Do D9-tetrahydrocannabinol concentrations indicate recent use in chronic cannabis users?

Do D9-tetrahydrocannabinol concentrations indicaterecent use in chronic cannabis users?add_2705 2041..2048

Erin L. Karschner1, Eugene W. Schwilke1, Ross H. Lowe1, W. David Darwin1, Harrison G. Pope2,Ronald Herning3, Jean L. Cadet3 & Marilyn A. Huestis1

Chemistry and Drug Metabolism1 and Molecular Neuropsychiatry,3 Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health,Baltimore, MD, USA and Department of Psychiatry, Harvard Medical School, and Biological Psychiatry Laboratory, McLean Hospital, Belmont, MA, USA2

ABSTRACT

Aims To quantify blood D9-tetrahydrocannabinol (THC) concentrations in chronic cannabis users over 7 days ofcontinuous monitored abstinence. Participants Twenty-five frequent, long-term cannabis users resided on a secureclinical research unit at the US National Institute on Drug Abuse under continuous medical surveillance to preventcannabis self-administration. Measurements Whole blood cannabinoid concentrations were determined by two-dimensional gas chromatography-mass spectrometry. Findings Nine chronic users (36%) had no measurable THCduring 7 days of cannabis abstinence; 16 had at least one positive THC �0.25 ng/ml, but not necessarily on the firstday. On day 7, 6 full days after entering the unit, six participants still displayed detectable THC concentrations[mean � standard deviation (SD), 0.3 � 0.7 ng/ml] and all 25 had measurable carboxy-metabolite (6.2 � 8.8 ng/ml). The highest observed THC concentrations on admission (day 1) and day 7 were 7.0 and 3.0 ng/ml, respectively.Interestingly, five participants, all female, had THC-positive whole blood specimens over all 7 days. Body mass index didnot correlate with time until the last THC-positive specimen (n = 16; r = -0.2; P = 0.445). Conclusions Substantialwhole blood THC concentrations persist multiple days after drug discontinuation in heavy chronic cannabis users. It iscurrently unknown whether neurocognitive impairment occurs with low blood THC concentrations, and whetherreturn to normal performance, as documented previously following extended cannabis abstinence, is accompanied bythe removal of residual THC in brain. These findings also may impact on the implementation of per se limits in drivingunder the influence of drugs legislation.

Keywords Blood, cannabinoids, cannabis, chronic use, driving, tetrahydrocannabinol.

Correspondence to: Marilyn A. Huestis, Chemistry and Drug Metabolism, Intramural Research Program, NIDA, NIH, Biomedical Research Center Suite200, 251 Bayview Boulevard, Room 05A-721, Baltimore, MD 21224 USA. E-mail: [email protected] 12 January 2009; initial review completed 16 March 2009; final version accepted 1 June 2009

INTRODUCTION

Cannabis, the most widely used illicit drug world-wide,exerts dose-related psychoactive effects when the pri-mary psychoactive component, D9-tetrahydrocannabinol(THC), interacts with cannabinoid receptors in the brain.THC binding to the CB1-cannabinoid receptor leads tothe ‘high’ experienced by cannabis smokers [1]. THC ismetabolized by hepatic cytochrome P450 enzymes 2C9and 2C19 to the equipotent monohydroxy compound [2],11-hydroxy-THC (11-OH-THC), and undergoes furtheroxidation to non-psychoactive 11-nor-9-carboxy-THC(THCCOOH) [3,4]. Whole blood 11-OH-THC concentra-tions are only about 10% of THC concentrations after

smoking cannabis, in comparison to nearly equivalentconcentrations after oral THC administration [5]. First-pass hepatic metabolism of orally administered cannabisgreatly reduces the bioavailability of THC by this route,but does produce increased 11-OH-THC concentrationsthat contribute to observed pharmacodynamic effects.While THC concentrations decrease rapidly after smokingcannabis, THCCOOH, the water-soluble metabolite, isdetected for longer periods of time in blood [6]. For bestinterpretation of blood cannabinoid concentrationsand an improved understanding of THC disposition inbiological matrices, quantification of THC, 11-OH-THCand THCCOOH is needed. In addition, predictive modelsto estimate time of last cannabis use within 95%

RESEARCH REPORT doi:10.1111/j.1360-0443.2009.02705.x

© 2009 Society for the Study of Addiction. No claim to original US government works Addiction, 104, 2041–2048

confidence intervals require concentration data forTHC and THCCOOH [7–9], and THC and 11-OH-THCconcentrations may suggest whether the route of admin-istration was smoked or oral.

Cognitive and psychomotor performance, such asthat related to driving a motor vehicle, may be impairedwhen under the influence of cannabis [10–15]. Anotherbody of data suggests that residual neuropsychologicaldeficits may persist in chronic cannabis users for days oreven weeks after last drug exposure [16,17]. Althoughthe mechanism of these residual cognitive and motordeficits is uncertain, it seems likely that they might beattributable to the persistence of cannabinoids in theblood and, by implication, in the brain.

One important reason for evaluating THC dispositionafter chronic cannabis exposure is that impairment issometimes inferred in forensic settings on the basis ofTHC concentrations in whole blood. In 15 states(Arizona, Delaware, Georgia, Illinois, Indiana, Iowa,Michigan, Nevada, North Carolina, Ohio, Pennsylvania,Rhode Island, South Dakota, Utah and Wisconsin) andseven European countries (Belgium, France, Finland,Germany, Poland, Sweden and Switzerland), per se legallimits are set for blood cannabinoid concentrations; ifwhole blood THC concentrations equal or exceed the legallimit, drivers may be convicted of driving under the influ-ence of drugs (DUID). However, do blood cannabinoidconcentrations reflect recency of cannabis exposure inheavy long-term cannabis users? Despite the importanceof this question, few studies have been conducted thatinclude observed abstinence and extended monitoringperiods [18,19]. To augment these limited data, we mea-sured serial blood cannabinoid concentrations during 7days of monitored abstinence in 25 of the heaviest,longest-term cannabis users volunteering for our clinicalstudies.

METHODS

Participants

Cannabis users were recruited by print, radio andtelevision advertisements. For the present research, wechose experienced cannabis smokers, aged 21–45 years,reporting multiple years of use, who exhibited a positiveurine cannabinoid immunoassay test greater than100 ng/ml prior to admission to the unit. Participantswere excluded from the study if they displayed clinicallysignificant medical conditions, including cardiovascular,pulmonary, neurological, endocrine, hematological orhepatic abnormalities. Participants provided voluntarywritten informed consent and the National Institute onDrug Abuse (NIDA) Institutional Review Board approvedthe study. Medical [physical examination, electrocardio-

gram (ECG), blood and urine chemistries] and psycho-logical evaluations, including self-reported drug use,histories were conducted. Participants resided on thesecure clinical research unit under 24-hour medical sur-veillance to ensure cannabis abstinence. Access to a uni-versal gym, exercise bicycle and a secure courtyard areafor recreation was provided. Meals were ordered from thehospital cafeteria and no diet or liquid restrictions wereimposed.

Study design

Whole blood specimen collection

Three ml whole blood was collected with an indwellingvenous catheter into sodium heparin BD Vacutainer®

tubes (Becton Dickinson, Franklin Lakes, NJ, USA) at thetime of admission and generally at 9 a.m. each day there-after for 7 days. Specimens were stored at -20°C untilanalysis.

Specimen analysis

Whole blood specimens were analyzed for cannabinoidsby modification of a validated two-dimensional gaschromatography-mass spectrometry (2D-GCMS) methodfor simultaneous THC, 11-OH-THC and THCCOOH quan-tification [20]. Briefly, proteins in 1 ml of whole blood wereprecipitated with 3 ml cold acetonitrile while vortexing.After centrifugation, the supernatant was decanted into5 ml sodium acetate buffer (pH 4.0), vortexed and appliedto conditioned 200-mg ZSTHC020® solid phase extrac-tion (SPE) columns (United Chemical Technologies, Inc.,Bristol, PA, USA). Columns were washed with 3 ml deion-ized water, 2 ml 0.1 N hydrochloric acid/acetonitrile(70 : 30 v/v), and dried by full vacuum for 20 minutes.Analytes were eluted with 1 ¥ 3 ml and 1 ¥ 2 mlhexane : ethyl acetate (80 : 20 v/v). Eluents weredried under nitrogen, reconstituted with 25 ml N,O-bis-(trimethylsilyl)trifluoroacetamide + 1% trimethylchlo-rosilane (BSTFA + 1% TMCS) for derivatization at 70°C for45 minutes.

Derivatized extracts were injected (3 ml) onto anAgilent 6890/5973 2D-GCMS equipped with cryofocus-ing and operated in electron impact/selected ion monitor-ing mode. 2D-GCMS provided effective separation ofanalyte from matrix, while cryofocusing significantlyenhanced signal. Split calibration curves (low 0.25–25,and high 25–100 ng/ml) were constructed withr2 � 0.990. Limits of quantification (LOQ) were0.25 ng/ml for THC and THCCOOH and 0.50 ng/ml for11-OH-THC. Intra-assay imprecision (n = 16 in four dif-ferent assays) was 1.5–3.7% for all analytes and inter-assay imprecision (n = 20) was less than 8.0%.

2042 Erin L. Karschner et al.


Data analysis

Statistical analyses were performed with SPSS forWindows, version 13.0 (Chicago, IL, USA) and MicrosoftExcel 2002 for Windows (Microsoft Corp., Redmond, WA,USA).

Body mass index (BMI) was calculated as:

BMI weight lb height in= × ( ) ( )[ ]703 2 2

RESULTS

Twenty-five frequent cannabis users (12 male, 13 female;84.0% African American, 8.0% Hispanic, 4.0%Caucasian, 4.0% American Indian; mean age, 26.2 �

4.5 years; median, 25.0 years; range, 21–38 years)completed the study. Race/ethnicity was defined bythe participant. Demographic and physiological charac-teristics of participants are reported in Table 1. Mostof the participants reported daily or near-dailycannabis use in the last 14 days. Mean duration of useaveraged 8.8 � 4.4 years, with mean age of first use of15.8 � 2.9 years. Mean time since last self-reported usewas 0.6 � 0.7 days. Every participant reported drinkingalcohol in the month prior to admission, and all but four(participants A, C, S and X) reported smoking tobaccowithin 2 weeks of admission. No participant reportedother illicit drug use during the 2 weeks prior to admis-sion, with the exception of participant M, who reportedusing cocaine on 1 day.

THC concentrations during 7 days of abstinence arereported in Table 1. Nine chronic users (36%) had nopositive specimens, despite the low LOQ, throughout the7-day abstinence period, similar to that documented afteracute cannabis exposure. Fourteen participants werepositive on admission (day 1) (Fig. 1) and four (28.6%) ofthese had THC concentrations above 1.0 ng/ml, the cut-off applied frequently to indicate DUID in the United States.Surprisingly, on the seventh day of monitored cannabisabstinence, 6 full days after entering the unit, six partici-pants’ whole blood specimens contained THC �0.25 ng/ml, with three �1.0 ng/ml. THC concentrations alwaysexceeded 11-OH-THC concentrations, with the exceptionof four participants’ specimens on day 1. A total of 20specimens were positive for both THC and 11-OH-THC,with only one positive for 11-OH-THC without THC. Allparticipants exhibited measurable THCCOOH concen-trations throughout 7 days of abstinence (Table 2).

Five participants, all female, were THC-positivethroughout the 7 days (Table 1). Among these five indi-viduals, THC, 11-OH-THC and THCCOOH concentrations[mean�standard error (SE)] on day 1 were 2.5 � 1.1,1.9 � 1.1 and 45.2 � 15.0 ng/ml, respectively. Day 7cannabinoid blood concentrations were 1.5 � 0.5,0.3 � 0.2 and 18.7 � 6.1 ng/ml, respectively.

For the first time, to our knowledge, negative wholeblood specimens were found interspersed between posi-tive samples (Table 1 and Fig. 2). Of the 16 participantswith positive specimens, two (12.5%) had THC concen-trations less than LOQ at admission, but at least one laterpositive specimen. Participants’ data displayed differentpatterns (Fig. 2). For instance, participant O’s blood wasTHC negative at admission and on day 3, but THC wasdetectable on days 2, 4, 5 and 6 (Fig. 2a). Participant U’sTHC blood concentrations decreased daily until no drugwas detectable on day 7 (Fig. 2b), while participantS’s THC concentrations were the highest obtained oneach day and exceeded 2.2 ng/ml for the entire 7 days(Fig. 2c).

We found no significant correlation between BMI andtime until the last THC-positive whole blood specimen(r = -0.2; P = 0.445).

DISCUSSION

We assessed whole blood THC concentrations in 25 fre-quent cannabis users living on a secure research unitwith 24-hour medical surveillance during 7 days of can-nabis abstinence. Some of these users displayed substan-tial whole blood THC concentrations after at least 7 daysof abstinence. This finding is in accordance with Johans-son et al., who found measurable THC in plasma of threemales up to 13 days after 60 mg smoked THC, adminis-tered over 2 days. [21]

There were few 11-OH-THC-positive specimens. OralTHC (Marinol) produces approximately equivalent THCand 11-OH-THC concentrations after first-pass metabo-lism in the liver [5,22]; but when cannabis is smoked,efficient gas exchange in the lungs distributes THC intosystemic circulation, yielding only about 10% 11-OH-THC. In contrast, THCCOOH was detectable in this studyin all participants for at least 7 days. This is consistentwith previous studies reporting prolonged THCCOOHurinary excretion in frequent cannabis users [23–26].

Whole blood THC concentrations were highly variableamong participants; nine participants had no quantifi-able THC at any time, whereas others displayed substan-tial THC concentrations even after 7 days. Variable THCrelease rates from tissue stores during abstinence mayaccount for these marked variations. It is interesting tonote that of the five participants with positive THC speci-mens on all 7 days, all were female. Although previousstudies have reported no differences in THC metabolism,disposition and kinetics between sexes [5], we havereported recently that the time to last positive THCCOOHin urine was 140 hours longer in 10 abstinent femalescompared to 12 males (P < 0.02) [27]. In the presentstudy, there was no significant difference in BMI betweenmales and females; however, females generally have more

THC concentrations in chronic cannabis users 2043


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adipose tissue than males and, possibly, these women hada greater THC body burden. Interestingly, participant S,who showed the highest THC concentrations, had thelowest BMI among the women in the study—suggestingthat factors other than BMI, such as neuroendocrineeffects, might account for differences between individualsand between the sexes. Although we did not assess bodyfat percentage among our subjects, this measure shouldbe considered in future excretion studies.

Clearly, cannabis produces impairment in neurocog-nitive and psychomotor skills necessary for safe driving;however, defining the relationship between THC bloodconcentrations and performance decrements has beenchallenging. Numerous early studies failed to find a sig-nificant increase in accident risk when cannabinoidswere present in blood or urine [28–33]. In some of these,the presence of the non-psychoactive THCCOOH metabo-lite, rather than THC, defined recent cannabis use. Otherlimitations included long intervals between accidentoccurrence and blood collection leading to false negativecannabinoid results, and limited numbers of casespositive for cannabis only. More recently, Drummer et al.conducted a study in 3398 fatally injured drivers todetermine the effect of cannabis on accident culpability[34]. Using a validated method of classifying drivers asculpable or non-culpable, the investigators found thataccident risk increased significantly in drivers with mea-surable blood THC concentrations (no LOQ provided)when compared to drug-free drivers [odds ratio (OR) 2.7,95% confidence interval (CI) 1.0–7.0]. When THC con-centrations were greater than 5 ng/ml, culpabilityincreased (OR 6.6, 95% CI 1.5–28.0). This OR is compa-rable to that observed with a blood alcohol concentrationof 0.15 g%.

Recent experimental laboratory research proposedimpairment limits of 2–5 ng/ml serum THC (approxi-mately 1–2.5 ng/ml whole blood) after observing behav-

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Figure 1 Detection rate percentages of D9-tetrahydrocannabinol(THC), 11-hydroxy-9-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in whole blood of participants (n = 25) over7 days of continuously monitored cannabis abstinence. Limits ofquantification (LOQ) for THC and THCCOOH 0.25 ng/ml and11-OH-THC 0.50 ng/ml

Table 2 Number and percentage of participants with whole blood 11-hydroxy-D9-tetrahydrocannabinol (11-OH-THC) �0.5 ng/mland 11-nor-9-carboxy-THC (THCCOOH) �0.25 ng/ml (n = 25). Common laboratory cut-offs of 1 and 2 ng/ml 11-OH-THC and5 ng/ml THCCOOH are presented. Mean, median and range of positive whole blood cannabinoid concentrations are included.

(a) 11-OH-THC

Day n � 0.5 % Mean � SD Median Range n � 1.0 % n � 2.0 %

1 7 28 0.5 � 1.3 0.0 ND–6.3 2 8 1 42 3 12 0.2 � 0.5 0.0 ND–2.4 2 8 1 43 3 12 0.1 � 0.3 0.0 ND–1.5 1 4 0 04 2 8 0.1 � 0.3 0.0 ND–1.3 1 4 0 05 2 8 0.1 � 0.2 0.0 ND–1.1 1 4 0 06 2 8 0.1 � 0.3 0.0 ND–1.1 1 4 0 07 2 8 0.1 � 0.2 0.0 ND–0.9 0 0 0 0

(b) THCCOOH

Day n � 0.25 % Mean � SD Median Range n � 5.0 %

1 25 100 20.2 � 21.4 15.4 2.8–91.7 19 762 25 100 14.8 � 16.6 11.7 1.1–69.3 16 643 25 100 10.1 � 12.7 7.3 0.8–54.3 14 564 25 100 8.4 � 10.0 5.0 0.6–36.4 12 485 25 100 7.5 � 9.7 3.4 0.6–35.7 11 446 25 100 7.2 � 10.4 4.0 0.4–45.1 10 407 25 100 6.2 � 8.8 3.1 0.4–36.5 8 32

SD: standard deviation.



ioral impairment in a majority of participants with serumTHC concentrations within the suggested limits in tasksrelating to driving skills [35]. Others suggested that aserum THC between 7 and 10 ng/ml (approximately 3.5–5.0 ng/ml whole blood) was indicative of impairment,similar to a blood alcohol concentration (BAC) of 0.05%based on a meta-analysis of multiple toxicological studies[36]. However, it is difficult to apply these limits in thefield because THC concentrations decrease rapidly aftercannabis smoking, even in frequent users, from high peakconcentrations (100–400 ng/ml depending upon theindividual, cannabis potency and smoking parameters) tolevels of 1–10 ng/ml in a few hours. The time required toobtain biological specimens after automobile or industrial

accidents often exceeds 3 hours, leading frequently tonegative THC tests. THC concentrations in the majorityof DUID cases are 1–2 ng/ml. If a 5 ng/ml whole bloodlimit had been the law, 77–90% of apprehended driversrecently using cannabis in Sweden from 1995 to 2004would not have been prosecuted [37]. Conversely, as thepresent study shows, some individuals may display con-centrations well over 1 ng/ml many days after last can-nabis exposure.

One limitation of our research is reliance on self-reports of drug use; reports of the recency and quantityof cannabis smoked often did not correlate with analyteconcentrations. However, this limitation would notimpact our principal finding that substantial blood THCconcentrations persisted for days in many chronic can-nabis users. Another possible limitation of the study isthat specimens were kept in long-term frozen storage forup to 5 years prior to analysis, raising the possibility ofdegradation of cannabinoids in whole blood specimens.For example, analytes may have adsorbed to polypropy-lene storage tubes [38], precipitated with blood proteinsor degraded in vitro. Whole blood cannabinoid stability isvariable [38–40]. A recent study [41] has observed can-nabinoid concentration decreases of a least 20% in spikedwhole blood stored at -20°C for 2 weeks, while a separatestudy [39] has observed no statistically significantdecreases after 6 months in fortified blood stored at-10°C. However, these factors could only have caused usto under-, rather than overestimate, cannabinoid concen-trations. Also, these stability studies were conducted inblank whole blood fortified with cannabinoids and notin authentic specimens that were collected and analyzedin the current study.

In summary, we found highly variable, but often sub-stantial whole blood THC concentrations in chronic can-nabis users for many days after last drug exposure. Thesefindings may be relevant for legislation employing THCconcentrations to define intoxication and accident culpa-bility. The findings also raise the intriguing possibility thatcannabis-associated cognitive and motor impairment,demonstrated in some individuals for many days after lastcannabis exposure [16,17,42–44], may be related to thepersistence of THC in the blood and, by implication, in thebrain [45]. If so, our findings suggest that residual neu-rocognitive impairment after days of abstinence might bequite variable among individuals, depending upon thepersistence and magnitude of THC in an individual’sbrain. These data suggest a potential mechanism for cog-nitive impairment after chronic cannabis use and providedata for evidence-based policy decisions on DUID. In sub-sequent studies, it will be invaluable to assess the associa-tion between neuropsychological performance andsimultaneous blood THC concentrations. If neurocogni-tive impairment is documented after at least 7 days of

a.

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Figure 2 Representative patterns of D9-tetrahydrocannabinol(THC) (�), 11-hydroxy-9-THC (11-OH-THC) ( ) and 11-nor-9-carboxy-THC (THCCOOH) (�) excretion. Participant O (a), U (b)and S (c) whole blood cannabinoid concentrations during 7 days ofcontinuously monitored cannabis abstinence



abstinence, the implementation of per se impairmentlimits may be valid.

Declarations of interest

None.

Acknowledgements

The authors would like to thank Allan J. Barnes for dataanalysis support and Kathleen Demuth, Janeen Nichelsand John Etter for clinical research assistance. Thisresearch was supported by the Intramural ResearchProgram of the National Institute on Drug Abuse,National Institutes of Health.

References

1. Huestis M. A., Gorelick D. A., Heishman S. J., Preston K. L.,Nelson R. A., Moolchan E. T. et al. Blockade of effects ofsmoked marijuana by the CB1-selective cannabinoid recep-tor antagonist SR141716. Arch Gen Psychiatry 2001; 58:322–30.

2. Perez-Reyes M., Timmons M. C., Lipton M. A., DavisK. H., Wall M. E. Intravenous injection in man ofdelta-9-tetrahydrocannabinol and 11-OH-delta-9-tetrahydrocannabinol. Science 1972; 177: 633–5.

3. Bland T. M., Haining R. L., Tracy T. S., Callery P. S. CYP2C-catalyzed delta9-tetrahydrocannabinol metabolism: kinet-ics, pharmacogenetics and interaction with phenytoin.Biochem Pharmacol 2005; 70: 1096–103.

4. Watanabe K., Yamaori S., Funahashi T., Kimura T., Yama-moto I. Cytochrome P450 enzymes involved in the metabo-lism of tetrahydrocannabinols and cannabinol by humanhepatic microsomes. Life Sci 2007; 80: 1415–9.

5. Wall M. E., Sadler B. M., Brine D., Taylor H., Perez-ReyesM. Metabolism, disposition, and kinetics of delta-9-tetrahydrocannabinol in men and women. Clin PharmacolTher 1983; 34: 352–63.

6. Huestis M. A., Henningfield J. E., Cone E. J. Blood cannab-inoids. I. Absorption of THC and formation of 11-OH-THCand THCCOOH during and after smoking marijuana. J AnalToxicol 1992; 16: 276–82.

7. Huestis M. A., Henningfield J. E., Cone E. J. Blood cannab-inoids. II. Models for the prediction of time of marijuanaexposure from plasma concentrations of delta-9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCCOOH). J Anal Toxicol 1992;16: 283–90.

8. Huestis M. A., Elsohly M., Nebro W., Barnes A., Gustafson R.A., Smith M. L. Estimating time of last oral ingestion ofcannabis from plasma THC and THCCOOH concentrations.Ther Drug Monit 2006; 28: 540–4.

9. Huestis M. A., Barnes A., Smith M. L. Estimating the time oflast cannabis use from plasma delta9-tetrahydrocannabinoland 11-nor-9-carboxy-delta9-tetrahydrocannabinol con-centrations. Clin Chem 2005; 51: 2289–95.

10. Ramaekers J. G., Berghaus G., Van Laar M., Drummer O. H.Dose related risk of motor vehicle crashes after cannabisuse. Drug Alcohol Depend 2004; 73: 109–19.

11. Block R. I., Farinpour R., Braverman K. Acute effects of

marijuana on cognition: relationships to chronic effects andsmoking techniques. Pharmacol Biochem Behav 1992; 43:907–17.

12. Chait L. D., Perry J. L. Acute and residual effects of alcoholand marijuana, alone and in combination, on mood andperformance. Psychopharmacology (Berl) 1994; 115:340–9.

13. Riedel G., Davies S. N. Cannabinoid function in learning,memory and plasticity. In: Pertwee R. G., editor. Handbook ofExperimental Pharmacology. New York: Springer; 2005, p.446–70.

14. Hall W., Solowij N. Adverse effects of cannabis. Lancet 1998;352: 1611–6.

15. Hampson R. E., Deadwyler S. A. Cannabinoids, hippocam-pal function and memory. Life Sci 1999; 65: 715–23.

16. Pope H. G., Gruber A. J., Hudson J. I., Huestis M. A.,Yurgelun-Todd D. Cognitive measures in long-term can-nabis users. J Clin Pharmacol 2002; 42: 41S–47S.

17. Pope H., Gruber A., Hudson J., Huestis M., Yurgelun-Todd D.Neuropsychological performance in long-term cannabisusers. Arch Gen Psychiatry 2001; 58: 909–15.

18. Peat M. A. Distribution of delta-9-tetrahydrocannabinoland its metabolites. In: Baselt, R. C., editor. Advances in Ana-lytical Toxicology II. Chicago: Year Book Medical Publishers;1989, P. 186–217.

19. Skopp G., Potsch L. Cannabinoid concentrations in spotserum samples 24–48 hours after discontinuation of can-nabis smoking. J Anal Toxicol 2008; 32: 160–4.

20. Lowe R. H., Karschner E. L., Schwilke E. W., BarnesA. J., Huestis M. A. Simultaneous quantification ofdelta-9-tetrahydrocannabinol (THC), 11-hydroxy-delta-9-tetrahydrocannabinol (11-OH-THC), and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) inhuman plasma using two-dimensional gas chromatography,cryofocusing, and electron impact-mass spectrometry. JChromatogr A 2007; 1163: 318–27.

21. Johansson E., Agurell S., Hollister L. E., Halldin M. M. Pro-longed apparent half-life of delta-1-tetrahydrocannabinolin plasma of chronic marijuana users. J Pharm Pharmacol1988; 40: 374–5.

22. Frytak S., Moertel C. G., Rubin J. Metabolic studies of delta-9-tetrahydrocannabinol in cancer patients. Cancer Treat Rep1984; 68: 1427–31.

23. Ellis G. M., Mann M. A., Judson B. A., Schramm N. T., Tash-chian A. Excretion patterns of cannabinoid metabolitesafter last use in a group of chronic users. Clin PharmacolTher 1985; 38: 572–8.

24. Dackis C. A., Pottash A. I. C., Annitto W., Gold M. S. Persis-tence of urinary marijuana levels after supervised absti-nence. Am J Psychiatry 1982; 139: 1196–8.

25. Cridland J. S., Rottanburg D., Robins A. H. Apparent half-lifeof excretion of cannabinoids in man. Hum Toxicol 1983; 2:641–4.

26. Goodwin R. S., Darwin W. D., Chiang C. N., Shih M., Li S.-H.,Huestis M. A. Urinary elimination of 11-nor-9-carboxy-D9-tetrahydrocannabinol in cannabis users during continu-ously monitored abstinence. J Anal Toxicol 2008; 32:562–6.

27. Darwin W. D., Herning R. I., Bolla K., Cadet J. L., Huestis M.A. A comparison by gender of urinary elimination of11-nor-9-carboxy-D9-tetrahydrocannnabinol (THCCOOH)in cannabis users. Paper presented at the Society of ForensicToxicologists Annual Meeting–2007, Durham, NC, 15–19October, 2007.



28. Terhune K. W., Fell J. C. The Role of Alcohol, Marijuana, andOther Drugs in the Accidents of Injured Drivers. Washington,D.C.: US Department of Transportation, 1982.

29. Terhune K. W., Ippolito D. L., Hendricks D. L., Michalovic J.G., Bogema S. C., Santinga P. et al. The Incidence and Role ofDrugs in Fatally Injured Drivers. October, 1-K1. Washington,D.C.: US Department of Transportation/National HighwayTraffic Safety Administration, 1992.

30. Williams A. F., Peat M. A., Crouch D. J., Wells J. K., Finkle B.S. Drugs in fatally injured young male drivers. Public HealthRep 1985; 100: 19–25.

31. Drummer O. H. Drugs in Drivers Killed in Australian RoadTraffic Accidents. Victoria, Australia: Victorian Institute ofForensic Medicine, 1994.

32. Hunter C. E., Loken R. J., Longo M. C. The prevalenceand role of alcohol, cannabinoids, benzodiazepines andstimulants in non-fatal crashes. S Austr Forensic Sci1998.

33. Lowenstein S. R., Koziol-Mclain J. Drugs and traffic crashresponsibility: a study of injured motorists in Colorado. JTrauma Inj Infect Crit Care 2001; 50: 313–20.

34. Drummer O. H., Gerostamoulos J., Batziris H., Chu M.,Caplehorn J., Robertson M. D. et al. The involvement ofdrugs in drivers of motor vehicles killed in Australian roadtraffic crashes. Accid Anal Prev 2004; 36: 239–48.

35. Ramaekers J. G., Moeller M. R., Van Ruitenbeek P., Theunis-sen E. L., Schneider E., Kauert G. Cognition and motorcontrol as a function of delta9-THC concentration in serumand oral fluid: limits of impairment. Drug Alcohol Depend2006; 85: 114–22.

36. Grotenhermen F., Leson G., Berghaus G., Drummer O. H.,Kruger H. P., Longo M. et al. Developing limits for drivingunder cannabis. Addiction 2007; 102: 1910–7.

37. Jones A. W., Holmgren A., Kugelberg F. C. Driving under the

influence of cannabis: a 10-year study of age and genderdifferences in the concentrations of tetrahydrocannabinolin blood. Addiction 2008; 103: 452–61.

38. Christophersen A. S. Tetrahydrocannabinol stability inwhole blood: plastic versus glass containers. J Anal Toxicol1986; 10: 129–31.

39. Johnson J. R., Jennison T. A., Peat M. A., Foltz R. L. Stabilityof delta-9-tetrahydrocannabinol (THC), 11-hydroxy-THC,and 11-nor-9-carboxy-THC in blood and plasma. J AnalToxicol 1984; 8: 202–4.

40. Wong A. S., Orbanosky M. W., Reeve V. C., Beede J. D.Stability of delta-9-tetrahydrocannabinol in stored bloodand serum In: Hawks R., editor. Analysis of Cannabinoids,Research Monograph. Rockville, MD: National Institute onDrug Abuse, p. 119–24.

41. Schwilke E. W., Karschner E. L., Lowe R. H., Gordon A. M.,Cadet J. L., Herning R. et al. Intra- and inter-subjectwhole blood/plasma cannabinoid ratios determined by2-dimensional, electron impact-gas chromatography, massspectrometry with cryofocusing. Clin Chem 2009; 55:1188–95.

42. Pope H. G., Yurgelun-Todd D. The residual cognitive effectsof heavy marijuana use in college students. JAMA 1996;275: 521–7.

43. Bolla K. I., Brown K., Eldreth D., Tate K., Cadet J. L. Dose-related neurocognitive effects of marijuana use. Neurology2002; 59: 1337–43.

44. Solowij N., Stephens R. S., Roffman R. A., Babor T., KaddenR., Miller M. et al. Cognitive functioning of long-term heavycannabis users seeking treatment. JAMA 2002; 287: 1123–31.

45. Mura P., Kintz P., Dumestre V., Raul S., Hauet T. THC can bedetected in brain while absent in blood. J Anal Toxicol 2005;29: 842–3.



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