Pharmacokinetics encompasses the absorption of cannabinoids following diverse routes of administration and from different drug formulations, the distribution of analytes throughout the body, the metabolism of cannabinoids by different tissues and organs, the elimination of cannabinoids from the body in the feces, urine, sweaty oral fluid, and hair, and how these processes change over time. Cannabinoid pharmacokinetic research has been especially challenging because of low analyte concentrations, rapid and extensive metabolism, and physio-chemicaI characteristics that hinder the separation of drugs of interest from biological matrices and from each other, and that lower drug recovery because of adsorption of compounds of interest to multiple surfaces. Mass spectrometric developments now permit highly sensitive and specific measurement of cannabinoids in a wide variety of biological matrices.
Cannabis sativa contains more than 421 different chemical compounds, including over 60 cannabinoids. THC is usually present in cannabis plant material as a mixture of monocarboxylic acids, which readily and efficiently decarboxylate upon heating. THC decomposes when exposed to air, heat, or light; exposure to acid can oxidize the compound to cannabinol, a much less potent cannabinoid Mechoulam et al. elucidated the structure of THC after years of effort in 1964, opening the way for studies of the drug’s pharmacokinetics. THC, containing no nitrogen but with 2 chiral centers in the trans-configuration is described by 2 different numbering systems, the dibenzopyran or Δ9, and the monoterpene or Δ’ system; the dibenzopyran system is used throughout this chapter.
Route of drug administration and drug formulation determine the rate of drug absorption. Smoking, the principal route of cannabis administration, provides a rapid and efficient method of drug delivery from the lungs to the brain, contributing to its abuse potential. Intense pleasurable and strongly reinforcing effects may be produced because of almost immediate drug exposure to the central nervous system. Bioavailability following the smoking route was reported as 2 to 56%, parry because of the intra- and inter-subject variability in smoking dynamics that contribute to uncertainty in dose delivery. The number, duration, and spacing of puffs, hold time, and inhalation volume, or smoking topography, greatly influence the degree of drug exposure. Many individuals prefer the smoked route, not only for its rapid drug delivery, but also the ability to titrate their dose. THC content of cannabis is higher now with improved horticulture and selection of higher-potency strains. In the United States, the mean THC content in 2003 seized cannabis specimens was 6.25%.
We used a continuous blood withdrawal pump to capture the rapid absorption of THC and formation of 11-hydroxy-THC (11-OH-THC) and THCCOOH during cannabis smoking.
The disposition of THC acid its metabolites after smoking a single placebo, 1.75% or 3.55% THC cigarette was followed in plasma over 7 days. THC was detected in the plasma immediately after the first cigarette puff (Figure 13-1). Mean ± SD THC concentrations of 7.0 ± 8.1 µg/L and 18.1 ± 12.0 µg/l were observed following the first inhalation of a low- (approximately 16 mg) or high- (Approximately 30 mg) dose cigarette, respectively. Concentrations increased rapidly, reaching mean peaks of 84.3 µg/L (range 50-129) and 162.2 mg/L (range 76-267) for the low- and high-dose cigarette, respectively. Peak concentrations occurred at 9.0 min, before initiation of the last puff sequence at 9.8 min.
Despite a computer-paced smoking procedure that controlled the number of puffs, length of inhalation, hold time, and time between puffs, there were large inter-subject differences in plasma THC concentrations because of differences in the depth of inhalation as participants titrated their THC dose (Figure 13-2). Mean THC concentrations were approximately 60% and 20% of peak concentrations 15 and 30 min post-smoking, respectively. Within 2 h, plasma THC concentrations were at or below 5µg/L. The time of detection of THC (GC/MS limit of quantification [LOQ] = 0.5 ug/L) varied from 3 to 12 h after the low-dose and from 6 to 27 h after the high-dose cannabis cigarette.
THC is readily absorbed because of its high octanol/water coefficient, estimated to be between 6,000 and more than 8 million by different technologies. Absorption is slower when cannabinoids are ingested with lower, more delayed peak THC concentrations. Dose, vehicle, and physiological factors such as absorption and rates of metabolism and excretion can influence drug concentrations in circulation.
Perez-Reyes et al. described the efficacy of 5 different vehicles used in the oral administration of THC in gelatin capsules. Glycocholate and sesame oil improved the bioavailability of oral THC; however, there was considerable variability in peak concentrations and rates of absorption, even when the drug was add stored in the same vehicle.
Oral bioavailability was estimated to be 6% by Ohlsson et al. Peak THC concentrations ranged from 4.4 to 11 ug/L and occurred 1 to 5 h following ingestion of 20 mg of THC in a chocolate cookie. Several factors may account for the low oral bioavailability of 4 to 20% (as compared to intravenous drug administration) including variable absorption, degradation of drug in the stomach, and significant first-pass metabolism to active 11-OH-THC and inactive metabolites in the liver.
Recently, there has been renewed interest in oral THC pharmacokinetics because of the therapeutic value of orally administered THG. In a study of THC, 11-OH-THC, and THCCOOH concentrations in 17 volunteers after a single 10-mg Marinol® capsule, mean peak plasma THC concentrations of 3.8 ug/L (range 1.1-12.7), 11-OH-THG 3.4 ug/L (range 1.2-5.6), and THCCOOH 26 ug/L (range 14-46) were found 1 to 2 h after ingestion. Similar THC and 11-OH-THC concentrations were observed with consistency higher THGCOOH concentrations. Interestingly, 2 THC peaks frequently were observed because of entero-hepatic circulation. The onset magnitude, and duration of pharmaco-dynamic effects generally occur later, are lower in magnitudes and have a delayed return to baseline when THC is administered by the oral as compared to the smoked route of administration.
In a recent controlled cannabinoid administration study of THC containing hemp oils and dronabinol, the pharmacokinetics and pharmaco-dynamics of oral THC were evaluated. Up to 14.8 mg of THC was ingested by 6 volunteers each day in 3 divided doses with meals for 5 consecutive days. There was a 10-day washout phase between each of the 5 dosing sessions. THC was quantified in plasma by solid-phase extraction followed by positive chemical ionization GC/MS. THC and 11-OH-THC were rarely detected in plasma following the lowest doses, 0.39 and 0.47 mg/day THC, while peak plasma concentrations of less than 6.5 ug/L THC, less than 6.6 ugly 11-OH-THC, and less than 43.0 ug/L THCGOOH were found after the 2 highest THC doses of 7.5 and 14.8 mg/day.
Interestingly, THCCOOH concentrations after the 7.5 mg/day dronabinol dose were greater than or equal to those of the high-potency 14.8 mg/day hemp oil dose. This could be because of the dronabinol formulation that afforded greater protection from degradation in the stomach because of encapsulation and perhaps, improved bioavailability of THC in sesame oil, the formulation of synthetic THC, or dronabinol. Plasma THC and 11-OH-THC concentrations fell below the method’s limits of quantification of 0.5ug/L by 25 h, while THCCOOH was still measurable for more than 60 h after the last dose of the higher concentration hemp oils.
THC concentrations decrease rapidly after the end of smoking because of its rapid distribution out of the blood into the tissues and metabolism in the liver. THC is highly lipophilic and initially taken up by tissues that are highly perfused, such as the lung, heart, brain, and liver. Less highly perfused tissues, including fat, accumulate drug more slowly as THG redistributes from the vascular compartment.
Chianti and Rapaka estimated that equilibration was reached between plasma and tissue THC approximately 6 h after an intravenous THC dose. With prolonged drug exposure, THC concentrates distribute into fat and may be retained for extended periods of time. It is suggested that fad acid conjugates of THC and 11-OH-THC may be formed, increasing the stability of these compounds in fat.THC’s volume of distribution (Vd) is large, approximately 10 L/kg, despite the fact that it is 95 to 99% protein-bound in plasmas primarily to lipoproteins.
More recently, with the benefit of advanced analytical techniques, THC’s steady-state Vd was found to be 3.4 L/kg. Few pharmacokinetic changes were noted during chronic administration, although average total metabolic clearance and initial apparent volume of distribution increased from 605 to 977 mL/min and from 2.6 to 6.4 L/kg, respectively. The pharmacokinetic changes observed after chronic oral THC could not account for the observed behavioral and physiologic tolerance that developed, suggesting that tolerance was because of pharmaco-dynamic adaptation.
Burstein et al. were the first to show that 11-OH-THC and THGCOOH were primary metabolizes of THC in rabbits and rhesus monkeys. The primary metabolic routes and metabolites of THC included hydrous on of THC at C9 by the hepatic cytochrome P450 enzyme system to produce the equipotent metabolite, 11-OH-THC (Refer to Figure 13-3). Cytochrome P450 2C9, 2CI9, and 3A4 are involved in the oxidation of THC HO. More than 100 THC metabolizes, including di- and tri-hydroxy compounds, ketones, aldehydes, and carboxylic acids, have been identified.
Although 11-OH-THC predominates as the first oxidation product, significant amounts of 8α-0H-THC and lower amounts of the 11-OH-THC, are formed. Much lower plasma 11-OH-THC concentrations approximately 10% of THC concentrations are found after cannabis smoking than after oral administration few. Cytochrome P450 2C9 is believed to be primarily responsible for the formation of 11-OH-THC, with peak concentrations occurring approximately 13 min after the start of smoking. Dihydroxylation of THC yields (8α-11-di-OH-THC).
Oxidation of the active 11-OH-THC produces the inactive metabolite, THCCOOH. THCCOOH concentrations gradually increase and are greater than THC concentrations 30 to 45 min after the end of smoking. Phase II metabolism of THCCOOH involves addition of glucuronic acid, and less commonly, sulfate, glutathione, ammo acids, and fatty acids via the C11 carboxyl group.
The phenolic hydroxyl group and C11 hydroxyl group may be targets as well. It is also possible to have 2 glucuronic acid moieties attached to THCCOOH, although steric hindrance at the phenolic hydroxyl group could be a factor. Addition of the glucuronide group improves water solubility facilitating excretion, but renal clearance of these polar metabolites is low because of extensive protein binding. No significant differences in metabolism between men and women have been reported.
After the initial distribution phase, the rate-limiting step in the metabolism of THC is its redistribution from lipid depots into blood. Lemberger et al. suggested that frequent cannabis smoking could induce THC metabolism. However, later studies did not replicate this finding.
Within 5 days, in total of 80 to 90% of a THC dose is excreted, mostly as hydroxylated and carboxylated metabolite. More than 20% is excreted in the feces, with approximately 20% eliminated in the urine LEO. Numerous acidic metabolites are found in the urine; many are conjugated with glucuronic acid to increase water solubility. The primal urinary metabolite is the acid-linked THCCOOH glucuronide conjugate, while 11-OH-THC predominates in the feces.
Terminal Elimination Ha/f-Lives of THCCOOH
Another common problem with studying the pharmacokinetics of cannabinoids in humans is the need for highly sensitive procedures to measure low cannabinoid concentrations in the terminal phase of excretion, and the requirement for monitoring plasma concentrations over an extended period to adequately determine cannabinoid half lives. Many studies utilized short sampling intervals of 24 to 72 h that underestimated terminal THC and THCCOOH half-lives. The slow release of THC from lipid storage compartments and significant enterohepatic circulation contribute to THC’s long terminal half-life in plasma, reported as greater than 4.1 days in chronic cannabis users. Iso-topically labeled THC and sensitive analytical procedures were used to obtain this drug half-life.
Garrett and Hunt reported that 10 to 15% of the THC dose is enterohepatically circulated in dogs. Johansson et al. reported a THCGOOH plasma elimination half-life up to 12.6 days in a chronic cannabis user when monitoring THCCOOH concentrations for 4 weeks. Mean plasma THCCOOH elimination halflives were 6.2 ± 0.8 and 6.2 ± 6.7 days for frequent and infrequent cannabis users, respectively. Similarly, when sensitive analytical procedures and sufficient sampling periods were used for determining the terminal urinary excretion half-life of THCCOOH, it was estimated to be 3 to 4 days. Urinary THCCOOH concentrations drop rapidly until approximately 20 to 50 ug/L, and then decrease at a much slower rate. No significant pharmacokinetic differences between chronic and occasional users have been substantiated.
Percentage TUG Dose Excreted as Urinary THCCOOH
An average of 93.9 ± 24.5 ug THCCOOH (range 34.6-171.6) was measured in urine over a 7-day period following smoking of a single 1.75% THC cigarette containing approximately 18 mg THC. The average amount of THCCOOH excreted in the same time period following the high dose 3.55% THC containing approximately 34 mg THC was 197.4 ± 33.6 ug (range 107.5-305.0). This represented an average of only 0.54 + 0.14% and 0.53 ± 0.09% of the original amount of THC in the low- and high dose cigarettes respectively. The small percentage of the total dose found in the urine as THCCOOH is not surprising considering the many factors that influence THCCOOH excretion after smoking.
Before harvesting, cannabis plant material contains little active THC. When smoked, THC carboxylic acids spontaneously decarboxylate to produce THC with nearly complete conversion upon heating. Pyrolysis of THG during smoking destroys additional drug. Drug availability is further reduced by loss of drug in the side-stream smoke and drug remaining in the un-smoked cigarette butt. These factors contribute to high variability in drug delivery by the smoked route. It is estimated that the systemic availability of smoked THC is approximately 8 to 24% and that bioavailability depends strongly upon the experience of the cannabis user. THC bioavailability is reduced because of the combined effect of these factors; the actual available dose is much lower than the amount of THC and THC precursor present in the cigarette. Another factor affecting the low amount of recovered dose is measurement of a single metabolite. Numerous cannabinoid metabolizes are produced in humans as a result of THC metabolism most of which are not measured or included in the percentage dose excreted calculations.
Cannabinoid Glucuronide Conjugates
Specimen preparation for cannabinoid testing frequently includes a hydrolysis step to free cannabinoids from their glucuronide conjugates. Most GC-MS confirmation procedures in urine measure total THCCOOH following either an enzymatic hydrolysis with β-glucuronidase or more commonly, an alkaline hydrolysis with sodium hydroxide. Alkaline hydrolysis appears to efficiently hydrolyze the ester THCCOOH glucuronide linkage.
Urinary Biomarkers of Recent Cannabis Use
Most investigators believed that little THC or 11-OH-THC was excreted into the urine. Significantly higher concentrations of THC and 11-OH-THC were observed when Escherichia coli β-glucuronidase was used in the hydrolysis method compared to either Helix Sumatra β-glucuronidase or base. THC and 11-OH-THC primarily are excreted in urine as glucuronide conjugates that are resistant to cleavage by alkaline hydrolysis and by enzymatic hydrolysis procedures using some types of β-glucuronidase. Kemp et aL demonstrated that β-glucuronidase from E. coli was needed to hydrolyze the ether glucuronide linkages of the active cannabinoid analytes.
Mean THC concentrations in urine specimens Mom 7 subjects collected after each had smoked a single 3.58% cannabis cigarette was 22ug/L using the E. coli β-glucuronidase hydrolysis method, while THC concentrations using either H. pomatia β-glucuronidase or base hydrolysis methods were near zero. Similar differences were found for 11-OH-THC with a mean concentration of 72 ug/L from the E. coli method and concentrations less than 10 ug/L from the other methods. The author suggested that finding THC and/ or 11-OH-THC in the urine might provide a reliable marker of recent cannabis use, but adequate data from controlled drug administration studies were not yet available to support or refute this observation. Using a modified analytical method with E. coli glucuronidase we have analyzed hundreds of urine specimens collected following controlled THC administration. We found that 11-OH-THC may be excreted in the urine of chronic cannabis users for more than 30 days, and THC itself for more than 24 days. Thus, finding either of these 2 analytes in urine is not a biomarker for recent cannabis use.
Interpretation of Cannabinoid Concentrations in Workplace Drug Testing
Urinary THCCOOH Concentrations
Detection of cannabinoids in urine is indicative of prior cannabis exposure, but the long excretion half-life of THCCOOH in the body, especially in chronic cannabis users, makes it difficult to predict the timing of past drug use. THC accumulates over time in fat depots in the body and is slowly released over time. In a single extreme case, one individual’s urine was positive at a concentration greater than 20ug/L by immunoassay up to 67 days after last drug exposure. This individual had used cannabis heavily for more than 10 years. However, a naive user’s urine may be found negative by immunoassay after only a few hours following the smoking of a single cannabis cigarette. Assay cutoff concentrations and the sensitivity and specificity of the immunoassay affect drug detection times.
positive urine test for cannabinoids indicates only that drug exposure has occurred. The result does not provide information on the route of administration, the amount of drug exposure, when drug exposure occurred, or the degree of impairment.
Total THCCOOH concentrations include both the free THCCOOH and THCCOOH-glucuronide concentrations that are obtained after alkaline or enzymatic hydrolysis. Substantial intra- and inter-subject variability occurs in patterns of THCCOOH excretion. THCCOOH concentration in the first specimen after smoking is indicative of how rapidly the metabolite can appear in urine. Mean first-urine THCCOOH concentrations were 47 ug/L ± 22.3 and 75.3 ug/L ± 48.9 after smoking one 1.75 or 3.55% THC cigarette, respectively. Fifty percent of the subjects’ first urine specimen after the low dose and 83% of the first urine specimens after the high dose were positive by GC/MS at a 15 ug/L THCCOOH cutoff concentration.
Thus, THCCOOH concentrations in the first urine specimen are dependent upon the relative potency of the cigarette, the elapsed time following drug administration, smoking efficiency, and individual differences in drug metabolism and excretion. Mean peak urine THCCOOH concentrations averaged 89.8 ± 31.9 ug/L (range 20.6 to 234.2) and 153.4 ± 49.2 ug/L (range 29.9 – 355.2) following smoking of approximately 15.8 and 33.8 mg THC, respectively. The mean times of peak Anne concentration were 7.7 h 0.8 after the 1.75% THC and 13.9 h ± 3.5 after the 3.55% THC dose. Although peak concentrations appeared to be dose related, there was a 12-fold variation between individuals.
THCCOOH Detection Windows in Urine
Drug detection time, or the duration of time after drug administration that an individual’s urine tests positive for cannabinoids, is an important factor in the interpretation of urine drug results. Detection time is dependent on pharmacological factors E.g., drug dose, route of administration rates of metabolism and excretion} and analytical factors (e.g., assay sensitivity; specificity, accuracy). Mean detection times in Urine following smoking vary considerably between subjects, even in controlled smoking studies where cannabis dosing is standardized and smoking is computer-paced. During the terminal elimination phase, consecutive urine specimens may fluctuate between positive and negative as THCCOOH concentrations approach the cutoff concentration. It may be important in drug treatment settings or in clinical trials to differentiate between new drug use and residual excretion of previously used cannabinoids.
After smoking a 1.76% THC cigarette, 3 of 6 subjects had additional positive urine specimens interspersed between negative urine specimens. This had the effect of producing much longer detection times for the last positive specimen. Using the 15 ug/L confirmation cutoff for THCCOOH currently used for most urine drug testing, the mean GC-MS THGCOOH detection times for the last positive urine specimen following the smoking of a single 1.75% or 3.55% THC cigarette were 33.7 ± 9.2 h (range 8 – 68.5) and 88.6 to 23.2 h (range 57-122.3).
Normalization of Cannabinoid Urine Concentrations to Urine Creatinine Concentrations
Normalization of the cannabinoid drug concentration to the urine creatinine concentration aids in the differentiation of new from prior cannabis use and reduces the variability of drug measurement because of urine dilution. Because of the long half-life of drug in the body, especially in chronic cannabis users, toxicologists and practitioners are frequently asked to determine if a positive urine test represents a new episode of drug use or represents continued excretion of residual drug. Random urine specimens contain varying amounts of creatinine depending on the degree of concentration of the urine. Hawks first suggested creatinine normalization of urine test results to account for variations in urine volume in the bladder.
Whereas urine volume is highly variable because of changes in liquid, salt and protein intake, exercise, and age, creatinine excretion is much more stable. Manno et al. recommended that an increase of 150% in the creatinine normalized cannabinoid concentration above the previous specimen be considered indicative of a new episode of drug exposure. If the increase is greater than or equal to the threshold selected, then new use is predicted. This approach has received wide attention for potential use in treatment and employee assistance programs, but there was limited evaluation of the usefulness of this ratio under controlled dosing conditions. Huestis and Cone conducted a controlled clinical study of the excretion profile of creatinine and cannabinoid metabolites in a group of 6 cannabis users who smoked 2 different doses of cannabis separated by weekly intervals.
As seen in Figure 13.4, normalization of urinary 1 THCCOOH concentration to the urinary creatinine concentration produces a smoother excretion pattern and facilitates interpretation of consecutive urine drug test results. A relative operating characteristic (ROC) curve was constructed from sensitivity and specificity for 26 different cutoffs ranging from 10 to 200%. The most accurate ratio (85.4% correct predictions) was 50%, yielding a sensitivity of 80.1%, a specificity of 90.2%, 5.6% false positive, and 7.4% false-negative predictions. For example, if the first urine specimen was 100 ng/mg and the second specimen was 55 ng/mg, this exceeds the cutoff of 50% for the ratio of the second specimen to the first times 100, and would indicate new cannabis use. Using the 50% criteria, we would accurately discriminate new drug use from residual drug excretion 85.4% of the time. If the previously recommended ratio of 150% was used as the threshold for new use, sensitivity of detecting new use was only 33.4%, specificity was high at 90.8%, for an overall accuracy prediction of 74.2%.
To further substantiate the validity of the derived ROC curve, urine cannabinoid metabolite and creatinine data from another controlled clinical trial that specifically addressed water dilution as a means of specimen adulteration were evaluated. Sensitivity, specificity, accuracy, percentage false positives, and percentage false negatives were 71.9%, 91.6%, 83.9%, 5.4%, and 10.7%, respectively, when the 50% criteria was applied. These data indicate selection of a threshold to evaluate sequential creatinine normalized urine drug concentrations can improve the ability to distinguish residual excretion from new drug usage. Generally, the 50% criterion is applied in treatment and workplace settings for occasional cannabis users. If serious punitive consequences can result, the 150% criterion may be selected. Although the model is frequently used for frequent cannabis users at low THCCOOH concentrations bless than about 30ng/mg, the model may be less accurate. Research is underway to devise a more accurate model for frequent cannabis users at low THCCOOH concentrations.
Urine Cannabinoid Concentrations after Chronic Daily Cannabis Exposure
The difficulty of investigating urinary excretion after chronic, daily cannabis use is the need to provide constant monitoring during abstinence to ensure that participants do not self-administer additional drugs. The Chemistry and Drug Metabolism Section of the National Institute on Drug Abuse has been studying the excretion of cannabinoids in chronic users in blood, plasma, and urine for more than 15 years. Chronic daily users resided on the closed NIDA research unit for up to 30 days under 24-h medical surveillance. Every urine specimen was individually collected and analyzed by GC/MS after alkaline hydrolysis and/or enzymatic hydrolysis with E.Coli , β -glucuronidase. 11-OH-THG and THCCOOH were measurable in urine of the heaviest users for more than 30 days at low-method limits of quantification (LOQs) of 2.5 and 5 ng/mL, while THC itself was detectable for up to 25 days. These data document the need for clinical research on the terminal elimination half-lives of cannabinoids in multiple biological matrices after chronic exposure.
Oral fluid also is a suitable specimen for monitoring cannabinoid exposure and is being evaluated for driving under the influence of drugs, drug treatment, workplace drug testing, and clinical trials. There are a number of difficult challenges for oral fluid drug testing for cannabinoids that have fostered research in this area by toxicologists and manufacturers. First, basic aspects of the methodology are being addressed: analytes of interest in oral fluid are being identified, the volume of oral fluid required for meeting screening and confirmation analyses sensitivity limits is being determined, the ability to collect reproducible oral fluid volume needs to be demonstrated, efficient extraction of cannabinoids from the collection device is a major problem because of strong adsorption of drug to the device, and means of adulterating oral fluid to produce false negative results is being investigated.
There also are many clinical aspects, including windows of drug detection after controlled cannabis administration and the potential for passive exposure from smoked drug, among many other factors. Oral fluid testing is still a work in progress, not yet approved for federally mandated drug testing, but clearly selected as the method of choice for driving under the influence of drugs testing programs. In Victoria, Australia, motorists are roadside-tested for recent methamphetamine, Ecstasy, and cannabis use by analysis of oral fluid. The effort has achieved good public support and is serving as an effective deterrent to impaired driving, despite shortcomings in the analytical performance of the testing devices. Oral fluid testing is an exciting and dynamic field with rapidly changing capabilities and increasing knowledge base.
The advantages of oral fluid testing as compared to urine testing for cannabinoids are the ease and noninvasiveness of specimen collection, ability to perform collection at the roadside, and under direct observation. Depending upon the cutoff concentration used, a positive oral fluid test may reflect more recent cannabis use than a urine test. Disadvantages include limited specimen volume, need for lower analytical detection limits, high adsorption of analytes to the collection device, shorter detection windows after drug use, potential contamination from smoked drug, and fewer data available from controlled drug administration studies to guide interpretation of test results. Adequate sensitivity is best achieved with an assay directed toward THC detection, rather than 11-OH-THC or THCCOOH, because of the much higher concentrations of parent compound in oral fluid. The oral mucosa is exposed to high concentrations of THC during smoking and serves as the primary source of THC in oral fluid. Although Hawks showed that only minor amounts of drug and metabolizes diffuse from the plasma into oral fluid, Huestis and Gone later found that shortly after cannabis smoking, plasma and oral fluid THC concentrations declined in a parallel fashion, indicating later equilibration between these compartments.
Figure 13-5 depicts excretion of THC in oral fluid and plasma and creatinine-normalized THCCOOH excretion in urine in 1 subject after smoking a single 3.55% cannabis cigarette. Early investigators reported that following intravenous administration of radio-labeled THC, no radioactivity could be demonstrated in oral fluid. In addition, it was thought that there was no measurable THCCOOH in oral fluid; Huestis and Cone found no measurable analyte for 7 days following cannabis smoking by GC/MS with a LOQ of 0.6 ug/L. Furthermore, no THCCOOH was measured in oral fluid from 22 subjects positive for THCCOOH in the urine. Oral fluid collected with the Salivette collection device was positive for THC in 14 of these 22 participants. Although no 11-OH-THC or THCCOOH was identified by GC/MS, cannabinol and cannabidiol were found in addition to THC.
With the advent of 2-dimensional GC/MS and cryo-focusing, lower LOQs of 2 p~/mL were achieved, enabling measurement of THCCOOH and THCCOOH-glucuronide in oral fluid. Moore et al. reported that in 153 specimens tested 66.4% were positive for both THC and THCCOOH; 14 (9.7%) were positive for THCCOOH only, and 27 (18.8%) were positive for THC only. THCCOOH was identified in 76.2% of cannabinoid-positive specimens, providing a good biomarker for cannabis use and a safeguard against potential passive exposure of cannabis smoke. Therefore, although THC is by far the cannabinoid present in greatest concentration, THCCOOH may be highly useful to monitor to preclude passive drug exposure.
There are fewer controlled cannabis administration studies evaluating the disposition of cannabinoids in oral fluid than in urine. Hours after smoking, the oral mucosa serves as a depot for release of THC into the oral fluid. Oral fluid THC concentrations were found to temporally correlate with plasma cannabinoid concentrations and behavioral and physiological effects, but wide intra- and inter-individual variation precluded the use of oral fluid concentrations as indicators of drug impairment. Less also is known of the windows of THC detection in oral fluid. After smoking cannabis, oral fluid cannabinoid tests were positive by GC/MS/MS for TRIG with a cutoff of 0.5 g/L for 13 ± 3 h (range 1-24). After these times occasional positive oral fluid results were interspersed with negative tests for up to 34 h.
Kauert et al. evaluated oral fluid cannabinoids in 10 subjects for up to 6 h after smoking a single 18.2 ± 2.8 mg (low)and 36.5 ± 6.6 mg (high) THC cigarette. THC concentrations m oral fluid were 900 ± 589 and 1,041 ± 652 ug/L (low and high dose, respectively) in the first sample collected at 0.25 h and decreased to 18 ± 12 g/L over 6 h with an elimination half-life of 1.5 ± 0.6 h. There was high inter-subject variability in oral fluid/serum ratios, making the prediction of drug impairment from these ratios unreliable. The authors concluded that the pharmacokinetics of cannabinoids in oral fluid was not yet satisfactorily understood.
There has been interest in oral fluid as a biological matrix for evaluating driving under the influence of drugs for many years. Peel et al. first tested oral fluid specimens from 56 drivers suspected of being under the influence of cannabis with the enzyme-multiplied immunoassay technique (EMIT) screening test and GC/MS confirmation in 1984.
They suggested that the ease and noninvasiveness of specimen collection made oral fluid a useful alternative matrix for detection of recent cannabis use. Oral fluid specimens also were evaluated in the European Union’s Roadside Testing Assessment (ROSITA) and Driving Under the Influence of Drugs (DRUID) Projects to reduce the number of individuals driving under the influence of drugs and to improve road safety. The ease and noninvasiveness of oral fluid collections reduced hazards in specimen handling and testing, and shorter detection window are attractive attributes to the use of this specimen for identifying the presence of potential performance impairing drugs. Detection of cannabinoids in oral fluid is a rapidly developing field; however, there are scientific issues still to be resolved. One of the most important is the degree of absorption of THC to oral fluid collection devices.
Recently, Pehrsson et al., and Walsh et al. reviewed the efficiency of a variety of oral fluid collection devices for oral fluid testing. Great advances have been achieved in the release of cannabinoids from the collection device into extraction buffers, there is improved consistency in the amount of extraction buffer included in each device, and devices function more reliably. A number of collection devices are now available for adequate monitoring of cannabinoids in oral fluid. Oral fluid testing is expected to increase greatly m the next decade, emphasizing the need for a scientific database to guide interpretation of results.
Cannabinoids in Sweat
Sweat testing is being applied to monitor cannabis use in drug treatment, criminal justice, workplace drug testing, and clinical studies. In 1989 Balabanova and Schneider utilized radioimmunoassay to first detect cannabinoids in apocrine sweat. Currently, there is a single commercially available sweat collection device, the PharmCheck patch, offered by PharmChem Laboratories in Texas, USA and only 1 laboratory offering commercial sweat test analysis. Generally, the patch worn for 7 days and exchanged for a new patch once each week during visits to the treatment clinic or parole officer. Theoretically, this permits constant monitoring of drug use throughout the week, extending the window of drug detection and improving test sensitivity. As with oral fluid testing, this is a developing analytical technique with much to be learned about the pharmacokinetics of cannabinoid excretion in sweat, potential re-absorption of THC by the skin, possible degradation of THC on the patch, and adsorption of THC onto the patch collection device. It is known that THC is the primary analyte detected in sweat, with little 11-OH-THC and THCCOOH. Several investigators have evaluated the sensitivity and specificity of different screening assays for detecting cannabinoids in sweat. Kintz et al. identified THC 4-38 ng/patch in 20 known heroin abusers who wore the PharmChek patch for 5 days while attending a detoxification center fit. Sweat was extracted with methanol and analyzed by GC/MS. The same investigators also evaluated forehead swipes with cosmetic pads for monitoring cannabinoids in sweat from individuals suspected of driving under the influence of drugs. THC, but not 11-OH-THC or THCCOOH, was detected (4 to 152 ng/pad) by electron-impact GC/MS in the sweat of 16 of 22 individuals who tested positive for cannabinoids in urine. Ion trap tandem mass spectrometry also has been used to measure cannabinoids in sweat collected with the PharmChek sweat patch with a limit of detection of 1 ng/patch.
To date, there is a single controlled THC administration study evaluating the excretion of cannabinoids in sweat. Huestis et al. evaluated THC excretion in 11 daily cannabis users after cessation of drug use. PharmChek sweat patches worn for 7 days were analyzed for THC by GC/MS with a LOQ of 0.4 ng THC patch. Sweat patches worn the first week of continuously monitored abstinence had THC above the United States Substance Abuse Mental Health Services Administrations proposed cutoff concentration for federal workplace testing of 1 ng THC/patch (Figure 13.6). Mean ± SEM THC concentrations were 3.85 ± 0.86 ng THC/patch. Eight of 11 subjects had negative patches the 2nd week and 1 produced THC positive patches for 4 weeks of monitored abstinence. Daily and weekly sweat patches from 7 subjects administered oral doses of up to 14.8 mg THC/day for 5 consecutive days were also tested. In this oral THC administration study, no daily or weekly patches had THC above the LOQ; concurrent plasma THC concentrations were all less than 6.1 ug/l. With proposed federal cutoff concentrations, most daily cannabis users will have a positive sweat patch in the 1st week after ceasing drug use and a negative patch after subsequent weeks, although patches may re-loan positive for 4 weeks or more. Oral ingestion of up to 14.8 mg THC daily did not produce a THC positive sweat patch test.
There are multiple mechanisms for the incorporation of cannabinoids in hair. THC and metabolites may be incorporated into the hair bulb that is surrounded by capillaries. Drug may also diffuse into hair from sebum that is secreted onto the hair shaft and from sweat that is excreted onto the skin surface. Drug may also be incorporated into hair from the environment. Cannabis is primarily smoked, prodding an opportunity for environmental contamination of hair with THC in cannabis smoke. Thorspecken et aL studied the deposition of cannabinoids in hair from environmental smoke and found that false-positive cannabinoid hair tests could be produced by in vitro environmental contamination of blank hair. Strands of hair were moistened with water, greased with sebum or sebum/sweat, or bleached or permed, and treated and untreated samples were exposed to cannabis smoke for 60 min. Aliquots of each hair specimen were analyzed unwashed and after washing with methanol, dichloromethane, or 5 g/L dodecyl sulfate in water. Cannabinoid concentrations in hair were determined by GC/MS and were dependent on the THC air concentration and hair pretreatment. Drug uptake was less in untreated than in pretreated hair, and was higher in damp and greased hair. External contaminants were completely removed by washing with methanol and dichloromethane in untreated hair only. Washing with dodecyl sulfate in water was insufficient in all cases. Expel sure of the hair to cannabis smoke yielded detectable cannabinoids with concentrations depending on the amount of THG in the air, hair care habits, and cosmetic treatment.
Basic drugs such as cocaine and methamphetamine concentrate in hair because of ionic bonding to melanin, the pigment in hair that determines hair color. The more neutral and lipophilic THC is not highly bound to melanin, resulting in much lower concentrations of THC in hair as compared to other drugs of abuse. Usually, THCi s present in hair at a higher concentration than its THCGOOH metabolite. An advantage of measuring THCCOOH in hair is that THCCOOH is not present in cannabis smoke, avoiding the issue of passive exposure from the environment. Analysis of cannabinoids in hair is challenging because of the high analytical sensitivity required. THCCOOH is present in the femtogram-topicogram per milligram of hair range. GC/MS/MS or 2D GCMS is required in most analytical techniques. A novel approach to the screening of hair specimens for the presence of cannabinoids in hair was proposed by Cirimele et al. They developed a rapid, simple GC/MS screening method for THC, cannabinol, and cannabidiol in hair that did not require derivatization before analysis. The method was found to be a sensitive screen for cannabis detection with GC/MS identification of THC recommended as a confirmatory procedure.
It is difficult to conduct controlled cannabinoid administration studies on the disposition of cannabinoids in hair because of the inability to differentiate administered drug from previously self-administered cannabis and the low rates of incorporation of cannabinoids into hair. Huestis et al. conducted a study of THC and THCCOOH in 53 hair specimens from 38 documented cannabis users. History of cannabis use was documented by questionnaire, urinalysis, and controlled, double-blind administration of smoked or oral THC. All participants had positive urine cannabinoid tests at the time of hair collection. The subjects completed a questionnaire indicating daily cannabis use (N = 18) or non-daily uses i.e., 1 to 5 cannabis cigarettes per week (N = 20). Additional hair specimens were collected following smoking of two 2.7% THC cigarettes containing a total of about 48 mg THC (N = 13} or multiple oral doses totaling 116 mg THC (N = 2). Cannabinoid concentrations in all hair specimens were determined by ELISA and GC/ MS. Pre- and post-dose detection rates did not differ statistically therefore, all 53 specimens were considered as one group for further comparisons. Nineteen specimens (36%) had no detectable THC or THCCOOH at the LOQ of 1.0 and 0.1 pg/mg hair, respectively (Table 13-1). Two specimens (3.8%) had measurable THC only, I4 (26%} THCCOOH only and 18 (34%) both cannabinoids. Detection rates were significantly different (P < 0.05 Fisher’s exact test) between daily cannabis users (85%) and non daily users (52%). There was no difference in detection rates between African-American (N= 40) and Caucasian (N = 13) hair specimens (P> 0.3, Fishers exact test). For specimens with detectable cannabinoids, concentrations ranged from 3.4 to >100 pg THC/mg and 0.10 to 7.3 pg THCCOOH/ mg hair. THG and THCCOOH concentrations were positively correlated (r = 0.38, P < 0.01), Pearson’s product moment correlation. Using an immunoassay cutoff concentration of 5 pg THC equiv./mg hair, 83% of specimens Mat screened positive were confirmed by GC/MS/MS at a cutoff concentration of 0.1 pg THCCOOH/mg hair.
There are advantages to monitoring drug use with ham testing, including a wide window of drug detection, a less invasive specimen collection procedure, and the ability to collect a second specimen at a later time. However, one of the weakest aspects of testing for cannabinoids in hair is the low sensitivity of drug detection in this alternate matrix. There are many scientific questions that remain to be answered, including the minimum dose required for detection, potential for external contamination from cannabis smoke, and whether there is a color bias for deposition of cannabinoids in hair; as is noted for strongly basic drugs such as cocaine and methamphetamine. Preliminary work appears to indicate that because of minimal binding to melanin in hair, THC concentrations in dark and light hair may be more equivalent than with other illicit drugs. Additional controlled drug administration studies are needed to improve the interpretation of cannabinoid hair tests.