 119 72 101* 129 164 123 28 176 144* 148* 325* 196 3 124 79 55 106* 105 97* 48 4 126 61 134 107 126 91* 196* 290 5 127 40 57 93 * 78 6 120 123 90 125 166* 205 215 7 121 13 14 21 10 8 5 8 121 19 4 18 10* 23 23 9 132 17 31 28 23 31 10 128 17 17 DD UD UD 11 128 9 ll 12 UD UD UD *Repeat cigarettes on subject demand DD = undetectable Table 4 Urine C-THC levels in occasional THC users 1-48 hours after smoking 900 mg THC cigarettes (2.2% A9-THC/cigarette) Urine C-THC’level (ng/ml) Prein- toxi- Postintoxication (hrs) Subject cation 1 2 3 4 6 8 12 24 48 B W. 5 33 18 17 33 39 23 15 3 2 K G. UD 3 17 2 16 2 3 -- S 3 R. B. 5 9 10 12 21 7 7 4 4 3 W. Z UD 7 18 21 9 S4 31 -- 5 UD L. G. 2 6 7 4 7 15 3 -- 7 T. T. 4 20 26 44 59 -- 29 14 —- 9 UD = undetectable 1—l 1'8 Urine A9-THC: None of four subjects had mea- suraBIe urinary A9-THC. This is consistent with previous work (Hollister 1974) de- scribing urine A9-THC levels to be far below present immune assay sensitivity. Urine C—THC: Assays of urine from 9 chronic users I, 2, 4, 6 and 8 hours after completion of the first standard cigarette are summar- ized in Table 3 and Figure 2. Preintoxica- tion levels varied from 28—123 ng/ml. Large C-THC increases occurred (Table 3) peaking at 2-4 hours after initial exposure. Unfortun- ately 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 A9-THC and C-THC levels did emerge. In occasional smoker subjects there was a 15-30 minute plasma A9-THC peak and a 30—60 minute C-THC peak after use of a single standardized marihuana cigarette. Im- portantly, A9-THC became almost undetectable in plasma 1-2 hours after exposure while sig- nificant amounts of C-THC persisted in circu— lation for several hours. This critical di- vergence of A9-THC and C-THC was even more obvious in subjects who had smoked 3 consecu- tive marihuana cigarettes. A9—THC was almost undetectable in all plasma samples four hours after the last cigarette had been consumed REFERENCES Agurell, 5., Gustafsson, B., Holmstedt, B., Leander, K., Lindgreen, J. E., Nilsson, 1., Sandberg, F. and Asberg, M., J. Ifianw. Phar- mac. 25, 554 (1973). Bernstein, 8., 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. Phanw. Sci. 62, 1211 (1973). .14 despite the prolonged (48 hrs) persistence of significant levels of C-THC. Urine from occasional marihuana subjects was negative or marginal for A9-THC while C-THC (60 ng/ml or less) was detected 1-48 hours after one standard cigarette. The pre—smoke values were zero (A9-THC and C-THC) for occa— sional smokers. However chronic smokers had significant pre-smoke levels (17-23 ng/ml) in addition to a vastly greater rise of urine CTTHC 2—4 hours after consumption of a single c1garette. Clearly, a single THC metabolite level or use of a significantly crossreacting antiserum OWarks, 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 ex- posures several days earlier. The failure to detect plasma A9-THC indicates unambiguously that marihuana was not smoked within the pre- ceeding hour, whereas detection of plasma C-THC in the absence of A9-THC indicates dis— tant exposure. A large controlled experi- mental population is now essential to corre— late A9-THC and C-THC (ratios) with individ— ual metabolic variants for behavior differen- ces. 1Samples were provided by the Department of Psychiatry, UCLA School of Medicine. Gross, S. J. and Scares, J. R., Nature 252, 581 (1974). Hollister, L. E., Kanter, S. L., Board, R. D. and Green, D. E., fies. Cbmm. Chem. Pathol. Phanwacol. 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 publication). Teale, J. D., Clough, J. M., 9151111, B. M., King, L. R. and Marks, V., fies. Comm. Chem. Pathol. Phanmacol. 11, 339 (1975). RADIOIMMUNOASSAY OF A9-TETRAHYDROCANNABIN0L Clarence E. Cook, Ph.D., Mary L. Hawes, B. A., Ellen W. Amerson, B. A., Colin 6. Pin, Ph.D., David Williams, B.A. Research Triangle Institute, North Carolina INTRODUCTION Since its introduction by Yalow and Berson (see Berson and Yalow, 1971), radioimmuno— assay (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 a1., 1973; Spector et al., 1973; Abraham, l971)1 In particular 5—dfamatic improvement in assay methodology for many steroid hor- mones has resulted from the application of radioimmunoassay to these substances. Due to its extreme sensitivity, RIA can per- mit the quantitation of substances present in concentrations as low as a few pg/ml of a biological fluid. The inherent selecti- vity of antibodies introduces a further ad- vantage since it often makes sample prepara- tion requirements minimal and assay method- ology simple and thus pennits the analysis of large numbers of samples. These obvious assets of RIA have naturally led to its consideration as a means for mea- suring blood levels of cannabinoid compounds. A valid RIA procedure for A9—THC would be useful in studying the phanmacokinetics of the drug. Furthernnre it is conceivable that l5 RIA methods could be used for the identifica- tion of THC and/or its metabolites, thus facilitating forensic investigations. There- fore a number of investigators have been interested in the development of RIA proce- dures for the cannabinoids. we believe it is fair to state that develop- ment of useful and simple RIA procedures for A9-THC has proven to be a relatively diffi- cult research problem. The difficulties in- volved in THC RIA development may be sum- marized 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 plas- tic--generally in preference to dissolving in an aqueous medium. (2) In plasma the compound is tightly bound, principally to a lipoprotein fraction (Klausner, gt_al., 1975). (3) It has proven difficult to obtain antibodies highly selective for Ag-THC vs its metabolites and various analogs. (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 analy— sis, a technique which has proven highly successful with certain other drugs (Specter 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 ob- tain. (6) High specific activity radioligand for cOmpetitive binding studies has been dif— ficult 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 (ini- tial dilution) is 1:500. If in the above— example t e 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 not . T, B, and N-tubes - Designation for differ- ent assay tubes. At end of assay, volumes in all tubes are equal. T tubes measure total radioactivity; N tubes measure non- specific radioactivity (activity not ad— sorbed by charcoal) and B tubes measure the amount of labeled drug bound to antibody in the absence (B6) or presence (B1) of added unlabeled compound (see Table l). 123tfl€3 1 DEFINITION OF ASSAY TUBES Tub- m Tub-Comma Unlabeled RIdiol' Com m: Bun-r Antiserum Charcoal + - + t - u + - + - + so + - + + + B; + + + + 4- Table 1. Definition of abbreviations for assay tubes 16 Materials Chemicals used in the work were reagent grade obtained from commercial sources. Bovine serum albumin was obtained from Sigma Chemi- cal Co., St. Louis, MO (crystallized and lyo- philized, 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 ob- tained from Dr. James Scares of the UCLA School of Medicine (Lot No. 62532; Gross et a1}, 1974) and from Dr. V. Marks thru the—— National Institute for Drug Abuse (Teale et al., 1974, 1975; SlSSY/30/9). Additional—— antiserum was prepared at Research Triangle Institute (see below). RIA buffer (0.1 M, pH 6.8) contained 16.35 g NazHPO4-7H20, 5.38 g NaHzPO4-H20, 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 tolu- ene, 1 liter Triton X-100 [purified by stir— ring for 30 min with 606 Tell-Tale Silica Gel (25 g/z) 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 (As—THC-SH; 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 di- luted for assays to 10,000 cpm/lO ul which gives ca. 100 pg A8-THC/lO 1.11. Dilutions in EtOH/HEG may be kept refrigerated for no more than 7-10 days. Radioligand decomposition becomes significant after that time. Stock solutions were tested frequently for decompo— sition by thin layer chromatography. Silica gel plates were developed in 100% benzene and the purity of the radioligand determined by radioscan. Ag-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% etha- nol and refrigerated. Dilutions were pre- pared in 50% ethanol/50% water. These dilu- tions may be kept refrigerated for approxi- mately 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, Land- steiner (see Landsteiner, 1962) showed that covalent bonding of a small molecule (hap- tenic component) to a protein gave an anti— genic substance and that antibodies formed to this antigen were capable of selectively binding the small molecule. He further es- tablished 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 metabo- lites, 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 selec- tivity. 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 rela— tion to the expected benefit. Depending upon their needs, then, it can be expected that different investigators will synthesize anti- gens in different ways. Most of the metabolic alterations of Ag-THC occur in the cyclohexene moiety with hydroxy- lation of the A9 compound occurring at the ll-and 8-positions along with conversion of the ll-carbon to a carboxyl group. More re- cently it has been reported that hydroxyla- tion can also occur in the amyl side chain of the phenolic ring (wall, 1975). A number of previously reported antigens are summarized in Figure l. Teale et a1. (1974, 1975), formed a hemi-ester linkageWFith the phenolic hydroxyl group and then coupled this compound to bovine serum albumin to form an antigen. Tsui 22 a1. (1974), report forma- tion of a hemisuccifiate from the phenolic hy- droxyl, and also formed an ether linkage at this position to give a carboxymethyl deriva- tive. The products were coupled to a variety of proteins as shown in Figure l. The pheno- lic group apparently undergoes no metabolic Figure 1 a. b. c. d hf a) CO-NH—BSAWIIIIJLIL, 1974) o) C0—CH26H200—NH—BSA (Ted-,et_al., 1975) c) co—cuzcnzco—NH—Pce (usa, sec, or PLL) ”whet—al., 1974,) d) CHch—NH—HSAhv soc) (nui,g_./,, 1974) a) N=N-©- CO-NH—PGG (HSA orSGG) (Twi,gt_al.,1974) f) N- CO—NH—KLH (Grooms—11L, 1974) (+ 4—5101mv) g) 10—l—9—NH—CO—NH—HSA (or PGG) (Tsui,o_t_al., 1974) Figure 1. Some positions through which Ag—T‘HC has been linked to protein alterations, but spatially the attachment to protein is relatively close to the metaboli- cally important cyclohexene ring. To avoid this problem Tsui et al. also prepared a 2— azophenylcarboxy derivative, a substitution employed by Gross et a1, (1974) as well. This substitution ffees the hydroxyl group for binding, although at the possible ex- pense of introducing a relatively immunodom— inant azobenzoyl moiety. Finally, Tsui et a1. (1974) also prepared a lO-iodo-Q-ureido Iihked 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 metaboli- cally reactive cyclohexene ring (although not from sites of hydroxylation on the amyl side chain). Also it was of interest to exam- ine the effectiveness of the flexible hydro- carbon 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 phenyl— butazone (Cook et aIT}_l975a). For synthetic reasons our initial—attempts dealt with the A8-THC analog as a substrate. Synthesis of the AB—THC antigen is shown in Figure 2. 5'-Carboxy-A8—THC labeled with Figure 2 SVNTHESIS OF A. — THC ANTIGEN OH O n 0 (9195 coon «- HO-N O H on on o o (b) 9 0 Dig), -C-0—N —‘ o (cups-c-uN-(Ium m 0 tv m mm mama/cup: (h) Bovin- mum albumin] diam - W (a) —. 1 Figure 2. Synthetic route to antigen based on 5'-carboxy—A8-THC tracer amounts of carbon-l4 in the carboxyl group (Pitt et E13: 1975) was converted to its N-oxysuccinimidoyl ester by reaction with N- hydroxysuccinimide and dicyclohexylcarbodi- imide. This active ester readily coupled with bovine serum albumin in a mixture of di- oxane and water to yield the desired antigen. Radioactivity measurements indicated the in— corporation of ca. 33 residues of A8—THC/ molecule of bovine serum albumin. We have found this mode of coupling to be a very useful one in certain circumstances. Where 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 homoge- nized with an equal volume of Freund's com— plete adjuvant. Immunization with 200 ug of antigen was carried out by intradennal tech- nique of vaitukaitus et al. (1971). This was followed after two weEEs_By another intrader- mal immunization (100 ug) and then at four week intervals subcutaneous booster injec- tions (100 pg of antigen) were given. Rab- bits 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. Anti- gen 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 AB-IHC 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 signifi— cant increases were observed on subsequent 18 booster administrations. A third rabbit even- tually achieved a titer (final dilution) of 1:2250 at the fourth bleeding. Titers gener— ally declined at the fifth and sixth bleed- ings. These titers compare quite favorably with those reported by others (Teale gt al., 1975; Gross et al., 1974) and demonstrate—{hat with this antigen the rabbit is a reasonable ani— mal to use for antibody production. A fur- ther comparison of the azobenzoyl-THC—derived antiserum obtained from UCLA and the anti- serum prepared from the amyl—linked antigen is shown in Figure 3. The affinity constants Figure 3 ANTISERUM CHARACTERISTICS UCLA RTI—RS—81 Affinity Comm 1.6 x 109 2.5 x 109 (for A3— THC) Std. Cum Characterinlu (Logit — log Plot) 50% Intercept 0.5 no 1.4 ng Linear Rang 0.3 — 4.1 ng 0.3 — 8 no Figure 3. Characteristics of two antisera as determined in this laboratory; UCLA (Gross, §E_§l:, 1974); RTI-RS-81-2 (this paper). are quite close. The slightly higher affi- nity of the A8—antiserum for A8-THC is under- standable. Due to the heterologous nature of the systems employed (A3-radioligand, unla- beled A9 and antiserum generated to either A8- or A§-), the standard curve characteristics are not similar for the two antisera. When standard curves are converted to the logit- log basis (Rodbard et al., 1969), the UCLA antiserum exhibits 5—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 conserva- tive number. If one defines sensitivity as the amount of A9-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. Antibody selectivity The avidity of the antisera for various me- tabolites and analogs of A9—THC was measured by determining the relative amount of com— pound required for 50% displacement of the initially bound radioligand. This is a re- latively crude measure of cross-reactivity. It is strictly valid only if the displace- ment curves for the two compounds in ques- tion are completely parallel and it does not take into account the possibility of small subpopulations of antibodies with high avi- dity 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 "/o CROSS-REACTION WITH ANTISERUM TO 0 STRUCTURE ‘ 0W“. \ 0" Oz O 0“ nm . a BOUND O { w O L O D O "N A a c IOOSG 100% loose m5 / (IOO) (:00) m 63 as 27| / (I00) C W, 94 I44 loz fig; 244 49 13 noon“ A‘) (47) 7 W" I57 (1 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, §t_al., 1974) _ — the cross—reactivity of three types of anti- sera, all measured in our own laboratory. Selectivity can be significantly influenced by the assay conditions. Therefore the values reported by the producer of the anti- serum are also given in the figure in aren- theses. The displacement ability of A -THC is taken as a standard at 100%. For reasons discussed later, the radioligand used was As-THC-3H. 19 A change which alters the tricyclic character of the molecule (see cannabidiol, CBD) es- sentially destroys binding to all of the an- tibodies. However, much more subtle changes can also have significant effects. In the case of the antibody to A8-THC, shift of the double bond from the A3— to the A -position results in a two and one-half fold decrease in cross-reaction. A similar (two—fold) de- crease is seen on comparison of the binding of A9- and A8-THC to antibody obtained from the other two antigens. Reduction of the cyclohexene double bond has no greater effect with the A8-antibody than does the shift to the A9—position and actu— ally somewhat increases the cross-reaction with the Ag-antiserum prepared from the azo- benzoyl antigen. Aromatization of the cyclo- hexene ring (to CBN) results in a four-fold decrease in cross-reaction as compared with A9-THC in the case of the azobenzoyl— and amyl-linked antigens. It has little effect on antibody to the 0-succinoyl antigen. Oxygenated C-ll metabolites cross-react rela- tively little with the antibody from the amyl antigen. Cross—reaction of the ll-hydroxy metabolite with the azobenzoyl antiserum is rather significant (49%) but the cross—reac— tion drops markedly when the ll-nor-Q-car- boxy 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 outstand- ing, selectivity for A9-THC vs a number of metabolites and analogs can 53 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 prob- lems in the development of an assay. Assay parameters General procedure.--The general assay proce- dure finally adopted is shown in Figure 5. Data leading to definition of the various conditions are presented in suceeding para- graphs. Radioligand.—-The sensitivity of a radioim- munoassay is very much a function of the specific activity of the bound radioligand. Thus, in general, one would prefer the high— est possible specific activity. Storage of such high specific activity compounds can often lead to radiation-induced decomposi- tion. The general experience of our labora- tory in the preparation and storage of such materials is that unlabeled substances which readily decompose through autoxidation, etc. are often particularly unstable when pre- pared radiolabeled and with high specific activity. This was found to be the case for Figure 5 GENERAL ASSAY FORMAT 0.1M phospham buffu, pH 6.3 Antiurum 3H.A91Hc ~1o,ooo com/10 "a Unlabeled Drug Total Volume — 0.52 ml Von-x 10 he Incubate 24 hn (1°C) 25% Chemo-l Susponsion (1.0 ml) Vortex 10 Inc Incubm 15 minutes Centrifuge 1200 u — 15 minutes Dom! Scintillation Fluid (‘0 ml) Count 1 Minute (Bound Fraction) Figure 5. General procedure for RIA of Ag-THC A9-THC which was prepared at a specific ac- tivity of ca. 50 Ci/mmole (Pitt et al., 1975) but had a very short shelftlifEI On the other hand, AS—THC-3H, prepared in simi— lar specific activity, has proven to be a quite stable entity. Its binding character— istics with the antisera raised to either A8— or A9—THC make it a useful radioligand. Hexahydrocannabinol also exhibits good cross-reactivity with the antisera but syn— thetic 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 A9-THC. This is illustrated in Figure 6. When SE: 5 ng of tritiated A8-THC in a small amount of ethanol was added to a normal RIA buffer (phosphate buffered saline) con- taining 0.1% bovine y—globulin as the pro— tein, very significant amounts of the THC were bound to the glass. This was illus- 20 Figure 6 BINDING OF THC T0 GLASS A'— THC 1 ——-b —> + BUFFER caususo GuAss 51% 41% Buffer + 0J%BGG Figure 6. Illustration of the binding to glass observed with THC trated by decanting the buffer into scintil— lation liquid. The glass tube was then bro- ken 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 re- sulted in much diminished adherence to glass and a better assay system. This is illustra- ted in Figure 7. Labeled THC was incubated with buffer containing either bovine serum albumin (BSA) or bovine y-globulin (BGG) plus varying dilutions of antiserum. The solu- tions 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 pro- teins. 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 con- taining the ECG and eventually the recovery Figure 7 EFFECT OF BUFFER PROTEIN 0N THC SOLUBILITV 3 I 8 I xAMLMWhmTTub-s 3‘ l S I L I I I I I I I 66 132 264 528 1060 Antiumm Dilution Figure 7. Variation of THC solubility as a function of antiserum dilution and buffer protein. Anti- serum of Gross, et al. (1974); BSA = bovine serum albumin; BCE';_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 pro- blem may be overcome by higher concentra- tions 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 an— tiserum by using bovine serum albumin in the buffer system. At the concentrations em- ployed, 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 crystal- lized 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) 2110 4220 8450 Figure 8 0 UCLA A nn—ns -III .2 I fiTIrRS»81~-3 700 . Z' 4:"..7?‘ BSA TVPE 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 (crystal— lized 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 in- sulin 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 gener- ally better results. Treatment of one of the less useful batches with charcoal to further remove fatty acids (Chen, 1967) gave some im- provement, 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 be— fore large amounts of antiserum are diluted in the buffer, (b) large batches should pre- ferably 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 inter- Figure 9 EFFECT OF CHANGES IN PROTEIN CONCENTRATION ON NET BINDING 50- E / % 3° ' , E, I I § 20 . I j! E I g C; H CRYSTALLIZED a 10 x An-A FRACT. v [FAF] 5 o 0-th PURIFIED -10 I I I l I 0.01 0135 0.1 0.2 0.5 In 5638A Figure 9. Net binding as a function of BSA concentration and type: A-—-A Sigma fraction V (<0.005% fatty acids); 0+H+o different lot of the above after second removal of fatty acids; o———o crystallized and lyophilized Figure 10 pH EFFECTS O.A —UCLA ..A auras—314 -..o 40 _ I'p an _ E U —l ‘1 I- O I— 20 __ B a? 10 _ 0 I I l I I I l I L 5 s 7 s o , v pHOFBUFFER 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—TRTI-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-8l—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 concen- trations (<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, radio- activity 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 con- siderable 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. 22 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 in— crease in apparent binding also appeared to occur at higher pH with the RTI antiserum, the n§t_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 condi- tions 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 equi- Figure 11 EFFECT OF INCUBATION TIME 50 ‘0 an ‘ n W 10 o l l I 1 l I I A) l I l ‘l 2 3 4 5 G 24 26 lmhlfion Tim- (4’) in Hr Figure 11. Percent of radioligand bound as a function of tune of incubation at 4°C Figure 12 EFFECT OF TIME ON CHARCOAL ADSORPT ION 60 ‘ #Antiuvum . Na Anus-rum" 40 3 3, k A #- E m .— 3 I [ I n n A l l 5 10 15 I!) 45 80 Incubation Tlmo (Min) Figure 12. Effect of tune on adsorption of radioligand by charcoal at 4°C. One ml of 2.5% charcoal suspension was added to incubation tubes con- taining 0.52 ml. Contents were mixed for 10 sec on a vortex mixer and allowed to stand 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 over- night period. Charcoal Treatment.--Since the charcoal ad- sorbant removes free radioligand, one might expect that exposure to charcoal would even- tually shift the binding equilibrium so as to remove essentially all of the label from the antibody. The rate at which this would hap- pen would be dependent upon the rate of bind- ing reversal, which in turn should be a func- tion of temperature. A finite time is also required to adsorb the free radiolabel and to effect its removal from weakly binding sub- stances such as protein. The best equilibra- tion 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 rsituation obtained. By 15 min the minimum percent bound was essentially reached. There- fore, in order to reduce the non-specific binding to its minimum, a 15 min time for standing with the charcoal is necessary. Other experiments on the time of mixing of the charcoal suspension with the incubation mix- ture 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 Vbrtex mixer gave the best results within a reason- able 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 re- sults. 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 ana- lysis 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. Whe— ther this is successful depends upon the amount of plasma which can be added to each assay tube and therefore upon the concentra- tion of the drug in plasma and the sensiti- vity of the assay itself. Plasma was added directly to T, B, and N tubes containing both the RTI and UCLA anti- sera and its effect on each was studied. As expected, T tubes showed no change in going from 0 to 60 ul of plasma in a total volume of 0.52 ml. Addition of 15 or more ul of Figure 13 ETHANOL — ETHER EXTRACTION PROCEDURE AND RECOVERIES 1mlplasma+2ml EtOH +5—100n9THC shake 20 min ultimo-1200 x g for 10 min pp! suparnaxam + 3 ml H20 + 3 ml E120 Shaka 20 min aqueous organic evaporate under N2 RedissoIva in Rodissolva in 50% HOH/Hzo 1 ml RIA buffer (84% recovery) (85% recovery) Figure 13. Procedure used for extraction of Ag—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 ul of plasma were only 60—70% that of the tubes containing no plasma. Further in- creases 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 will— ing to ignore the possibility of intersubject variation in effects on the assay. This lat- ter point will be considered later. For pharmacokinetic studies, assays at levels below 20 ng/ml are required. Solvent extrac— tion to remove protein and other interferen— ces must ap arently be used to analyze low levels of A :THC. Some care must be used in the choice of solvents since small amounts of certain solvents can have a significant in- fluence on the assay. For example, the addi— tion of only 1 pl 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 ab- sence ofpfhe isoamyl alcohol. Therefore traces of this relatively non-volatile sub- stance remaining from an extraction process may have a very significant effect upon assay results. Ethanol has somewhat less of an effect. In- deed we routinely add up to 10 ul of ethanol to each assay tube in preparing the standard curve. Addition of 20 pl more ethanol redu— ces binding to about 75% of the initial value. Nevertheless, extraction of A9—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 ex- traction process used is shown in Figure 13. Plasma is treated with twice its volume of ethanol and precipitated material is centri- fuged out. Aliquots of the supernatant may be taken for analysis at this point. We tested the possibility of utilizing this su- pernatant as has been reported by Teale gt g. (1975). Since the plasma/ethanol extract has a defi— nite 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 dif- ferent 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 analyzed 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 thesemconditions it was possi- ble to add sufficient of the ethanol extract to get down to levels of 5 ng/ml with reason- able 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 accu- racy of analysis of samples from plasma C was Figure 14 COMPARISON OF ASSAY RESULTS FROM SPIKED PLASMA SAMPLES AFTER EtOH PRECIPITATION OF PROTEIN Figure 15 STD. CURVES FROM SPIKED PLASMA SAMPLES (EtOH PRECIPITATION; E120 EXTRACTION; RECONSTITUTED IN BUFFER) INITIAL SLOPE 50% CORRELATION BINDING (LOGIT — LOG) INTE RCEPT COEFFICIENT Percent of Analyses with < 20% Error ' SM Cum 35% —3.54 0.59 ng 41.99 (Buffer) Pl 4' 5014!? um A 32% —2.88 0.31 In 4.98 asma Series Standard Curve Employed + 50ml .mA 32% 4.34 0.11 nu —o.9s Analyzed A g g D _ sm. Cum 45% 4.06 0.59 on 4.99 A + 50 ull am 3 35% 4.50 0.71 ng —0.99 B + 5014! um C' 38% —2.36 0.71 no —0.96 C 9 501.42 "(not D 39% —2.49 0.88 no 4.99 D . Figure 15. Figure 14 Comparison of standard curves generated in Accuracy of analysis when extracts from one plasma spiked with Ag—THC are analyzed by means of standard curves generated from extracts of other plasma samples. Antiserum from UCLA (Gross, et 31., 1974) 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 v5 standard curve C, where only one out of £135 samples fell within the 20% limits. If one requires accuracy within :l0%, results are much worse. we believe this study leads to two conclu— sions: (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 A9-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 con— centrations than the A —THC itself. Tb further purify the plasma material before analysis, we then followed a procedure which was described to us by Scares (private com- munication, 1975) and which consists of the last part of Figure 13. The ethanol extract is treated with water and ether. After 25. equilibration, ether is removed and evapora- ted. 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 lasma samples spiked with 5-100 ng/ml of A -THC and carried through this procedure. Adding 50 ul (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. CONCLUSIONS (1) The work we have done leads us to be- lieve that there are factors which can be extracted from normal plasma under the conditions described which will in- terfere with the assay. Since these 3 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 eval- uating the data obtained in such a man— ner. Care must also be taken in ob- taining plasma samples and storing them to avoid inadvertent addition of such potentially interfering materials as plasticizers, etc. The effect of freez- ing and storage on the stability of the THC and on the formation of interfering substances needs to be further evaluated. (2) The sensitivity of the assay to condi- tions such as buffer composition and plasma materials makes interlaboratory comparisons difficult. Standardized techniques and materials are therefore a pressing need. REFERENCES Abraham, G. E. in biological materials. 1974, Suppl. 183, 1-42. Radioimmunoassay of steroids Acta Endocr., Berson, A. S. and Yalow, R. S. Current knowledge of basic concepts in immuno— logy and their clinical application. R. A. Good and D. W. Fisher (Eds.), IMmunobioZogy. Sinauer Associates, Stan- ford, CN, 1971: 287-293. In 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 di- phenylhydantoin 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. PharmacoZ., 1973, 5: 767—774. .2 6. (3) Radioimmunoassay has not yet lived up to its potential in the analysis of cannabinoid substances. The sensiti- vity 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 avail- able for the steroid hormones yet been re- ported for A9-THC. Since factors such as titer and sensitivity can appar- ently 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 develop— ment of antisera to a variety of antigens and in a variety of animals are all valid approaches to further work. (4) The reasonable results obtained by im- munization of animals with a conjugate prepared from 5'-carboxy-A8—THC have encouraged us to undertake the synthe- sis of shnilar antigens based on 5'- carboxy-Ag-THC. Wbrk along these lines is in progress and can be reported at future meetings. 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 radioimmuno- assay. Clin. Pharmacol. Therap., 1975b, 18: 742-747. Gross, S. J., Soares, J. R., ang, S. L., and Schnster, R. E. Marihuana metabolites measured by a radioimmune technique. Nature, 1974, 252: 581-582. Klausner, H. A., Wilcox, H. G., and Dingell, J. V. The use of zonal ultracentrifuga- tion in the investigation of the binding of A9-tetrahydroca1mabinol by plasma 1ypo- proteins. Drug Metab. Disp., 1975, 3: 314-319. Landsteiner, K. The specificity of serologi- cal reactions, Revised Edition. Dover Press, Inc., New York, 1962. Pitt, C. G., Hobbs, D. T., Schran, H., Twine, C. B., Jr., and Williams, D. L. The syn- thesis of deuterium, carbon-l4 and car- rier-free tritium-labeled compounds. J. Label. Comp., 1975, _: _-_ Rodbard, D., Bridson, W. and Rayford, P. L. Rapid calculation of radioinmnmoassay results. J. Lab. Clin. Med., 1969, 74: 770-781. Skelly, D. S., Brown, L. P. and Besch, P. K. Radioilmnmoassay. Clin. Chem, 1973, 19: 146-186. Spector, S., Berkowitz, B. Glynn, E. J. and Peskar, B. Antibodies to morphine, bar- biturates, and serotonin. Phamacol. Revs., 1973, 25: 281-291. ,2 7. Teale, J. D., Forman, E. J., King, L. J. and Nbrks, V. Production of antibodies to tetrahydrocaxmabinol as the basis for its radioimmunoassay. Nature, 1974, 249:~ 154-155. Teale, J. D., Forman, E. J., King, L. J., Piall, E. M. and Marks, V. The develop- ment of a radioimlmmoassay for caimabi- noids 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. A9- Tetrahydrocalmabinol-protein conjugates. Can. J. Biochem., 1974, 52: 252-258. Vaitukaitus, J., Robbins, J. B., Meschlay, E. and Ross, G. E. A method for producing specific antisera with small doses of immimogen. J. Clin. E‘ndocr., 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. DETERMINATION OF THC AND ITS METABOLITES BY EMIT® HOMOGENEOUS ENZYME IMMUNOASSAY: A SUMMARY REPORT G.I.. Rowloy, Ph.D., 1'- A. Armstrong , C.P. Crowl, W. M. Eimstod, W. M. Hu, Ph.D., .l.K. Kam, R Rogers, Ph.D., R. C. Ronald, Ph.D., K.E. Rubonuoin,Ph. 0., 3.6. Sheldon, E. r. Ullmon, PI‘I.D. 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 general- ity of the technique and have devel— oped 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, Crowl 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 281 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 inhi- bition 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 determ- inant 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 avail- ability 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 spectrophoto— metric measurement. The mechanism of antibody inhibition of morphine con- jugates of MDH had previously been studied and an immunoassay for mor— phine was developed (Rowley, Rubenstein, Huisjen, and Ullman, 1975) which permitted detection of 92x10‘ molar morphine in the assay mixture. SYNTHESIS OF THE THC HAPTEN Synthesis of the THC hapten, III, that was used for producing the com- ponents of the assay is illustrated below: 0“ 1) 03,-78° ———————a 0 2) Zn/HOAc I 14 o nocn2 cozn 0H 14 OH uuzocnz cozn o 11 III Ag’ll—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-Clh-0-carboxy- methylhydroxylamine in anhydrous refluxing methanol to yield the desired acid, III, in 61% yield. PREPARATION OF THC ANTIBODY Acid III was conjugated to bovine y- globulin via its N-hydroxysuccinimide ester, IV, prepared by condensation with N—hydroxysuccinimide using the condensing reagent, N—dimethylamino- propyl—N'-ethyl carbodiimide hydro- chloride in anhydrous dimethylforma- mide. 0 l4 NOCH2 COZN OH O 0 IV Subsequently, the dimethylformamide solution of "active ester" was added to a buffered solution of the protein containing 30% dimethylformamide co— solvent at pH 8.5. The protein was exhaustively dialyzed to remove non- 29 covalently bound THC residues. The number of bound residues Was deter- mined by scintillation counting of the radiolabeled products to yield conju- gates containing 21 and 32 THC resi- dues per protein molecule in two separate preparations. Antibodies were obtained from sheep by immuniz- ing 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. Non— covalently bound THC residues were removed either by exhaustive dialysis or by gel chromatography on Sephadex 6-25. The number of bound radio- labeled THC residues and the residual enzyme activity of each conjugate was determined (Figure 1). Enzyme ‘ activity of the conjugates decreased sharply with increasing sub- stitution 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 I 100 l 1 I I l I t E 80 - — a ’2’ I E E 60- . - E g E 140 - — Z O ,_. o E . E 20— _ Lu CL 0 I l I I I I 0.0 2.0 4.0 6.0 8.0 10.0 12.0 CONJUGATED THC RESIDUES PER ENZYME Addition of excess THC the conjugates reduced activity. The maximum the presence of excess antibody was only slightly affected by the animal source. Maximal inhibition was, however, directly dependent on the antibody to their enzymatic inhibition in 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 IMMUNOASSAY FOR THC AND ll-NOR-Ag— THC-9—CARBOXYLIC ACID THC—MDH conjugate, which was maximally inhibited by THC antibodies (4.2 THC residues), was chosen for the immuno- heavily (12.2 residues) the antibody assay. Stability of the conjugate was induced inhibition decreased to 35%. 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. Figure 2 The conjugate readily adsorbed on glass and plastic measuring devices thus preventing quantitative and . reproducible transfer of dilute solu- Wfi — tions. Such adsorption is reminis- cent of reported adsorption of THC itself to glass and plastics (Garrett and Hunt, 1974). Triton X-405 was found to prevent troublesome adsorp— tion of THC in radioimmunoassays for THC (Teale, Forman, King, Piall, and Marks, 1975). We likewise found that NO I I I I 1 I adsorptignbofiTHC-MDH cznjuggtg :3; revente y ncorporat on o . a0 23 “fl RD &0 1&0 110 griton X—405 in dilute enzyme solu- tions. we _ 60 a _ 80 “ - T PERCENT INHIBITION BY THC ANTIBODY . MY WMWMEDTKIEEDWSPRE M The effect of varying the ratio of antibody to enzyme is given in Figure 3. Upon titration of the conjugate Figure 3 200 I I I I I I I 1:250 DILUTION $656J ANTIBODY 180 _ 1:450 DILUTION THC-ENZYME CONJUGATE _ (1.96 x 10‘9 M CONJUGATE IN ASSAY) 3 1m ‘ ' a 2 E g 1140 ~ — a D: & Té 120 — _ >< <1: <3 100 - ~ m — — 60 I I I; l I VI I MICROLITERS ANTIBODY 30' with antibody, the enzyme activity at first dropped off rapidly and assymp- totically 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 ethan- olic THC solutions to the assay mixture (Figure 4). In the assay procedure 1 ul 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 ll-nor-Ag-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 cross— ’ reactive behavior of our antibody both to THC and to V is different from that exhibited by antibodies raised by others against O—carboxy- methyl-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 BOT» ' 1 500— 10.0 ng/ml g 950— 2 E E lIOO—I 0H s . C 0 O x 350- :] C02H (4/ OH 300%; 0.5 1-0 A / 0 \I 250 I I I r I 9.5 x 10'10II 3.2 x 10-99 9.5 x 10-991 3.2 x 10‘8” 9.5 x 10-814 LOG CONCENTRATION 1 31 for THC and its metabolites, and could also be used to assay either THC or V if these compounds are first separated by an extraction prooedure. 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. $1, 967. Garrett, E. R., and Hunt, C. A. (1974) J. Pharm. Sci. g;, 1056. Grant, J. D., Gross, S. J., Lomax, P., and Wong, R. (1972) Nature, New Biol. 236, 216. R0w1ey, G. L. Rubenstein, K. E., Huisjen, J., and Ullman, E. F. (1975) J. Biol. Chem., 250, 3759. Rubenstein, K. E., Schneider, R. S., and Ullman, E. F. (1972) Biochem. Biophys. Res. Comm. 31, 846. 32 Enzyme immunoassays for THC and its metabolites in urine are presently being developed and a serum assay is under investigation. Schneider, R. S., Linquist, P., Wong, E., Rubenstein, K. E. and Ullman, E. F. (1973) Clin. Chem. 12, 821. Teale, J. D., Forman, E. J., King, L. J., Piall, E. M., and Marks, V. (1975) J. Pharm. Pharmac. 21 465. Ullman, E. F., Blakemore, J., Leute, R. K., Eimstad, W., and Jaklitsch, A. (1975) Clin. Chem. $1, 1011. Van Vunakis, H., and Levine, L. (1974) in Immunoassays figr Drugs Subject to Abuse (Mulé, S. J., Sunshine, 1., Brande, M., and Willette, R. E., eds.) pp. 23-25, Ohio. CRC Press Inc., Cleveland, SEPARATION AND SENSITIVE ASSAY OF THC IN BIOLOGICAL FLUIDS BY HPLC AND GLC Edward R. Garrett and C. Antho College of Pharmacy, University of Flor' a Gainesville, Florida Hunt ABSTRACT HPLC systems were developed to permit quanti- tative separation of A9-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 un- modified compound and by electron capture GLC of pentafluorobenzoylated compound. Reverse phase HPLC elution, with 47% acetonitrile in water, and nonnal phase HPLC with 25% chloro- form in heptane, separated tetrahydrocannabinol from ll-hydroxy-A9-tetrahydrocannabinol and other monohydroxylated tetrahydrocannabinols. These systems also purified stock solutions of tetrahydrocannabinol from accompanying con- taminants. The various monohydroxylated tetrahydrocannabinols were resolved from each other in normal phase, 80% chloroform in hep- tane. The Asand A9-tetrahydrocannabinols were separable in normal phase with 5% tetrahydro- furan in hexane. The GLC analysis of penta- fluorobenzoylated tetrahydrocannabinol had a sensitivity of 1 ng/ml of plasma with an esti- mated 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 33 between the liquid scintillation and electron capture GLC assays of the HPLC separated A9- 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 tetrahydro— cannabinol 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 tetrahydro- cannabinol 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 time- dependent degree of adsorption could be mini- mized by prior treatment of all glassware with an organic solution of a silyl reagent. In the case of organic solutions, the tetra- 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 signif- icant increase in GLC background from inter- facing 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 pharmacokinetic studies were contem— plated in both dogs and humans over longer periods of time so that fasting would be impractical, such interferences were antici- pated that would lower the analytical sensi— tivity. 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 moni- toring 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 pro- vided the proper sensitivities. This paper presents HPLC techniques to separate tetra- hydrocannabinol from various cannabinoids and endogenous materials of biological fluids with subsequent analysis by various appro- priate methods. RESULTS AND DISCUSSION Purification and Reproducibility of Collection from Injected Ethanolic solutions of AgyTetra— hydrocannabinol on HPLC The 1“C- A9 -tetrahydrocannabinol used as supplied by NIDA was reported low in radio- labelled contaminants by TLC. However, reverse phase HPLC (Fig. 1) revealed the presence of a labelled contaminant. The major peak, I, contained A9 -tetrahydrocan- nabinol ( > 96%) and A8 -tetrahydrocannabi- n01 (= %), quantified by flame ionization Figure I 25 20 2 Q. 9 I5 X o. ‘o — no 5 o o z 4 s 3 IO :2 I4 RETENNON VOLUME (mu Figure 1. Reverse phase HPLC of stock 1”0— A9—tetrahydrocannabinol. Zhe total radio— activity per eluate fraction is plotted vs retention volume (curve A). Peak I was ll‘C— Ag-tetrahydrocannabinol and peak II was an un- known radioactive contaminent. Curve B is a plot of the mean UV detector response (arbit- rary units) vs retention volume and is displac— ed relative to A by the solvent volume between the detector and the collection point. Collec— tion between volumes g_and b_(peak retention volume t 30%) for curve B recovered 92.5% of the radioactivity in this case. Column: Bond- apack 018; 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 tetrahydro- cannabinol 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. Figure 2 I 45 40 t G. U X r P - L0 .05 F o 64* 3 s 9 I2 us In L3fi MILLILITERS Figure 2. Normal phase HPLC of stock 1”C—A9- tetrahydrocannabinol. The total activity per eluate fraction is plotted vs milliliters of eluate. Peak I was 11+C-A9-tetrahydrocannabi- nol and peak II was an unknown radioactive contaminant. Collection between volumes g_and Q_recovered 97.5% of the radioactivity in this case. Column: p-Porsil; eluent: 20% chloro- form 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, recon- stituted in ethanol and re-analyzed on the 'same HPLC system, only the single peak I was observed. All the stock A9 -tetrahydrocan- nabinol used herein underwent this purifica- tion 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 :_l.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 I3< T d 1 C 9' E .. l l I D 4 c > z 9 E -L w T E 5‘- a I l 1 l l 45 47 49 5| %ACETON|TRILE-WATER Figure 3. The retention volumes for the peak amounts of Ag-tetrahydrocannabinol (O) and IJ-hydroxy-Ag—tetrahydrocannabinol (()) for reverse phase HPLC are plotted vs solvent com- position. 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 radio- activity vs retention volume. The fact that the retention volume of peak I and the appropriate collection ranges for 98% recovery of injected labelled tetrahydro- cannabinol were sensitive to solvent composi- tion (Fig. 3) necessitated the prior estab- lishment of an appropriate collection range for each newly prepared batch of solvent by radiochemical analysis of the collected HPLC fractions of previously purified 11+C— A9 -tetrahydrocannabinol. The retention volume and the appropriate collection range increased with decreasing solvent polarity (less % acetonitrile) due to peak spreading (Fig. 3). Table I TABLE I - Recovery of Radioactivity of "‘C- A‘Ll'etrallydrocannaltlinola from Reverse Phase HPU: Table 2 TABLE [I - Recovery of Radioactivity of I"C- ’-Tetrahydroca:mbinola from Norm] Phase HP Elut ing 10'“ CM Average Average Solvent nb Injectedc l0"‘ cm Percent (t acetonitrile Collectedd Recovered in water) 4.006 3.813 95.18 8.012 7.688 95.95 5“ 12.018 11.86 98.7 NNNN 16.024 15.824 98.75 491 4 16.024 16.760 98.33 478 4 16.024 15.804 98.63 n6 10’3 cm Average Percent lnjectedc 10'3 cmd Recovered“ Collected 5 0.1420 0.1344 94.7 (6.58) 3 0.7110 0.689 97.04 (3.0) 3 1.423 1.377 96.82 (4.9) 2 1429 13.615 95.88 (4.15) 2 71.02 69.77 98.27 (1.13) 451 4 16.024 15.441 96.36 3 142.06 137.81 97.05 (1.65) Overall Avg. 20 . 97.57 ; 0.699 aThe stock solutions of the material had been purified previously by HPLC. bHullbers of replicates. cInjections of 2. 4. 6 or 8 ul were taken from the same ethangl solution of 1"C- A9-tetrahydrocamwhinol: a x 103 CPM/us and 2.003 x 10“ CPM/ul. For each solvent the collection range (Fig.3) was constant. All samples were corrected for back- ground counts. eStandard error of the mean where 3.07 is the standard deviation. 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 radio- activity 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 evi- dence of these contaminants was demonstrated when the GLC background significantly varied when distilled water from various sources was collected after reverse phase chroma- tography, extracted as if A9 ~tetrahydrocan- nabinol were present, then treated by the derivatization procedure and analyzed by electron capture GLC. HPLC Separation of A9-Tetrahydrocannabinol from Selected Cannabindids and Metabolites Tetrahydrocannabinol, cannabinol and cannabi- diol were resolved by the normal phase HPLC system (Fig. 4). If 98% of the tetrahydro- cannabinol 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 Total (n - 18) 96.32 L l.ozsf “The stock solutions of the material had been purified previously by HPuL' 5Eluting solvent: 25% chloroform in heptane. c15 ul of n replicates of each solution were Anjected. The specific activity of 1"0 AB-tetrahydrocannabinol was 142 . Each sallqzle was corrected for b§ckground counts. 'eThe parentheses contain standard deviations as percent of mean. Standard error of the mean where 4.23 ls the standard deviation. diol relative to tetrahydrocannabinol in- creased as the percent of chlorofonn 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 tetrahydrocan- nabinol on this system. They were resolved from each other with a more polar solvent, 80% chloroform in heptane (Fig. 5). A9 -Tetrahydrocannabinol and ll-hydroxy- A9 -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-ll-hydroxy- A9 -tetra- hydrocannabinol and 1')!C— A -tetrahydrocan— nabinol. as and A9 -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 tetrahydrocan- nabinols collected under peak I (Figs. 1 and 2) could be further separated by this system. Figure 4 THC CID CIN UV DETECTOR RESPONSE l2 IO 0 s 4 2 o umurso Figure 4. Normal phase HPLC separation of cannabinoids. The UV detector response (arbi- trary units) is plotted vs retention time after injection (inj.) of a mixture of cannabidiol (CED), Ag—tetrahydrocannabinol (THC) and canna- binol (CBN). The amounts of A9-tetrahydro- cannabinol and cannabidiol injected were approxh imately twice that of the cannabinol. Column: u-Porsil; eluent: 20% chloroform in heptane at 1.5 ml/min. Figure 5 C B CBN UV DETECTOR RE S PONSE IO C 6 4 2 0 MINUTES Figure 5. Normal phase HPLC separation of A9— tetrahydrocannabinol metabolites. The UV de- tector response (arbitrary units) is plotted vs retention time after injection (inj.) of a mixture of cannabinol (CBN), 11-hydroxy-A9- tetrahydrocannabinol (C), 8u- and 8B-hydroxy- A9—tetrahydrocannabinol (B and A, respectively). Column: u-Porsil; eluent: 80% chloroform in heptane at 1.5 ml/min. Table 3 TABLE III - Percent Recoveriesa of 1“C- A9-Tetrahydrocamabinol in the Heptane Extract of Plasma, in the Collection of the Proper Fraction of the Heptane Extract Separated by HPIL and the Overall Recovery after Both Extraction an Capture-GIL and Scintillation Analysis d HPLC Collection as Monitored by Both Electron ”C- Ag-Tetrahydrocannabinolb, n_g/_2 ml plasma Extracted by Heptane, ’6 Extract HPLC Overall Recover-yd, % Collectedc, % Scintillation Scintillation GLC Scintillation GLC 2.25 90.2 i 4.7 93 92 84 1 8 83 1 32 22.5 90.3 1 2.5 as 97 so :_6 as 1 12 225 90.9 1 8.5 97 89 88 1 4 81 1 7 Overall Average 1 std. error 90.6 + 0 7 92 93 84 s 2 84 + 5 aGiven as the mean from 4 separate plasma samples 1 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. b142.47 CPM/ng. Two ml of plasma was extracted with 15 ml heptane and 14 ml was analyzed for total 1"C. CQuotient divided by the extraction efficiency (0.91). Additional studies were conducted for 26 ng 1"C- A9-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 1 3% (standard error) respectively. tient of amount recovered corrected for volumes of extract used and amo—lmt added, which is the product of the extraction efficiency and the collection efficiency. 57 Ejyect of HPLC Separation on GLC Analysis of A9 Tetrahydrocannabinol in Plasma An equal amount of A9 tetrahydrocannabinol and ll-hydroxy- A9 -tetrahydrocannabinol in 2 m1 of dog plasma was extracted and sepa- rated from a majority of the extracted com- ponents by reverse phase HPLC. The reduc- tion 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 (Fi . 6). Fugure-Z; A: Bow. HPLC a: MOI! HPLC éo I I0 15 HETENHON TIME: M|NUTES Figure 6. GLC chromatograms (flame ionization detection) of an extract of 2 ml of plasma containing A9-tetrahydrocannabinol, I (200 ngfiml), before (A) and after (B) reverse phase HPLC separation of both cannabinoids over a range of predetermined collection volumes (Fig. 1). One ul of 18 ul of extract was in- jected into the GLC prior to HPLC (A). fen ul of 18 ul of the extract was injected into the HPLC; the collected fraction was reconsti- tuted in 10 ul of chloroform and 1 ul was in— jected into the GLC (8). Peak III is the sol- vent; peak IV is an unknown from plasma. HPLC: column, Corasil 018; eluent, 51% aceto- nitrile in water at 1.5 ml/min. GLC: column, 5' x 2 mm 1.9% 0V-225 at 2450 with a N2 flow of 24 ml/min. and an attenuation of 8 x 12-12 fbr both chromatograms. The initial base lines and injection times fbr both chromato- grams are superimposed for comparison. 38 The normal phase HPLC with 20% chloroform in heptane could separate A9 -tetrahydrocannabi- n01 from monohydroxylated metabolites and from 11-hydroxy-A9 -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 7 “I: J.-_—-—— - - 'n' -r‘” {f \‘ DETECTOR RESPONSE —0 (Minutes) RETENTION TIME Figure 7. GLC (electron capture) analysis of derivatized samples. The chromatograms repre- sent: A) the injection of 1 of 300 ul from a derivatiaed extract of 2 ml of blank plasma without HPLC purification: B) the injection of 1 of 200 pl of the final solution of deriv— atized compound which was 70% of the total amount from 2 ml of dog plasma containing 200 ng/ml Agtetrahydrocannibinol pentafluoroben- ‘zoate (THC) are appropriately labelled. Iypi- cal estimated base lines are shown. GLC con- ditions: 6’ 0V 17 column, 2250; detectorg 2800; injector, 2550; N flow, 45 ml/min; attenuations 4 x 10-9 (i and B) and 8 x 10—10 (C). Plasma samples obtained from dogs administer- ed A9 —tetrahydrocannabinol solutions intra— venously were analyzed by the electron cap- ture 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 cannabi- diol pentafluorbenzoate and no significant amounts of cannabinol or cannabidiol could be detected as metabolites of A9 —tetra- hydrocannabinol in the dog. Thus, either compound, when purified, should serve as an appropriate internal standard in pharmaco- kinetic studies. However, since cannabinol has been reported as a minor metabolite (McCallum 1973; McCallum et a1. 1975; Widman et a1. 1974), cannabidiol pentafluro— benzoate was chosen as the internal standard. It must be realized that cannabinol is known to be a contaminent of degraded A9 -tetrahy— drocannabinol (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% 0V 225 was supplanted by a longer CV 17 packed column to be consistent with the data in the literature accumulated for the reso- lution of the cannabinols (Fetterman and Turner 1972; Turner and Hadley 1973; Turner et a1. 1974). Efficiency, Reproducibility and Sensitivity of the Steps in the Radiochemical and Electron Capture Assay of 1l+C-A9-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 A9 -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 radiochem- ical analysis (83.7 :_l.8% standard error) and by electron capture GLC analysis (84.0 1 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 39 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 monohydro- xylated or 11-hydroxy-A9 ~tetrahydrocannabi- nol metabolites in plasma. A slight overlap of their HPLC peak areas with tetrahydrocan- nabinol would have occurred if the larger collection ranges had been used. A similar stud of the reproducibility of collection of ”C-Ag -tetrahydrocannabinol in plasma assayed by liquid scintillation after extraction and reverse phase HPLC was also conducted. The amounts recovered were pro- portional to the amounts injected (Fig. 8) and the HPLC recovery efficiency of the drug in the heptane extract in this case was 95.7% Fugure 8 50 z ‘2 E ._ if so Ill :3 O U 3i :2 5‘ .a U I. 3: °" l0 |:0 30 50 EXFERIUENT‘L CONCENTRATION IN PLASMA ng/ml Figure 8. Recovery of 1l+C-A9—tetrahydrocanna- binol calculated as concentration in plasma corrected for an extraction efficiency of 91% against the experimentally prepared concentra— tions. 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 collec— tion was analyzed by liquid scintillation. The slope, 0.957, is the HPLC collection efficiency for Ag-tetrahydrocannabinol and was typical of the values obtained. The vertical bar is the range for i one standard deviation (n=4). Eguivalency of Radiochemical Analyses of 1”C- A ~Tetrahydrocannabinol and GLC Electron Capture Detection of Derivatized Material after Normal Phase HPLC in Dog Plasma during Pharmacokinetic Studies The plasma of a dog intravenously administered solutions of 1"C-A -tetrahydrocannabinol was monitored with time after heptane extraction by both radiochemical analysis and electron capture GLC of the derivative of the appro- priately 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. Figure9 :03 '0 2 :9 (0 D a ‘5 — 0 g 6 \4 filo-:- 00 ° - 3 v - o O s O- 2 4 68.8 8? I2 l4 I6 5 E u 0 3' is s g c o §Io5 — . S I . E j 8 o E O 3 _ F - o . o mq ‘ o o - o o .064............... o 60 |20 I80 240 300 340 Minutes Figure 9. Semilogarithmic plots of fraction of the Ag-tetrahydrocannabinol 0.1 mg/kg dose per ml of plasma against time for dog A plotted from the liquid scintillation analysis of the total 1"C collected as A9-tetrahydrocannabinol on normal phase HPLC ((3) 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 IO Io” 90 o E 3 ' e a 8 3 4 v0 — 9 g _ CD \ ’ o 3 : 0 o 8 0 j 4 6 B IO l2 I l4 3 _0 E .9. B ‘8 E LL ; 105— 8' 2 00 ‘2’ ’ “0 C . ‘2. fi 00 o f, O c o o l . O (h < .06 . . . . . o . OJ 0 ICC 200 300 400 500 600 700 Minutes Figure 10. Semilogarithmic plots of fraction of the A9-tetrahydrocannabinol 2.0 mg/kg dose per ml of plasma against time for dog A plotted from the liquid scintillation analysis of the total 1“c collected as A9-tetrahydrocannabinol on normal phase HPLC ((3) 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 mg). A statisti- cal analysis of the apparent differences between the tetrahydrocannabinol assays at a given time from both analytical methods (Figs. 9 and 10) showed no significance. This demonstrated that all of the recovered radioactivity from the HPLC separation pro- cedure could be assigned to the A9 —tetra- hydrocannabinol assayed specifically by electron capture GLC and thus no significant amounts of radiolabelled metabolites were in the collected HPLC fractions. It can be concluded that A9 -tetrahydrocan— nabinol 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; assur- ance 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, Emperientia, 31, (5), 520 (1975). M. Widman, M. Nordquist, S. Agurell, J. E. Lindgren and F. Sandberg, Biochem. PharmacoZ., 23, 163 (1974). R. S. Fetternmn and C. E. Turner, J. Pharm. Sci., 61, 1476 (1972). C. B. Turner and K. Hadley, ibid, 62, 251, 1083 (1973). C. E. Turner et a1., ibid, 63, 1872 (1974). 41 recovered is all that is necessary since it is directly proportional to the total drug concentration. If unlabelled tetrahydrocan- nabinol in a solution of plasma were ana- lyzed, the calculated recovery of known amounts of labelled tetrahydrocannabinol added either to plasma prior to extraction or to heptane extract subsequent to extrac- tion would permit calculation of the extrac- tion and/or HPLC collection efficiencies for that particular biological sample. These known efficiencies would permit the calcula- tion of the original plasma concentrations. If a labelled 1 C-A9 -tetrahydrocannabinol were used in pharmacokinetic studies, extracted and separated on the HPLC, a trit- ium 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 acknowl— edged. DETERMINATION OF A9-TETRAHYDROCANNABINOI. IN HUMAN BLOOD SERUM BY ELECTRON CAPTURE GAS CHROMATOGRAPHY David C. Fonimoro Ph.l>. Chester M. Davis, Ph.D., Alec H. Horn Texas Research Insfitute of Mental Sciences Houston, Texas The application of electron capture gas chromatography (ECGC) to the determination of A9-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 modi- fications of the method which now permit quantitation of A9—THC in human blood at concentrations approaching that attainable with the animal subjects. Although the cannabinoids do not possess high affinities for thernml electrons, it is a relatively simple matter to form poly- halogenated derivatives of these compounds ,4 2. in quantitative yield to which the electron capture detector responds with excellent sensitivity. The limit of detection for the heptafluorobutyrate or pentafluoropropio- nate esters of A9-THC is approximately one picogram (Fenimore, 1973). Because the levels of A9-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 1973L 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 prepa— ration, or both. One approach toward eliminating the inter- fering components is to achieve a relatively pure isolate prior to gas chromatographic analysis through sample preparation in- volving 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 neces- sary resolution to enable the determinations of A9-THC in human blood serum. DESCRIPTION OF THE GAS CHROMATOGRAPHIC APPARATUS A schematic diagram of the dual column-dual oven gas chromatograph is shown in Figure l. Oven N° 1, containing a conventional packed column, is temperature programmed while oven N° 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 I [_ I (TVEHH 1 CARRIER I GAS | “1° [EiPfiART . ECID COLD TRARI I I H | CA::IER I l | GAS I I ‘ Haven 2 | H | | COLD TRAP _____ JL__J 1 ug) by flame ionization. The transferred effluent is further separated by the capillary column and detected by a micro- volume electron capture detector (Fenimore, 1971). This system provides the means to elimi- nate three major problems encountered in high resolution ECGC. First, temper- ature programming, which is usually not feasible with EC procedures because of 43 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. 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 con- sists 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 temper- ature 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% 0V—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 7180°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-llO (Applied Science Lab, State College, Pennsylvania). Nitrogen carrier gas flow rate is 4 cc/ min. Column temperature is 190°C. Ad- ditional 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 l Curie scandium tritide foil (U.S. Radium Corp., Panorama City, California) and is operated at a 1000 usec. 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 extrac- tion and derivatization procedure is shown in Figure 2. All solvents are '44 Figure 2 BLOOD SERUM 1 ML. AQUEOUS FRACTION HEXANE'ISOAMYL ML. 1- ADD INTERNAL STANDARD 1.5% ISOAMVL ALCOHOL 10 ML. HEXANE ‘ HEXANE FRACTION POOLED HEXANE EXT. TAKE. T0 DRYNESS UNDER NITROGEN. PLACE UNDER vncuun son 15 MIN. ADD 2 ML. H 0, 3 ML. PENTANE. VORTEX mx AND CENTRIFUGE PENTANE FRACY 0N DRV UNDER N... ‘ DISSDLVE IN 100 “L PENIANE TMA SOLUTION. ADD 50 “I. PFPA. REACT 1 HR. Dav UNDER I12. DISSOLVE IN 20 .L ISO-OCIANE SOLUTION FOR G.C. reagent quality or better and are re- distilled 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 Ag-THC using pentafluoropropionic anhydride (PFPA) in the presence of trimethylamine. The final volume of sample prior to gas chromatog- raphic analysis is 20 ul of which 5 D1 is taken for each sample injection. EHHHHHiI FRACTION DISCARD The internal standard is hexahydrocanna- binol (HHC) prepared by hydrogenation of AB—THC and purified by preparative scale thin layer chromatography. For most de- terminations 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 A9- 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 inter- fering substances originating in the blood serum or introduced to the sample during preparation. This latter source of con- tamination can, at least, be controlled by exercising the usual precautions gener- ally recommended for electron capture de- terminations, i.e. use of glass-distilled solvents, use of small total solvent vol- ume where evaporative concentration is required, re-distillation of derivatizing reagents, scrupulous cleanliness of glass- ware, and avoidance of all plastic mate- rials with the exception of polytetra- fluorethylene 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 chroma— tographic 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 parti- tioning manipulations prior to gas chro- matographic separation. 0n 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 A9-THC—PFP and were found to have no significant contribution to solute loss even at the limit of de- tectability. It is for these reasons that reliance was placed on gas chro- matographic separation rather than ex- tractive techniques for reduction of interference. In even the most meticulous extraction procedures involving recovery of com- pounds 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 approxi- mate those of the compounds in question as closely as possible. The deuterated A9-THC employed by Agurell in mass frag— mentographic assays (Agurell, 1973) cer- tainly approaches the ideal, but, of course, cannot be differentiated from unlabeled A9-THC by electron capture procedures. Hexahydrocannabinol was there- fore selected as having similar properties to A9-THC while retaining good chromatog- raphic separation. 4 5'. 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 A9‘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 A9- THC were added to random sera samples, and the ratio of detected peak heights of A9—THC 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 A9-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 approxi- mately 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 Figure 3 Figure 4 I' F "NC “NC THC THC In 20 3'0 4'0 5 lo 2'0 30 43 TIME (min.) TIME(min.) Figure 5 30* E BLOOD SERUM LEVELS \ 3 A9- THC U : .- ml o i 3 s s TIME (Hours) 46. limited only by the rapidity with which the primary temperature-progranmed colmm can be serially operated. With automatic sampling and computor 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. PharmacoZ.25:554 (1973). Fenimore, D.C., Freeman, R.R., and boy, 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., Arm. NJ. Acad. Sei.191:l42v (1971). McCallum, N.K., J. Chromatogr. $072.11: 509 (1973). Rosenfeld, J.J., Bowins, B., Roberts, J., Perkins, J., and Macpherson, A.S., Anal. Uhem.46:2232 (1974). 47 Although this re ort concerns the de- termination of A -THC only, the tech— niques and instrumentation should be applicable with minor modification to quantitation of other cannabinoids and metabolites of these compounds in bio- logical samples. DETECTION AND QUANTIFICATION OF TE'I'RAHYDROCANNABINOL IN BLOOD PLASMA Agnew Ohlssonl, Jan—Erik lindgron2'3, Kurt Loandorz, Stig Agurolll'3 1Faculty of Pharmacy, University of Uppsala , Uppsala 2Department of Toxicology, Karolinska lnstitutet, Stockholm 3Astra l'aikemedelAB, Siidert'alje , Sweden INTRODUCTION During the last few years much effort has been spent on developing methods for the identification of Cannabis users by ana- lysing different body fluids. Also, tech- niques for the quantitative determination of Al-tetrahydrocannabinol (Al-THC) and re- lated compounds have long been desired. Recent developments in this area have been summarized in a review by Grlié (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. '48 It must be emphasized that the definition of what a 'satisfactory method" is will vary with the goal of the users. For epidemio- logical studies of Cannabis use,simple screening procedures - where a high reliability may be traded for rapidity and expense - will probably be satis- factory. In forensic toxicology, the requirement for unequivocal identifica- tion 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 quantita- tive procedure. These considerations would suggest that eventually different procedures will be used for the determi- nation 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 (CC-MS) instruments are available in a limfied Scientific community, a mass spectrometric method is obviously less "sophisticated“than in a country where such instruments are scarce. Radioimmuno assay procedures for cannabi- noids in blood and urine have been ex- tensively investigated (cf. Gross et al., 1974; Teale at aZ., 1975). Both authors utilized goat antiserum obtained after immunization with conjugates of Al-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 2.9. 7-hydroxy-A1- THC and cannabinol (CBN) but not with cannabidiol (CBD). As pointed out by Teale at al. (1975) this cross-reactivity has disadvantages but also advantages in 6.9. epidemiological studies. Another immunological approach - free radical immunoassay - has been tested by Cais at al. (1975). 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 49 sensitivity in the detection step. Feni- more at 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 Al-THC and related compounds. Garrett and Hunt (1973) described another EC-based procedure using the pentafluoro- benzoate ester. Later, the same authors (Garrettand 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 quanti- tative determinations of Al-THC and Ae-THC in the blood of Cannabis smokers (Agurell at aZ., 1973, 1974). These methods are based upon the purification of THC extrac- ted from blood plasma by liquid chroma- tography 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 de- tection of Al-THC in humans after smoking. The latter authorsused trimethylanilinium hydroxide foron column methylation of the phenolic group and relied on the trideu- teromethyl 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 deriva- tives should be mentioned (Loeffler et aZ., 1975). volume present in detail a mass spectro- Wall and co-workers in this metric procedure for the determination of THC in humans after Cannabis smoking. Of course, the crucial test for any method is its performance (sensitivity, speci- ficity, capability) when applied to the actual problem in man - this is unfortun— ately lished. often lacking in the papers pub- 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. METHODS Cannabinoids Synthesis of olivetol-d7. The synthetic (1975) was used with the following modifications in order procedure of Pitt et al. to increase the amount of deuterium incor- porated into the molecule (Fig. l). 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 (l) (Buchta & Andree, 1959, 1960). l was then reduced with LiAld4 in ether at room temp. for 2 h. The complex was de- graded with dZO to insert one more d in the side chain. The isolated compound, with the hydrogen atom of the alcohol group ex- changed for deuterium, and the catalyst tris(triphenylphosphine)rhodium chloride was stirred in benzene under d2 atmosphere for 16 h at room temp. The 5-(3,5-dimethoxy- phenyl)pentan-l,1,2,3,4,S-d6-l~ol was then reacted with PBr3, LiAld4 and BBr3 according to Pitt at al. (1975) to yield olivetol-d7 (over-all yield from 3,5-dimethoxybenzal— dehyde 24%). Deuterium content according to mass spectrometry, m/e (180-187): do 2%, d1 2%, d2 2%, d3 3%, d4 3%, d5 9%, d6 30%, d7 100%. CBD—d7. Olivetol-d7 (160 mg) was condensed with (+)-trans-p-mentha-2,8-dien-l-ol in benzene using traces of oxalic acid as de- scribed 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) CED-d7 was obtained. Deuterium content according to mass spectrometry (m/e 314-321): do 2%, d1 2%, d2 2%, d3 3%, d4 3%, d5 9%, d6 30%, d7 100%. Al-THC-d7. Olivetol-d7 was condensed with (+)-trans-p-mentha-2,8-dien-1-ol using 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 A6- and A1- isomers. The purified (94% by GLC, 37 mg) AerHC- 7 showed the following deuterium content (m/e 314-321): do 2%, d1 2%, d2 2%, d3 3%, d4 3%, d5 9%, d6 30%, d7 100%. §§§:Q7. Ae-THC-d7 was treated with two equivalents of chloranil and traces of p-toluenesulfonic acid in refluxing dry toluene for 5 h (Mechoulam et aZ., 1968). The solution was chromatographed on a si- lica gel column with toluene eluent and further purification by TLC yielded 96% pure CBN-d7. The isotope distribution was the same as in Al-THC-d7; this in contrast to dehydrogenation methods using sulphur which causes extensive deuterium scramb- ling. Figure I OCH3 OCH3 OCH3 / 3 3 CH3O A/I CH3O / H O OCH 1) UN DAIEther °CH3D 3 2)I32()1) P Bfl§ 3) (V3P)3 RhCl/Dz CH3O DD 2) UN D4ITHF CH30 OH BBP3 D D HO D D D D Olivetol-d7 7 6 OH OH OH OH 5 9 1o A'-THC A6- THC \M do R= 1H 3” 5:. d2 = ><\A D d3 - D VVfD D D “7 = M Fig. 1. Scheme fbr synthesis of olivetoZ-d . Formulas of cannabinoids and deuterium containing internal standards. Olivetol-d3 was prepared as described by Pitt at al. (1975). Compound 1 (see olivetol-d7) was hydrogenated in the pre- sence of Pd and the product reduced with LiAlda. Subsequent reactions with PBr LiAldA and BBr 3’ 3 giving olivetol-d3 were as described for olivetol-d7. Al-THC-d3 and CBN-d3 were synthesized as described for the d7-analogues. Since larger amounts were prepared Al-THC-d3 was separa- ted from Ae-isomer on a column of Florisil using 2% ether in light petroleum as sol- vent. Al-THC-d3 was 952 pure according to GLC and the deuterium content (m/e 314*317: d0 2%, d1 6%, d2 12%, d3 100%. CBN-d3 eluted with 2-102 ether in light petroleum from the Florisil column was 95% pure according to GLC with a deuterium content (m/e 310-313): do 2%, d1 5%, d2 15%, d3 100%. Olivetol-dz. The methyl ester of 3,5- dimethoxybenzoic acid was reduced with LiAld4 in tetrahydrofuran and the re- sulting alcohol was reacted with PBr3. (See olivetol-d7). The bromide crystal- lized from ether-light petroleum was mixed with Cu(I)I in ether and butyl lithium was added drop-wise to the mixture (Petrzilka at al., 1969). The obtained 0,0-dimethylolivetol-l',l'-d2 was distilled at 110° c, 0.15 mm Hg and showed the following deuterium content (m/e 180-182): d0 0.3%, d1 42, d2 1002. After demethylation with hydriodic acid (cf. Agurell et aZ., 1973) olivetol-d2 was purified on a silica column with methanol-chloroform of increasing pola- rity as eluent (from 32 methanol). CED-mg, Al-THC-cé, CBN-cé. <31m~d2 was synthesized as described for the d7- analogue and crystallized from hexane. The CED-d2 was 97% pure by GLC and showed a deuterium content (m/e 314-316): do 8%, d1 12%, d2 1002. Al-THC-d2 was prepared as Al-THC-d3 and more than 952 pure by GLC: Deuterium content (m/e 314-316): d0 10%, d1 20%, d2 1002. CBN-d2 was prepared from A6- THC-d2 by dehydrogenation with sulphur at 260°C (Petrzilka at aZ., 1969). The deuterium content (m/e 310-312): d0 12%, d1 46%, d2 100%. 6— _ n . A THC dz. The synthe51s of this compound (98.5% pure) was described previously (Agurell et al., 1974); deuterium con- tent (m/e 314, 318): do 1.5%, d4 1002. Non-labelled cannabinoids. (Al-THC, Ae-THC, CBN, CBD) were synthesized accor- ding to standard procedures and care- fully purified and dried before use. Stock solutions were maintained in ethanol (1-5 mg/ml, 4° c, stored in the dark). EEE was carried out on a Varian 2100 FID gas chromatograph using 2 mm (i.d.) x 180 cm glass columns with 2% SE-3O ultraphase on Gas Chrom Q (100-120 mesh) at 250°C. Analysis of THC in blood plasma The following procedure was used for A6- THC (Agurell et aZ., 1974). Analogous procedures utilizing the proper deutera~ ted internal standard and mass numbers are applicable for Al—THC, CBN, and GED. The procedure for Al-THC has been pub- }lished (Agurell et aZ., 1973). 52 Blood samples. Blood samples (10 ml) were collected as desired in heparinized‘tubes after smoking of the A5-THC sample. Plasma is obtained by centrifugation and stored in silanized glass tubes at -200C 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 (As-THC-da) dissolved in 50 ul ethanol. The plasma sample is extracted three times with an equal volume of light petroleum containing 1.52 iso- pentanol in a glass stoppered centrifuge tube. After centrifugation the light petroleum is drawn off and the combined organic extracts evaporated under nitro- gen at 50°C almost to dryness. This ex- tract 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) con- taining Sephadex LH-20 and eluted with light petroleum-chloroform-ethanol (10:10:l) 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 AG-THC was deter- mined by calibration with ng amounts of AG-THC-3H (Fig. 2). The calibration can also be carried out by GLC analysis of 53 Figure 2 cpm AG-THc-3H l I 1 AtTHc CBN can I l | 25 so 35 ml Fi . 2. EZution pattern fbr AG—THC-3H, Al—THC, CB and CBN from a Sephadex LH-20 column. the concentrated fractions after appli- cation of 10 ug 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 ul conic vial (Reactivial, Pierce) dried, and finally dissolved in 10-15 ul 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% 0V-l7 on Gas Chrom CLP 100/120 mesh. Temperatures were in the column 180-2100, flash heater 250°, and source 2900. Helium was carrier gas (25 ml/min) and typical retention times are: CBD 3.4 min, Ae-THC 4.0 min, Al-THC 4.5 min, CBN 6.1 min. For mass fragmentography a multiple ion detector was added (Elkin 6t aZ., 1973) and used at 50 eV. For AG-THC, the mass spectro— meter was set to continously record the intensities of m/e 314 (molecular ion of non-labelled As-THC) and m/e 318 (internal standard, A6-THC-d4)as well as m/e 299, 303 as shown in Fig. 3. Figure 3 SWLE N0. 5 2] “RUINS PERIOD 2 SWLE PERIOD 5 DEL‘lfl-S—THC HNDH'I‘ETEHNRL S‘IHNDMD S CNHNNEL uzlsux 5:1. 11nt 33110 1 3:» 5:7 9-)9333 2.53153 2 3:9 233 , 13533 3 233 276 o 17:33 :.1s‘ax 9 ace 6: 9.12333 .2sy513 ma 0" 96_. 0 two 31: u' 299 u- 0 ° 0 . . on mc mu‘ no: N. 1 u_ .J E g 2% m 3 a "L I I l 1 l 2 3.2 9.9 5.5 5.8 S fiEiENTJDN TXHE Fig.5. Mass fragmentation of AG-THC-d as internal standard (m/e 518, 303) and AS-THC (m/e 314, 299) from purified plasma extract. Standard curves were prepared by adding known amounts of A6-THC (0.5-100 ng/ml) to blank plasma samples and carrying out 4). The correctness of the standard curves were the described procedure (Fig. checked during the day. Figure 4 . 5. § i f E :2. 4- % ‘3 i i ST E | & E , l- 2 s s a n :0 .0 so 41% no A'-TNC mm Fig. 4. Standard curves for As—THC (AB-THC) in plasma. 0-1— ng/mZ (left), 10-100 ng/mZ (right). (.AB-THc-d4) 54 RESULTS AND DISCUSSION Principles The described method for the determination of AS—THC (cf. Agurell et aZ., 1974) in blood is basically identical to the one published for Al-THC (Agurell et aZ., 1973). The method is based upon the addi- tion of the proper deuterated internal standard (As-THC-dA) due to the blood plasma sample. After extraction with light petro- leum, the AG-THC containing fraction is purified by liquid chromatography on a Sephadex LH-20 column. The pertinent fraction containing A6-THC (absorbed by smoking a spiked Cannabis sample) and AG-THC-d4 (added as internal standard) is collected. The relative amounts of the two compounds are determined by mass frag- mentography monitoring the molecular peaks (m/e 314 and 318, respectively; Fig. 3.). As subsequently discussed, this method can also be used for the analysis of other canna binoids, such as THC:s of different mole- cular weight, CBD, and CBN. However, at present less information is available with regards to these latter compounds. 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. Reference cannabinoids __.____________________ In all methods pure cannabinoids are ne- cessary for identification and calibra- tion purposes. Since cannabinoids are often rather unstable and, with few ex- ceptions, 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 at aZ., 1974) followed by careful drying and storage in ethanol solution at 40 in the dark provides cannabinoid standard solu- tions of satisfactory stability. The syntheses of the deuterium labelled compounds were accomplished by condensation of suitable deuterated olivetols and (+)- trans-p-mentha-Z,8-dien-l-ol according to standard methods (Mechoulam at aZ., 1976) - Fig. l. AG-THC-d:; The synthesis of this internal standard was described previously (Agurell at aZ., 1974). The label is looated in the 55 1"- and 2"-positions of the pentyl side chain (Fig. 3). Other deuterated cannabinoids. A synthesis of Al-THC—d4 was reported earlier (Agurell at al., 1973). paper the syntheses of olivetol-d7, In the present olivetol-d3, and olivetol-dz - mainly based upon the procedures of Pitt at al. (1975) - are outlined. From these inter- mediates.correspondingly labelled Al-THC, CBD, and CBN can be prepared as exemplified in Fig. l. The sensitivity in the final mass frag- mentographic assay may be limited by the amount of non-labelled (do) compound present in the deuterated internal standard. This interferes with the non-labelled canna— binoid present in the plasma. Thus, we have tried to minimize this interference by pre- venting exchange reactions, increasing the number of hydrogens substituted with deu- terium, and by limiting the amount of in- ternal 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 expected1 d7-containing Al—THC, CBD, and CBN showed, together with Al-THC-d3, the least conta- mination with dO-analogues (2%). This is in the same range as found for Ae-THC-d4 (1.52; Agurell at aZ., 1974). The mass spectra of Al-THC-d7 and non- labelled Al—THC are shown in Fig. 5 together with Al-THC-d3. Inca u 330 nfle 330 nfle 300 300 200 250 200 250 150 .— Em. m. 5% mwmqwg 8w Dram? >F§$m$ Si >F§$m$ mo _ O 6 2 O 6 2 330 In/e 1‘50 200 250 300 100 Extraction and purification Ae-THC and its Al-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 AI-THC, GED, or CBN are analogous. The extraction procedure for AS-THC, as revealed by experiments with As-THC-afl, is quite efficient and the recovery after both extraction and liquid chromatography is usually over 80%. Also. early studies on Al-THC showed recoveries of 70 i 6% (s.d.) after the column purification (Agurell et al., 1973). The Sephadex LH-20 separation is essential in removing interfering lipids and meta- -6a,7-diOH —- Bar-0H - 6 -OH A'-THC — 7.%H - ULOH - 3"—0H _ 6-O- - Al-THC ‘ - 7-0H CBN - 5”-OH - 4”- 0H - 3”-OH -2WOH - CBN - 2n_o- I l l l l l l l l l l l J O 100 200 ml Fig. 6. derivatives on Sephadex LH—20. bolites before mass fragmentography. The elution volume for e.g. A5-THC is stable for months if the column is operated at low const. temp. (120C) 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 sys- tems can be automated (Sippell et aZ;, 1975). Over 902 of the AS—Tuc peak is eluted in a 5 ml volume (Fig. 2) but a 7-8 ml fraction is collected. As a pre- caution ca. 3-4 ml on each side of the peak is collected and stored. We have investigated the elution patterns of different cannabinoids and their meta- bolites on the Sephadex LH-20 column (Fonseka et al., 1976). This is shown in Fig. 6. Figure 6 .. 5"-0H _ 7-OH 5 — 3"-0H A -THC —1"-OH _ 2n_ 0“ - As-THC - 3n.o_ _ 2&0- l I l l l l I l I l l l l O 100 200 ml -7-OH - 5"-0H - 1."- OH - Sci-OH _ sp-OH — 3”-OH — 1"-0H - 2"- OH — 6-0- L-cao llllllllllll O 100 200 ml CBD Elation patterns of Al-THC, A6—THC, 031v, and GED and their mono-oxygenated Abbreviations as exemplified by the Al-series 6-0 = 6-oxo—A1—THC', 3"-0H= 3"—hydroxy-A1-THC, 60L, 7-dihydroxy-A1;=THC, etc. 57 The complexity of cannabinoid metabolism in man and animal is indeed great and the re- quirements for specificity when assaying a certain cannabinoid are consequently con- siderable. To give an impression of the complexity, we have in a review (Agurell at aZ., 1976 b) listed 35 metabolites of Al—THC, AB-THC, CBN, and GED, 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 at aZ., 1976) should be added. Mass fragmentography The sensitivity achieved in the quantifica- tion of Ae-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 AG-THc-d4 for the analysis of AG-THC in blood plasma from four male, casual Cannabis smokers who had smoked 8 mg of A6—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 AG-THC to blank plasma samples and carrying out the described procedure. Peak heights of AS-THC-d4 (m/e 318) were plotted against known amounts of As-THC (Fig. 4). Since the same ratios were obtained simply by mixing known amounts of A6-THC and AS-THC-dh, usually such standard curves were used. The correctness of the Stan- dard curves should be checked occasionally during the day of analysis. 58 There are advantages as well as disadvan- tages in the use of deuterium labelled analogues as internal standards. They are carriers for the minute amounts of canna- binoids present in biological samples and, being almost identical to the ana- lyzed compound they can be added to the original plasma sample and will then com- pensate 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 Ae-THC can be used down to 0.3 ng/ml. As pointed out (see diseussion on "Reference cannabinoids") the sensitivity is partly limited by the small amount of Ae-THC-d0 present in the deuterated internal standard. Thus, we have tried to eliminate dO-contamination in the internal standards for Ae—THC, Al—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 Al—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. Al-THC, the kinetics of a particular Al-THC dose can be followed in spite of high background levels of non-labelled Al-THC (Fig. 7a). The d7-labelled Al—THC is then used as internal standard (Fig. 7b). Al-THC and GED can both be determined in the same mass fragmentogram using the same channels - m/e 314 and 2.9. 321 for inter- nal standard - whereas CBN is simultaneously assayed using m/e 310 and 313 (Fig. 7c). Figure 7a SAMPLE No.1 WAITING PERIODI SAMPLE PERIOD IO CHANNEL MASS HEIGHT RET. TIME RATIO I 314 2 317 555 2.55999 1 10C)[— A 8C)-— 6()-— 4O - 20 —— 317 2' z 314 ‘2 U) o I I I I I 1 3 5 7 9 11 RETENTION TIME Fig. 7 a. Mass fragmentation of purified plasma extract containing Al-THC (0.9 ng/ml plasma, m/e 314) and Al-THC-d3 as internal standard (m/e 317). Column temp. 210%. So far we have not developed assays for any of the possibly important Al-THC meta- bolites, e.g. 7-hydroxy-A1-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 mono- hydroxylated in the side chain can be assayed 15_ 9 Figure 7b SAMPLE N0. 2 VDI/AITING PERIOD I SAMPLE PERIOD I0 CHANNEL MASS HEIGHT_ RET. TIME RATIO I 3“. 210 2.53999 .656666 2 317 273 2.51999 .866666 3 321 315 2.47999 I I()O[— B 8C)'— 60 :f/L 314 40 — 20 317 .1 '4 Z 9 321 (n o I I I I I I 3 5 7 9 II RETTEFITHDN TIN‘E Fig. 7 b. Muss fragmentogram of A1—1%KL Al-THC-d , and A -THC—d7 (ca. 5 ng/ml plasma of each). Column temp. 210Pc. as their trimethylsilyl ethers using speci- fic 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). Figure 7c SAMPLE NO. 24 WAITING PERIOD 1 SAMPLE PERIOD 10 CHANNEL MASS HEIGTH RET.T|ME RATIO 1 314 453 4.06000 .628294 2 310 186 503999 .257975 3 313 135 4.02000 .187239 4 316 249 2.93999 . .345353- 5” 317 721 4 1 10()-— (I 8()-— 6C)-— 314 4(3 —- 1 f 310 ' 313 20 — ‘ 316 .J g 317 9 U) 0 l l l I 1 3 5 7 9 11 RETENHON'HME Fig. 7 0. Mass fragmentograms of CBD (m/e 314) and internal standard CBD—d (m/e 316, retention time 3.0); Al-THC (m/e 314) and internal standard A1~THC-d3 (m/e 317, re- tention time 4.0); and CBN (m/e 310) and internal standard GEN-d5 (m/e 513, retention time 5.0). Column temp. 2200 C. Plasma levels The plasma levels of A6-THC in man after smoking 8 mg AG-THC - of which about half is absorbed in the lungs - are shown in Fig. 8. Immediately after smoking high values (>100 ng/ml)of As-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 sensitivityof 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 Ae-THC levels with physiological and psychological effects (Agurell et aZ., 1974) but not enough to establish a true elimination phase. Lemberger and co—workers (1971) have estimated elimination phase half— lifes in man of 1—2 days. Garrett and Hunt (1976 b) have shown that theJterminal half-life of Al-THC in the dog is reached only slowly. They also found that the return of Al-THC from the tissues is the rate determining step of the drug elimination process after the initial distribution and metabolism. Similar plasma levels of Al-THC as for AS-THC (Fig.8 ) have earlier been found in man by us (Agurell et aZ., 1973) and Rosenfeld et a1. (1974) using mass frag- mentography, and by e.g. Galanter et a1. (1972) using Al—THc—lkc. Wall and co- workers (1974) have published plasma levels of both Al—THC, CBD, and CBN and certain metabolites as well as subjective psychological effects after i.v. admini- stration of the labelled drug. Hence, the sensitivity requirements for the determination of AerHC 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 sensitiviw ty with sufficient specificity is perhaps limited to the mass fragmentographic tech- ng/ml figure 8 200 100‘ 50 1O 0.5 ‘\‘7 -l I l I IAL l l l L O 15 3O 45 60 2 4 6 8 MIN HOURS Fig. 8. Plasma levels of Ae—THC after smoking 8.3 mg AG—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 Al-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 3.9. :61 steroids and other drugs also has to be ascertained. The capacity of the mass fragmentographic technique is more limited than the radio- immuno assay procedure, the main time re- quiring step being the liquid chromatography 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., Custafsson, B., Holmstedt, B., Leander, K., Lindgren, J.-E., Nilsson, I., Sandberg, F. & Asberg, M. (1973). J. Pharm. Pharmac. 25, 554-558. Agurell, S., Levander, S., Binder, M., BaderrBartfai, A., Gustafsson, B., Leander, K., Lindgren, J.-E., Ohlsson, A. & Tobisson B. (1974). Phanmacokinetics of AB-THC in man afrer 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. 62 purification. With the non-automated system now in use, a technician can process about ten plasma samples per day. This might be improved by automation, by high preSSure liquid chromatography or by using the double extraction technique of Rosenfeld et al. (1974). 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, 1626F1641. Buchta, E. & Andree, F. 3111. (1959). Ber. 92, Buchta, E. & Andree, F. (1960). Ber. 93, 1349. Cais, M., Dani, 5., Josephy, Y., Modiano, A., Gershon, H. & Mechoulam, R. (1975) F335 55, 257-260. Elkin, K., Pierrou, L., Ahlborg, U.G., Hohmstedt, B. & Lindgren, J.-E. (1973) J. Chromatogr. 813 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. Phann. Sci. 62, 1211-1214. Garrett, E.R. & Hunt, C.A. (1976 a). Separation and sensitive analysis of tetrahydrooannabinol in biological fluids by HPLC and GLC. To be published. Garrett, E.R. & Hunt, C.A. (1976 b). Pharmacokinetics of A9~THC in the dog. To be published. Grlié, 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). Ewami- nation of the metabolites of Al-tetrahydro— cannabinol in mouse liver, heart and lung .63 by combined gas chromatography and mass spectrometry. In press. Lemberger, L., Axelrod, J. & Kopin, I.J. (1971). N.Y. Acad. Sci. 191, 142-152. Loeff1er, K.0., Green, D.E., Chao, F.C. (1975). Proc. west. 18, 363—368. & Forrest, I.S. Pharmacol. Soc. McCallum, N.K. 11, 509—511. (1973). J. Chromatogr. 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). Hel J 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.,i Perkins, J. & McPhersson, A.S. (1974). 'Anal. Chem. 46, 2232-2234. Sippell, W.G., Lehman, P. & Hellman, G. (1975) J. Chromatogr.108, 305-312. Tea1e, 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 oannabinoids in man. Presented at Conference on the pharmaco- logy of Cannabis, Savannah, Dec. 3-6, 1974. In press in Pharmacology of Canna- bis, Eds. S. Szara & M. Brande, Raven Press, New York, USA. A METHOD FOR THE IDENTIFICATION OF ACID METABOLITES OF TETRAHYDROCANNABINOI. (THC) BY MASS FRAGMENTOGRAPHY Marianne Nordqvistl, Ion—Erik Lindy-on“, Stig Agurolll'3 1Faculty of Pharmacy, University of Uppsala, Uppsala 2Department of Toxicology, Karolinska Institutet, Stockholm 3Astra Lakemedel AB, Siidertilje INTRODUCTION Plans to identify Cannabis users by the detection of Al-THC in urine had to be abandoned after the reports by Lemberger at al. (1970, 1971). Their experiments with humans given 11+C-Al-THC i.v. and (1974) showed that the drug is almost completely meta— later work by Wall et a1. bolized and the metabolites excreted mainly via faeces. Most of the urinary radioactivity derived from acidic meta- bolites. The contents of unchanged Al-THC were less than 0.02% of the administered dose. Only small amounts of 7-hydroxy—A1- THC, the major metabolite of AI’THC in vitro, were indicated in the urine by TLC (Lemberger at al., 1970). In contrast, , Sweden 64 the faecal contents of 7-hydroxy-A1-THC are high, about 20% (Lemberger et aZ., 1971; Wall et al., 1974). The low amounts of Al-THC and also 7-hydroxy—A1—THC ex- creted 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, 197A) with or without introduction of additional hydroxyl group(s). The Al-THC-7-oic acid has been identified as a major metabolite in urine, faeces, and plasma from humans given Al-THC or 7-hydroxy-A1-THC i.v. (Wall et aZ., 197A). A method for detection of AI-THC or 7- hydroxy-Al-THC in urine or in plasma samples taken more than 12 h after the inhalation of a normal occasional dose of AI-THC wOuld have to be extremely sensi- tive. The plasma level of unchanged drug is than less than 0.3 ng/ml, which is the limit of our method for detection of THC. 7—Hydroxy-A1-THC, 6B-hydroxy-A1-THC, 6a—hydroxy-A1-THC and 6,7-dihydroxy-A1- THC are all minor metabolites in plasma udthconcentrations much lower than that of the parent compound at all times,accor- (1974). On the other hand, the amount of Al-THC-7-oic acid in ding to Wall at al. plasma is almost equal to that of Al-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 Al-THC-7-oic acid is identified in man so far. The major ones isolated from rabbit urine - 1"- and 2"-hydroxy-A1-THC- 7-oic acid (Burstein et al., 1972) and 4",5"-bisnor-A1-THC-7,3"-dioic acid Nordqvist et aZ.,l974) could also be ex- pected to be formed in man by further hydroxylation and oxidation of 7-hydroxy- Al-THC. The unidentified part of metabolites in human plasma, urine, and faeces classified (1974) are probably similar to the hydroxylated as more polar acids by Wall et a1. 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 Al-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 - 55 mass spectrometry (GC-MS) equipment is available. It is related to our method for detection of THC in blood plasma (Agurell et aZ., 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 Al-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 AS-THC-7-oic acid (Mechoulam et aZ., 1973) dissolved in 25 ul ethanol was pipetted into a glass stoppereicentrifuge tube containing 2.5 ml human plasma. It was equilibrated and 2.5 ml 1 M citrate-EC] 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 pl 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. Column chromatography. The resulting the extraction with equal volume of diethyl residue was transferred to a jacketed ether (7.5 m1 x 1). Sephadex LH-20 column (1 x 55 cm, Vo = 17 ml) using three consecutive 200 111 Figure I portions of the elution solvent light petroleum-chloroform-ethanol (10:10:l). At the elution‘rate 0.2 ml/min and the temperature 120 the methyl ester of A6- THC-7-oic acid was eluted between 37 and 47 ml. This fraction was collected in a 10 m1 conic tube. The solvent was evapo- rated under nitrogen, the residue dissolved in ethanol and transferred to a 1.0 ml COOCH; conic vial, where it was stored at 40 in a "”5 the dark until analysis. 2 0 O Silylation. On the day of analysis the methylated extract was dried under nitrogen, dissolved in 25 pl dry acetonitrile, mixed r with 10 ul silylating agent N,O-bis-(tri- 4‘5 methylsily1)acetamide] and kept at 50-600 374 4 4 for 10 min. It was then dried again under ‘ g \AU\J 430 g nitrogen and redissolved in 25 ul acetoni- 0 9 . 6 w trile. l l l l | J l l I l l l l l l O 2 4 6 8 O 2 4 6 Mass fragmentography. The silyl ether of MIN MIN Ae-THC-7-oic acid methyl ester was subjected Fig. 1. Mass fragmentograms of the aiZyZ ethers of the isomeric THC-7-oic acid methyl to mass fragmentegraphy (3% SE—3O Gas Chrom esters from purified plasma extracts. 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 RESULTS AND DISCUSSION fimtherdetails, see previous paper in this volume (Fig. 1). The partition of 3H-AG-THC-7-oic acid (pre- pared by acid catalyzed exchange) between Analysis of THC-7-oic acid in urine buffer solutions of different pH and an organic phase (diethyl ether, light petro- The procedure described above for analysis leum With 1'51 isopentanol, and toluene, of human plasma can also be applied to respectively) was studied to determine the urine. In a preliminary study we equilibrated combination of pH and extraction solvent 100 ng 3H‘A6"THC‘7'01C acid f°r 20 min With ~giving the best recovery. Diethyl ether 5.0 ml human urine and added 2.5 m1 1 M . ‘proved tobe the most efficient solvent citrate-HC1 buffer to maintain pH 4.1 during (log D = 2.2, pH 4.1). The recovery from 66 plasma of added 3H-Ae-THC-7-oic acid (in the range of 50-200 mg) was over 852 after extraction with a double’volume of ether. The recovery from urine was over 90% using equal volumes. The major in viva metabolite Al-THC—7-oic acid is ex- pected to behave almost identically. The diazomethane was effective in esteri- fying the As-THC-7-oic acid but the corresponding acid metabolite of Al-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 As-THC-7-oic acid was determined by calibration with ng amounts of tritia- ted compound. From earlier experiments Al-THC is known to have an elution volume slightly higher than that of As-THC. In O§c/OCH3 _ OTMS 100, o D 8() . 0 6C) 4‘) 2() I ’ I 0 ii I I II VB V3 1()() 8() 6C) 41() our study the delayed elution of the ester of Al—THC-7-oic acid, in comparison to its Ae-isomer, had to be compensated for by a wide fraction of collection. Total recovery of 3H-As-THC-7-oic acid in plasma after extraction, methylation, and column purification was around 70%. The silylated methyl ester of AG—THC- 7—oic acid is not fully separable from the Al-isomer on the conventional GLC columns tested (SE—30, SE-SZ, 0V-17, JXR, XE—60). When not silylated, the methyl esters were resolved but that of the Al-isomer partly decomposed on the column. Thus, the silylated methyl ester of Ae-THC-7-oic acid was used as an external standard for the Al-isomer since both compounds have very similar retention times but different intensities in the mass fragmentograms (Fig. 2, 3). The As-isomer can also serve as a reference for semiquantitative estima- Figure 2 430 (M‘) 374 371 415 l h i r I 350 400 mle 371 430 (M’) 415 20 255 303 l ILL, C) 1%? | I II I h I l l I ‘ 200 250 300 350 400 nfle Fig. 2. Mass spectra of the ailyl ethers of As- (upper panel) and Al-THC—7-oic acid methyl ester (lover panel). 67 Figure 3a SAMPLE No.1 WAITING PERIOD 1 SAMPLE PERIOD 10 DELTA-1-SYRA CHANNEL MASS HEIGHT RET.TIME RATIO 1 415 570 4J5000 522935 2 374 109 4J8000 1 3 430 564 4.18000 517431 100 7 8C) —- Ix 60 _ I 4C)-— /\ 415 ~.—-“___._.P 7 20 __ 3 4 .1 § 0 430 63 o l I I I I 1 3 5 7 9 11 RETENTION TIME Fig. 3. a. The silyZ ether of Al-THC-7—oic acid methyl ester (m/e 430, 374, 415). tions of Al-THC-7-oic acid in plasma or urine. The possible concomitant occurrence of A6~ THC-7—oic acid in plasma or urine samples from smokers should be of little significance since A6~THC at most occurs in 2% of the amount of A1~THC in Cannabis (Ohlsson et aZ., 1971). There is only a limited need for an accurate quantitation method for A1- THC-7—oic acid since pharmacological test with Rhesus monkeys (Edery et al., 1971) have shown that the isomeric AG-THC-7-oic Figure 3b SAMPLE PERIOD‘ 10 SAMPLE NO. 2 WAITING PERIOD 1 DELTA-S-SVRA CHANNEL MASS HEIGHT RET,TIME RATIO 1 415 75 A 9.84251E-2 2 374 762 402000 I 3 1.30 690 4.02000 .9055” 100 — B so — so — 4o — “A 415 20 b 2' ~———} I 374 Z ‘2 “J 430 U) o I I I I I I 3 s 7 9 11 RETENTION TIME Fig. 3. b. The siZyZ ether of Alamo—7-0% acid methyl ester. acid is inactive in doses up to 10 mg/kg i.v. If necessary, the deuterium labelled Al-THC-7-oic acid can be synthesized as an internal standard according to methods described by Pitt and Wall (1974) and Pitt et aZ., (1975). There are no studies on the urinary excretion rates of Al-THC-7-oic acid in humans after Cannabis smoking. However, the studies on Al-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. ACKNOWLEDGMENTS The support of the Medical Research Council was appreciated. Mass spectro— metry was supported by grants to the Department of Toxicology. Reference acids were kindly provided by Drs. R. Mechoulam and M. Wall. REFERENCES Agurell, S., Gustafsson, B., Holmstedt, B., Leander, K., Lindgren, J.-E., Nilsson, I., Sandberg, F. & Asberg, 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). Ebamination of the metabolites of Ale tetrahydrocannabinol in mouse liver, heart and lung by combined gas chromato- graphy and mass spectrometry. In press. 69 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, 3., 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 9 471—473- Ohlsson, A., Abou-Chaar, C.I., Agurell, 8., Nilsson, I.M., Olofsson, K. & (1971). Bulletin on Nar— cotics, 23, 29-32. Sandberg, F. Pitt, C.G., Fowler, M.S., Sathe, S., Srivastava, S.C. & Williams, D.L. (1975) J. Am. Chem. 500., 97, 3798-3802. Pitt, C.G.& Wall, M.E. (1974). Synthesis of radiolabeled cannabinoids and meta- bolites. 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. 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 extrac- tion procedures, on the TLC fractionation of individual in viva metabolites, and on the development of an automated GC/MS quantita- tive procedure. For the first two aspects, we have relied heavily upon 1"C-studies. In California it is almost impossible to admini- ster ll'C-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 snnilar (Figures 1 and 2), so most of our early development efforts em— ployed Rhesus specimens. EXPERIMENTAL Baboon feces Extractions: Fecal specimens, although of no direct practical importance, were use- ful for orientation and for methods develop- Figure 1 Cumulative Urinary Excretion ol Orally Administered Radioactive Ag-THC 25— Rhesus Monkey —-— Man (Lemberger,ET.AL,. N.E,J. 20 — -— z I/’ Med., 216.385.1972) 0 15a / a ‘5 / Baboon ,_ 3 10— I x I, I 5‘/ Squirrel Monkey 0 I I i I I i l 2 3 4 5 6 Day Figure 1. Comparative urinary excretion of Ag—THC and its metabolites in man and three primates. 70 Figure 2 RADIOACTIVITY EXCRETED IN FECES 100‘} 80- 3‘, Squirrel Monkey 8 Baboon 3 so 5" Human a ,..--—---* E if” g 40‘ I” Rhesus Monkey < LL 0 x / 20‘ I, I / ‘1 i é 5 i 5 é i é 5 n h b m m TIME AFTER LABELED Ag- THC (DAYS) Figure 2. Comparative cumulative fecal excretion of parent drug and metabolites after oral administration of AS-THC in man and three primates. (Human data from Lemberger et al, 1971) ment because they contained copious quanti- ties of the parent drug and its less exten- sively 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 ll’C—Ag-THC was administered to chroni- cally THC-dosed baboons, 24—hour fecal col- lections 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 ex— tracted 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 11). 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 a2, 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 Table 1 Continuous Liquid/Liquid Extractions of Soxhlet-Extracted Residues Petroleum Ether (neutral pH) 80.2% Diethyl Ether (pH 10) 5.2 Diethyl Ether (pH 2.5) 6.4 Total Recovery 89.8% Figure 3 , <——— Ongm +——Solvent from 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 Ag-THC; a moderately polar diffuse region (9%) in the approximate position of mono-hydroxy-AQ-THC's; and some highly polar compounds near the origin (l§%) (Figure 3). A corresponding non-radioactive petroleum ether extract - obtained from feces collected on the day prior to radio-label dos- ing - 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 Aero- Graph Model 8500) equipped with a 25 cm NHZ- bonded 10 u silica column and an experimental filter fluorometer detector (Figure 4) Figure 4 Micropak-Si-‘O NH 2 214 '1a' Dana (‘00 ul) 1 ml/min How Rate k Henna: B: 2% Moon in 01202 5% B lo 100% B at 1%lmin 3‘" min 50mV FullScnlo mum-(cum -A9 -mc o '4 a 1’2 $6 25 Minutes 24 32 36 40 44 2 ul Dans Camabinoid Std. 1 mllmin Flow Rate A: Hum: R 2% MeOH in (mm; Siam me at 1%min ‘A'lmin 200mV FollScate Figure 4. High pressure liquid chromatog- raphy of Dansylated Fraction I-A from petro- leum ether extract of baboon feces (top) and mixture of Dansylated standards (bottom). (Loeffler et a1, 1975). From this chromato- gram, it can be seen that I-A had a major com- ponent with the same retention time as A9-THC. Minor components at the approximate retention times of CBN and GED were also present. Gas chromatography/mass spectrometry: A Similar aliquot of Band l-A was Silylated with BSTFA and analyzed by GC/MS (Figure 5). The automated GC/MS report (the inset in Figure 5) shows that Ag-THC and CBN were abundantly present (2.02 ug and 544 mg, respectively), but that little, if any, of the common mono- and di-hydroxylated or carboxy'metabolites were present in this TLC band. On our instrument, the automated GC/MS anal- yses 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 72- Figure 5 oLm/n: “an“: than 71-29. min“: 2x13.“ w w. :32 Lg”; 4.15: .. rum. ;-.. Danna-mt was an: .2zo Catlin: =r I: . .7 .3 it n: .. .efl 27 275 2 arv s.1::- 2 RECORDER RESPONSE 3 8 9 12 MINUTES 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 m1 nitrogen/min.; 275°C.) Inset: Auto- mated GC/MS analytical data. Shaded areas indicate retention time intervals during which positively identified components were scanned. eight characteristic ions from the spectrum of [lg-THC were present in the proper inten- sity ratios (and at the correct retention time) and that nine such ions were recorded for CBN. As an illustration, the five high-~ est mass ions from the Ag—THC contracted spec— trum were mnitored as a function of time (Figure 8) and it can be clearly seen that the GC peak is entirely hormgeneous with res— pect to A9 --THC. The ion currents reveal that the typical pattern was already recognizable at about 10 ng. , The quantities of A9 —THC and CBN in these in— dividual compound assays represent a total daily output of 1.48 mg and 390 ug, respec— tively. The first of these figures indicates that about 8.5% of the total A -THC which was administered was excreted as metabolized 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 a1, 1975) , is truly a meta-- Figure 6 OLFAX/GC ANALYSIS TS'207 T10295 ’HEXANE EXTRACT of PP“ 039 FEGESI 6-238 0F FRACTION X-A- DiLTH-9-THC +905 QTY 3-38EO B CONF l02 RT Bl MASS INTENSITY 67 0 2-223- 7 93 C 6J16E; 3 13! 5.685- 9 196 865255 9 219 83585; 9 23] 6&32E‘ 9 246 1:295; S 336 [£49Eh 8 3‘3 2.78E‘ 3 37! BJOSEE 3 PLOT Y 08 NTY 00F DECADESII Fs-2.225- 7 O l 2 3 fl 5 6 7 8 9 IO 0- ¢ 9 #- .n-o §_---.----, 67Co>>>h>>>>>>:>>>s>>>>>>>>>>>>>>>>>)>>>»>>>>>>>>>>>>> 93CQ>>>h>>>>>>>a>> [81 0> [96 +>> 2I9 9” 231 0’ 2&6 ¢>>> 333 Oi>> 343 o>>>>>> 313 o>>>aaa>>>>>>>>>>>a O 9 9 Figure 6. Single compound GC/MS analysis of Fraction I-A of petroleum ether extract of baboon feces indicating 3.08 ug of Ag-THC with a Confidence Index of 102 at a retention time of 81 secs. Note that only 2 ions (m/e 67 and 93) out of 10 are contaminated. 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 Iminm of about 3% CBN in the A9-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 metabo- lites of Nana, CBN, c131), and 11-Ho-A9-1Hc, has shown that orally administered CBN is very extensively metabolized (Kanter, 1975) . 73 Figure 7 OLFAK/GC ANALYSIS TS-209 Tl-298 >HEXANE EXTRACT 0F PPA :33 FECEI: 3.23% OF FRACTION I-A- CANNABINOL +Pos QTY 8.385- 1 cour |36 RT 139 MASS INTENSITY 165 C 7-68E- 9 I78 6.'84£'- 9 238 9J36E- 9 295 léanfii 3 313 2-952- 3 323 7&6853 9 367 ggg75; 7 368 8&235- 8 369 2&2453 8 382 1.0423 8 PLOT Y on N1Y 50F DECADES-l FS-2.27E- 7 a l 2 3 4 5 6 7 8 9 1a o----§----¢----¢----¢----¢----¢----+----+----+----+ O Q 165c+>> 178 +>> 238 4>> 295 §>>> 313 +>>>>> 323 #>> 367 #>)>>>>’>>>>>>>>>>)>>>>>>>>>>>>>>>)>>>>>>)>)>>>>>>> 365 ¢>>>>>>>>>>>>>>>s>> 369 ¢>>>a> 382 +>> ¢ 4' + figure 7. Single compound GC/MS analysis (Confirmation mode) of Fraction I-A of petro- leum ether extract of baboon feces indicating 0.808 mg of CBN with a Confidence Index of 136 at a retention time of 109 secs. (Only m/e 165 is not present in the required rela— tive abundance for pure CBN.) Thus it seems unlikely that more than half of the "administered" CBN should be recoverable from the feces in a single fraction ._ espe— cially after all the processing this sample received - and therefore it would appear that CBN is a genuine metabolite of A9 -THC. The remaining four bands derived from the petro— leum ether extraction contained none of the eight compounds in our test panel. The continuous ether extraction at pH 10 (Fraction II) contaide 54 ug ll-HO—Ag-THC (total daily output) and 210 ug cannabinol-ll-- oic acid (providing further evidence for the metabolic origin of CBN) , but the presence of A°«~’I'HC-ll--oic acid could not be unequivocally demnstrated. The ether extract obtained at Figure 8 2; one cannot: I a: Prom: nq-mc m; 5:4: MM 1 : rs-na n-zvs -| us DELu-9-THC/Yns :/ 0/16 "new M-nl: RUN l :4 rs >| u rs-2.aa:-a rs-z.Iu:-n i-Zl s»: 5-: ”mum I n rvn sen: -|u runs a nnLu-y-mc/ms :1 Nu OPWTDR 1 a: "com 139-": "as salt mu 1 J Is-HI nuns >1 ua Dunn-mums rs-I .uI-I l J I s o 1 o i la . qt ......_........................................ ... 3000009.. -»--.. ...... ”pun".....»»».n.»n»n».n»» n-p». ........ .n..-»..n ..... n». ......... ,,. Figure 8. Top: Single ion mass chromatogram (m/e 386) for 1 pg Ag-THC. Center: S-Mass selected ion recOrd for 1 ug A9 -THC (5 high- ...»n» ........ ,,.,, ........... » n»»» ....... n... .....;..ooo‘..oo.‘o..o.3.o...3.oo.. 3 . v 5 u :u »» .oo...o:o~o~.:o....,..... v v est masses used in the automatic assay pro- gram). Bottom: Same as center, but with 3-decade logarithmic presentation. pH 2.5 (Fraction III) contained 93 ug of 88,11- dihydroxy—A9 --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 sys- tem (Universal Monitor Corp., Pasadena, CA) which utilizes Professor McLafferty's "Proba- bility Based Matching" (PBM) algorithm (McLafferty et al, 1974) . In the FEM pro— cedure, reverse search 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. 74‘ Probability based matching McLafferty's PBM technique was specifically designed to mnitor individual compounds in mixtures by "resolving" their merged mass spec- tra 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 automa- tically ascertain whether or not any particular mass spectral line (fragment ion) is contami— nated by the siszltaneous presence of an impu- rity. Such judgments are possible because of one particular characteristic physical property of mass spectra; when two or more different compmmds contribute an identical molecular weight fragment ion to a merged spectrum. the 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 bbtching concerns the manner of specific ion recording and the pro— cessing 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 mea- sure a set of ions which are particularly characteristic of the target compound. A prin— cipal advantage of this concept is that a com- plete 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 ac- curately 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 a1, 1975). It then automatically computes the relative intensities and com— pares the resulting ratios with the stored library spectrum in order to detect any con- taminated 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. A11 contaminated lines and those which are near background intensity are ig- nored in subsequent calculations. Confidence Indices: From the remaining un- contaminated 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 prob- ability 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 Vlscore) is a combination of the uniqueness of that particular mass number (frequency of occurrence in McLafferty's library of 18,800+ compounds) and the rela- tive abundance of that particular ion in the entire mass spectrum of that specific com- 75 pound (Pesyna et a1, 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 auto- matically make such judgments without being experienced in the interpretation of mass spectra. Quantitation: All of these criteria (and m5r5:—WEIEH_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 signi- ficant aspect of PBM quantitation is that the computer automatically selects the un- contaminated ion which represents the lowest quantity of the target compound (based upon the required relative abundances) and there- fore 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 conven— tional 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 con- tracted 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 con- ventional 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 Figure 9 2/ 8/76 OPERATOR : 23 PROGRAM D9-THC TYPE scmz RUN v 2 TS=197 TI=298 >a.7s us DELTA-Q-THC/THS MASS INTENSITY 246 3.76E-12 339 8.225-12 A 343 2.26E-12 R 371 1.115-11 3 386 1;81E-11 Q b 246 3.665-11 C 332 8.52E-ll E 343 1;945A18 m 371 3.842-18 E 386 3;z45510 8 246 5.445-1o 5 333 8.52E-lfl ' 343 1.582- 9 371 3.84E- 9 386 2.483- 9 246 2.435- 9 339 3.485; 9 343 7.335- 9 371 1.655; 8 386 7.882- 9 246 3.285- 9 333 4J26Ei 9 - 343 9.9212- 9 MaXImum 371 2.3412- 8 Cycle 386 1.395- 8 246 2.725- 9 338 3.48E¥ 9 343 S;24E- 9 371 l-78E- 8 Figure 9. Representative digital data recorded during elution of 0.75 ug Ag-THC GC peak. (Inten51ties are in amperesé e.g. m/e 371 in maximum cycle is 2.34 X 10 A.) 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 currentsfi and at the bottom is a logarithmic presenta- tion 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 dis— cerned at a very low level of signal - i.e., at less than 10 ng). Selection of specific fragment ions The PBM reverse search technique is such a powerful pattern matching procedure that Figure 10 3-4 an 2- \\\\\\\ 1. 03- o SECONDS Figure 10. Plot of typical digital data 76 (like that of Fig. 9) for 0.9 ug A9-THC. almost any set of ions selected from the spectrum of a compound will give acceptable qualitative and quantitative results. How- ever, for sets of compounds having the simi— larity of the cannabinoids, especially where high sensitivity is desired, selection of the optimum mass lists requires careful considera- tion 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 de- tectable 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. Figure 11 at 1/70 wanton o I: II 5/" ormrn‘! a u I! and ”In!” I I) D!MW‘ km: "PI sun Mum v-I'nc "I: l:— Vloum 0-": V0: 5:” W! I I VSIIII 'll-SOI I". l I 'S'III 'IISII I“! l I isllll Tl-JEI >5sz and” or "A us run. I." or Mum: I-A- mm: um“ or ”a I” "cu- l." or rucnm I-A- mm: “TIM! a! ”A ”I "an: I.“ or run”: x-A. is-Ia-L-I l‘s-I-Ml-l 75-3.: -: 1-) ha K-I u u o :n can». In a . u. nun-pun. .. ..oo.oon.oo-uo.o- nun-u». in nu». an «nun-n...» o n: on» I‘ll at u».- au on»...- nun-unn-nun-nun...- n-u-nnnnnn-n-u.nun-un-nun-sun. .- n.»- ”spun-nun»: unnunnnn..n». ..... ”nun-”nun nun»...-unn.-.n-nnunnnn-nn- ..... pun-nnun-”nun."-n...» .... "nun-unnun-pn .nnnn-nnunu... .- nun-nun.» "nun-un- NJ ”I'D... In “DD-pubI-hn-II. o Ill 0' 3" I“ on nun-n an coup-n»- o I“ h 3" In a I" u “I u” an oer-pp»- o u -. n...0....ooco:-oooo:oo.oo3...oo3.aoon'ooo~o'.oogo‘.o.o.too... Figure ll. Top: Mass chromatogram for Ag-THC tion). Bottom: Same as center, but with 1n Fractlon I-A of petroleum ether extract 0f 3-decade logarithmic presentation. (5-Highest baboon feces. Center: S-Ion selected ion masses used in automatic assay program.) record for the same sample (linear presenta- The closer the challenge comes to the reten- fore even PBM was unable to produce unique tion time of the target, the more critical it assays for the individual compounds. Accord- is to select contaminated or weak ions. Thus ingly, a single program was produced which for ll-hydroxy- and 8a, ll-dihydroxy-Ag-THC/ was labeled ”SB—hydroxy-Ae-THC," but which TMS, the retention times differed by less than was in reality an 88- and/or 8a—hydroxy-A9— 1% and it became more important to screen THC assay. against the challenging compound than to screen for the target. Because their spectra Responses Of the PBM algorithm are quite different this could be accomplished, but only by sacrificing some sensitivity. The The perfornmnce of the PBM algorithm can best pair 8d, ll—dihydroxy- and 8B, ll-dihydroxy- be understood by looking at the manner in which Ag-THC/TMS had similar spectra but differed it responds to specific single compounds. If markedly in retention time so that they could the Confidence Index is monitored as a func- be easily differentiated by GC alone. Un- tion of sample size, a decreasing value is fortunately, a different situation was encoun— observed with decreasing quantity of sample tered in the pair 8a—hydroxy— and 88-hydroxy- (Figure 12). This is due primarily to two A9-THC/TMS which have nearly identical spectra effects. At the upper end of the plot, it and nearly identical retention times; there- decreases due to a "dilution" correction be- 77 Figure 12 120' 100— AQ-THC/TMS ‘ \ ,_ 80- 23 x _ a) ‘O E 60 - (D O C a) _ f9 8 4o - L) Threshowfor 20 __ reliable identification I - or;7" AB-THC/TMS I I I I l 4] 001 002 005 OJ 02 05 to Quantity Injected (pg) Figure 12. Effect produced on PBM Confidence Index by varying the quantity of pure Ag-THC injected. cause it is assumed that low ion currents are frequently the result of dilution with impur- ities 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 ap— proach background levels, and are dropped from consideration because they are too weak to measure reliably. Numerically, K is a binary logarithmic repre- sentation of the probability of an accidental (random) match of the observed quality be- tween the unknown spectrum and the library spectrum. A rough approximation of the signi- ficance of the K—scores is that ZKth spectra, chosen at random, would have to be compared with the library spectrum before one encoun- tered a spectrum which would match as well as does the experimental s ctrum. Thus a K of 10 means roughly that 2 ° (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 equivo- cal, 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 Ae-THC, when used to chal- lenge the pattern for the 59-THC program, gives a K-score of 11, more than 100 points lower than does Ag-THC (the target compound). Differentials in K-score are likewise used to establish the "thresholds for reliable iden- tification” for each program. Somewhat arbi- trarily, this threshdld is set at 10 units greater than the maximum K-score generated by any known challenging compound or control spe— cimen (i.e. a "safety" factor of 1,000 ). >In the case of Ag-THC shown in Figure 12, the Confidence Index threshold for reliable iden- tification was set at 21, indicating that one can be reasonably certain that it is Ag-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 dis- tinguish 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 immediate- ly 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 im- mediately apparent by inspection. Along with the computation of the Confidence Index, the PBM algorithm provides a quantita- tive 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 es- sential parameters for the Olfax algorithm Figure 13 0 g Q COMPOUND : 5 h‘ a: o 9 MONHORED h a m 7 2 I 9 9 a: m D a: .' . g 0 i < ' 9 9 a ' 4 e 2 3 O : 1 2 T COMPOUND d E é ; £ 3 c g INTRODUCED o o to z: m an d: o DELTA-B-THC i 81 116 8 TA-- __. __ DEL 9 THC as ‘09 10 144 7 CANNABINOL as “1 W 12 127 10 83-HO-D9-THC 119 W W 0 122 9 11-HO-09-THC T1 —“9 —‘55 . . i fl i EA'“ 0' H0 154 155 197 aeuathHo .3. JEL .3. 163 197 254 9JHC4b0m .E. .EL _2_ 198 253 306 CBNdbom .fl. JEL 261 308 Figure 13. Confidence Indices and retention times for nine cannabinoids. Reported as K _~M(sec for 1 ug samples. Column tempera- ture 275°C.; nitrogen carrier flow rate 30 ml/min.) Quantity Found (0) 0.1 Figure 14. Table 2 OLFAX II ASSAY PROGRAMS Figure 14 01 Quantity Injected (pg) 1.0 Quantitation reported by the mass spectrometer (PBM algorithm) as a function of the quantity of A9-THC actually injected into the GC. COMPOUND TITLE IKTITSILEE1 2|}lhIslél7l8‘9llO 9 In/e 371 )1.) 330 2% 231 219 196 181 9) 67 A -THC/TMS DELTA-Q-THC 21 210 v 1A 11 11 8 8 8 6 7 3 3 m/e 367 )68 369 582 32) 310 295 £38 175 165 Cannebinol/‘X'HS CANNABINOL 21 210 v151h15131112997“ 9 m/e 393 )71 386 369 391 330 328 286 196 107 88-50—11. -mc/ms 88-HO-D9-THC 20 210 v1312111110910752 9 m/e 371 38L. )hl 329 )0} 289 265 75 73 #5 ll-HO-A mac/ms 11-H0-D9-THC 2; 210 v 15 12 11 10 8 8 B 5 6 2 m/e 359 371 )“1 330 317 )0} 26" N5 129 9} 8a.11-(HO) -A9-THC/TMS 8A,11-DI-HO 31 210 3 v 9 11 9 7 9 6 6 2 3 2 III/a 569 383 371 3‘0) 539 503 293 22} 1‘1 129 83.114110) -A9-1'Hc/ms 89,11-DI-Ho 23 210 3 v1212121010776~2 m/e 371 355 305 303 299 297 289 265 296 75 Ag—THC-ll-Oic Acid/ms 9-mc-11-oxc 23 225 v 1L. 11 7 7 6 7 6 7 6 L. m/e 353 350 321 509 296 299 235 220 219 Cannabinol-ll-oic Ana/ms cau-n-oic 27 225 v 11 10 10 7 6 6 6 s 6 Table 2. Confidence Index "thresholds for reliable identification" (KT), molecular sep- 79? arator temperatures (TS), and line-values (V) for the ions (m/e) used in the 8 cannabinoid assay programs. Figure 15 MY- URN (n! 5.5) Extract with hexane Adjust to [ii 12 llexano soluble Extract with other (ll) AdJIllt to pll 2.5 Ether soluble Extract with other (E-I) Ether hueluhle Extract with ethyl acetate Ether soluble Hash with 3% Hal-[005 I 2.5 other “J not to beard) I lthyl acct-to Iolnblo Dar-ct wit A NallCO: inlolublc ((1 (3-1:) Aqueou- Ethor Iolublo (dilurd) (E-III) Figure 15. Extraction scheme used for B-glucuronidase / aryl sulfatase hydrolyzed As-THC urines indicating derivation of the H, §;l 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 biologiu cal specimens, no great practical value Can be derived from their analysis. Therefore, we have concentrated our efforts upon investiga- ting urine, which would appear to be the most practical biological specimen, provided that some metabolite could be identified and moni- tored which follows the time course of physi- cal impainnent due to THC. (we have some‘pre- lbninary 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 a1, 1972; Melikian et a1, 1973). This scheme separated the total radioactivity in enzyme-hydrolyzed Rhesus urine into six approximately equal frac- tions: Hexane extractable, H (non-polar neu- trals); ether (pH 12) extractable, §;l (weakly polar neutrals); ether (pH 2.5) extractable, E-II and E-III (weakly polar acids); ethyl ace- tate (pH 2.5i extractable, EA (moderately polar acids); and tetrahydrofuran—(pH 2.5) extract- able, THF (highly polar acids). Figure 16 shows the relationships between the sizes of these fractions in hydrolyzed, as well as un- hydrolyzed, urines. The actual values are given in Table 3. Subsequently, this procedure 80 Figure 16 100 80 - Non-polar nouncl: ' Weakly-polar neutral: Weakly-polar acids «“77- Modorchly-polor acid: m Highly-polar acids - losiduo 60 40 20 Unhydvolynd Hydrolyud Figure 16. Distribution accordin to polarity (extractability) of Ag-THC metabo ites in viva (hydrolyzed and unhydrolyzed Rhesus urine). Non-polar neutrals at neutral pH weakly polar neutrals at pH 12 Weakly polar acids pH 2 ' Nbderately polar acids ethyl acetate at pH 2 Highly polar acids extractable with tetra- hydrofuran at pH 2. extractable with hexane extractable with ether extractable with ether at extractable with Table 3 Extraction Efficiencies of Various Solvents (Percent of total counts extractable from Rhesus monkey urine) Hydrolyzcd Urine Solvent and pll Unhydrolyzed Urine Hexane O . 196 6 . 796 (natural pH) Ether 0. 9% 11 .096 (1)" 12) Ether 47 . 8% 48 . 396 (PH 2) Ethyl acetate 26.8% 15.296 (pl! 2) Tetrohydrofuran 23 .996 14 . 6% (p11 2) Residue l .496 l .596 Total 100.9% 97.1% was refined by resolving the weakly polar acids (the largest single fraction) into strong and weak acids according to their solubility in NaHC03 (E-III and E-II, respectively)(Kanter et a1, 1974). Most of our efforts have been concentrated on the three least polar fractions, H, E-I, and E-II, which contain the parent cannabifioids and the mono- and di-hydroxys, as well as the ”ll—oic" acids. Standards do not distribute cleanly between these categories; Ag-THC and 11-hydroxy-A9-THC, for instance, distribute in the ratio of about 4:1 between the hexane frac- tion and the ether at pH 12 fraction (E-I . 8,11-Dihydroxy-A9-THC, on the other han , 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 fractiona— tion scheme very extensively to urines obtain- ed from human volunteers after oral administra- tion of pure standards of Ag-THC, CBN, CBD, ll-HD-Ag-THC, singly and in combinations (Hollister, 1973; Kanter, 1975). They have observed many interesting phenomena which can best be summarized briefly as: 1) the exis- tence of a multitude of drug-related metabo- lites; 2) very few matches with available standards; and 3) the extreme persistence (10- 15 days) of highly polar metabolites after a single administration of drug. Hi h dose 5 ecimens: As part of our own work u51ng these same procedures, our group was very fortunate to be able to obtain urine spe- cimens from subjects used in some unusual experiments last spring at the University of California Langley Porter Clinic in San Fran- cisco (Jones et a1, 1975). These subjects received the extremely high oral doses of 210 milligrams of A9-THC per day. The urine was hydrolyzed with B-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 "nega- tive"), 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 A9-THC at 10 ng/ml (Figure 18). Since this positive identification was achieved with only two ions, a more conventional mass chromato- gram 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 A9—THC. 81 In addition to the AQTHC, 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—UTB ng/ml, which probably was due to a slight carry-over from the hexane fraction, and a fairly strong positive for 8a,ll-dihydroxy-A9-THC at 24 ng/ml in the screening run (Figure 21). When frac- tion E-I was re-run in the single compound mode,_it'no longer showed a definite positive although the 8u,ll-dihydroxy-A9-THC was a near- miss 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 pos- itives for A9-THC (K=24), A‘TTHC-ll-oic acid (K=94) and CBN-ll-oic acid (K=37)(Figure 22), but only the THC acid could be unequivocally confinmed (Figure 23). The A9-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 inten- sity ratios do, in fact, correspond to those of authentic A9-THC-ll-oic acid (bottom). One aspect of PBM quantitation should be stres- sed in connection with the concentration of Ag-THC-ll-oic acid which was measured. The PBM algorithm provides an upper limit to the pos— sible quantity present. From this we must con- clude 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 A9-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. Thereforzthe A -THC- ll—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 Ag-TTE-ll-oic acid per se is definite- ly a minor component. Moderate dose 5 ecimen: An examination of a slightly more typical urine specimen was car- ried out on some extracts provided to us by Saul Kanter of Hollister's group. These frac- tions, H, E-I, and E-II, were extracted from the urifiés‘Ef an indiv1dual before and after he received a single oral dose of 30 mg of Ag-THC; specimens were collected 12 hours prior to drug intake and during the interval of 12 to 24 hours post-drug. Figure 17 Figure 18 OLFAZC/GC ANALYSIS TS=236 “-295 . magma EXTRACT :9}: a) or URINE Ill-l3: FRACTION :: mos-z 5 :-.1.. DELTA-9-THC +POS QTY 4-SIJE- 2 CONF 24 RT 32 own/ac ANALYSIS - - -.-. than 71.395 MAJS lNTLJS ITY mm: DKTIALT (PH 8) or van-II. 52:43: 1' 5 ML. 231 C**3.z4E-IB 246 C**6.92£5IZ DELTA-94»: NEG My 1.115- I com I: RT SA 333 C AoSZE-IZ CAI-mum“ P05 an .oaz- 2 com 23 In 137 - sa-no-ng-mc ‘mzn cry 2.“;- 2 com' H :21- IIs 3‘3 G'zSEfi'lfi n-Ho-m-ruc NEE m'rv I.sz:- I sour III 17 I33 37] l-flBE- 9 BAIIl-Dl-HU NEG an huh a cow ll RT ”2 BB-ll-Dl-Ho Hi6 an ma:- 2 com I: RT [13 . 9-mc-H-Mc use an IIIoz- I cum I5 RT 225 PLOT ‘I’ OR I-J'I'Y w GEN-II~OXC an; an 3.55:. a sour I2 n-r 272 [OF DECADES-l (I) § FS-loflbi- 9 U) u (I a l 2 3 4 5 6 7 S 9 l6 5 I-.--.----.----.----.---_._---I----.-_--I----I----. & 0 O o a 67 0 CE 93 0 l8] 0 196 it 219 + 231*¢>>>>>>>:>>>>>>> 246:4»>>>>>>>)>>>>>>>>>>>>>>>>>>>>>>>> aaac+>>>>>>>>>>>>>>>>>>>>>>> 343 o>>>>>>>>>>>>>>>>>>>>>>>>>>>>> 371 9>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Figure 18. Single compound GC/MS analysis (Confirmation mode) of Fraction H from hydro- , . lyzed human urine (same as Fig. I7) indicating 3 6 9 ‘2 45.4 ng of Aa-THC in 5 ml urine with K=24 at a WNUES retention time of 82 secs. Note that the com- . .puter elected not to scan the lowest 5 masses Figure 17. FID Chromatogram of silylated because it knew that they would be too close non-polar neutrals (5) fraction of human urine to background levels and that the computer (enzyme hydrolyzed). GC conditions same as judged 3 of the remaining ions to be contam- Fig. 5. Inset: Automated GC/MS analytical inated, i.e. their relative abundances were data (Autoscan mode). too high (CIf. pattern in Figs. 8, 9 and 11). Figure 19. I: —‘ * A I I I f‘ A A A i I I I I I I I I I I: L‘ A A A A A A I I I I I I I I 27 | A A A A A A A A A :3 I A A A A A A A A A H . I I I I I I I I I l— ") + A A A A A A A A A l) A A A A A A A A A A 'g A A A A A A A A A A I. A A A A A A A A A A 'i A A A A A A A A A A .I I I I I I I I I I I I I I I I I I I I I .. I I I I I I I I I I I') A A A A A A A A A A _ I I I I I I I I I I l \3 A A A A A A A A A A "1 A A A A A A A A A A A A .. I I I I I I I I I I I I I I I I I I I I I I I I I.) A A A A A A a A A A A A A "I 0 4 A A A A A II A A A A A A A I . I I I I I I I I I I I I I (If I A -\ A A A A A A A A A A A A A L: l A A A A A A A A A A A A A A A I D A A A A A A A A A A A A A A A 3):. r 9 A A A A A A A A A A A A A A A A A A n?.|C\D I A A A A A A A A A A A A A A A A A A I.M. . I I I I I I I I I I I I I I I I I I I {) a A A A A A A A A A A A A A A A A A A A A Inn“, A A A A A A A A A A A A A A A A A A A II n A A A A A A A A A A A A A A A A A A A Indzn I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I “>41,“ A A A A A A A A A A A A A A A A A A A A It” . I I I I I I I I I I I I I I I I I I I I c. Nl— < 4- A A A A A A A A A A A A A A A A A A A A :1 II I; I A A A A A A A A II A A A A A A A A A A A 1. ”,5 A A A A A A A A A A A A A A A A A A A A a p..‘ A A A A A A A A A A A A A A A A A A A A I. A A A A A A A A A A A A A A A A II A A A A r: o — A A A A A A A A A A A A A A A A A A A A A -J I A A A A A A A A A A A A A A A A A A A A A u I I I I I I I I I I I I I I I I I I I I I I :1 G A A A A A A A A A A A A A A A A A A A A A A A A A ' 3 A A A A A A A A A A A A A A A A A A A A A A A A A c E) ¢QO§009++§§+O0§OOQ+¢+Q§+§§§§§+§§4*9+§00§+*0§++549+§¢++ I _ _ _ _ _ _ i l _ _ _ _ _ _ _ _ _ _ _ I - _ _ _ - m I~ r~ h n r~ I~ r- I~ P I— I— r~ r~ r— I~ I~ h I~ I~ t~ I~ h h h I~ 'A I') l 1") I1 {'1 l') 5" n I') (’7 I") (7 fl ('1 H n f7 f1 f1 5’) ('1 ('3 ('7 f') K') Figure 19. Single ion Qn/e 371) mass chro Note that this ion becomes cleanly measurable matogram for same sample as Figs. 17 and 18. at about 5 ng. 82 Figure 20 ULFAX/EC ANALYSIS TSI229 Tl-SZI NZEKAHE EXTRACT (PH 3) 0r URXEw'E iH¢|3l FRACTION H F3011 5 ML- CANNABXNOL OPOS QTY S-ZUL- 2 COM" 23 RT Il'a MASS INTENSITY 175 cu! .22}:- 233 Cal .1165- 295 c.-3.aaa- :na Etta-335- 323 Cttl -465' 367 loAflE- 365 7-285- 369 CttS-Ufli- 3E2 C'tz-GAE- coma-00000 PLOT Y OR 147‘! 50F DECAD£S=1 FS-I dam-2» 8 55 l o---—¢---- o o 165 o l75t¢>>>> 235so>>>>> 295a»»»»»» aldto>>>>>5>x>>> 321t§>>>>> 367 ODE/000000000000000000DO'IOUDUUOUUUOOOODUUUUU‘JuGOUDOO J63 1.:»>>>>»»»>»)»n»»> 369ao>>>>>xs>>>>>>>>>>n>> 3512t0)>)>>>>>>) e on u or u- 60 4 Figure 20. Confirmation mode GC/MS analysis of Fraction 1-! 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 oLrAX/ac ANALYSIS Ts-flu n-JII ’l-l stun mucr an I!) or s m. WINE u-IJ DiLTA-Q-THC N“ “V 2.3.5- 2 Cour II in 2H; mumuot was in? 3.555- : cour as n In 1' an an o-uz- : cont 9 111' In In an! s-ul- 3 can u ll'l' Isa was on |.1u‘.- I Cour u 1" Iss In an I-IIE- I cm n n :93 um n" 1-Ill‘ a com I! a": 251 In WWI-3710. can! I! In an RECORDERPRESSURE 0 so I00 150 200 230 300 350 SECONDS Figure 21. FID Chromatogram of silylated weakly polar neutrals (E-I) fraction of hmnan urine (enzyme hydrolyzed? GC conditions same as Fig. 5. Inset: Automated GC/MS anal- ytical data (Autoscan mode). Figure 2 2 uLnx/sr.‘ «mun; 8 § 25 0 3 6 9 12 MINUTES 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 OLFAX/GC ANALYSIS TSI2¢7 71-296 >ETHER EXTRACT (PH 2) OF URINE lH-ISI FRACTION E-II FROM 5 ML- 9-THC-ll-OXC OPUS QTY 1.3950 a CONF 52 ET 227 HASS INTENSITY 75 C 2-535- 7 2A6 G ASZAE; 9 265 C 4-365- 9 289 C AJAAEF 9 297 GJZSE: 9 299 C A-IZE- 9 333 éilfli- 9 365 C 4.205- 9 355 7.882- 9 371 2-46Ei 8 PLOT Y OR NTY IOF DECADESI2 FS-2o53E- 7 E-2 E-l E3 c -"5 "f- a 5 + v 7SC§>>>>>>>>>>’>>>>>>>>>>>>>)>’>>>>>>>>>>>>>>)>>>>>>)>>>>>>>>>>> 2460¢>>>o>> 26509>>>>>>>> 239C+>’>>>>> 291 +>>>»»»» 299C¢>>>>>> 353 +>>>>>>>>>>> 35569>>>>>> 355 ¢>>>>>>>>>t>>>>>> 311 o>>>>>>>>>>>>>>>>>>>>>>>>>>>>> s Figure 23. Ag-THC-ll-oic acid/TMS analysis of Fraction E-II (same as Fig. 22) showing 1.39 ug/S- ml Hydrolyzed urine with K=52 at a retention time of 227 secs. All 10 lines were scanned, but 6 were contaminated. Figure 24 Figure 25 Figure 24. Top: Selected ion record of A9- THC-ll-01c acid GC peak from Fraction E-II (same sample and quantity as Figs. 22 and 23). Bottom: Same kind of selected ion record of 0.75 ug of authentic As-THC-ll-oic acid. 501 45- “\X 4m i- 35- 3 2 30- 3 '6 Ta 25- $ 20- u- 10- 5- \‘ I 2 5 4 E I 7 a 9 PH Figure 25. Effect of pH on extractability of Aa-THC in viva metabolites from unhydrolyzed Rhesus urine. We found a maximum quantity of about 50 ng of A9-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/lO mg creatinine, The E—I fraction (weakly polar neutrals) gave a very—marginal confirmation for 88—H0-A9-THC at a level of about 60 ng/lO mg creatinine and a suspiciously high reading of nearly 0.6 ug of Ba, ll-dihydroxy-Ag-THC per 10 mg creatin- ine with a K-score of 35. The E—II fraction (weakly polar, weak acids) from this specimen, surprisingly, failed to give a positive for AS—Tf-IC-ll-oic acid. Unhxdrolyzed urines: Four years ago, we re- ported that an appreciable proportion of the total counts in Rhesus urine could be extracted with ether, even without prior hydrolysis (Forrest et a1, 1972; Melikian et a1, 1973). This extraction was highly pH-dependent with the curve's inflection point being near pH 4 84 (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 L ley Porter Clinic Study, we ex- tracted anot er specimen with ether before and after treatment with enzyme at pH 5.5. Unfor— tunately this pH was far from the optimum pH for removal by ether, but the results are interesting, nevertheless. A highly signifi- cant Confidence Index (K=68) for Ag-THC-ll-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 AS-THC-ll-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. Figure 26 Figure 28 DLFAXIEC M‘LYSIS T5459 Tl-Ill iiTKER EXTRACT (PH 5-5; =LYRD'E. BEFORE LVZY'NE KYDROLYSISI DEL?A-9-THC MEG can! a 111' as “my“ "MU“; CANNAIHIOL NEG CDIJF 2 RT ”5 EB-X0-39-?HC RULE CONT I! 11' HS v- 5.5) :i- ujugzx .\."?E Il-HO-D9-THC NEE CON? 6 ".T It! BA-ll-Dl-NO NEG EDNF I/l 1T [.5 IE: I l -DI~HO NEG C0517 J 111' ”A E'THC-lI-OXC OPGS CDHF 62 '“" 9:25 :TY Io>5;- I CBN'II'OKC COS-17 9 ’IT 273 NY «nEIE- 3 ”‘I’V J-SJL- 2 lt‘.‘ 2-662- 3 w “Mama” “a Hymna. w Mull-Dl-nfl REG Th hnle- E z V-TM-ll-DIC «LG 2‘" l-IJE- l {DENIM-015 NLJ L‘TY blfio 3 8 (I) W I I I“ g g o g 8 a: I: 8 I O 0 I“ K o a e 9 ,2 0 3 6 9 12 MINUTES ““NUTES Figure 26. .Silylated ether extract (pH 5.5) Figure 28. Silylated ether extract (pH 5.5) of human urine prior to enzyme hydrolysis. 0f human urine after enzyme hydrolysis. Inset: Autoscan ana1y51s. Inset: Autoscan analysis. 1““m” M" “I FESTIZ. .:.....' W'"=3"W°"":=" Figure 27. Top: Selected ion record of urine at ii of A9—THC-ll-oic acid. Bottom: 0.75 pg of authentic Ag-THC-ll-oic acid. Ether extract of unhydrolyzed human urine at Center: E-II Fraction of hydrolyzed human RT of Ag-THC-ll-oic acid. .85 Figure 29 OLFAX/GC ANALYSIS TS-237 TIIJOI >ETHER EXTRACT (PH 5-5) H- URINE! FROM 5 ML- AFTER ENZYME HYDROLYSXSI BAnll-DI-HO OPOS QTY l-2BEO G CONF 35 RT [56 MASS INTENSITY 93 63:1.l4E- 129 Cttlcl75- 145 Ctt26725- 265 6 4-962- 3a3 7. 25:: 3I7 C 5468i: 333 cute-92:- 339 A-AAEH 341 C 6&8OE- 37I 4-80E- 00000000310 PLOT Y 0R NTY IOF DECADES-l FS'I-I7E- 3 fl 1 2 3 A 5 6 7 8 9 ID o---- o~ --* A --§ .VH“. “H“. + . 93:0»>>>>>>>>>>>a>>>>>>>>>>>>>>>>>>>»>>>>>>>>>>>>>>:> l29t¢>>>>>»>>>>>>>>>>>>>>>>>>>>>>>>a>>>>>>>>>>>>y>>>>>> 14530>>>>>>>>>>)> 2656¢>>>>>>>>a>>>>>>>>>>>> 363 o:>>>>>>>>>>t>>>>>>>>>>a>>t>>>>> 3I7C§>>>>>>>>>>>>>>>h>>>>>>>> 339t§>>l>>’>>>>>>> 339 ¢>>>>>)>>>>>>>>>>>>> 34|C§>>>>>>>>>>>>>>>>>>a>>>>>>>>>> 31| 0>>>>>>>>>>>>>>>>>>>> o + 9 Figure 29. 8a,ll-D1hydroxy-A9—THC/TMS analysis of ether extract of human urine after enzyme hydrolysis showing 1.20 ug/S ml with K=35 at a retention time of 150 secs. Three ions are uncontaminated. This selected ion record also shows that one ion Om/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 previ- ously discussed enzyme hydrolyzed urine, and 1 the top record is authentic Ag-THC—ll-oic acid. Figure 28 shows the results from the post- hydrolysis ether extraction. Only 8a,ll—di- hydroxy—A9-THC appeared to be present, but its identification was very marginal (K=32) although it was slightly better in the confir- mation mode (K=35) (Figure 29). 86 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 Ag-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 signifi- cant K-scores (60-80); the ether-extractable neutral fraction contained 8a,ll-dihydroxy- AS-THC (25-150 ng/ml) with low confidence and sometimes a suggestion of 88-hydroxy-A9-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 Ag-THC-ll-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 metabo- lites of As-THC are consistently observed and A9-THC per se is seen and quantitated with a degree of certainty which requires only slight improvement. Since the Ag-THC determinations were not entirely satisfactory, suggestions for future efforts aimed at improving this particular assay might include partially shift- ing 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 FEM 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 5 ectro- meter itself. Also, since 11-hydroxy-A -THC was not encountered in any urine samples, the 8a,ll-dihydroxy-A9-THC program should be modi- fied 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-A9-THC. In all of the work described here, a standard test panel of TMS derivatives of Ag-THC and seven of its metabolites was employed. Assay programs for additional meta- bolites, such as the side—chain hydroxylated compounds described by Professor Agurell, could easily be added to the panel if appro- priate reference samples were made available to us. The Olfax method, in which GC retention time screening is combined with the FEM mass spec— trum matching technique, appears to be a very promising procedure for the highly specific quantitative analysis of cannabinoids be- cause it automates and makes practical the best features of mass fragmentography. It cannot do anything which would not be pos- sible using conventional GC/MS systems, but its degree of automation provides an advan- tage in speed and convenience while retaining the inherent specificity of mass fragmento— graphy. The extractions employ solvents and apparatus which are available in any labora- tory, the labor and analysis time have been minimized, and the final assay uses a com- mercially 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. 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 Cali- fornia (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-ll-oic acid and Robert H. Hertel of Uni- versal Monitor Corporation for valuable discus- sions during the course of this work. The very able technical assistance of Fu-Chuan Chao and Kay 0. Loeffler is gratefully acknowledged. Finally, most special thanks are due to Duane P. Littlejohn and the Universal Monitor Corpora- tion for the loan of a prototype Olfax II GC/MS instrument system, without which this work would not have been possible. 87 REFERENCES Abramson, F.P. (1975) Anal. Chem. 51, 45-49 Ellman, G.L. (1975) Personal communication Forrest, I.S., Green, D.E., and Wflrsch, M.S. (1972) Proc. Fifth Intern. Cong. on PharmacoL Green, D.E., Killam E.E.K., Loeffler, K.O., Chao, F;C. and Forrest, 1.8. (1975) Pharmacologist 11, 268 Hertel, R.H., Green, D.E. and Strauss, P.A. (1975) Free. 26th Pittsburgh Conf. on Anal. Chem. and AppZ. Spectr. Hollister, L.E. (1973) Ehperientia 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. Phanwacol. 9, 205-213 Kanter, S.L. (1975) Personal communication Lemberger, L., Tamarkin, N.R., Axelrod, J. and Kopin, J. (1971) Science 113, 72-74 Loeffler, K.O., Green, D.E., Chao, F-C., and Forrest, 1.8. (1975) Proa. West. PharmacoZ. Soc. l§, 363-368 McCallum, N.K., Yagen, B., Levy, S. and Mechoulam, R. (1975) Emperientia 31, 520- 521 “— McLafferty, F.W., Hertel, R.H. and Villwock, R.D. (1974) Org. Mass Spectrom. 2) 690-702 Melikian, A.P., Green, D.E., Skinner, J.L. and Forrest, 1.5. (1973) Proc. West. Phannacol. Soc. 19, 234-239 Pesyna, G.M., McLafferty, F.W., Venkataragha- van, R., and Dayringer, H.E. (1975) Anal. Chem. 31, 1161—1164 Widman, M., Nordqvist, M., Dollery, C.T. and Briant, R.H. (1975) J. Pharm. Pharmacol. 21, 842-848 QUANTITATION OF A9-TETRAHYDROCANNABINOI. IN BODY FLUIDS BY GAS CHROMATOGRAPHY/ CHEMICAL IONIZATION -MASS SPECTROMETRY Ruthanne Derrick and Roger Foltz Battelle, Columbus Laboratories Cmumbua Ohm As part of a program for the develop- ment of methodology for quantitation of cannabinoids in body fluids, we have validated an extraction and GC/ CI-M analysis for the determination of A ~THC in plasma. The procedure has proven suitable for the analysis of relatively large numbers of sam- ples, yet has sufficient sensitivity a d selectivity for analysis of A -THC concentrations as low as 0.5 ng/ml in plasma. Using this proce- dure, we are currently analyzing a variety of body fluid samples from different sources submitted by out- side researchers. Previous work at Battelle and other laboratories indicated that substan- tial clean-up of the body fluid ex— tract was necessary prior to GC/MS analysis in order to achieve adequate sensitivity. Although several clean- up procedures have been used success- fully, many of these are cumbersome and not well suited for analysis of 88 large numbers of samples. The pro- cedure we are currently using is a modification of the RTI solvent ex- traction procedure (Rosenthal, 1975). The analysis scheme is essentially an organic solvent extraction and clean- up, concgntration and derivatization of the A —THC, followed by quantita— tion 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 buffgr solution and a known amount of A -THC—d3 internal stan- dard. The mixture is vortexed to assure complete mixing, then extract— ed twice with hexane. The combined hexane extract is successively wash- ed with 0.1 g NaOH and 0.1 E 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 be— low 50 C. The tube is rinsed with pentane and the pentane solution transferred auantitatively to a Reacti-Vial( . After removal of the pentane, BSTFA [N,O-bis-(trimethyl- silyl)-trifluoroacetamide] with 1% TMCS is added, and the vial is heat- ed for a minimum of 30 minutes at 75 C. The concentration of Ag-THC is deter— mined using the technique of select— ed ion monitoring (SIM) with a GC-MS. A 6 ft x 2-mm glass column packed with 3% OV-l7 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 moni- tored are m/e 387, the protonated molecule ion of the trimethylsilyl ether (Ag-THC-TMS) and m/e 390, the protonated molecule ion of the TMS ether of thg trideuterated internal standard (A —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 sBrength for a specific quantity of A -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 com- ponents of body fluid extracts. Also, the relative intensity of the M—2 peak is significantly lower when ammonia is used rather than methane. There- fore, the A -THC-d —TMS'will make less of a cogtribugion to the MH+ ion current of A —THC-d -TMS. Figure 1 shows the methane agg methane-ammonia CI mass spectra of A -THC-TMS, while Figure 1 '00 73 El 3,, AA? 386 50 303 343 o ‘Jl'thmu. I l. 1.. M' .Lnl J. i'. . I >~Ioo :°: MH+ 8 CICH 38? £2 4 £1 50 37: .‘é’ E3 030., .v -1 . W]. 0:100 MH+ CICH4+NH3 387 50 o SOIOO I50 200 250‘ 300 350 400 M/e Figure 1. Cbmparison of mass spectra of Ag—THC-EMS Table 1 M d.WL OSi (CH-:93 Can =386 “I. of Sample Relative Response Per I n'zation _ . oMlaihod m/e Monitored Ion Current Unn Weight of Ag-THC E1 386 (M?) 7 50 c1 (CH4) 387 (MH‘) 24 20 c1 (CH4+NH3) 387 (MH‘) 6? IOO c1 (N2+NH3) 387 (MH‘) 6? 80 TABLE 1. RELATIVE RESPONSES FOR PROMINENT IONS IN THE EI AND C: MASS SPECTRA 0F A9—THC—TMS Table 1 compares the response per unit weight of A -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 con— tributions to the MH ion currents by other components of the body fluid extracts. Chemical ionization with methane gives several prominent+frag- ment 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 im- portant 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 temp- erature programming of the GC column. 90 Quantitation of the A9-THC is achieved by monitorgng ion masses associated with the A —THC-TMS and the deuterat— ed internal standard, and measuring the peak height ratios of the ion9 currents. The actual amount of A — THC is determined by comparison of the ratio to a standard curve estab- lished at the time of each set of analyses. The standard curve is pre- pared by simply mixing gliquots of sgandard solutions of A -THC and A -THC-d , 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 pgak height ratio of 0.0074 or a A —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 Figure 2 THE-THEE STD. CLEVE. 11-24-75 TDTH_FEIGH 1.733 use L __ . l- SPf’PLE FHJSTFNH’L‘: HT. .592 I l I .w .51 1.8 S¥fLE NL/STHIIHD ”L l ‘1 1 1-61 2-13 Figure 2. Calibration curve fbr analysis of Ag-THC-TMS Table 2 9 Sample Ht./Standard Ht. Relative A -THC Experlmental Standard Number of Added Theoretical (Average) Deviation Determinations 96.8 mg 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 deviation at any given peak height ratio has typically been less than 20, even for the lower concentrations. The relative stan- dard 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 3 9 l‘. RELATIVE STANBARD DEVIATIONS FOR POINTS ON CALIBRATION CURVE (44.1 NC of A —THC—d ADDED TO EACH) differences in the Ag-THC values assigned to a sample by using differ- ent 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. Table 3 summarizes the precision and accuracy data for a sgries of analyses. Known quantities of A -THC and A -THC- d were added to l-ml portions of plasma. The final volume of each derivatized extract was about 25 ul. ana Injections into the GC were between 2 agd 4 ul. The actual quantity of A -THC injected on column is there- fore approxgmately one-tenth of the amount of A -THC added to each ml of plasma. Figure 3 is a typical com— puter plot of an extract of 1 m1 of plasma containing 1 ng of A —THC. In this example the peak in the m/e 387 ion current curve corresponds to about 100 pg of A -THC-TMS injected on col- umn. The corresponding peak in the m/e 90 ion current curve corresponds to A —THC—d concentration of 44.1 ng/ ml. The m/ 390 ion current signal is attenuated by a factor of 10 relative to the m/e 387 ion current signal. 330:3 we figure 3 THC.|-2Pn BIO-23 . 1— 390.3 387.3 / k 'I'IIrmHIIVIrIrI‘rTrI'TTTTTTFrmI 6 a: nu 1s: Figure 3. Computer plot of ion currents for m/e 387 and 390 corresponding to 1 NG/ML of A9-ch and 44 NG/MZ of A9-THC—d3. The m/e 390 ton current curve is attenuated by a factor of 10. Table 3 Ag-THC Ag-THC Relative Added Measured Standard Number of Percent (mg/ml) (Average) Deviation Determinations 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% o ‘ -o.13(a) 15.0% 8 - (a) The average of the measured weights corresponded to a value below the zero intercept of the calibration curve. 9 TABLE 3. PRECISION AND ACCURAC DATA FOR MEASUREMENT OF A -THC ADDED TO PLASMA (44 NC OF A —THC-d INTERNAL STANDARD) 92 3 ADDED PER ML 0F PLASMA AS Figure 4 12-10—78 TH: FRO‘1 PLRSMH. TOTF-L FEIG-iT -151 .103 SWPLE l-fi-ISTPNHWJ HT. 051 I 1 -00 .as SHVPLE WJSTPNJHRD NT. l l I‘O "6 '2‘ Figure 4. Plot of data points from SIM analysis of extracts of l-ML plasma samples containing 0 to 9. 7 [VG of A9—THC and 44.1 N0 of A9-TI-IC-d3. _Finally, Figure 4 plots data points corresponding to 50 separate SIM deter- minations on 2 sets of extracts from plasmas to wgich known amounts of Ag—THC and A —THC"d were added. The A —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 re- searchers. These have included rat, .93 monkey, and human plasma samples with volumes in §he range of 0.25 to 2.0 ml. Levels of A -THC in the samples have been typically in the range of 5-50 ng/ ml of plasma, although gamples contain- ing concentrations of A -THC as high as 400 ng/ml of plasma have been ana- lyzed. The actual procedure for ana- lyzing the unknowns is as previously described.To increase our confidence in the integrity of the results for any given set of samples, spiked plasma s mples containing a known amount of A -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, par- ticularly at the lower levels tion, 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 pro— cedure presently being used has p oven suitable for the analysis of A —THC in plasma to concentrations as low as 0.5 ng/ml and can be conve- niently used for relatively large numbers of samples. Experimental Section The plasma sample (usually N1 m1) is transferred to a 16-ml culture tube using a calibrated pipet and the vol- ume is recorded. To this is added 1.0 m1 of pH 7.0 buffer solution and 100 p1 of 441 ng/ml solution of A -THC-d in ethanol. The mixture is vortexed for 30 seconds. The plasma is then extracfied with 5 ml of Distilled-in— Glass 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 suc- cessively 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 rinsfid with 1 ml of Distilled-in-Glass pentane and the pentane solution transferred quafititatively to a 1.0- ml Reacti—Vial The pentane is removed under a stream of nitrogen at room temperature. The sides of the vial are washed down with 100 pl of pentane, and the pentane is again evaporated. To the vial is added 25 ul of BSTFA with 1% TMCSIR)The vial is capped using a Teflon liner and heated for 2 hours at 75 C. The concentration of A9-THC is deter- mined 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-l7 on 100/120 mesh Gas Chrom Q is temperature programmed In addi- 94 from 180 to 280 C at loo/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 pres- sure of ammonia in the ion source is 100 u. The flow rate of methane is 20 ml/min giving a partial pressure in the ion source of about 700 u- 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 be— tween injections. For every 5 unknowng, a spiked sample containing 9.7 mg A -THC/m1 of plasma is prepared by addigg 100 pl of a 97 ng/ml solution of A -THC in ethanol to 1 ml of human plasma. This is extracted in the same manner and at the same time as the unknowns. is established at the time of each set of analyses by adding 100 pl of the 441 ng/ml A -THCF d solution in ethanol and 100 ul f ode of various concentrations of A - THC i§)ethanol directly to a Reacti— Vial . A series of four such standards with do/d3 ratgos ranging from 0—1 for ~50 ng of A -THC—d are prepared. The ethanol is removgd at below 40 C, and the THC derivatized in the same manner as the unknowns: 25 ul 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 chromato- graphic column is used isothermally at 205 C. The values for the ratio of d /d are used to prepare a standard c rv . 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 sul- furic and nitric acid solution, rins- ed successively with tap water, dis— tilled water and methanol, and finally dried in an oven at >100 C. Aliquots of standard A9-THC and A9- THC—d3 solutions are measured out using a lOO—ul Eppendorf pipet with disposable tips. Syringes used for measuring out BSTFA or injecting samples into the GC—MS are cleaned in methanol. The hexane and pentane usfid are high—purity, Distilled-in—Glass solvents. REFERENCE D. Rosenthal, Second NIDA Tech— nical Review on Mass Spectro— scopY, Oak Brook, Illinois, October 22, 1975. 95 The concentration of the Ag-THC—d stock solution in ethanol was detgr- mined by comparison with a primary standard of A —THC in ethanol using SIM analysis. HPLC-MS DETERMINATION OF A9-TETRAHYDROCANNABINOL IN HUMAN BODY SAMPLES Jimmie L. Valentino, Ph.D., Paul J. Bryant, Ph.D., Paul I.. Gutsholl, M. 5., Owen H.M. Gan, 3.5., Everett D. Thompson, 3.5., Hsion 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 infor— mation on the societal use of mari— juana, in particular, comes from question-response type surveys. Pre— cise quantitatiVe data obtainable via body specimen analysis has been un- available due mainly to the lack of an accurate biological assay tech— nology for the chemical constituents found in cannabis. The marijuana commonly smoked in this country contains four principal con— stituents, viz., A9—tetrahydrocanna- binol (Ag-TEET, cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC) (Doorenbos, gt 21., 1971). One of these constituents, A9-THC is be- lieved to be responsible for the psy— chomimetic properties of marijuana (Edery, g; 31., 1971). Likewise some of the physiological responses in man have been shown to change following smoking of cigarettes containing A - THC and these correlate with plasma levels of A9-THC and its metabolites 96 (Galanter, et a1., 1972). However, such studies—were accomplishable only by administering radiolabeled A9-THC to the study subjects and following its deposition in accessible body fluids. More recently there have been a num- ber of methods proposed for determi- nation of A9—THC in blood plasma. A glc procedure which utilized an elec- tron capture detector was detailed and reported to be capable of detect- ing 0.5 ng per 1 ml of blood plasma (Fenimore, gt a1., 1973). This me— thod which required precolumn deriva- tization and a dual oven apparatus was not used to assay the blood plas— ma of actual cannabis smokers. An- other reported method employed glc- mass fragmentography with d2—A9-THC as the internal standard (Agurell, gt 31., 1973). This later method was employed to analyze 5 m1 samples of blood plasma from three volunteers each of whom smoked a cigarette con- taining 10 mg of Ag-THC. A chromato- graphic elution of the blood plasma on a Sephadex column was required prior to glc-ms analysis. Blood sam- ples were taken from the volunteers at 0, 0.2, 0.5, l and 2 hours follow- ing smoking. Peak levels of Ae-THC were found to be 19-26 ng/ml at 10 minutes following smoking. The lev— els declined to 5 ng/ml or less at 2 hours. Another method which has been reported to be capable of detecting A9—THC is based upon radioimmunoassay procedures (Teal, gt al., 1974 and Gross, gt al., 1974). The method reported herein makes use of a high pressure liquid chromatog- raphy-mass spectrometry (hplc-ms) technique for determining ng/ml quan— tities of A9-THC in human'body speci- mens. Problems associated with di- rect coupling of the hplc to the ms have been circumvented by collecting fractions of the mobile phase eminat- ing 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-A9— THC for controlling extraction effi— ciency, as a marker for collection of the hplc effluent and as a conven- ient internal standard for ms quan- tification. Thus by this method the body specimen has enough d3-A9—THC added to allow its detection by a u.v. spectrophotometer connected to the output of the hplc. Once the d3-A9-THC is detected, fractions of the mobile phase eluant are collect- ed and introduced to the ms via the direct insertion probe for quantifi— cation. This method was then vali- dated in various human body samples over the concentration range of l- 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 A9—THC in eleven male vol— unteers during a 24 hour period fol- lowing smoking of a marijuana cigar— ette. Also the new procedure has been used to determine the presence of AngHC in exhaled breath and sa- liva of volunteers following mari- juana smoking. Similarly, a corre- lation has been made, using the me- thod, between the blood plasma, bile and brain levels of A9-THC in human post—mortem specimens. The method has also been used to analyze human blood samples from a A9-THC aerosol inhalation study on a blind basis. 97 EXPERIMENTAL High pressure liquid chromatography All hplc analyses were conducted on a Varian 8520 gradient elution liq- uid chromatograph utilizing a Varian 635M spectrophotometer set at 273.7 mu as the detector. The column was a 10 u silica gel(Varian Si—10), 25 cm x 2 mm (i.d.). A gradient elu— tion program was developed using hep— tane and methylene chloride. For a satisfactory separation of the canna- binoids 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 gra— dient program. Only those lots of heptane and methylene chloride which gave minimum absorbance in the region of 260-280 mu were used. The gradient elution program, devel— oped 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 95:58 heptane:methylene chloride mixture, thereby regenerat- ing the column. A solvent flow rate of 120 ml/hr was used for all deter- minations. By employing these con— ditions A9—THC as well as d3-A9-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 mari— juana, CBD, CBN and CBC were found to have retention times of 4.4, 4.6 and 5.6 minutes, respectively. The amount of d3—A9-THC added to the body sample was sufficient to allow u.v. detection of A9—THC (labeled plus unlabeled) as it eminated from the column. A 10 cm "zero dead vol— ume" stainless steel tube was at— tached to the flow cell of the spec- trophotometer to facilitate collec- tion 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 reso- lution, double-focus mass spectrome- ter. A new ion-counting technique was developed in conjunction with the peak matching accessory which provided for a rapid comparison be- tween data from the internal stan— dard (d3-A9-THC, mass 317) and the assayed compound (Ag—THC, mass 314). Each sample from the hplc was intro- duced into the ms via the direct in— sertion probe. The instrument was initially focused exactly on the 317 (d3—A9-THC) mass signal, then through the action of the peak matching unit, and with the high resolution capabil- it , alternately focused to the 314 (A —THC) mass signal, cf., Figure 1. Figure 1 Liquid Sample (5 Incl) ii Direct Insertion Probe i Mass Spectrometer W Peak Matching Acces. / \ 317 314 \ I Standard Register A Date Register B Ion Counter V Date Processor —— Plotter 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 SSRlllO dedicated computer. This unit performs a summation of the num- ber of ion events occurring in both mass peaks (314 and 317) and stores these in two registers. Thus a run- ning total of ions detected from the 317 internal standard and the un— known amount of the 314 mass are 98 Figure 2 Ion Counting Cycle Counting Period Counting Period IRA= 0.067 sec. Ml. = 317 AMU 'RB' 0.067 sec. M/e = 314 AMU Fifty Cycles 50 50 z Ion Counts (317) 2 Ion Counts (31‘) ‘RA = 1 me - 1 T = 50 IRA + 50 ‘RB =6] uc. l One Dita Point 1 10 x T - 67 no. 500 Counting Cvdu 1,000 Counting Periods L Ten Data Point: 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 ex- perimentation 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 hits of data for comparison and quantification. With time the sample is depleted but the ratio based on the internal stan- dard remains linear and provides for dependable quantification as shown in Figure 3. The curve shown in this figure was obtained from a Hewlett— Packard 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 A9-THC (mass 314) is determined by multiply- ing this ratio times the known amount of internal standard added. In actual practice the d3—A9-THC em— ployed contained a small amount of undeutrated A9-THC (mass 314). A ms determination of this amount of mass 314 was made by analyzing 10 samples of 1.6 ug d3-A —THC which had been added to 1 m1 of blood plasma by the Figure 3 2.5 I I <— Slope=%314=0.76 3 M a i E 2 o o I I 05 Typical data summation ova: 67 g seconds for humn plasma containing g 1.0 ng of A9—THc and 1.6 ug o! 3 d3—139—THc § L5“ 1.00 I I : l to L5 19 25 an as Ion Counts x105 for Mass 317 Table 1 Table 1. Precision and Accuracy in Recovery of A9-THC Added to Human Plasma. Added Found,a ng/ml ng/ml n iRSD RE 0 11.3 (9.35-13.6)b 10 11.15 —- l 1.2 (0.6-1.5) 3 43.3 20.0 5 4.5 (4.0-4.7) 3 9.0 -11.12 10 10.5 (9.5-12.4) 10 10.0 5.0 20 20.0 (19.1-21.1) 3 5.14 0.0 40 41.2 (39.6-42.5) 3 3.58 3.0 60 62.1 (61.3-62.8) 3 1.26 3.5 80 82.8 (74.9-90.7) 3 9.55 3.5 100 103.7 (99.3—106.0) 3 3.65 3.7 Average percent recovered 103.8 a Average (range) of n determinations. This value represents the amount of unlabeled A9-THC in 1.6 ug of d3—A9-THC. Other found values have been corrected for this amount. method described above, cf. Table I. This background value for A9—THC (mass 314), inherently present in the internal standard, is subtracted 99 from the total amount of A9-THC re- corded to give the actual found value. Figure 4 120 100 nq o! A 9-1’HC Iound 40 Mass swam (humiliation av Ag-THC addod «7 human plun- 20 40 50 60 ng o! A 9—THC add-d to 1 ml plasma Methods of blood plasma analysis All glassware used was silinized us— ing a reported method (Garrett and Hunt, 1974). Whole blood was cen- trifuged at 2600 rpm for 20 minutes to obtain blood plasma. To 1 ml of blood plasma was added 1.6 ug of d3- A9-THC followed by three repetitive extractions with 2 m1 of petroleum ether for each extraction. The ex- tracts were combined and evaporated to dryness under nitrogen at room temperature. The resultant residue was reconstituted in 300 pl of hep— tane and the entire solution injected into the hplc. A 100 ul wash of hep- tane was used on the vessel which contained the extracts and it was al- so injected into the hplc and the gradient elution program begun as described above. When the peak for A9—THC was noted on the recorder, the eluant was collected in a silinized screw cap vial. Collection of the eluant was synchronized with the re— corder tracing such that equal amounts were obtained from either side of the symmetrical peak. In general, approximately 1 m1 of eluant was col- lected for each Ag-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 pl 100 volume. A microsyringe was used to transfer this solution in two por- tions to a 5 ul gold cup. The solu- tion 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 consti— tuted the control samples. Each sam- ple contained some background amount of A9—THC (mass 314) since the 1.6 pg of d3-A9-THC, added to each plasma sample, contained an average of 11.3 ng of A9—THC (mass 314), cf. Table I. To demonstrate the reproduci— bility and accuracy of the devel— oped method, pooled human plasma samples were analyzed to which known amounts of Ag—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 A9—THC (mass 314) is present in the d3-A9-THC used as the internal standard. Methods of breath analysis For human breath collection a stan- dard face mask (Welch model 7500— 30G) was modified by placing a 2.5 cm diameter port directly in front of the mouth. 0n the interior of the mask was placed a 4.5 cm (o.d.) by 2.0 cm thick rubber ring of suf— ficent flexibility to firmly hold a specially molded polyethylene fil- ter. 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 dis- posable examination gloves. After breath collection, each sample was placed in 10 ml of methanol in a silinized beaker and then ultra- sonicated for 30 minutes. To each extract was added 1.6 ug d3-A9-THC and the solution was evaporated to dryness at room temperature, under nitrogen. The residue was recon- stituted in 300 ul of heptane and analyzed via hplc-ms as described above. Figure 5 ii Flapper Valve (allows air out only) Filtered Air Foam 0/ /'\ // Air In Method of saliva analysis Saliva was collected from each vol- unteer in silinized collection tubes and immediately frozen until analy- sis. To 100 pl of saliva was added 1.6 ug of d3-A9—THC followed by ex- traction 3 times with 2 ml of petro- leum ether for each extraction. The extracts were combined and evapor- ated to dryness under nitrogen at room temperature. The residue was reconstituted in 300 pl of heptane and analyzed by hplc-ms as discussed above. Method of bile analysis Bile taken at post—mortem examina— tion was frozen until analysis. To 100 pl of the bile was added 1.6 pg of d3—A9-THC and 1 ml of pH 7.0 buf— fer. 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 pl of heptane and analyzed by hplc— ms as discussed above. Method of brain analysis Brain samples from the cerebrum ta— ken 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 ug of d3—A9-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 tem— perature. Marijuana smoking studies Eleven healthy male volunteers be- tween 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 medi- cal 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: electrocardiogramrchest x-ray, creatinine, BUN, LDH, SGOT, alka— line phosphatase, blood sugar, cal— cium, phosphorus, bilirubin, total protein, albumin, cholesterol, uric acid, hematocrit, hemoglobin, platelet count and prothrombin time. 102 All subjects were moderate marijua- na smokers. Each was requested not to smoke marijuana for 2 days prior to reporting for the study. The subjects were brought into the hos— pital 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 fore— arm vein and 5 ml of blood was with— drawn and placed in a silinized hep- arin 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 sam- ple. Each subject was then allowed to smoke one marijuana ci arette' which contained 10.8 mg A -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, l, 2, 3, 4, 12 and 24 hours. Each blood sample was handled as des— cribed above. At the same time in— tervals saliva and breath samples were taken from each subject. A9—THC aerosol and oral administra- tion studies Two normal male volunteers, who met the same physical criteria listed for the marijuana smoking volunteers, were used. Each subject was admin- istered an aerosol spray which con— tained 10 mg of A9—THC. -Blood sam- ples were taken from each subject at 0, 0.25, 0.5, l, 1.5, 2.0, 3.0, 4.0, 5.0 and 6.0 hours. Samples were sup- plied 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 ana— lyzed 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 A9—THC since prior to the present work there were not many analytical methods which offered the selectivity and sensi— tivity 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 A —THC to be selectively separated from the other cannabinoids present in mari- juana preventing an erroneous anal- ysis with the other mass 314 canna— binoids, Viz., CBD and CBC. Also when the m§_analysis is performed the high resolution capability of the ms prevents any possible confu- sion of compounds, i.e., only A9—THC will have the correct mass number. Use of the stable isotopic'form of A9-THC in the developed method allows for a control of extraction efficien— cy as well as a marker for peak col— lection and quantification. Thus the d3-A9-THC was added to the plasma prior to extraction and gave a con— trol for any losses which might oc— cur during extraction or chromato- graphic 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 multi— ple extractions is to introduce to the ms a sufficient amount of the stable isotope for accurate quanti— fication while not using excessive amounts of the isotope for the inter— nal standard. The other important use of the d3-A9—THC was to permit the total amount of A9—THC (both la— bled and unlabled) to be detectable by the u.v. spectrophotometer at- tached to the output of the hplc since A9—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 A -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 A9- THC in their control samples. This is readily explainable since all sub— jects 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 quantifi- cation 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 A9-THC (ng/ml) found in human blood plasma following marijuana smoking. TIME (hrs.) SUBJECTS 0 .25 .5 2 3 4 12 24 D.L. 3.6 23.0 10.1 12.2 7.1 0.9 3.5 2.7 0 B.N. 1.2 39.0 11.7 3.8 0.9 O 0.7 0 1.7 B.B. 1.6 34.1 23.3 16.8 1.4 1.1 0 2.0 0.9 D.J. 0 66.7 19.7 11.8 3.0 3.8 4.3 0 3.1 F.R. 4.2 38.4 20.1 6.8 8.4 2.8 6.3 0.6 0.9 B.W. 0.4 57.9 24.8 14.8 0.7 2.4 6.2 5.8 2.5 W.Z. 1.2 39.7 19.7 6.8 5.3 4.9 2.0 0 0 R.B. 5.7 43.7 18.9 27.6 6.7 4.4 2.5 0.1 3.4 L.G. 7.6 15.3 39.9 8.1 2.1 11.1 7.5 9.7 2.5 T.V. 11.3 34.6 16.5 10.1‘ 5.1 4.2 2.3 1.6 2.8 K.C. 4.7 20.6 22.4 14.5 8.4 7.7 4.7 5.0 2.5 MEAN 3.8 37.5 20.7 12.1 5.2 3.9 3.6 2.5 1.9 Table 3 Table 3. Blood Plasma Levels of A9-THC a Time (mi?) - 9 Code No. Follow1ng A —THC ng of A —THC SUBJECT 1 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 SUBJECT 2 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 10stb ORAL 3 09 0.1 2322220221” 4 5 300g lostb 6 300e o 0 7 300e 0.0 8 of 0 4 9 0g 0.0 10 0f 4.0 a Code number of sample as supplied by Dr. Tashkin. b Laboratory accident resulting in sample loss. 2 Sample broken in transit. All subjects were confined in the hospital ward for 4 days prior to receiving the oral formulation. Receiving oral CBN. Receiving oral CBD. Receiving oral A9-THC. 104 LOO-hm each subject may have varied. More important, however, was the fact that a positive and statistically meaning— ful level of A9-THC could be deter- mined 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 at— tempt was made to accurately control the amount of saliva exudated. Thus any attempt to quantitatively asSign levels of A9-THC per unit volume would be meaningless. However, it was observed by analyzing the saliva samples that a positive, statistic- ally significant level of A9-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 Ag—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 A9-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 con— fined for 4 days prior to the col- lection of their control samples. Also as shown in Table III, other cannabanoids were being administered which could have conceivably inter- fered with the A9-THC determination. However, the hplc pro ram success- fully separated the A -THC fraction as was verified by correlation fol- lowing the blind sample assays. For 105 both studies there was a good corre- lation between the physiological states recorded following administra— tion and the Ag—THC levels measured later by means of the quantification technology reported in this paper (Tashkin, 1976). CONCLUSIONS A method was developed for quantita- tively determining A9-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 psaliva of human subjects following smoking of a marijuana cigarette. Results from these studies demonstra- ted that during the early time inter— vals following marijuana smoking the levels of A9—THC measured are of suf— ficient magnitude to be clearly dis- cernable from the amount of A9-THC (i.e., 11.3 ng) added in the internal standard. The newly developed method has also been used to study the relationship between Ag-THC levels in blood plas- ma, bile and brain of post-mortem samples. Such data has revealed that Ag-THC levels are higher in brain and bile than in blood plasma. Data was also accumulated on blood plasma samples which came from sub- jects receiving either an aerosol formulation of A9-THC or oral formu— lations of CBD, CBN and A9—THC. Analysis of these samples on a blind basis provided further validation of the new technology and gave results which were consistant with the ob— served physiological responses. 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 per- formed there. This work was performed under con- tract DOT HS 4 00968 from the Depart- ment 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., Holm- stedt, B., Leander, K., Lindgren, J., Nilsson, I., Sandberg, F., and Asberg, M.: J. Pharm. Pharmacol., 2_5,554 (1973)? Doorenbos, N.J., Fetterman, P.S. I Quimby, M.W. and Turner, C.E.: E-X- Acad. Sci., 191,3 (1971). Edery, H., Grunfeld, Y., Ben—Zvi, Z., and Mechoulam, 191,40 (1971). R.: §.!. Acad. Sci., Fenimore, D.C., Freeman, R.R., and Loy, P.R.: Anal. Chem., 42,2331 (1973). 106 Galanter, M., Wyatt, R.J., Lemberger, L., Weingartner, H., Vaughan, T.B. and Roth, W.T.: §gi., 176,934 (1972). — Garrett, E.R. and Hunt, C.A.: Pharm. Sci., £3,1056 (1974). J. 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. ANALYTICAL METHODS FOR THE DETERMINATION OF CANNABINOIDS IN BIOLOGICAL MEDIA M.E. Wall, Ph.D., 1'. M. Harvey, Ph.D., J. 'l'. Bursoy, Ph.D., D. R. Brine, 3.5., D. Rosonthal, Ph.D. Research Triangle Institute. North Carolina ABSTRACT A pharmacokinetic Study of the blood plasma levels in mag of A ~tetrahydrocannabinol, ll-hydroxy-A -tetrahydrocannabinol and canna- binol has been carried out by means of com- bined 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 chro- matography. 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 non- labeled drug present in plasma. Blood samples were obtained at periodic intervals up to 24 hours from volunteers receiving 4-5 mg Ag-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 chro- matography could be omitted. In all cases calibration curves were obtained from repli- cate analyses of spiked plasma containing L07 the internal standard and various quantities of the cannabinoid under analysis. A typical biphasic elimination of the drug was observed with rapid elimination of A9-THC from the blood over a period of 40 min followed by a much slower elimination up to 24 hours. 9The experimental data show that ll—hydroxy-A - THC is found in the plasma in quantities only of about one-twentieth go one-twenty- fifth the values found for A —THC Cannabinol was not found in significant quantities. Good agreement was obtained between the mass _ spectral analyses and the thin layer chroma- tography or electron capture gas-liquid chromatographic procedures. INTRODUCTION In recent years there has been a great in- crease in interest in the pharmacology, metabolism and biodisposition of the cannabi- noids [for recent reviews cf. Mechoulam, Paton and Crown, Wall (1975) and Wall, et al. (1975)} Until recently quantitatiEn—Bf the various cannabinoids in blood, urine, feces and other biological tissues could be carried out only by means of the use of appropriately radiolabeled analogs of the cannabinoids under study (Wall, et a1. (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 canna- binoids are not used, the need for the devel- opment of non-radiolabeled quantitative methodology for certain key cannabinoids has become increasingly apparent. In addition, the use of radiolabeled thin layer chromatog- raphy techniques, while useful in initial studies, lacks sufficient accuracy in the sense that when biological extracts are studied, separation of A —THC from cannabinol and ll-hydroxy-Ag-THC from other monohydroxy— lated analogs is poor. If such interfering substances are present in considerable quan- tity, one would obtain erroneously high values. This will increasingly be the case when one is analyzing biological materials obtained from marihuana smokers which contain A9-THC, cannabinol, cannabidiol, and 11- hydroxylated analogs of these compounds. Quantitative gas liquid chromatography com- bined 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 separa- tive powers of glc and the inherent sensi- tivity 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 con- cept has been applied to many drugs and re- cently by Agurell, Holmstedt and co-workers to the determination of A9-THC in blood plasma. We wish to present in this paper methods for the quan itative determination by combined glc-ms of A -tetrahydrocannabinol (Ag-THC), ll—hydroxy-Ag-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 A9—THC by the intravenous infusion method of Perez-Reyes gt a}: The volunteers were kept under medical supervision for 24 hours in the Clinical Research Unit of the Univer- sity 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 centri- fugation, 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 cannbinoids under study as both car- riers 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 cannabi- noids and their deuterated analogs used in these studies are shown in Figure 1. All of Figure 1 0111 5. o nzcuzcnzcuzcxz 9 1 A— ~ . . . a THC. n 112 HJ. R1 u z 9 1 1 - - _ . . . . 1 n] A THC, x u}, R! a. :12 H3 9 A .1“ . . . . c C PFP. R R2 H3, R1 OCCFZCF :r 3 \ 2- 11-nydroxy-A9—rnc; n - uzou, x1 - n, n2 - n3 _ _ '-2 - 9— ~ - - - 2 b 11 Hydroxy 5 H; A mc, R H2011, R1 )1. x2 M3 CH3 cu3 0 CH CH CH CH CR 22221 CHCHCFCHCH ZI'ZZ] 3. CEN: R - H, R1 - H3 2 , _ _ 2 b 5'- 113—63". R H, R1 H c CEN-FFP; R - OCCFZCF 3 3 «a llexahydro-CEN; R - H b )Iexahydro-CBN»PFP; R - uncut"J 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. Synthetic methods for the various deuterated cannabi- noids utilized in these studies have been presented by Pitt, §£_§l. 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 quan- titative data. In general in working with cannabinoids exposure of samples or extracts 1 0‘8 to light or air should be minimized. All solvent evaporations should be conducted '2 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% [MCS in toluene. Extraction and Purification Prior to Analysis by GLC-MS in Ei Mode.—-When the mass spectro- meters were operated in this mode the molecu- lar ions or charged fragments utilized for the quantitative analysis of underivatized canna- binoids 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 carry- ing out a preliminary cleanup by Sephadex LH- 20 chromatography prior to the glc-ms step. The methods which are pr sented are for the combined determination of A —THC (19‘), ll-hydroxy—Ag-THC (21), and cannabinol (3a). The methods, of course, are equally utifizable for the determination of individual consti— tuents. Deuterated internal standards (cf. , Figure l) were added to a sample of 3.0 ml of cold (not frozen) plasma as follows: £33 150.0 ng; 2b, 15.0 ng; and 3b, 15.0 ng. Each internal standard was added—in 15-30 111 ethanol. Following addition of each internal standard the plasma sample was st1rred for 3-5 seconds in a vortex agitator and then subjected to sonication (Cole-Farmer ultra- sonic cleaner) for the same time. The plasma samples (contained in a screw capped centrifuge tube) were then extracted 4 times with 6.0 m1 petroleum ether Cbp 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, evapo- rated in vacuo at room temperature and freeze dried overnifit to remove water and isoamyl alcohol. The dried residue was dissolved in a minimal volume of petroleum ether/chloro- form/ethanol (10:10:1) and chromatographed on 1 x 40 cm water jacketed Sephadex LH—20 columns at 26°C in the same solvent mixture. 'IWenty—seven m1 of column eluant were col- lected and discarded. Seven ml of eluant were then collected as the fraction contain— ing A9-THC. The next 8 ml of eluant were collected as the CBN-containing fraction. Thirty-eightml of column effluent were then collected and discarded. Finally, 17 ml of eluant were collecteg as the frac ‘on con— taining ll-hydroxy—A -THC. The A -THC and CBN fractions were evaporated to dryness and dissolved in 35 ul hexane. The ll—hydroxy— A9-THC fraction was evaporated to dryness 109 under vacuum and heated with 75 ul of Regisil (BSTFA + 1% 1048) in a closed vial at 110° for 3 boars. The reagent was removed in vacuum and tl'e residue dissolved in 20 ul hexane. Extraction Prior to Analysis by GC-MS in CI Moder—In this mode of operation A9-THC and CBN were determined as their pentafluoropro- pionate ester derivatives, Fig. 1, E and 3_c, respectively, using the PFP ester of hexahydrocannabinol, g as tl’e internal standard for both analyses. It was found that endogereus plasma constituents dwjdfl interfere at the molecular ion positions util— ized which were in the range of m/e 455-465. - Hence preliminary purification of the extracts was, not required. To a sample of 2.0-3.0 muofplasmawereaddedtheinternal standard hacahydrocannabinol g, 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 072ml hexane, 50 ul pyridine/benzene (5:95), and 50 pl 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 flen backwashed with an additional 0.2 ml hexane. The hexane artracts were combined and evapo- rated under nitrogen. The derivatized extracts were dissolved in 20 pl hexane. Gas chrcmatography conditions I.KB-GI.C-l~B.—-With this instrument we utilized 2% OV-17 on Chrcmosorb W—HP, 80-100 mesh in 3' or 6' x l/4" glass columns, temp. 2 0°C, the former being used for ll-hydroxy-A -THC- bis-‘IMS ether; the latter for both [8ng 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 reten- tion times of 4-6 minutes were observed for each cmpound. Fimligan-GLC-NS.——mly A9-'I'HC and CBN were analyzed on this instrument in the ei mode. We used 6' glass columns containing 1% SE—30 on 100/120 mesh Chrcmosorb W—HP, column tanperatures 2002-230°; He flow 30—35 ml/min. In the ci mode, which was used only with pentafluoropropionate derivatives, tie same column was used at 180°. Mass spectrometry mly basic operating details are presented in regard to the ms-canpiter systens described below. For a much more detailed report cf. Ibsenthal g a_1_. LKB 9000 GLC-NS.--'Ihis instth is of the magnetic sector type and was operated at 70 evintheeimode. The instrumentwas fitted with a peak matching accessory, modified fran an instrument described by Klein, which per- mits detection of very low levels of compounds by the technique of accelerating voltage alternation (AVA) (Hammer, 1968 and Agurell , 1973). For A9-THC the mass spectrum was set to focus alternately on the ions l'_l_L/_e_ 314 and 317 which correspond to the molecular ion of the canpound and its trideuterio analog. For CBN the molecular ions were m/g 310 and 313. For analysis of 11-hydmcy—Ag-Jmc as the bis- tns-ether, the strong M—lo3 ion (Wall, et a1., 1970, Wall, $31., 1976) at 11/53 371 E 374 respectively was selected. The AVA accessory used with the mass spectrcmeter scans through each peak in turn; a hardware counter determines the area under the peaks eachtimetheyarescanned. Theratioofthe selected response (either area or peak height) for the mo ions in question is the value used to determine the quantity of unknown cannabinoid in the plasma. Appropriate cali- bration curves (Fig. 2, 3) are constructed by adding variable anounts of cannabinoid to a fixed quantity of corresponding trideuterio analog in human plasna. Extraction, prelimi- nary purifcation, and glc are carried out as described above, Analysis by GEE-IVS-EI. Finnigan 3000-GlC-NS.--‘I'his instrument is a quadrupole mass spectraneter and was operated at70evintheein‘ode. Itsscanisoontrolled by a dedicated PDP-12 oanputer 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 Holecular or fragment ions described in the previous section. In the ci mode isobutane was used as the car- rier gas at a pressure of 400 u; A9—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 Illlll III}! III} I III NG A"THC/|'l PLflSm 1 1 Figure 2. 3 LICB 9000-e1 Plasma Calibration Curve for Ag-JTHc \ Figure 3 V, o, D » smu- Run an Btikunl Du)- Jill] I III] I III] II HREH RflTIO NHTURHL/DEUTERHTED , ..1 1 1 i 1' z 10 10 lo 10' N0 NflTURFIL THC flDDED/SGNG DEUTERHTED me Ill . VI 1 C Figure 3. Finnigan 3300-ei Plasma Calibration Curve for Ag-THC 110 TLC-radiolabel procedure The volunteer subjects (described in Clinical protocol) all received 100 uCi of tritium labeled A9-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, §t_al, (1975). Electron capture—glc procedure In this procedure Ag-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 instru- mentation utilized was constructed at RTI by Dr. Edo Pellizzari as a modification of the design reported by Fenimore, §t_§l: 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 detectoris sensitivity to column bleed. In the reported dual CEIUmn-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 pre- column without disturbing the base-line re- sponse of the ec detector. The instrument which we have assembled in our laboratory consists of two separately con- trolled ovens CVarian 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 con- sists 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-ZOO capillary tubing. A water supply at 4°C is provided through the 1/2 in tubing for cooling purposes. ' Figure 4 Coolant N: capillary trap l T Carrier gas ‘ (B column) GLC Column 3 \\\\\%\&\§ >\\\ ‘ / / ///////l////// \ w / x . / cu: A—a \\ Z/ / //.—GLC B \\\\ . L__ /// / / GLC column A ;:\\ : k~-——- VALVE ECD c_.__, / \\ 1.4 // Amffffier A }/ B \\\ I/// / I:/ I___.::: n d Recorder \ . \ \ \ // l’ I /// / :E Recorder A 3,1,, \ FID // / 3:“ / / 5:; B \ aw \ Zry/M // //' i Variable restrictor Figure 4. Dual Gas Chromatographic Systan lll 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 tem- perature 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 capillar- ies. A linear ec amplifier (Hewlett Packard, Avondale, Pennsylvania) and a miniature ecd developed in our laboratories (Pellizzari, 1974) are employed in this dual gas chromato- graphed system. Because of the low nanogram levels of cannabi- noids found in human plasma, it was decided to use the electron capture detector to allow maximum sensitivity in the cannabinoid analy- sis. 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 sepa- rated on a ten-foot column of 2% SE30 on Supel- coport (80/100 mesh) at 205°C with a carrier gas (N ) flow rate of 15 ml/min. Column effluefit was collected at the retention times of HHC and A9-THC and transferred for quantita- tive analysis to a six-foot column of 2% OVZZS on Supelcoport (100/120 mesh) at 178°C with ‘a carrier gas (5% methane - 95% argon) flow rate of 9.5 ml/min. RESULTS Plasma calibration curves for Ag-THC obtained with the IKE and Finnigan mass spectrtueters in the ei node are shown in Figures 2 and 3; the calibration curve obtained with the Finnigan instrument in the ci nude is shown in Figure 5. In each case a linear calibra— tion curve was obtained in range 1-100 nano- grams A9-THC/ml plasma. Detection of A9-THC below the lower limit could be obtained (de— tection limits 0.1). 0.5 Ngfinl of plasma is regarded as the minimal concentration at Figure 5 I III; I III] I III I III l 0 NB A‘-THC/'t PLfiSffl Figure 5. ~Finnigan 3300-ci Plasma Calibration Curve for Ag-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. Fbr the Finnigan in ei mode, the crngarable values were 0.034 and 0.9994, re- spectively. In the ci mode of the Finnigan mass spectrrnemer 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-e1. 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 esti- mation for the LKB and Finnigan instruments was respectively 0.05 and 0.078; the corre- lation coefficients in each case were 0.998. Preliminary data for CBN determined in the ci node as the PFP ester indicate that similar results are to be expected. The plasma calibration curve for ll—hydroxy—A9JTHC—bis- uns\O a " <\ \ i s- I? 2 III 41 2‘ E 3 E LO E as» 3 cs /'\ as 8 0.. a: (12. A x’ A A no 006 40 no 2 so» 40 so 0 so so ’To‘cT’ Minum Figure 9. Plasma Levels of Ag-THC, 11-Hydroxy-A9-THC and CBN Found Over a 24-Hour Period in Human Plasma from Volunteers Receiving A9—THC by IV Administration Figure 9 presents the average values with standard error obtained for A9-THC, ll-hydroxy— A9-THC and cannabinol from plasma of male volunteers receiving A9-THC by intravenous infusion. The measuranents covered a 24 hour period. A9-THC values obtained with the LKB- 9000 ei source were in close agreement with the data obtained on the Finnigan 3300-ci source. other data not presented here also show close agreement between the LKB and Finnigan instruments when both were in the ei mode. A9—THC values increased rapidly during the first 10—20 minutes, the peak values in the range of 50-60 ng/ml coinciding with the maxi- mal psychcmimetic activity. A typical biphasic elimination pattern was noted; the A9—'I‘HC plasna levels decreased rapidly between 15-40 minutes and then fell at a much slower rate. With a particular group of volunteers (3 sub— jects) levels after 24 hours were between 3-5 ng/ml. Spot checks at lower levels utilizing the Finnigan MID program confirmed tha the substance being evaluated was indeed A -THC and not instrument "noise". Figure-10 compares the results obtained frcm the average of four subjects, analyzed by ms— ei 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 agreenent for most points over the whole curve. In the case of ll-hydrwty—A9—IHC 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 A9-THC, falling to a level of 1.0 ng/ml in 60—90 minutes and 0.5 11ng after 24 hours. The values for CBN are con- sidered unreliable as obtained by the ei technique since for the most mrt the levels were well below 1.0 ng/ml and show no consis- tent pharnecokinetic pattern. DISCUSSICN The basic objective of this investigation was to establish sensitive methodology, which would not depend on radiolabsling for the quantitative estimation of A —'IHC, its primary metabolite (Wall, 1970), ll-hydroxy—Ag—THC and cannabinol, whighhasbeenreportedtobea metabolite of A -'mC in the rat (McCallum, 1975) . This objective has been realized, utilizing glc-ms with a variety of techniques and instmments. 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. 114 Figure 10 so. 7 \ 0—0 m goon, AVA, 2x ‘ D~---D 11: Analyst- § v—-—V nlc Analysil 60- run- n; A ant/m minute- Figure 10. A9-THC Found in the Plasma Following Intra- venous Administration of 5 mg A9-THC, Average of 4 Subjects Figure 11 m, Jinn (mi pkaem.‘ so mu nl A94“ (m1 whom: rFigure 11. Least-Squares Best Lines Comparing All Data Obtained by Bach No Methods of Analysis Aes- . *1? ’ 5.1"" e Choice of instrument TWo crnpletely different types of mass spec- traneters 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 pennitted mea3urenent of the ratio of the peak area of the unknown as canpared with that of the internal standard. The other was a relatively new (1974) quadru— pole ms, the Finnigan 3300 which was interfaced to a PDP-12 Ctnputer. 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 iden- tical linear range and quite similar stand error of estimation. Figure 9 gives pharmaco- kinetic 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 appro- priate 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 uti— lized for all of the ei studies. It is possi- ble 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 fog this purpose and was used in ci studies of A -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 115 mode. In our studies we have found no par- ticular 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 A -THC and cannabinol in the region of m/e 310-320. This endogenous inter- ference cafindt be removed by glc techniques alone but requires a preliminary "clean up", . utilizing Sephadex LH-20 chromatography. This method which was described by Agurell §t_al. for A -THC and extended by us to mixtures of A9-THC, ll-hydroxy-Ag-THC and CBN, works well for the former two compounds but is time consuming. In addition, there is danger of some conversion of A9-THC to CBN because of the longer exposure period. All of these problems are avoided when the cannabinoids are converted to the corresponding pentafluoropro- pionate esters. The molecular ions are then in the region of m/e 455-465 and no interfer- ence was found after extraction of plasma with hexane, derivatization and glc-ms determina- tion The method at present has been applied to Ag—THC and CBN which can be extracted with non-polar solvents. Whether more polar canna- binoids which require more polar solvents can be successfully analyzed without prior purifi- cation by the ci technique is an open question at this time. Metabolic and phannacokinetic data The development of sensitive and accurate glc- ms methodology permitted a preliminary study in man utilizing these techniques for the precise determination of A9-THC, ll-hydroxy- A9-THC and CBN in plasma. Previously we have made an extensive stud (Wall, gt al,, 1975) of the metabolism of A -THC in man using radiolabeled tracers and thin layer chromatog- raphy. 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 A9-THC from CBN, and in the case of ll-hydroxy-Ag-THC, would not permit separation from.other monohydroxy-metabolites which might be present (Wall, et al., 1973; Wall, et a1. 1975). The data in Figure 9 for A9TTHC—are quite comparable to pharmaco— kinetic data obtained in earlier studies (Wall, et al., 1975). In both instances a biphasiE—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 5 -60 ng/ml. After 24 hours, 3-5 ng/ml of A -THC were still found in the plasma. Our results for ll-hydroxy-Ag-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 A - Tl-KI to 0.5 ng/ml after 24 hours. Although A -THC is readily converted to ll-hydroxy—Ag- THC in the liver (Wall, et al., 1975) only small quantities find theirTiay into the blood. Our interest in CBN was aroused by reports from McCallum (1975) and McCallum, et fl. (1975) which indicate that CBN might—be a transitory metabolite found at very eagly time periods after administration of A -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 sane general plemecokinetic pattern as A9-THC in man (Wall, 1975) , we must con- ACKNOWLEDGMENTS These studies were conducted with the support of the National Institute on Drug Abuse under contracts HSM-41—71-95 and ADM- 45—74-109. We wish to thank Mario Perez— Reyes, 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 assis- tance. REFERENCES Agurell, S., Gustafsson, B., Holmstedt, B., Leander, K., Lindgren, J.-E., Nilsson, 1., Sandberg, F. and Asberg, M. Quantitation of A9-tetrahydrocannabinol in plasma from cannabis smokers. J. Pha/un. Pha/lmac” 1973, 25: 554-558. Fenimore, D. C., Free , R. R. and Loy, P. R. Determination of A -tetrahydrocannabinol in blood by electron capture gas chromatog- raphy. Anal. Chem, 1973, 45: 2331-2335. Hammar, C.—G. and Holmstedt, B. Mass fragmen— tography identification of chlorpromazine and its metabolites in human blood by a new method. Amt. Mathew, 1968, 22: 532- 548. clude that CBN can be disregarded in terms of its importance as a metabolite in man. Omparison of gc—ms with other procedures As shown in Figureleand 11, the gc-ms pro—- cedures show reasonable agreenent in the case of A9-’I'HC with data obtained by two indepen- dent procedures, involving respectively thin layer chrcmatography of radiolabeled cannabi- noids, and a double glc-electron capture procedure- In preliminary studies on canna- binol levels of subjects who received A9—THC, good agreerent was famd between the mass spectrcnetric glc-Ci nethod and electron cap— ture glc. In each case no cannabiml could be found. Studies on ll-hydrmty—Ag-THC levels by the glc-electron capture method are in progress. Initial attempts to use high pressure liquid chromatography as another method indicate that the sensitivity by this procedure with current detectors is of the order 10 ng/ml. Hence at present hplc techniques do not have requisite sensitivity. FOOTNOTE :LResearch Triangle Institute Contract HSM- 42-71—95. Appropriately qualified investi- gators 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. Klein, P. D., Hauman, J. R. and Eisler, W. J. Gas chromatograph-mass spectrometer-accel- erating voltage alternator system for the measurement of stable isotope ratios in organic molecules. Anal. Chem, 1972, 44: 410—493. Lemberger, L. The metabolism of the tetra- hydrocannabinoids. In S. Garratini, F. Hawking, A. Golden and I. Kopin (Eds.), Advances in Phalunacoflogy and Chmothe/Lapg. Academic Press, New York, NY. 1972: 221- 251. McCallum, N. K. The effect of cannabinol on Al-tetrahydrocamabinol clearance from the blood. Expat/Lama, 1975, 31: 957—958. em ‘ ..~ .y me.- McCallum, N. K., Yagen, B., Levy, S. and Mechoulam, R. Cannabipol: a gapidly formed metabolite of A - and A -tetra— hydrocannabinol. Expedientéa, 1975, 31: 520-521. Mechoulam, R. (Ed.), Ma/ulhuana. Academic Press, New York, NY, 1973: 1-409. Paton, W. D. and Crown, J. Cannabié and £12 denivaxivea. Oxford University Press, London, England. 1972: 1-198. Pellizzari, E. D. High resolution electron capture gas-liquid chromatography. .J. Chaomatog., 1974, 92: 299-308. Perez-Reyes, M., Timmons, M., Lipton, M., Davis, K. and wa l, M. Intravenous injec- tion in man of A -tetrahydrocannabinol and ll-OH-A -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. Labez. 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 spectrometEy methods for the determination of A -tetra- hydrocannabinol in plasma. Biomed. MaAA Spec., in preparation. 117 wall, M. E. Recent advances in the chemistry and metabolism of the cannabinoids. In V. C. Runeckles (Ed.), Recent AdvanceA in Phytochemibtay. Plenum Publishing Co., New York, NY, 1975: 29-61. Wall, M. E. and Brine, D. R. Application of mass spectrometry to structure of metabo- lites of A -tetrahydrocannabinol. Sum- maries, International Symposium on Mass Spectrometry in Biochemistry and Medicine. Milan, Italy, 1973, p. 52. Wall, M. B., Brine, D. R. and Perez—Reyes, M. Identification of cannabinoids and metab- olites in biological materials by combined gas-liquid chromatography-mass spectrom- etry. In G. G. Nahas (Ed.), Maaihuana: Chewing, Biochenulbay, and CW E66ect4. 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. Braude and S. Szara (Eds.), Phanmacalogy 06 Maaihuana. 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 biolggical activity of several metabolites of A - tetrahydrocannabinol. J. Am. Chem. Soc., 1970, 92; 3466-3468. LIST OF CONTRIBUTORS Quantitation of Cannabinoids in Biological Fluids by Radioimmunoassay Arleen R. Chase, Paul R. Kelley, Alison Taunton-Rigby Collaborative Research, Inc. 1365 Main Street waltham, Mass. 02154 Reese T. Jones Langley Porter Neuropsychiatric Institute University of California San Francisco, Ca1. 94143 Theresa Harwood Drug Enforcement Administration 1405 Eye Street, N.W. washington, D.C. 20537 Separate Radioimmune Measurements of Body Fluid A9~THC and 11-nor-9-Carboxy-A9-THC. Stanley J. Gross, M.D. and James R. Scares, Ph.D. Department of Anatomy, School of Medicine University of California, Los Angeles Los Angeles, Ca1. 90024 Radioimmunoassay of A9-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 Triang1e Park, North Carolina 27709 Determination of THC and its Metabolites by EMBTbebmogeneous Enzyme Inmunoassay: 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, Ca1. 94304 Separation and Sensitive Analysis of Tetrahydrocannabinol in Biological Fluids by BPLC 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 Ag-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 s '. mans...» “in. Detection and Quantification of Tetrahydrocannabinol in load Plasma Agneta Ohlssonl, Jan-Erik Lindgren2 3, Kurt Leander , Stig Agurell1 3 Faculty of Pharmacy, University of Uppsala BMC, Box 579, 8-751 23 Uppsala 2Department of Toxicology Karolinska Institutet, S-104 01 Stockholm 60 3Astra Lakemedel AB, 8—151-85 SSdertilje, Sweden A Method for the Identification of Acid Metabolites of Tetrahydrocannabinol (THC) by Mass Fragmentography 2 3 Marianne Nordqvistl, Jan-Erik Lindgren , Stig Agurell1 3 Faculty of Pharmacy, University of Uppsala BMC, Box 579, 8-171 23 Uppsala Department of Toxicology Karolinska Institutet, S-104 01 Stockholm 60 3Astra Lakemedel AB, 53151-85 deertilje, 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 Ag-Tetrahydrocannabinol in Body Fluids by Gas Chromatography/Chemical Ionization— Mhss Spectrometry Ruthanne Detrick and Rodger L. Foltz Battelle Memorial Institute 505 King Avenue Columbus, Ohio 43201 HPLC—MS Determination of A9—Tetrahydrocannabinol in Human Body Samples Jhmnie 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.0. Box 12194 Research Triangle Park, North Carolina 27709 119 National Institute on Drug Abuse 2 earcn monograph series Each monograph in the NIDA Research Monograph series is for sale from the following sources: 1 Findings of Drug Abuse Research An annotated bibliography of IVIMH and [VIDA- supported extramural grant research 1967-74 Volume 1: Stock #017—024—0467; p. 384; $7.00 Volume 2: Stock #017—024-0466-9; pp. 377; $5.05 Order from: Superintendent of Documents U.S. Government Printing Office Washington, 0.0. 20402 2 Operational Definitions in Socio-behavioral Drug Use Research 1975 Editors: Jack Elinson,Ph.D. and David Nurco,Ph.D. Task Force articles proposing consensual defin- itions of concepts and terms used in psycho- social research to achieve operational compara- bility. Order by PB number: PB #246 338 Papercopy: $6.75; Microfiche: $2.25. Order from: NTIS (National Technical Information Service) (1.5”. 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