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J Anal Toxicol. 2012 July; 36(6): 405412. Published online 2012 May 15. doi: 10.

1093/jat/bks044 PMCID: PMC3523952

Psychomotor Performance, Subjective and Physiological Effects and Whole Blood 9Tetrahydrocannabinol Concentrations in Heavy, Chronic Cannabis Smokers Following Acute Smoked Cannabis
David M. Schwope,1 Wendy M. Bosker,2 Johannes G. Ramaekers,2 David A. Gorelick,1 and Marilyn A. Huestis1,* Author information Copyright and License information Go to:

Abstract
9-tetrahydrocannabinol (THC) is the illicit drug most frequently observed in accident and driving under the influence of drugs investigations. Whole blood is often the only available specimen collected during such investigations, yet few studies have examined relationships between cannabis effects and whole blood concentrations following cannabis smoking. Nine male and one female heavy, chronic cannabis smokers resided on a closed research unit and smoked ad libitum one 6.8% THC cannabis cigarette. THC, 11-hydroxy-THC and 11-nor-9-carboxy-THC were quantified in whole blood and plasma. Assessments were performed before and up to 6 h after smoking, including subjective [visual analog scales (VAS) and Likert scales], physiological (heart rate, blood pressure and respirations) and psychomotor (critical-tracking and divided-attention tasks) measures. THC significantly increased VAS responses and heart rate, with concentration-effect curves demonstrating counter-clockwise hysteresis. No significant differences were observed for critical-tracking or divided-attention task performance in this cohort of heavy, chronic cannabis smokers. The cannabis influence factor was not suitable for quantifying psychomotor impairment following cannabis consumption and was not precise enough to determine recent cannabis use with accuracy.

These data inform our understanding of impairment and subjective effects following acute smoked cannabis and interpretation of whole blood cannabinoid concentrations in forensic investigations. Go to:

Introduction
An estimated 125203 million persons aged 1564 worldwide smoked cannabis at least once in the previous year (1), with 16.7 million Americans and 18.1% of individuals aged 1825 years smoking cannabis in the previous 30 days (2). Additionally, cannabis is the most common illicit substance detected in blood and oral fluid of nighttime drivers (3). Although impairment cannot be assumed from drug presence, detection windows in these matrices are relatively short for less than daily cannabis smokers (46), increasing impairment probability following a positive test. The primary psychoactive chemical in cannabis, 9-tetrahydrocannabinol (THC), is metabolized in vivo to several phase I metabolites, most prominently the equipotent 11hydroxy-THC (11-OH-THC) and non-psychoactive 11-nor-9-carboxy-THC (THCCOOH) (78). Several studies have investigated cannabinoid metabolism and urine, plasma and oral fluid pharmacokinetics following acute and chronic oral, smoked and intravenous THC administration (914). Recently, we investigated whole blood cannabinoid pharmacokinetics with direct liquid chromatography tandem mass spectrometry (LCMSMS) analysis for the first time, providing valuable data regarding cannabinoid detection in this matrix following cannabis smoking (15). THC is detectable in plasma within seconds after the first puff of a cannabis cigarette, with dose-related tachycardia, subjective high and conjunctival injection (9, 10, 12, 16). Cannabis impairs psychomotor performance, cognition and driving ability in both driving simulators and on-the-road driving tests (1725). Hunault et al. examined cognitive and psychomotor effects following smoked cannabis containing up to 69 mg THC and observed significant, dose-related impairing effects following paced smoking in primarily occasional cannabis smokers (26). Papafotiou examined cannabis-induced impairment in a driving simulator in cannabis smokers with unknown histories after one week of abstinence; significant impairment was observed 80 min but not 30 min after smoking a 1.74 or 2.93% THC cannabis cigarette (27). However, Ramaekers et al. studied effects of cannabis smoking (13% THC, 500 g/kg) in heavy (>4 days/week) and occasional (1x/week) smokers and found significant impairment in tracking performance, divided attention, and inhibitory control in occasional, but not heavy smokers (28). The cannabis influence factor (CIF), described by Daldrup as a predictor of cannabisinduced impairment, is calculated as molar blood [THC] + [11-OH-THC] divided by [THCCOOH] (29). A CIF > 10 was proposed to predict cannabis-induced impairment analogous to a 0.11 g/dL blood alcohol concentration. Menetrey et al. examined this model following controlled oral cannabis decoction (hemp extracted into hot milk) administration (30), yet none, to our knowledge, examined this following controlled cannabis smoking.

The present study examined relationships between whole blood cannabinoid concentrations and pharmacodynamic effects in heavy, chronic cannabis smokers. Cannabis-induced effects were determined in critical tracking and divided attention task performance, subjective and physiological measures, as well as the CIF, following cannabis smoking. These data should facilitate interpretation of whole blood cannabinoid results, often the only specimen available in driving under the influence of drugs (DUID), accident and other forensic investigations. Go to:

Materials and Methods


Participants
Participants provided written informed consent for this National Institute on Drug Abuse Institutional Review Board-approved protocol and this study was performed in accordance with the Declaration of Helsinki. Inclusion criteria were cannabis use at least twice monthly for three months before study entry, positive urine cannabinoid test, 1845 years old, normal cardiac function and veins suitable for intravenous catheter placement. Clinically significant medical or psychiatric disease, history of clinically significant adverse event associated with cannabis intoxication, current interest in drug abuse treatment, pregnancy or nursing, or blood donation in the previous 30 days were exclusionary. Participants were heavy (>4 days/week) and chronic (>2 years) cannabis smokers admitted to the secure research unit 1520 h before dosing; no cannabis use restrictions were enforced prior to admission. All participants underwent an immunoassay-based urine drug screen (iScreen, Instant Technologies, Inc, Norfolk, VA) on admission and were negative for all substances other than THC. Cigarette smoking was permitted up to 30 min before and after 90 min following THC dosing.

Smoked cannabis administration and blood collection


The NIDA Chemistry and Physiological Systems Research Branch supplied cannabis cigarettes containing 6.8 0.2% THC, 0.25 0.08% CBD and 0.21 0.02% CBN (w/w). Mean cigarette weight was 0.79 0.16 g, yielding total THC, CBD and CBN content of 54, 2.0 and 1.7 mg per cigarette, respectively. Participants smoked a single cigarette ad libitum for 10 min while seated with legs elevated, and remained in this position during the data collection period. Whole blood and plasma were collected on ice into sodium heparin blood tubes via indwelling intravenous catheter 0.5 h before and 0.25, 0.5, 1, 2, 3, 4 and 6 h after the start of smoking. Blood collected for plasma was centrifuged (1,600 x g, 15 min) and plasma separated within 2 h.

Cannabinoid analysis
Cannabinoids were quantified by a previously validated LCMS-MS method (31). Briefly, 1.5 mL acetonitrile was added to 0.5 mL specimen to precipitate proteins. After mixing and centrifugation, the supernatant was diluted and subjected to solid-phase extraction (SPE).

Extracts were evaporated, reconstituted and chromatographed; detection was via electrospray ionization. Imprecision was < 10.5% coefficient of variation (CV), recovery was > 50.5% and bias within 13.1% of target across the linear range. Limits of quantification (LOQ) were 1.0 g/L for THC, 11-OH-THC and THCCOOH.

Subjective scales
Visual analog scales (VAS) were presented on a computer screen 0.5, 0.25, 0.5, 1, 2, 3, 4 and 6 h after the start of smoking. Participants indicated the magnitude of Good Drug Effect, High, Stoned, Stimulated, Sedated, Anxious and Restless on a 100mm line anchored with not at all and most ever. Position on the scale was converted to a percentage between 0 and 100. 5-point Likert scales for Difficulty concentrating, Altered sense of time, Slowed or slurred speech, Body feels sluggish or heavy, Feel hungry, Feel thirsty, Shakiness/tremulousness, Nausea, Headache, Palpitations, Dizzy and Dry mouth or throat were presented on a computer screen immediately following VAS. Participants selected the response best characterizing their condition: 1, none; 2, slight; 3, mild; 4, moderate; or 5, severe.

Cardiovascular measures
Blood pressure (systolic and diastolic), heart rate and respiratory rate were measured before and after smoking at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5 and 6 h.

Cannabis influence factor


CIF was calculated as 100 ([THC] + [11-OH-THC])/[THCCOOH] (29, 30) for all whole blood and plasma samples; analytes detected below LOQ were considered to have 0.0 nmol/mL.

Impairment assessments
The critical tracking task (32) measures a participant's ability to control a displayed error signal in a first-order compensatory tracking task. Error is displayed as a horizontal deviation of a cursor from the midpoint on a horizontal, linear scale. Compensatory joystick movements null the error by returning the cursor to the midpoint. The frequency of cursor deviations, and, therefore, its velocity, increases as a stochastic, linear function of time. Control is lost at the point where the compensatory response lags the cursor's last movement by 180 degrees. The response frequency at this point is defined as the critical frequency or lambda-c (c). The test includes five trials. The average of the middle three scores (i.e., deleting the highest and lowest) is taken as the final score. This 2-min task was performed before smoking and 1.5, 3 and 5.5 h after starting smoking. The divided attention task (33) measures a participant's ability to divide attention between two simultaneous tasks. The primary task is the same as the critical tracking task

described previously, with the exception that the velocity of the error signal is kept constant at 50% of the participant's optimal performance (c/2). Tracking error is measured by the absolute distance (mm) between the cursor's position and the center of the scale. The secondary task involves monitoring 24 single-digit numbers (09) displayed in the four corners of a central screen that change asynchronously every five seconds. The participant is required to remove his/her foot from a pedal-switch as quickly as possible any time the target numeral 2 appears. Mean absolute tracking error (mm), number of correct detections (hits) and number of control losses are the primary performance measures. This 15-min task was performed before smoking and at 1.6, 3.1 and 5.6 h after starting smoking. Participants were trained before the study session to achieve stable task performance and minimize practice effects. Critical tracking task training continued until participants performed with < 10% variance from the average measured over three trials. The divided attention task was practiced for 12 minutes, regardless of final performance.

Calculations and statistical analysis


SPSS 15.0 for Windows (SPSS, Chicago, IL) was utilized for statistical evaluations. GLM repeated-measures ANOVA were conducted to investigate changes in participants VAS scores over time, with a Huynh-Feldt epsilon correction applied to counter sphericity violations. Bonferroni post-hoc testing determined differences between specific time points. Wilcoxon signed-rank tests evaluated differences between whole blood and plasma CIF values at each time point. Areas within concentration-effect curves were calculated in a manner similar to Galeazzi et al. (34) by subtracting the total area under the lower limb of the curve from that under the upper limb, determined by the trapezoidal rule. P values 0.05 were considered significant. Other statistical calculations were performed with GraphPad Prism 5.2 for Windows (GraphPad Software, La Jolla, CA). Go to:

Results
Samples
Participant demographics and self-reported smoked cannabis histories are detailed in Table I. Ten participants (nine male, one female) completed the protocol. Age ranged from 18.545.7 years and BMI ranged from 18.132.0 kg/m2. Mean (standard deviation; SD) self-reported joints smoked in the previous 14 days was 11.7 (2.2), with a range of 10 168 joints. Only one scheduled blood sample (Participant I at 0.25 h) was not collected, due to catheter dislodgement.

Table I Demographics and Self-Reported Cannabis Smoking Characteristics for 10 Adult Cannabis Smokers*

Subjective effects following smoked cannabis


Cannabis smoking significantly increased Good drug effect [F(2.37, 21.36) = 17.6, P < 0.001], High [F(2.78, 25.02) = 26.03, P < 0.001], Stoned [F(2.94, 26.49) = 13.29, P < 0.001], Stimulated [F(3.25, 29.30) = 8.54, P < 0.001] and Sedated [F(4.70, 42.34) = 3.35, P = 0.014], but did not significantly change Anxious [F(4.82, 43.35) = 1.15, P = 0.349] or Restless [F(2.55, 22.91) = 2.58, P = 0.086] VAS scores. Mean blood concentration-effect curves for each of the seven VAS scales are shown in Figures 1AH. For all scales, observed peak THC blood concentration and peak VAS scores occurred 0.25 h after starting smoking, at the time of the first blood collection. Blood THC concentrations decreased rapidly, although subjective effects persisted. Starting approximately 1 h after smoking, subjective effects decreased linearly through 6 h, with THC concentrations changing more slowly during this period. This pharmacodynamicpharmacokinetic relationship described a counterclockwise hysteresis for all VAS measures, demonstrating the lack of correlation between blood concentrations and observed effects until after the initial distribution phase.

Figure 1. Median (inter-quartile range) whole-blood THC concentrations (A) and mean (SEM) hysteresis plots for seven VAS scales (BH) following smoking of a 6.8% THC cannabis cigarette (n = 10). VAS scores are calculated as change from baseline (0.5 ... While group mean blood concentration-VAS relationships yielded consistent hysteresis effects, concentration-effect curves for individual subjects varied substantially (Figures 2AL). All participants (except I) reported peak subjective High between 66 and 85; with peak whole blood THC concentrations at the time of these responses ranging from

13 (Participant A) to 63 g/L (Participant L). There was no apparent relationship between hysteresis areas and age or self-reported frequency or chronicity of cannabis intake.

Figure 2. Individual (n = 10) hysteresis plots for High VAS scales following smoking of a 6.8% THC cannabis cigarette. VAS scores are calculated as change from baseline (0.5 h). Samples collected at 0.5, 0.25, 0.5, 1.0, 2.0, ... Five-point Likert scales displayed varied effects. Participants reported significant increases compared to baseline for Slowed or slurred speech [F(2.10,18.9) = 4.35, P = 0.026], Feel Hungry [F(5.86, 52.7) = 22.0, P < 0.001], Feel thirsty [F(5.37, 48.3) = 19.2, P < 0.001], Shakiness/tremulousness [F(1.81, 16.3) = 4.19, P = 0.037] and Dry mouth or throat [F(6.34, 57.0) = 20.6, P < 0.001], with no significant increases observed for other Likert scales.

Cardiovascular measures
Cannabis smoking significantly increased heart rate only at 0.5 h [ F(1, 9) = 11.99, P = 0.007]. Diastolic blood pressure decreased significantly only from 0.5 to 1 h, [F(1, 9) = 8.95, P = 0.015]. Systolic blood pressure and respiratory rate were unaffected at all times during the session.

Cannabis influence factor


CIF increased substantially immediately following cannabis smoking, reaching a median peak (range) of 150 (93250) in whole blood and 170 (79270) in plasma 0.25 h after starting smoking (Figure 3). CIF then rapidly decreased, although the rate of change lessened over time. Median (range) whole blood CIF was 13 (0.032) 4 h after smoking and 8.0 (0.027) 6 h after smoking; plasma CIF was 14 (6.934) at this final observation. Six-hour plasma CIF was significantly higher than baseline [F(1, 9) = 19.24, P = 0.002], although whole blood CIF was not [F(1, 9) = 4.35, P = 0.067]. No significant differences were observed between plasma and whole blood CIF at any time other than 6 h (T = 2, P = 0.037).

Figure 3. Mean (SEM) CIF and VAS High score following ad libitum smoking of a 6.8% THC cannabis cigarette (n = 10). VAS score is calculated as change from baseline (0.5 h). CIF calculated from molar whole blood concentrations as 100 ...

Critical tracking task/divided attention task


Cannabis smoking had no significant effects on critical tracking task performance in this population of almost-daily, chronic cannabis smokers (Figure 4). For the divided attention task, significant differences were observed between baseline and 3 h, F(3, 27) = 2.4, P = 0.022 for the number of correct identifications (hits) in the secondary peripheral stimuli task, although no significant differences were observed 1.5 h after smoking. No significant effects due to cannabis smoking were observed for control losses, reaction time or tracking error.

Figure 4. Mean (SEM) values (n = 10) for lambda-c in the critical tracking task (CTT) and tracking error, hits and reaction time in the divided attention task (DAT) as a function of time after smoking ad libitum a single 6.8% THC cannabis cigarette. Samples collected ... Go to:

Discussion
The present study characterizes acute cannabis effects over 6 h after smoking a 6.8% THC cigarette containing approximately 54 mg THC in a sample of heavy, chronic cannabis smokers. Robust subjective and cardiovascular effects were observed, while psychomotor performance changes were modest. THC potency was selected to closely represent current mean illicit cannabis potency in seized US drugs, which increased from 3.4% in 1993 to 8.8% in 2008 (35). Thus, these data provide insight into the pharmacodynamics and pharmacokinetics of the higher-potency cannabis currently prevalent in the US. As expected, smoking a single cannabis cigarette significantly increased positive effects such as Good drug effect, High, Stoned, Stimulated and Sedated, while having no significant effect on negative effects such as Anxious or Restless. The frequency and chronicity of cannabis smoking in our population may have minimized negative effects and adverse events.

When VAS subjective effects were compared to simultaneously collected whole blood THC concentrations, counter-clockwise hystereses were observed. Cocchetto et al. (36) first detailed hysteresis following cannabis smoking, indicating a delay in the onset of subjective effects as compared to plasma concentrations. Barnett et al. (37) also observed a counter-clockwise hysteresis for heart rate and THC plasma concentrations, and Chiang et al. (38) described differences in hysteresis areas for oral, smoked and intravenous THC based on differences in administration route or cannabis potency. In 1993, Cone and Huestis detailed hysteresis for How much drug effect do you feel? (measured by VAS) with multiple measurements of subjective effects and plasma concentrations obtained with a continuous blood withdrawal pump during cannabis smoking (10, 39). Data from the present study also documented a counter-clockwise hysteresis starting immediately after the end of smoking, and during the initial distribution and elimination phases. Counter-clockwise hystereses are typically observed for drugs with a larger volume of distribution, drugs with active metabolites, or an indirect mechanism of drug action (40). Highly lipophilic THC, with 2- or 3-compartment pharmacokinetics and an equipotent metabolite (11-OH-THC), is a likely candidate for counter-clockwise hysteresis. Here, for the first time, we report hystereses for whole blood THC concentrations and several VAS scores, improving our understanding of subjective THC effects following ad libitum smoked cannabis. Yet, as demonstrated in Figures 2AL, inter-individual variability is high for these concentration-effect curves. Hystereses may vary substantially depending on dose, experimental setting or prior chronicity or frequency of use (40). Significant impairment differences were observed in tracking and divided attention tasks in frequent and occasional users following 500 g/kg smoked cannabis (28). The authors suggested tolerance development to acute THC-induced impairment in the heavy smoker group, similar to the findings of Hart et al. (20). In the present study, we found minimal performance changes in critical tracking and divided attention tasks 1.55.5 h after smoking 700 g/kg (range 4801,000) THC (Table I). Most participants in the present study smoked cannabis almost daily for a mean (SD) of 7.2 (5.3) years, with several self-reporting numerous smoked joints per day. These findings support those reported by Ramaekers et al. and Hart et al., documenting significant subjective response and minimal impairment in driving-related psychomotor tasks in chronic daily cannabis smokers. Additional research is needed to determine whether tolerance develops to the impairment of complex driving skills in chronic daily cannabis smokers compared those who use cannabis occasionally. Menetrey et al. investigated whole blood CIF following a cannabis decoction in milk containing 45.7 mg THC (30). A mean peak CIF of approximately 50 was achieved, decreasing to approximately 18 after 5.5 h, and 5 after 24 h. The present study examined CIF in whole blood and plasma following smoked cannabis intake, yielding peak median CIF of 150 and 170, respectively. CIF in whole blood and plasma were similar (Figure 3), with absolute CIF decreasing much more quickly than subjective high. This was expected, because blood cannabinoid concentrations decreased much more rapidly than subjective response (Figure 1AH). Because we observed minimal impairment on tracking and divided attention tasks through 4 h after smoking, this model does not appear suitable for quantifying impairment following cannabis consumption in heavy, chronic cannabis smokers. Additionally, four participants had whole blood CIF > 10 in the baseline sample

0.5 h before smoking despite residence for more than 12 h on the closed clinical unit, precluding CIF as a definitive marker for recent cannabis intake. As described by Schwope et al. (15), Mareck et al. (41), and Kelly et al. (42), detection of minor cannabinoids, CBD, CBN or THC-glucuronide may be beneficial in identifying recent cannabis intake. Our data indicated that these markers occurred during the period of strong subjective effects and potential impairment. Unfortunately, the frequent lengthy delay between cannabis smoking and sample collection during DUID and other investigations may preclude this approach, because concentrations may fall below current limits of quantification (15). Concentration-effect curves for whole blood THC and subjective effects produced counterclockwise hysteresis following ad libitum smoking of a single 6.8% THC cannabis cigarette. Despite whole blood THC concentrations of 1363 g/L 15 min after the start of cannabis smoking, little psychomotor impairment was observed, although there were robust cardiovascular and subjective responses. These data suggest differential tolerance to the psychomotor, but not other effects of THC in heavy, chronic cannabis smokers, as previously reported by others (20, 28). CIF did not accurately predict impairment or subjective response following a single cannabis cigarette, but a high value ( 30) may suggest recent cannabis intake. These data advance our understanding of whole blood pharmacodynamic-pharmacokinetic relationships following cannabis smoking in heavy, chronic cannabis smokers. Additional research is needed to provide insight into pharmacodynamic-pharmacokinetic relationships in occasional cannabis smokers, because impairment may differ with less frequent exposure. Go to:

Acknowledgments
We acknowledge Erin Karschner and Karl Scheidweiler for technical assistance in manuscript preparation and the NIDA Intramural Research Program clinical research team, especially Rebecca Price, John Etter, Janeen Nichels and Daniel Lipstein, for clinical support. This research was supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health. Go to:

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Forensic Sci Int. Author manuscript; available in PMC 2012 October 10. Published in final edited form as: Forensic Sci Int. 2011 October 10; 212(1-3): 247251. Published online 2011 July 20. doi: 10.1016/j.forsciint.2011.06.028 PMCID: PMC3413259 NIHMSID: NIHMS309333

Postmortem redistribution of 9tetrahydrocannabinol (THC), 11-hydroxyTHC (11-OH-THC), and 11-nor-9carboxy-THC (THCCOOH)


Michael G. Holland,1,2 David M. Schwope,3 Robert Stoppacher,4,5 Shane B. Gillen,5 and Marilyn A. Huestis3 Author information Copyright and License information The publisher's final edited version of this article is available at Forensic Sci Int Go to:

Abstract
Introduction
Postmortem redistribution (PMR), a well-described phenomenon in forensic toxicology for certain drugs, can result in increased central blood concentrations relative to peripheral blood concentrations. 9-tetrahydrocannabinol (THC), the primary psychoactive component in cannabis or marijuana, is the illicit substance most commonly implicated in driving under the influence of drugs (DUID) cases and fatally-injured drivers. No investigation of PMR of THC in human blood has been reported to date.

Methods
Matched heart and iliac postmortem blood specimens were collected from 19 medical examiner cases (16 Males, 3 Females) with positive cannabinoid urine immunoassay screens. THC, its equipotent metabolite 11-hydroxy-THC (11-OH-THC) and nonpsychoactive metabolite 11-nor-9-carboxy-THC (THCCOOH) were quantified by twodimensional gas chromatography-mass spectrometry with cryofocusing, with 0.5 ng/mL limits of quantification (LOQ) for all analytes.

Results

10 cases had quantifiable THC and 11-OH-THC; THCCOOH was present in all 19. Median (range) heart:iliac blood ratios were 1.5 for THC (range: 0.33.1); 1.6 for 11-OH-THC (range: 0.32.7); and 1.8 for THCCOOH (range: 0.53.0).

Discussion
Cannabinoids, in general, exhibited a mean and median central: peripheral (C: P) concentration ratio of less than 2 following death. A trend was observed for greater PMR with increasing postmortem interval between death and sampling. To our knowledge, these are the first data on THC PMR in humans, providing important scientific data to aid in the interpretation of postmortem cannabinoid concentrations in medico-legal investigations. Keywords: Postmortem cannabis, marijuana Go to: redistribution, THC, tetrahydrocannabinol, cannabinoids,

Introduction
Postmortem redistribution (PMR) is a phenomenon in postmortem toxicology that has been recognized for over two decades. PMR is a process whereby drugs can diffuse from contiguous tissues with higher drug concentrations into the surrounding blood, and the concentration of drugs at certain anatomic sites (usually cardiac or central blood) can differ significantly from their antemortem concentrations. {[1][2][3], [4], [5], [6]}. While the likelihood of a specific drug exhibiting PMR is dependent on many factors, detecting PMR requires investigators to sample post-mortem blood from both central and peripheral sites. For drugs demonstrating PMR, diffusion occurs from tissue stores in cardiac, lung, and liver tissue; as well as gastric contents, along the concentration gradient into contiguous central blood in the heart or large vessels (vena cava, aorta, subclavian, etc). Drug properties, including lipophilicity (octanol:water partition co-efficient or Log P), charge, pKa and volume of distribution (Vd), influence the degree of PMR, as do host factors such as body fat composition, age and nutritional status {[1][2][3], [4], [6]}. Body temperature and degree of decomposition, time interval since death, body position, and body handling (causing movement of central blood) also may affect the degree of PMR {[5][3], [4]}. Multiple drug classes including tricyclic antidepressants [7], cardiovascular drugs[3], and some drugs of abuse such as cocaine [2] display well-documented PMR, although reports are often hindered by small sample sizes (often limited to only 35 cases) and/or high variability. Many drugs subject to PMR are basic (pKa > 7) and highly lipophilic, with Log P >0.5, and with large apparent Vd{[1][2][3], [4], [5],[6]}. For those drugs that exhibit PMR, central blood samples (obtained from cardiac chambers) can have 35 times greater drug concentrations than simultaneously obtained peripheral (femoral, iliac,) blood samples. The heart:femoral or central:peripheral (C:P) ratio is a common PMR quantifier; when this ratio is substantially greater than unity, the drug is considered to exhibit PMR. 9-tetrahydrocannabinol (THC), the primary psychoactive component of cannabis or marijuana, with a pKa of 10.6, is actually an acidic phenolic compound, but is quite

lipophilic, with a high Log P of 5.648 [8] and a high Vd (414 L/kg) [7] and would be expected to demonstrate detectable PMR. Surprisingly, even though cannabis is the most commonly abused illicit drug and is often found in postmortem examinations, there are currently no published reports describing THC PMR (or lack thereof) in humans. As THC overdoses are exceedingly rare and almost never a direct cause of drug-overdose deaths, no lethal THC concentration in humans is described. Except in the setting of trauma resulting from drug-induced impairment, THC measurement in post-mortem specimens has little impact on the ultimate determination of cause and manner of death by medical examiners, and this fact is likely responsible for this relative scarcity of information. The major reference textbook in forensic toxicology regarding postmortem blood levels does not discuss the possible PMR of THC as it does for most other drugs [7]. Karch presents only a theoretical discussion of THC PMR based on its physical properties and the difficulties with interpretation of postmortem THC concentrations [9]. Drummer estimates THC PMR as most likely low to moderate, although no supportive data are presented[2]. While little information is available regarding THC PMR in humans, some recent studies investigated this phenomenon in animals. Brunet, et al. studied postmortem redistribution of THC utilizing the Large White Pig model[10]. Fifteen pigs were administered intravenous THC and euthanized two hours later, with blood and tissue samples collected at autopsy (0, 6, 15, 24 and 48 h post-mortem). Cardiac (central) and suprarenal inferior vena cava (authors considered peripheral) blood specimens were analyzed for THC (LOQ 0.5 ng/mL). The authors concluded that THC in this species is subject to PMR as C:P ratios increased with increasing postmortem interval. They also concluded that no collection site adequately reflects perimortem THC concentrations. Another study conducted by Hilberg, et al. studied PMR of a number of different drugs and compared human cases to experimental animal models[11]. In two human cases where THC was examined, postmortem femoral (obtained one day postmortem) to perimortem heart blood (obtained within one hour of death) ratio (i.e. peripheral to central ratio) for THC of 2.8 in one case and postmortem femoral (2 days postmortem) to perimortem neck blood (one hour postmortem) ratio of 0.4 was obtained in a second case. In the animal portion of the study, the authors reported THC concentrations in rats administered 30 mg THC by gastric tube. They compared antemortem heart blood THC concentrations to postmortem heart blood, postmortem suprarenal vena cava blood and postmortem vitreous humor THC concentrations. Mean (SEM) ratios of postmortem heart blood concentrations to postmortem vena cava blood concentrations were 1.9 (0.4). Although no fatalities from cannabis (or THC) overdose have been reported, cannabis use is an important public health issue. Numerous studies indicate THC is the most common substance found in DUID cases worldwide [12] [13]. Determining impairment due to THC is more complex than for ethanol, since impairment cannot be inferred by THC blood concentrations alone, especially for chronic cannabis smokers who may have detectable THC in whole blood for up to 7 days after last cannabis intake, when no longer impaired [14] However, given the degree of THC involvement in DUID and its implications on traffic accidents and fatalities, assigning culpability is increasingly important in many jurisdictions [13,15,16]. Therefore, this research was initiated to determine whether THC

exhibits PMR in humans. We postulate that the parent drug THC, being the most lipid soluble, would exhibit the most PMR. Since more water soluble drugs and metabolites generally have smaller Vd and generally exhibit less PMR, we also postulate that the more polar THC metabolites 11-OH-THC and THCCOOH would exhibit less PMR than the parent THC molecule. Go to:

Materials and Methods


Specimen Collection
Nineteen cases were selected from consecutive adult deaths falling under the jurisdiction of the Onondaga County Medical Examiners Office (OCMEO), Syracuse, NY, from July 2009 through October 2009. Cases were chosen if an autopsy examination was to be performed and if a postmortem urine immunoassay (Status DS, LifeSign, LLC, Somerset, NJ) was positive for cannabinoids. During autopsy, 1015 mL cardiac (central) and iliac vein (peripheral) blood was collected in appropriately-labeled Vacutainer sodium fluoride blood tubes (BD, Franklin Lakes, NJ). Bloods were frozen at 60 C and shipped frozen to Chemistry and Drug Metabolism, National Institute on Drug Abuse IRP (Baltimore, MD) for analysis. 20 control cases were selected in which a postmortem urine drug screen was negative for cannabinoids; these were utilized for analytical method validation of cannabinoids in postmortem blood. Central and peripheral control samples were collected, stored and transported similarly to study samples. In three cases, antemortem whole blood specimens (K2EDTA preserved) were available from referring hospitals. The dates and times of collection for these specimens were recorded.

Whole Blood Cannabinoid Analysis


A previously validated method[17] was utilized with slight modifications to complete postmortem blood analyses. Blank postmortem blood (0.5 mL) and blank whole blood (0.5 mL) were pipetted into culture tubes to which working calibrator solutions were added to produce final calibrator concentrations of 0.5, 1.0, 2.5, 5, 10, 25, and 50 ng/mL of all three analytes (THC, 11-OH-THC and THCCOOH). Working quality control (QC) solutions were added to produce final QC samples of 0.7, 2.0, 20 and 40 ng/mL that were analyzed in duplicate in each batch. 25 L deuterated THC, 11-OH-THC and THCCOOH internal standards (5 ng) were added to all calibrators, controls and authentic postmortem specimens, and proteins precipitated with 2 mL cold acetonitrile added in 0.5-mL increments while vortexing. After centrifugation (10 min, 3000 rpm), supernatants were decanted, solid phase extraction performed (United Chemical Technologies (UCT) Clean Screen ZSTHC020) and analytes quantified by 2-dimensional gas chromatography-mass spectrometry (2D GCMS) as previously described [17]

Method Validation

Whole blood cannabinoid method development and validation were previously reported [17]. The current postmortem method was validated for linearity, limit of detection (LOD) and limit of quantification (LOQ), analytical recovery and intra- and inter-assay imprecision. Four replicate QC samples were analyzed during 4 separate validation runs (prior to analyzing case specimens) for a total of 16 replicates. During actual case specimen batches (after validation was completed), duplicate QC samples were included to monitor ongoing assay accuracy and precision. Analytical recovery (acceptable within 80%120% of target concentration) and intra- and inter-assay imprecision (acceptable if 20% CV) from 16 replicates of quality control samples at 0.7, 2, 20 and 40 ng/mL for all three analytes, assayed in 4 separate batches. Transition peak area ratios for QC and authentic specimens were required to be within 20% of the mean peak area ratios for calibrators of each respective analyte to be considered acceptable. Coefficients of determination (r2values for the calibration curve; utilized to evaluate the fit of the linear regression to the empirical data.) were 0.990. LOD and LOQ were empirically determined by assaying decreasing analyte concentrations. LOD was the lowest calibrator concentration, with retention time within 2% of calibrator mean, acceptable chromatography, acceptable qualifier ion ratios, and signal-to-noise ratio 3:1. LOQ was the lowest calibrator meeting LOD criteria and quantifying within 20% of target. LOD and LOQ were 0.25 ng/mL and 0.5 ng/mL, respectively, for all analytes. Analytical recovery and inter- and intra-assay imprecision were within a priori specifications ( 20%). Potential endogenous matrix interference was investigated by analysis of 20 matched central and peripheral blank postmortem whole blood specimens.

Postmortem Validation
Dilution integrity, a measure of quantification accuracy after diluting a high-concentration specimen with blank matrix, was investigated for postmortem specimens up to a 1:4 dilution for THCCOOH (n = 6 each). Median (range) difference from undiluted specimens was 3.0% (13.2%7.3%), indicating acceptable dilution integrity up to a 1:4 dilution with blank blood for this analyte. Other analytes could not be investigated due to low initial concentrations and/or insufficient specimen volume. Reanalysis of some positive PMR specimens (central THC n = 4, peripheral THC n = 5, central 11-OH-THC n = 10, peripheral 11-OH-THC n = 6, central THCCOOH n = 19, peripheral THCCOOH n = 8) was performed to assess reproducibility. Individual quantifications differed by a median (range) of 3.6% (0.3%9.4%) THC, 2.8% (0.0%26.7%) 11-OH-THC and 1.4% (0.1% 11.0%) THCCOOH, indicating a lack of bias and acceptable analytical precision. All repeated specimens originally quantifying below LOQ (n = 12 across all analytes) quantified below LOQ during repeat analyses.

Statistical Analyses
Statistical calculations were performed with SPSS 15.0 for Windows (SPSS Inc, Chicago, IL). A Wilcoxon signed-rank statistical test (median values) was performed for cases where

there are both valid central and peripheral concentrations to determine potential differences (P < 0.05) between central and peripheral blood concentrations (n = 10, 10 and 19 for THC, 11-OH-THC and THCCOOH, respectively). As data were non-normal, linear regression analyses were performed to characterize the relationships between postmortem interval and C:P ratios. P values < 0.05 were required for statistical significance. Go to:

Results
Specimens
Matrix interference from putrefactive or in vitro breakdown products markedly affected chromatography in some specimens analyzed 210 months after collection. It was necessary to repeat analyses or utilize reduced specimen volume to achieve acceptable results for some postmortem specimens. Two specimens could not be quantified for THC and one for 11-OH-THC due to matrix effects that resulted in poor internal standard recovery, even after multiple quantification attempts. Table 1 shows overall statistics for quantification results in each matrix.

Table 1 Numbers and percentage of specimens having quantifiable 9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) concentrations from the 19 postmortem specimens

Cannabinoid PMR
Median (range) C:P blood ratios were 1.5 (0.33.1), 1.7 (0.32.7) and 1.8 (0.53.0) for THC, 11-OH-THC and THCCOOH, respectively (Figure 1), suggesting modest postmortem redistribution to the central blood for all three cannabinoids following death. Wilcoxon signed-rank testing yielded no significant difference between median central and peripheral blood concentrations for THC (T = 10.0, p > 0.164, r = 0.494) and 11-OH-THC (T = 9.5, p > 0.164, r = 0.487), although there was a significant difference between central and peripheral concentrations for THCCOOH (T = 39.0, p < 0.026, r = 0.517). Interestingly, cases #13 and #15 contained higher concentrations of all analytes in peripheral blood as compared to central blood, with case #13 C:P ratios 0.5 for THC and 0.8 for THCCOOH; and case #15 C:P ratios 0.3 for THC, 0.3 for 11-OH-THC, and 0.5 for THCCOOH. These cases contributed to the high variability observed in these data.

Figure 1 Central: Peripheral Ratio A trend toward positive correlation for time interval from death to blood collection and central: peripheral ratio for all analytes was observed (Figure 2). R2 values: THC, 0.0866; 11-OH-THC, 0.0863; THCCOOH, 0.0514. However, for all analytes, slopes were not significantly different than zero, with slopes (95% CI) of 0.028 (0.0540.110) for THC, 0.030 (0.0490.109) for 11-OH-THC and 0.019 (0.0240.062) for THCCOOH.

Figure 2 C:P Ratio vs Post-Mortem Interval

Antemortem Specimens
Antemortem whole blood specimens were quantified for cases #5, #8 and #11 as shown in Table 2. In all cases, antemortem concentrations were greater than or equivalent to postmortem concentrations. In case #5 antemortem concentrations for THC and 11-OHTHC were quite low and similar to postmortem levels; however, THCCOOH (28.0 ng/mL) was more than twice the highest postmortem concentration (12.4 ng/mL). Conversely, in case #11 antemortem THC and 11-OH-THC were within 20% of postmortem concentrations, but THCCOOH was 49.5% higher (160 vs 107 ng/mL)

Table 2 Demographics and postmortem and antemortem 9-tetrahydrocannabinol (THC), 11hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) concentrations.

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Discussion
A drug is consider to exhibit post-mortem redistribution when its C:P ratio is substantially greater than unity. Here we report median C:P blood ratios between 1.5 and 2.0 for THC, 11-OH-THC and THCCOOH, suggesting modest PMR for all analytes and confirming the prediction for low to moderate redistribution as postulated by Drummer[2]. The relatively wide ranges and variability of C:P ratios is typical of PMR findings of many other drugs[7] and is illustrative of variables that can affect these results such as cause of death, postmortem interval and tissue breakdown. Other drugs subject to PMR, such as tricyclic antidepressants, cardiovascular drugs and cocaine, typically demonstrate increased redistribution with increased postmortem interval[18][19]. Here we report a similar trend for cannabinoids, consistent with these other drug classes. As the postmortem interval increases and decomposition progresses, additional drug is released from central cavity organs, resulting in C:P ratio increases. Although slopes here were not significantly different from zero for any analyte, we believe this results from the high variability inherent in C:P ratios. Further study is warranted to substantiate these preliminary findings. We postulated that parent THC would exhibit increased PMR compared to its more polar metabolites. Our results did not support this hypothesis, as PMR was similar across all analytes. Of particular interest was the finding that the three antemortem specimens contained higher concentrations than post-mortem specimens for THC, 11-OH-THC and THCCOOH with the greatest difference observed for THCCOOH. The interval from antemortem specimen acquisition to time of death was approximately 4.5 and 5.5 h in cases #5 and #8, respectively. This would have allowed for further metabolism, tissue distribution or excretion in the time between blood collection and death, and could explain lower postmortem concentrations. However, in case #11, the antemortem specimen was obtained minutes prior to death, yet the antemortem THCCOOH concentration was still approximately 50% higher than the postmortem concentration. This case presented the highest postmortem (and antemortem) concentrations of any in the current study, with approximately 60 ng/mL antemortem THC. Concentrations of this magnitude indicate recent cannabis intake, likely within 0.1 3.1 hours of blood collection according to predictive models[20,21]. In the two human cases described by Hilberg, perimortem and postmortem blood THC concentrations were assayed. In one case, perimortem heart blood was obtained < 1 hour after death, and was compared to autopsy femoral blood one day later, and the postmortem femoral concentration to perimortem heart blood concentration was 2.8, likely demonstrating PMR. However, the other case had a ration of perimortem THC neck vein blood concentration to femoral blood concentration (from autopsy two days later) of 0.4, indicating the post-mortem blood THC concentration was more than twice the peri-mortem value. In addition, the rat data from the same study showed a post-mortem vena cava blood THC to antemortem heart blood THC concentration ratio of 0.6, indicating antemortem blood THC nearly twice the concentration of post-mortem.[11]. If this trend of higher THC concentrations in ante-mortem blood than in post-mortem can be confirmed in future cases, it may have significant implications for future work in determining antemortem impairment using post-mortem blood concentrations and could have major

implications in DUID cases: the possibility of falsely high post-mortem samples causing erroneous impairment conclusions would be negated. In addition, further studies could possibly focus on whether current formulae for estimating impairment and time of last cannabis use could be applied to post-mortem THC and THC-COOH levels. The low numbers of antemortem values (n = 3) render these results preliminary; further study and confirmation are needed. However, the feasibility of finding significant numbers of cases where antemortem blood is drawn moments before death is unlikely. Another possible explanation for higher antemortem values is the potential difference in analyte stability between the K2EDTA-preserved antemortem blood specimens and the NaF-preserved postmortem blood specimens. In one study, THCCOOH and the esterlinked THCCOOH-glucuronide demonstrated time- and temperature-dependent concentration changes during fortified plasma and urine storage[22]. However, in another study, 1 month of either room temperature or 4C storage yielded no concentration differences in fortified THCCOOH whole blood samples stored in various blood collection tubes (including NaF and EDTA) [23]. It should be noted these were fortified, not authentic, specimens and did not include THCCOOH-glucuronide. Further research regarding analyte stability, especially THCCOOH-glucuronide, in these matrices is warranted as matrix-induced stability differences could confound interpretation. The detection window of cannabis impairment is poorly defined. After acute cannabis consumption in occasional cannabis users (less than daily use), impairment is suggested for at least 4 to 8 h, with some indicating possible residual effects for as long as 24 h[24]. Specific concentrations are frequently meaningful when correlated with hours since last cannabis use. Extensive work by Huestis et al showed that in living volunteers who were less than daily cannabis smokers, time since last cannabis use is reliably predicted from plasma THC and THCCOOH concentrations. Predictive models were developed to estimate this time window and are often helpful in determining whether impaired driving had occurred.[2527]. It is currently unknown whether these models would be valid for impairment determination when using postmortem THC and THCCOOH concentrations for several reasons. First, impairment studies correlating driving performance and cannabinoid blood concentrations are conducted with live participants. Second, performance studies typically utilize plasma or serum, not whole blood as is used for postmortem analyses. While it has been demonstrated that average plasma: whole blood ratios for THC are approximately 0.5 in antemortem specimens, because THC does not partition well into erythrocytes [7], [28], [29]; documenting this ratio in postmortem specimens is more problematic. Third, it was previously unknown how postmortem whole blood concentrations correlated with antemortem concentrations, and most importantly, prior to this study, it was unknown whether THC exhibited PMR. Go to:

Conclusions

THC and its metabolites 11-OH-THC and THCCOOH undergo only modest PMR, much less than expected based on the lipophilic nature and the high Vd of the cannabinoids. Average C:P ratios for all analytes were less than 2.0. There was a positive trend (albeit not statistically significant) of increasing PMR and postmortem interval between death and blood draw observed for all analytes. Go to:

Acknowledgments
The authors would like to thank the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health and SUNY Upstate Medical University Department of Emergency Medicine and the Onondaga County Medical Examiners Office for supporting this research. Additionally, the authors would like to thank Allan Barnes and Karl Scheidweiler for technical assistance. Go to:

Footnotes
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