DOT HS 804 009 DEVELOPMENT OF A LOW COST PORTABLE FLUOROMETRY TECHNOLOGY AND QUANTIFICATION OF CANNABINOIDS IN BODY FLUIDS J. L. Valentine P. J. Bryant P.L. Gutshall 0. H. M. Gan B. H. C. Niu P. Psaltis University of Missouri-Kansas City 2411 Holmes Street Kansas City, Missouri 64108 Contract No. DOT HS-4-00968 Contract Amt. $69,651.00 ^^EaS OF TRANS^4 'z W 4rES OE 00' APRIL 1919 FINAL REPORT Document is available to the U.S. public through The National Technical Information Service, Springfield, Virginia 22161 Prepared For U.S. DEPARTMENT OF TRANSPORTATION National Highway Traffic Safety Administration Washington, D.C. 20590 This document is disseminated under the sponsorship of the Department of Transportation in the interest of information exchanca. The United States Govern­ ment assumes no liability for its contents or use thereof. Technical Report Documentation Page 1. Report No. 2. Gooernmeni Accession No. 3. Recipient's Catalog No. DOT HS-804 009 4. Title and Subtitle - S. Report Date Development of a Low Cost Portable Fluorometry Tech- May 4, 1977 nology and. Quantification of Cannabinoids in, Body 6. Performing Organization Code Fluids 8. Performing Organization Report No. 7. Author's) J.L. Valentine, P.J. Bryant, P.L. Gutshall, O.H.M. Gan, B.H.C. Niu, and P. Psaltis 9. Performing Organisation Nome and Address 10. Work Unit No. (TRAIS) University of Missouri-Kansas City 241.1 Holmes Street 11. Contract or G rant No. Kansas City, Missouri 64108 USDOT HS 4 00968 :13. Type of Report and Period Covered 12. Sponsoring. Agency Nome and Address Final Report June 28, 1975-June 28, 1976 14. Sponsoring Agency Code 13 Supplementary Notes 16 Abstroct Tec h no l ogy was d eveloped for determining o9 -tetrahydrocannabinol (I) and it ' s major metabolite.ll-nor-o9-tetr.ahydrocannabinol-9-carboxylic acid (II) in human blood plasma utilizing high pressure liquid chromatography (hplc)-ultraviolet (uv) detection. The methods were found to be capable of determining 10 ng of I or II in 1 ml of human plasma. A third type of assay method was developed for identifying in human saliva one of the major constituents of marijuana, cannabinol (III). Results indicated that III photolytically converted to a highly' fluorescent compound in the absence of air. Using this technique, a limited study of marijuana smoker's saliva and saliva from non-marijuana smokers to which I and III had been added was conducted. This study demonstrated that 1 ng/ml of III can be detected in saliva. 17. Key Words 18. Distribution Statement. Marijuana, hplc, fluorometry, blood, Document is available to the public saliva through the National Technical Information Service, Springfield, Va. 22161 19. Security Clossif. (of this report) 20. Security Clossif. (of this page) 21. No. of Pages 22. Price Unclassified Unclassified Form DOT F 1700.7 (8-72) Reproduction of form and completed page is authorized METRIC CONVERSION. FACTORS . -- _ .- ._ _ . e - - n .?: .- ?a Approximate Conversions lo Metric Measures .. --- -.. - -- .. Approximate Conversions from Metric Mea,surrs - .- - .- - ?4 . - .. Symbol . When Yon Know Mulriplv by Te ftnl Srmb~l - :7. Symbol When You Know Multiply by To Find Symbol .-.- - - N -.-. m -- - .. LENGTH -- .- F; LENGTH - . - m mtlfonplers 0.08 tnches n - E ~n, CP~I~VP(PI- 0.4 anches n -. mete,< 3.3 (QCI It n lncher '2.5 cent\rrators c W' - - f ,.. wtcrc fl feel 30 CPntlllYIIIlF ..,. 4 .-- - - 1.1 yard* ~d - ... i-. h~lnretprs, 0.6 miles mi . 0.9 m+.tt,rr - vd yards - - . m, mtl~s 1.6 ~J~~>PIPIF i -- -- - r- -- -. - .- --. - . - -..-- AREA AREA -- - -c -- . - P - - . .-. c".' quare centemete,s - - 0.16 square ~nches tn2 In2 square ~nches 6.5 SQ!$~CPrnnt~~r?!ers CT* - -. 5 n,. square wlers - :: 12 square vards rd 112 square feet 0.09 sqst~reret~rs m- -- square k~lareters ' - -- kd mq2 - - 0.4 squat* milts yd2 square vards 0.8 ~q,.dr*-l*rs 7- -- --- -f 11 ,1 [email protected] 2.5 . - --.- acres n.,> square rt~lrs 2.6 suu,>re k,im->eters kvL - - - . - ------ acres 0.4 h?rl.treq ha - .-- - - 0 -- ---- - - - -..- MASS (weight) -- ---:.___- MASS (weight) .- --- 9 -- -- - - - r( grams 0.035 wnce- 02 P* 01 ounces 28 grams 9 . - - - --- kr( kbloqrams 2.2 mds !b patnds 0.45 koloqrams hq - - . Ih - - - lwrnes ilOOO kg) , 1 .I sh& tons shwl tons 0.9 IO~IIPS I - -=-. I - - - .. - .. .. 12000 Ibl b -. -- --.-. 0 -- . C VOLUME -- - -*. VOL'JME - -..- . - - - --- 0, ' -- -. .- - - mt mill~ltters 0.03 flu~dumces 1. oi ISP teaspoons 5 mtll~llters T l - - ,- -- - - I Tbsp tablespoons 15 mlll,l,ters rn 1 --- -- OJ tilers 2.1 pjnts' c' 11 oz Iluld ounces 30 n~all~ltlers m 1 w -- >-.- I liters 1.06 quarts u: 9--- c CLIPS' 0.24 lrlers I -- -z- r- I l~fets 0.26 gallons -- - 7' PI pints 0.47 lst~rs I - -- v3 ctlhlc rnetcrs cnlhic lee! - - -- I 35 , - - .. ql qtmrts 0.95 l~l~ft I -- --.- . cuh8r wters 1.3 cuhlc yards ,t3 - --.- i~al gallons 3.8 I-t~rt I - -- - -- fti cub~~feet 0.03 ctch~c!I-#,I,,~S - .- ". -- ----- -. vd3 cubic yards 0.76 cuhlc nr'.tcrs v1 b. -- --- Lo -- -.- TEMPERATURE (exrct) - -.- - - . - --.. .- TEMPERATURE .(exact! -- 4 -I --- --- C Celsrus 9.5 llhen Fahrenhsct , - -- lamperat\tre temo~ralure . - - -..~ .-. .- add 321 . ' F Fahrenhett 5 9 latter Celsaus C - .--- - C -- --. temperat~~te s~htr~~cling twnpvratlore - -- --- or - .- - -_ . 321 -- .-= ____- F . 32 98-6 .RP .-- - c4 - --- -- -49 -0 4 0 8 0 120 I60 203 I --. --- : , : . , , , , , , \ , , , ,. , ! I ., I , L. .. ..ic ". ., .,,, . 4 , -- -= .-. t4; ./I ;'.' 411'11 '~'i'1' 'I' It2 4 . 0" , -? 2 - .... - - 60 -, ,.d" ,,?,,,Ib .,,. , ,,,.. , . ' : 3.8 ,.,,: .,,, i i . ii. I..,. - -... - .- - 60 -2t> V CV 1;; - . -- 37 C -- 5 OC SUMMARY Technology was developed for determining 09-tetrahydrocannabinol (A9-THC) and its major metabolite 11-nor-49-tetrahydrocannabinol-9-carboxylic acid (9­ C02H-69-THC) in human blood plasma utilizing high pressure liquid chromatography (hplc) and ultraviolet (uv) detection. The method developed for analysis of 69-THC was accomplished using an extraction of 1 ml of plasma with petroleum ether followed by normal phase hplc analysis. With the method, the lower prac­ tical limit of quantification was found to be 10 ng/ml, but a detection limit of S ng/ml was readily achieved. Thus the method would be useful for quantifying 69-THC in the one-hour period following marijuana smoking. The precision and accuracy of the method was comparable to the previously developed hplc-ms methodology. For. the analysis of 9-C02H-A9-THC, an hplc-uv method was developed utilizing a reverse phase column. Although the lower practical quantification limit was found to be 1.0 ng/ml of plasma, the plasma levels determined in.several marijuana smokers were found to give a response greater than that observed for a 100 ng/ml standard. Mass spectrometry was used to demonstrate that the quantified peak in the hplc analysis was consistant with the mass spectrum of 9-C'0.2H-a9-THC. The developed method was used to determine the plasma levels of 9-CO 2H-ATHC in both marijuana smokers and patients receiving G9-THC. Results indicated that 9-CO 2H-o9-THC does not give a smooth plasma decay curve as does 09-THC and that levels remain quite high for 24 hours following marijuana smoking. This assay method may be of practical value in identifying a marijuana user. A third type of assay method was also developed during the study for one of the major constituents of marijuana, viz. cannabinol (CBN). Spectral studies with the various cannabinoids indicated that CBN could be photolytically converted to CBN I if air was excluded and to CBN II and CBN III in the presence of air. Of these photolytic compounds, CBN I proved to be highly fluorescent. An assay iii Summary .Page 2 was developed for saliva which allowed CBN present from marijuana smoking to be detected. A limited study was conducted using saliva from both marijuana smokers and nonsmokers as well as the saliva from nonsmokers to which 1 ng/ml of CBN was added. The results indicate that a marijuana smoker can be.identified if at least 1 ng/ml of. CBN is present in the saliva. All three of the methods developed during the study represent an advance in reducing the complexity, time and expense in assaying for marijuana use. In the case of a9-THC and 9-CO2H-A9-THC, the developed method is somewhat more involved since quantification is the major objective. With the CBN assay, the procedure is not lengthy but gives only qualitative information as to marijuana use. All three analysis utilize comparatively simple techniques. Although not all clinical laboratories. are equipped with hplc instruments to perform the A9-,THC and/or 9-CO7H-d9-THC assays, there. would be the. universal ability to perforn the CBN assay since a readily available fluorometer is used. iv The authors are F ateful to the National Institute on Drug Abuse for additional financial support of this work. v Table of Contents (Text) Introduction 1 Experimental A.­ Ultraviolet Studies of the Cannabinoids . ... 1 B.­ Fluorescence Studies of the Cannabinoids . 2 C.­ Design and Construction of a Fluormetric Flow Cell . 5 D.­ Analysis of A -THC in Human Plasma Using Sephadex-LH-20 and Hplc . 5 E.­ Analysis of 0 9 -THC in Human Plasma Using HPLC-UV . ... 7 9 F. Analysis of 9-CO 2H-p -THC in Human Plasma Using HPLC-UV . 7 Results and Discussion A. Hplc-Fluorescence Studies . .­ . 8 B.­ Hplc-Ultraviolet Studies for p9-Tetrahydrocannabinol . 10 9 C.­ Hplc-Ultraviolet Studies on 11-nor-p -tetrahydrocannabinol-9­ carboxylic Acid .