Abstract
Oral cannabis products (a.k.a. “edibles”) have increased in popularity in recent years. Most prior controlled pharmacokinetic evaluations of cannabis have focused on smoked cannabis and included males who were frequent cannabis users. In this study, 17 healthy adults (8 females), with no cannabis use in at least the past 2 months, completed 4 double-blind outpatient sessions where they consumed cannabis brownies containing Δ9-tetrahydrocannabinol (THC) doses of 0, 10, 25 or 50 mg. Whole blood and oral fluid specimens were collected at baseline and for 8 h post-brownie ingestion. Enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC–MS-MS) were used to measure THC and relevant metabolites. In whole blood, concentrations of THC and 11-hydroxy-THC (11-OH-THC) peaked 1.5–2 h after brownie consumption, decreased steadily thereafter, and typically returned to baseline within 8 h. Blood concentrations for 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) and THCCOOH-glucuronide were higher than THC and 11-OH-THC and these metabolites were often still detected 8 h post-brownie consumption. Women displayed higher peak concentrations for THC and all metabolites in whole blood compared to men, at least partially owing to their lower body weight/body mass index. Detection of THC in oral fluid was immediate and appeared to reflect the degree of cannabis deposition in the oral cavity, not levels of THC circulating in the blood. THC concentrations were substantially higher in oral fluid than in blood; the opposite trend was observed for THCCOOH. Agreement between ELISA and LC–MS-MS results was high (i.e., over 90%) for blood THCCOOH and oral fluid THC but comparatively low for oral fluid THCCOOH (i.e., 67%). Following oral consumption of cannabis, THC was detected in blood much later, and at far lower peak concentrations, compared to what has been observed with inhaled cannabis. These results are important given the widespread use of toxicological testing to detect recent use of cannabis and/or to identify cannabis intoxication.
Introduction
Cannabis is more accessible today than at any other point in modern human history as a result of policy reforms that have expanded its legalization. In the USA (as of 11 May 2020), cannabis is permitted for medicinal purposes in 33 states and the District of Columbia and for nonmedicinal (“recreational”) purposes in 11 states. Several countries outside of the USA also permit medicinal (e.g., Australia) or nonmedicinal (e.g., Canada) use of cannabis. In many of the locations where cannabis use is permitted, there is now a retail cannabis marketplace which contains a vast array of cannabis products that can be administered via various routes (e.g., inhalation via smoking or “vaping”, oral consumption, topical application, etc.).
Oral cannabis products, or “edibles”, have become a particularly popular method for cannabis administration. Indeed, recent survey studies indicate that between 30 and 47% of adult ever-cannabis users have consumed cannabis edibles (1, 2). Some cannabis users perceive cannabis edibles to be healthier and to provide stronger drug effects than smoking cannabis, and these perceptions increase the appeal of these products (3). Cannabis users may also prefer cannabis edibles because they can be used more discreetly than smoking cannabis (3, 4). Some cannabis users report that drug effects following consumption of cannabis edibles are often unpredictable (i.e., either too weak or too strong) (3), which is likely due to the fact that concentrations of Δ9-tetrahydrocannabinol (THC) are often labeled inaccurately for these products (5, 6). Because drug effects stemming from consumption of cannabis edibles are often unpredictable and delayed relative to cannabis inhalation, there is also a greater risk for acute overdose from these products (7, 8).
Due to the increased availability and use of a variety of cannabis products, there is a growing need to accurately detect recent cannabis use and/or cannabis intoxication in the workplace, at the roadside and in other settings. Presently, urine and blood are the primary biological matrices used to detect recent cannabis use or to infer intoxication, although there is a growing interest in using oral fluid as an alternative biological matrix for these same purposes because it can be collected easily and noninvasively (9). THC, the principal psychoactive constituent of cannabis, or metabolites of THC, are typically the main targets of such biological testing. For example, in many legal jurisdictions, blood THC concentrations are used to help infer whether an individual was driving while intoxicated following traffic stops. Importantly, however, there is great uncertainty as to whether extant biological cutoffs for detection of recent cannabis use/intoxication are appropriate for oral cannabis products, given that prior controlled studies have predominantly focused on the pharmacokinetics of inhaled cannabis.
To date, only a handful of studies have characterized the pharmacokinetics of oral cannabis in blood and/or oral fluid in a controlled fashion (10–14). Niedbala et al. (10), Swortwood et al. (12) and Newmeyer et al. (13) compared the pharmacokinetic profiles of THC and relevant metabolites in blood and/or oral fluid between one oral cannabis dose [~20–25 mg THC (10); ~ 50.6 mg THC (12,13)] and a similar dose of inhaled cannabis; Wachtel et al. (14) conducted a similar comparative study but with two doses of smoked and oral cannabis (~8.4 and 16.9 mg THC). In a study recently conducted in our laboratory, participants were assigned to receive a single oral cannabis dose containing either 10, 25 or 50 mg THC, and blood/oral fluid pharmacokinetics, as well as numerous pharmacodynamic outcomes (e.g., subjective drug effects, cognitive/psychomotor performance) were assessed (11). Collectively, these studies provided several noteworthy insights. First, maximum concentrations (Cmax) of THC in blood are substantially lower and delayed (i.e., greater time to maximum concentration; Tmax) following oral consumption of cannabis compared to inhalation, though Cmax concentrations of the primary THC metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) are higher, with longer detection windows, for oral cannabis (11, 13). Second, subjective drug effects and cannabis-induced impairment often persist well after quantitative blood and oral fluid THC concentrations return to zero (11). Lastly, there is considerable inter-individual variability with respect to oral cannabis pharmacokinetics in both blood and oral fluid (10–13).
Despite the scientific knowledge gained from these prior studies, various methodological limitations suggest additional controlled pharmacokinetic evaluations of oral cannabis are needed. For instance, Niedbala et al. (10), Swortwood et al. (12) and Newmeyer et al. (13) included moderate to heavy cannabis users who occasionally exhibited elevated cannabinoid levels at baseline due to cannabis use prior to study entry, partially obscuring the acute pharmacokinetic profile of the study dose for those participants. These two studies were further limited by the inclusion of a single study dose and predominantly male participants. Notably, though multiple oral cannabis THC doses were administered in our prior oral cannabis study, each individual participant only received a single dose (11). Each prior study was also generally limited by a small sample size. The present study builds upon prior studies by utilizing a within-subjects crossover design whereby study participants received four separate oral cannabis doses (0, 10, 25 and 50 mg THC) on outpatient visits completed at least 1 week apart. This design allowed us to better control for the considerable inter-individual variability in cannabinoid absorption and elimination following oral cannabis consumption, as each participant served as their own control. The present study also included almost an equal number of men and women who had not used cannabis for at least 2 months prior to study entry. This manuscript reports THC and THC metabolite pharmacokinetics (e.g., Cmax and Tmax values), as assessed via qualitative (enzyme-linked immunosorbent assay; ELISA) and quantitative (liquid chromatography–tandem mass spectrometry; LC–MS-MS) methods. Sensitivity, specificity and agreement between ELISA and LC–MS-MS results is also reported. Outcome measures are presented with all participants included and divided by males and females to facilitate sex comparisons.
Methods
Study design and procedure
Study volunteers were recruited via media advertisements and word of mouth. Interested individuals first completed a short telephone interview. Those who appeared eligible were invited to complete a screening visit at the Johns Hopkins Behavioral Pharmacology Research Unit (BPRU) where participants completed a physical examination that assessed cardiovascular health (via a 12-lead electrocardiogram), major organ and musculoskeletal systems and general appearance, as well as self-report assessments that assessed participants’ demography, medical history and recent use of drugs and alcohol [via the Timeline Follow Back (15)]. At screening, blood, urine and breath samples were also collected for routine chemistry, hematology and serology analysis, assessment of recent use of alcohol or illicit drugs and pregnancy testing (females). In order to be considered eligible, participants were required to be healthy, as determined via the above procedures, provide urine/breath samples that tested negative for recent use of cannabis, other illicit drugs and alcohol (at screening and prior to each session) and to have not used cannabis (according to self-report) for at least 1 month prior to participation; females also had to test negative for pregnancy at screening and prior to each session. This study was approved by the Institutional Review Board (IRB) of Johns Hopkins University School of Medicine (IRB00035394) and was conducted in accordance with ethical standards established in the Helsinki Declaration. All participants received compensation for their time and provided written informed consent prior to participation.
All participants completed four, double-blind, outpatient sessions in which they consumed a cannabis brownie that contained either 0, 10, 25 or 50 mg THC. All study procedures were conducted at the BPRU and each experimental session lasted approximately 8.5 h. THC dose order was counterbalanced and randomly assigned to each participant. Sessions were separated by at least 1 week to allow for satisfactory drug washout between sessions. At the beginning of each session, participants provided a urine sample which was tested for recent drug use and pregnancy status (for females) and completed an alcohol breathalyzer (positive results for any of these tests precluded study participation). After consuming a standardized low-fat breakfast (toast and jam), an intravenous catheter was placed in the nondominant arm of each participant to allow for repeated blood sampling. Next, participants consumed one cannabis brownie within 5 min under study staff supervision (they were not allowed to eat other snacks for at least 30 min after dosing to reduce variability in drug absorption across participants). Blood and oral fluid samples were collected prior to brownie consumption and for 8 h thereafter. Pharmacodynamic assessments (e.g., subjective drug effects, cognitive/psychomotor performance) were also collected throughout the study sessions and these results are reported separately (this manuscript is currently under review elsewhere).
Outcome measures
Blood specimens
Whole blood samples were obtained at baseline and 0.17, 0.5, 1, 1.5, 2, 3, 4, 5, 6 and 8 h after the end of the 5-min brownie consumption period in each experimental session. Once collected, specimens were mixed by inversion and aliquoted into two 5 mL plastic cryotubes. Blood specimens were stored at −60°C before being shipped, frozen on dry ice, to the Immunalysis Corporation (Pomona, CA) for analysis.
ELISA and LC–MS-MS methods were used to analyze all blood samples. Blood samples were tested with the Cannabinoids Direct ELISA Kit (Immunalysis, catalog # 205) using the manufacturer’s recommended procedure which specified a cutoff concentration of 10 ng/mL for THCCOOH. Cross-reactivities for this assay (as listed in manufacturer’s brochure) are: THCCOOH (100%), 11-nor-9-carboxy-Δ8-THC (110%), Δ9-THC (21%), Δ8-THC (45%), 11-OH-THC (<5%), 8–11-dihydroxy-Δ9-THC (<5%), cannabinol (CBN) (<5%) and cannabidiol (CBD) (<5%). The manufacturer communicated to us that, at a 10 ng/mL cutoff concentration, the cross-reactivity for THCCOOH-glucuronide is 5% for this assay.
Blood samples were also tested for THC, 11-OH-THC, THCCOOH and THCCOOH-glucuronide concentrations using LC–MS-MS analysis; these analytic methods are described in detail elsewhere (16). As described in our prior studies (11, 17), THC, 11-OH-THC and THCCOOH concentrations were extracted from blood by solid-phase extraction (SPE); THCCOOH-glucuronide was extracted by liquid/liquid extraction (LLE). The limit of quantitation (LOQ) for all analytes in blood was 1 ng/mL and the upper limit of linearity (ULOL) was 100 ng/mL. Whole blood control samples were prepared from Cerillant solutions (Round Rock, TX) at target concentrations of 2 and 20 ng/mL.
Oral fluid specimens
Oral fluid samples were collected at the same time points (detailed above) as whole blood. These samples were collected by expectoration into glass culture tubes (Thermo Fisher Scientific, Waltham, MA, 16 × 100 mm, #14–959-35AA). The inside surface of the collection tubes was silanized with Sylon-CT™ (Sigma-Aldrich, St Louis, MO, USA, #33065-U), rinsed with methanol, and dried prior to specimen collection. Participants were not permitted to eat or drink anything for the 10 min before providing each specimen. Participants were given 5 min to produce each specimen. Once collected, all samples were wrapped in para-film and stored refrigerated until they were shipped overnight to Immunalysis Corporation (Pomona, CA) for analysis. All specimens were tested within 30 days of collection.
As with whole blood, oral fluids specimens were analyzed using ELISA and LC–MS-MS methods. Oral fluid samples were tested with the Saliva/Oral Fluids Cannabinoids Direct ELISA Kit [Immunalysis, catalog # 224; see also (18)]. As per the manufacturer’s recommended procedure, a cutoff concentration of 4 ng/mL for THC was used. Cross-reactivities for this assay (as detailed in the manufacturer’s brochure) are: THC (100%), Δ8-THC (66.7%), CBN (4%), CBD (50%) and conjugated-THC (25%). The Ultra-Sensitive Cannabinoids Direct ELISA Kit was also used to test oral fluid samples; a cutoff concentration of 0.05 ng/mL for THCCOOH was used per the manufacturer’s recommended procedure. As described in the manufacturer’s brochure, cross-reactivities for this assay are: THCCOOH (100%), 11-nor-9-carboxy-Δ8-THC (125%), Δ9-THC (10%), 11-OH-THC (33%), CBN (<0.25%) and CBD (<0.25%).
THC and THCCOOH concentrations were also quantified in oral fluid using LC–MS-MS [see Coulter et al. (16) for description of methods]. For THC, the LOQ and ULOL for LC–MS-MS analysis were 1 and 100 ng/mL, respectively. For THCCOOH, the LOQ and ULOL for LC–MS-MS analysis were 0.02 and 0.1 ng/mL, respectively.
Study drug
Cannabis used in this study was obtained from the National Institute on Drug Abuse (NIDA) Drug Supply Program. Two batches of cannabis were used: an active batch and a placebo batch. The active cannabis batch contained 11% total THC (i.e., sum total of THC plus tetrahydrocannabinolic acid, THC-A); 0.1% CBD, and 0.8% CBN. The placebo cannabis batch was cannabis plant material from which the cannabinoids had been extracted using ethanol. The Johns Hopkins BPRU Pharmacy prepared and dispensed the cannabis brownies. Preparation of the brownies consisted of the following steps: (1) the cannabis was ground into a fine powder with a food processor, (2) the cannabis was baked at 250°C so that THC-A would decarboxylate to THC and (3) the decarboxylated cannabis, measured to achieve the target THC dose, or 250 mg of placebo cannabis was mixed with brownie batter, made in accordance with manufacturer instructions (the same amount of ingredients such as water, eggs, oil etc. was used to prepare each brownie). Each brownie was baked individually (in the same size baking tray) in order to ensure accurate dosing and that each brownie would be the same size (precisely weighed amounts of ground cannabis were included in each brownie). Subsequent independent testing confirmed that THC-A was fully decarboxylated to THC using these procedures and that target doses of THC were achieved for each study dose.
Data presentation and analysis
Demographic information and quantitative LC–MS-MS results are presented using descriptive statistics. Sensitivity, specificity and agreement between ELISA testing and confirmatory LC–MS-MS testing were calculated for THC (for both whole blood and oral fluid results) and for THCCOOH (for oral fluid only). These analyses were only conducted for active cannabis conditions (i.e., the placebo condition was omitted). For whole blood THC, the screening cutoff for ELISA testing was 10 ng/mL and the confirmatory cutoff was 2 ng/mL. For oral fluid, ELISA screening cutoffs were 4 ng/mL for THC and 0.05 ng/mL for THCCOOH and confirmatory cutoffs were 2 ng/mL for THC and 0.05 ng/mL for THCCOOH. Test results were categorized as either: true positive (TP; ELISA response ≥ cutoff concentration and LC–MS-MS positive), true negative (TN; ELISA response < cutoff concentration and LC–MS-MS negative), false positive (FP; ELISA response ≥ cutoff concentration and LC–MS-MS negative) or false negative (FN; ELISA response < cutoff concentration and LC–MS-MS positive). Sensitivity, specificity and agreement were calculated as follows: sensitivity (100 × [TP/(TP + FN)]), specificity (100 × [TN/(TN + FP)]) and agreement (100 × [(TP + TN)/(TP + TN + FP + FN)]).
Results
Participants
A total of 23 individuals completed the screening visit (and provided informed consent) and 17 of these individuals were deemed eligible and went on to complete all study procedures. Demographic and select substance use information for the 17 study completers (9 males, 8 females) are presented in Table I. Upon study entry, an average of 856 days had passed (SD = 2032; range 60–7665) since participants had last used cannabis. At screening and prior to each session, all participants provided urine samples which tested negative for cannabis and other drugs, which suggested compliance with presession abstinence requirements.
Table I.
Participant Characteristics
| Number or mean (SD) | |||
|---|---|---|---|
| Total (N = 17) | Males (N = 9) | Females (N = 8) | |
| Race (# Caucasian) | 11 | 7 | 4 |
| Age (years) | 25.4 (5.3) | 27.0 (6.4) | 23.6 (3.3) |
| Height (in) | 68.2 (5.1) | 71.4 (3.5) | 64.6 (4.0) |
| Weight (lbs) | 169.8 (35.8) | 191.2 (27.4) | 145.7 (28.6) |
| BMI (kg/m2) | 25.5 (3.9) | 26.3 (2.6) | 24.6 (5.0) |
| BMI (Median) | 25.4 | 26.4 | 22.5 |
| Days since last cannabis use | 856.1 (2032.1) | 1421.7 (2734.9) | 219.8 (128.7) |
| Tobacco cigarettes smoked per day | 0.4 (1.7) | 0.8 (2.3) | 0 (0) |
Whole blood and oral fluid results
Complete quantitative and qualitative whole blood and oral fluid results for each participant are presented in Supplementary Table I for the nonplacebo experimental conditions. Table II displays mean Cmax and Tmax values and Table III displays means (and individual ranges) for time to first detection for THC and THC metabolites in whole blood and oral fluid. Figures 1 and 2 present mean whole blood/oral fluid cannabinoid concentrations at baseline and for 8 h after cannabis brownie consumption for each THC dose.
Table II.
Mean Maximum Concentrations (Cmax), Time to Maximum Concentrations (Tmax) and Individual Ranges of THC and THC Metabolites in Whole blood and Oral Fluid Following Consumption of Cannabis Brownies
| Dose (mg) | THC Cmax (ng/mL; range) | THC Tmax (h; range) | 11-OH-THC Cmax (ng/mL; range) | 11-OH-THC Tmax (h; range) | THCCOOH Cmax (ng/mL; range) | THCCOOH Tmax (h; range) | THCCOOH- GLUC Cmax (ng/mL; range) | THCCOOH- GLUC Tmax (h; range) |
|---|---|---|---|---|---|---|---|---|
| All participants (N = 17) whole blood | ||||||||
| 10 | 0.8 (0–4) | 1.7 (0–4) | 1.4 (0–4) | 1.9 (0–5) | 12.6 (6–27) | 2.7 (1.5–5) | 14.1 (0–39) | 5.1 (0–8) |
| 25 | 3.4 (0–9) | 1.9 (0–5) | 4.0 (1–10) | 2.2 (1–5) | 32.8 (17–62) | 3.5 (2–6) | 50.2 (5–91) | 4.9 (2–8) |
| 50 | 5.9 (0–18) | 1.6 (0–3) | 6.5 (3–23) | 1.9 (0.5–4) | 49.6 (17–148) | 2.6 (1.5–4) | 73.9 (8–211) | 5.4 (4–8) |
| Oral fluid | ||||||||
| 10 | 104.6 (11–414) | 0.2 (0.2–0.2) | N/A | N/A | 0.1 (0–0.5) | 5.0 (2–8) | N/A | N/A |
| 25 | 246.9 (9–667) | 0.2 (0.2–0.5) | N/A | N/A | 0.2 (0–0.5) | 5.1 (2–8) | N/A | N/A |
| 50 | 638.4 (322–1196) | 0.3 (0.2–1) | N/A | N/A | 0.6 (0.2–1.2) | 4.6 (2–8) | N/A | N/A |
| Men (N = 9) whole blood | ||||||||
| 10 | 0.1 (0–1) | 1.0 (0–1) | 0.8 (0–1) | 1.6 (0–3) | 12.0 (6–21) | 2.7 (1.5–4) | 12.7 (0–39) | 5.6 (0–8) |
| 25 | 2.6 (0–8) | 1.8 (0–5) | 3.1 (1–5) | 2.1 (1–5) | 28.9 (17–42) | 3.8 (2–5) | 34.4 (5–71) | 5.0 (2–8) |
| 50 | 3.4 (0–9) | 1.8 (0–3) | 4.7 (3–10) | 2.2 (1–4) | 41.4 (22–72) | 2.8 (1.4–4) | 60.4 (8–103) | 5.0 (4–6) |
| Oral fluid | ||||||||
| 10 | 125.1 (11–414) | 0.2 (0.2–0.2) | N/A | N/A | 0.2 (0–0.5) | 5.6 (5–8) | N/A | N/A |
| 25 | 234.6 (9–667) | 0.2 (0.2–0.5) | N/A | N/A | 0.2 (0–0.4) | 5.3 (2–8) | N/A | N/A |
| 50 | 506.4 (322–682) | 0.3 (0.2–0.5) | N/A | N/A | 0.6 (0.2–1.2) | 4.6 (2–8) | N/A | N/A |
| Women (N = 8) whole blood | ||||||||
| 10 | 1.5 (0–4) | 1.8 (0–4) | 2.0 (0–4) | 2.1 (0–5) | 13.4 (6–27) | 2.8 (1.5–5) | 15.6 (9–27) | 4.6 (4–6) |
| 25 | 4.3 (1–9) | 2.1 (0.5–3) | 5.0 (2–10) | 2.3 (1–4) | 37.3 (27–62) | 3.3 (2–6) | 69.0 (28–91) | 4.8 (3–6) |
| 50 | 8.8 (2–18) | 1.5 (0.5–3) | 8.6 (3–23) | 1.6 (0.5–3) | 58.9 (17–148) | 2.3 (1.5–3) | 89.1 (34–211) | 5.9 (4–8) |
| Oral fluid | ||||||||
| 10 | 81.5 (17–196) | 0.2 (0.2–0.2) | N/A | N/A | 0.1 (0.03–0.1) | 4.5 (2–6) | N/A | N/A |
| 25 | 260.9 (57–555) | 0.2 (0.2–0.5) | N/A | N/A | 0.2 (0.1–0.5) | 4.9 (4–8) | N/A | N/A |
| 50 | 786.9 (358–1196) | 0.3 (0.2–1) | N/A | N/A | 0.6 (0.2–1.2) | 4.8 (3–8) | N/A | N/A |
Note: N/A = not available for that biological matrix.
Table III.
Mean (Range) Time to First Detection of THC and THC Metabolites in Whole Blood and Oral Fluid Following Consumption of Cannabis Brownies
| Dose (mg) | Blood THCCOOH ELISA (cutoff = 10 ng/mL) | Blood THC LC–MS-MS (LOQ = 1 ng/mL) | Blood 11-OH-THC LC–MS-MS (LOQ = 1 ng/mL) | Blood THCCOOH LC–MS-MS (LOQ = 1 ng/mL) | Blood THC-COOH- GLUC LC–MS-MS (LOQ = 1 ng/mL) | OF THC ELISA (cutoff = 4 ng/mL) | OF THCCOOH ELISA (cutoff = 0.05 ng/mL) | OF THC LC–MS-MS (cutoff = 0.5 ng/mL) | OF THCCOOH LC–MS-MS (cutoff = 0.5 ng/mL) |
|---|---|---|---|---|---|---|---|---|---|
| All participants (N = 17) detection time (h) to first positive | |||||||||
| 10 | 1.7 (1–3) | 1.1 (0.5–2) | 1.6 (0.5–5) | 0.8 (0.5–1.5) | 2.5 (1–4) | 0.2 (0–0.2) | 0.2 (0.2–0.2) | 0.2 (0.2–0.2) | 2.9 (0–6) |
| 25 | 0.9 (0.5–2) | 1.0 (0.5–3) | 1.0 (0.5–3) | 0.6 (0–1) | 1.7 (1–4) | 0.2 (0–0.5) | 0.3 (0.2–2) | 0.2 (0.2–0.2) | 2.6 (0.2–8) |
| 50 | 0.7 (0.5–1.5) | 0.9 (0.5–3) | 0.6 (0.5–1) | 0.5 (0–0.5) | 1.3 (1–3) | 0.2 (0.2–0.5) | 0.2 (0.2–0.5) | 0.3 (0.2–1) | 1.5 (0.2–8) |
| Men only (N = 9) detection time (h) to first positive | |||||||||
| 10 | 1.9 (1–3) | 1.0 (1–1) | 1.6 (1–3) | 0.8 (0.5–1.5) | 2.6 (1.5–4) | 0.2 (0–0.2) | 0.2 (0.2–0.2) | 0.2 (0.2–0.2) | 1.6 (0–4) |
| 25 | 0.9 (0.5–1) | 0.9 (0.5–1) | 1.0 (0.5–2) | 0.7 (0.5–1) | 1.9 (1.5–4) | 0.2 (0–0.5) | 0.4 (0.2–2) | 0.2 (0.2–0.2) | 3.0 (1–8) |
| 50 | 0.8 (0.5–1.5) | 1.1 (0.5–3) | 0.6 (0.5–1) | 0.5 (0.5–0.5) | 1.4 (1–3) | 0.2 (0.2–0.2) | 0.2 (0.2–0.2) | 0.2 (0.2–0.5) | 1.8 (0.2–8) |
| Women only (N = 8) detection time (h) to first positive | |||||||||
| 10 | 1.4 (1–3) | 1.2 (0.5–2) | 1.6 (0.5–5) | 0.8 (0.5–1.5) | 2.4 (1–4) | 0.2 (0.2–0.2) | 0.2 (0.2–0.2) | 0.2 (0.2–0.2) | 4.2 (2–6) |
| 25 | 0.9 (0.5–2) | 1.1 (0.5–3) | 1.0 (0.5–3) | 0.5 (0–1) | 1.4 (1–3) | 0.2 (0.2–0.2) | 0.2 (0.2–0.2) | 0.2 (0.2–0.2) | 2.1 (0.2–5) |
| 50 | 0.6 (0.5–1) | 0.7 (0.5–1.5) | 0.6 (0.5–1) | 0.4 (0–0.5) | 1.2 (1–1.5) | 0.2 (0.2–0.5) | 0.2 (0.2–0.5) | 0.3 (0.2–1) | 1.2 (0.2–3) |
Figure 1.
Quantitative mean (+SEM) whole blood concentrations of THC, 11-OH-THC, THCCOOH and THCCOOH-glucuronide.
Figure 2.
Quantitative mean (+SEM) oral fluid concentrations of THC, 11-OH-THC, THCCOOH and THCCOOH-glucuronide.
For whole blood, THC and 11-OH-THC concentrations followed a similar time course. That is, on average, both THC and 11-OH-THC were first detected approximately 0.5–1.5 h after brownie consumption, reached peak concentrations between 1.5 and 2 h, and declined steadily thereafter. At each active THC dose, Cmax values for blood THC and 11-OH-THC were also comparable (see Table II). After consumption of the 10 mg THC brownie, 10 participants (i.e., Participants #15, #35, #36, #37, #38, #39, #40, #41, #46 and #50) had no detectable levels of THC in their blood and 11-OH-THC was also not detected for two of these individuals (Participants #36 and #50; Supplementary Table I). Moreover, THC was not detected in the blood of several participants after consumption of the 25 mg (Participants #36 and #39) or 50 mg THC brownies (Participant #39; Supplementary Table I). Mean Cmax and Tmax values for blood THCCOOH and THCCOOH-glucuronide concentrations were higher compared to THC and 11-OH-THC (Table II) and these metabolites were often detected at the final blood draw time point, 8 h after brownie consumption (see Supplementary Table I). Cmax and Tmax blood concentrations were higher for THCCOOH-glucuronide compared to THCCOOH at each active THC dose (Table II). Blood concentrations for THC and each THC metabolite increased in a dose-orderly manner.
For oral fluid, THC concentrations peaked at the 10 min post-brownie consumption time point and declined sharply thereafter (Figure 2). THC was detected much earlier in oral fluid than in blood. Cmax concentrations for THC in oral fluid were higher than those observed in whole blood. Mean time to first detection was similar for oral fluid THC across ELISA and LC–MS-MS methods. Following brownie consumption, THCCOOH was generally first detected after 1.5–3 h and peaked between 3 and 5 h. For several participants, THCCOOH was detected at the final time point, 8 h after brownie consumption (see Supplementary Table I). Oral fluid Cmax concentrations for THCCOOH were substantially lower than those observed for THC and also much lower than THCCOOH concentrations in whole blood (Table II).
Sensitivity/specificity and agreement between ELISA and LC–MS-MS
Sensitivity, specificity and agreement results between ELISA screening and confirmatory LC–MS-MS testing for whole blood THC and oral fluid THC and THCCOOH are summarized in Table IV. For whole blood and oral fluid THC, there was high agreement between ELISA and LC–MS-MS results. Specificity, or the ability to accurately detect true negative results, was slightly worse for oral fluid ELISA THC testing compared to whole blood (i.e., 97.0 vs 86.1%). For oral fluid THCCOOH, agreement, sensitivity and specificity between ELISA and LC–MS-MS were generally worse due to a higher number of false positive and false negative results for this analyte.
Table IV.
Comparisons of Immunoassay Responses (ELISA) to Confirmation Analyses (LC–MS-MS) in Blood and Oral Fluid Following Consumption of Cannabis Brownies
| Blood THCCOOH ELISA (cutoff = 10 ng/mL) vs THCCOOH LC–MS-MS (confirmation = 2 ng/mL) | Oral Fluid THC ELISA (cutoff = 4 ng/mL) vs THC LC–MS-MS (confirmation = 2 ng/mL) | Oral Fluid THCCOOH ELISA (cutoff = 0.05 ng/mL) vs THCCOOH LC–MS-MS (confirmation = 0.05 ng/mL) | |
|---|---|---|---|
| All participants (N = 17) | |||
| #True positive (%) | 387 (69.0) | 255 (48.6) | 183 (34.1) |
| #True negative (%) | 131 (23.4) | 217 (39.8) | 164 (30.9) |
| #False positive (%) | 4 (0.7) | 35 (6.4) | 136 (25.8) |
| #False negative (%) | 39 (7.0) | 14 (2.7) | 35 (6.5) |
| % Sensitivity | 90.8 | 94.8 | 83.9 |
| % Specificity | 97.0 | 86.1 | 54.7 |
| % Agreement | 92.3 | 90.6 | 67.0 |
| Men only (N = 9) | |||
| #True positive (%) | 198 (66.7) | 133 (48.0) | 104 (37.0) |
| #True negative (%) | 71 (23.9) | 114 (39.7) | 78 (27.7) |
| #False positive (%) | 0 (0.0) | 17 (5.9) | 71 (25.3) |
| #False negative (%) | 28 (9.4) | 10 (3.6) | 20 (7.1) |
| % Sensitivity | 87.6 | 93.0 | 83.8 |
| % Specificity | 100.0 | 87.0 | 52.3 |
| % Agreement | 90.5 | 90.1 | 66.7 |
| Women only (N = 8) | |||
| #True positive (%) | 189 (71.6) | 122 (49.2) | 79 (31.0) |
| #True negative (%) | 60 (22.7) | 103 (39.9) | 86 (34.5) |
| #False positive (%) | 4 (1.5) | 18 (7.0) | 65 (26.3) |
| #False negative (%) | 11 (4.2) | 4 (1.6) | 15 (5.8) |
| % Sensitivity | 94.5 | 96.8 | 84.0 |
| % Specificity | 93.8 | 85.1 | 56.9 |
| % Agreement | 94.3 | 91.1 | 67.3 |
Note: 0 mg THC condition was not included in these analyses.
Sex differences
Figure 3 displays whole blood and oral fluid cannabinoid concentrations by sex following consumption of the 50 mg THC cannabis brownie. Overall, in each nonplacebo cannabis brownie condition, women exhibited higher concentrations of THC and all THC metabolites in whole blood than males. Notably, of the 10 aforementioned participants who had no detectable levels of blood THC and/or 11-OH-THC following consumption of the 10 mg THC cannabis brownie, only two of these individuals (i.e., Participants #35 and #41) were female (Supplementary Table I). Despite Cmax differences, Tmax values for cannabinoids in whole blood did not differ systematically by sex (Table II). Moreover, mean times to first detection for THC (both ELISA and LC–MS-MS) and THC metabolites in whole blood were largely similar for males and females.
Figure 3.
Quantitative mean (+SEM) whole blood and oral fluid cannabinoid concentrations divided by sex for 50 mg THC dose.
For oral fluid, females again had higher Cmax concentrations for THC at the 50 mg dose, but this trend was not observed at the 10 or 25 mg THC doses (Table II). THCCOOH oral fluid concentrations were similar for males and females. Time to first detection for oral fluid cannabinoids was similar for men and women.
Description of adverse events
One male participant (#15) vomited twice after consumption of the 50 mg THC cannabis brownie (once 2.25 h after ingestion and again 3 h after ingestion). Another male (#36) vomited 2.20 h after ingesting the 25 mg THC cannabis brownie. Given the delay of more than 2 h prior to emesis in both cases, it is not believed that this significantly impacted study outcomes, as food and liquid are generally emptied from the stomach within 2 h of ingestion (19). Moreover, pharmacokinetic data for these individuals were generally in line with other participants who did not vomit following brownie ingestion (see Supplementary Table I).
Discussion
Toxicological examination of THC or metabolites of THC in human bodily fluids is commonly used to test for recent cannabis use or identify instances of cannabis intoxication in the workplace, at the roadside and in many other important settings. Given the recent proliferation of oral cannabis products (a.k.a. “edibles”) and expansion of the legal cannabis market, the pharmacokinetics associated with oral cannabis consumption need to be further elucidated; this information can help refine biological standards for determining recent cannabis use and/or intoxication. The present study examined the acute pharmacokinetic profile of four doses of oral cannabis (0, 10, 25 and 50 mg THC) in human blood and oral fluid. Participants included healthy adults who had not used cannabis for at least 2 months prior to enrollment. This study improved upon earlier controlled evaluations of oral cannabis by including almost an equal number of men and women (8 women and 9 men), multiple doses of THC (including a placebo condition) and utilizing a within-subjects design whereby all participants received each study dose.
There were several findings in this study that were consistent with prior controlled examinations of oral cannabis. First, detection of all cannabinoids in blood following oral cannabis consumption was delayed (with lower Cmax concentrations), relative to what has been observed following cannabis inhalation (17, 20). Second, all blood cannabinoids increased in a dose-orderly fashion following oral cannabis consumption and blood THC and 11-OH-THC levels were lower, with shorter detection windows, compared to THCCOOH and THCCOOH-glucuronide (11, 13). Third, concentrations of THC peaked sooner, and were far higher, in oral fluid compared to whole blood, and THCCOOH oral fluid concentrations were very low overall for all study doses (11, 12). Conversely, there were some noteworthy differences between this study and prior pharmacokinetic evaluations of oral cannabis, including our previous between-subjects study which administered either 10, 25 or 50 mg THC cannabis brownies to a similar participation population (11). The range of Cmax concentrations were larger in the present study for the 25 mg (range: 0–9 ng/mL) and 50 mg THC conditions (range: 0–18 ng/mL) compared to the same doses in our prior study (25 mg: range 3–4 ng/mL; 50 mg: range 1–5 ng/mL) (11). However, the ranges of Cmax values for blood THC we observed in the 50 mg THC condition were similar to those reported by Newmeyer et al. (13) following consumption of 50.6 mg THC cannabis brownies by moderate (3.6–22.5 ng/mL) cannabis users. This variability in blood THC concentrations is at least partially due to THC undergoing significant first-pass metabolism to 11-OH-THC and THCCOOH (21) and indeed other studies have estimated the bioavailability of THC to be low (between 4 and 20% approximately) when orally administered [for review, see (21)]. As with blood THC, the range of Cmax concentrations for 11-OH-THC and THCCOOH in blood was also higher in the present study compared to our previous study at both the 25 and 50 mg THC doses (11).
In the present study, women tended to exhibit higher blood cannabinoid concentrations compared to men following consumption of brownies containing equal doses of THC. These higher concentrations were at least partially because women weighed less [and had lower body mass index (BMI) scores] than men on average (see Table I), but pharmacokinetic data from certain individual participants suggest other factors may have contributed to these differences. For example, male participants #36 and #37 weighed less and had lower BMI scores (both 25.4 kg/m2) compared to female participants #41 and #53 (BMI scores of 28.7 and 35.2 kg/m2, respectively), but the two females still displayed higher blood THC Cmax concentrations at one or more active THC doses (see Supplementary Table I). Future studies should consider recruiting male and female participants matched according to BMI and other relevant characteristics (e.g., age) to determine whether there are other mechanisms that contribute to sex differences in cannabinoid pharmacokinetics.
In many jurisdictions in the USA and abroad, individuals suspected of driving under the influence of cannabis are required to provide a biospecimen (e.g., blood or oral fluid sample) and concentrations of THC and/or THC metabolites detected in the sample are used to help infer whether the person will be charged with a criminal offense. For instance, some US states consider individuals to be impaired from cannabis if they have blood THC concentrations of 5 ng/mL or higher while driving (e.g., Colorado and Washington, while other states (e.g., Arizona and Pennsylvania) have stricter, “zero-tolerance” policies that consider someone to be intoxicated if they have any detectable levels of THC or THC metabolites in their system. Other countries (e.g., Australia, Belgium and France) perform roadside oral fluid testing to identify cannabis-impaired drivers. In the present study, for two oral cannabis doses known to produce marked cognitive/psychomotor impairment (i.e., 25 and 50 mg THC) (11), blood THC concentrations did not exceed 5 ng/mL in 20/34 sessions and THC was not detected at all in some instances. Moreover, detection of THC in oral fluid appeared to be largely driven by deposition of cannabis in the oral cavity and not necessarily circulating blood THC levels. This suggests that factors such as the manner/speed with which a person eats or drinks an oral cannabis product, or how the product is intended to be consumed (e.g., swallowed whole versus chewed), may impact oral fluid THC detection. Overall, findings from this study may have important implications for jurisdictions which utilize blood or oral fluid cutoffs for inferring cannabis intoxication.
This study had a few limitations. First, the collection window for biospecimens in this study (i.e., 8 h) was too short to characterize the full pharmacokinetic profile in blood and/or oral fluid for some cannabinoids. For example, THCCOOH and THCCOOH-glucuronide remained present in whole blood at the final collection point (i.e., 8 h post-dosing), for the majority of participants. This limited window also reduced our ability to determine the time to last positive specimen for the various cannabinoids/biological matrices. Second, one type of cannabis was used in this study (i.e., THC-dominant). Future studies should examine the pharmacokinetics of CBD-dominant and “hybrid” (i.e., contains approximately equal proportions of THC and CBD) forms of cannabis following oral consumption. Third, only one type of cannabis edible product (i.e., brownies) was used, which had a high fat content. Future research should characterize the pharmacokinetics of other types of edibles (e.g., gummies, drinks) with specific emphasis on elucidating the extent to which the fat content of the product impacts cannabinoid absorption, elimination, etc., when the dose of THC is held constant. Fourth, the methods used to prepare cannabis brownies differ from some commonly used preparation methods. In this study, cannabis plant material was preheated (to decarboxylate THC-A to THC) and then mixed with brownie batter and baked. An alternative preparation method for infusing cannabis into food involves cooking cannabis plant material in an oil or butter, straining the plant material from the resulting liquid, and then using the oil/butter in preparation of foods such as brownies. The use of plant material, rather than infusing into butter or oil, in this study was selected to avoid additional analytical steps required to ensure accurate dosing of the final drug product. Future studies using butter/oil preparations are needed to determine whether alternative cannabis edible preparation methods produce different pharmacokinetic/pharmacodynamic results. The limited sample size and inclusion of only infrequent cannabis users are also limitations, as these factors limit the overall generalizability of our findings.
Conclusion
Consumption of oral cannabis dose-dependently increased concentrations of THC and THC metabolites in healthy male and female participants. Peak blood cannabinoid concentrations were generally lower, and substantially delayed, compared to what has been observed in prior evaluations of inhaled cannabis. Females exhibited higher blood cannabinoid concentrations compared to males. While these higher concentrations were at least in part due to the lower body weight/BMI of female participants, there may have been other factors which contributed to the observed inter-individual differences in cannabis pharmacokinetics. Oral fluid THC concentrations appeared to be strongly influenced by direct contact of cannabis with the oral cavity, and did not reflect systemically bioavailable levels of THC. As legal access to cannabis becomes more ubiquitous, the pharmacokinetics and associated pharmacodynamics of cannabis will need to be further elucidated across different product types, administration methods and cannabis chemovars in diverse participant populations.
Funding
This research was funded by the Substance Abuse and Mental Health Services Administration (SAMHSA) and National Institute on Drug Abuse (NIDA; Grant T32-DA07209).
Supplementary Material
Acknowledgments
We thank the support staff of the Johns Hopkins University Behavioral Pharmacology Research Unit for outstanding contributions to the implementation of this study. We also thank Dr. Christine Moore and Ms. Cynthia Coulter at Immunalysis, Inc., support staff at RTI International, and all the individuals involved in the NIDA Drug Supply Program.
Conflicts of interest
Dr. Vandrey has served as a paid consultant for Zynerba Pharmaceuticals and Canopy Health Innovations, Inc., and had received honoraria for serving on the scientific advisory boards of FSD Pharma and Present Life Corporation. The remaining authors have no conflicts of interest to declare.
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