Cannabis is one of the most commonly used illicit drugs worldwide.1 It is a flowering plant that contains over 500 known compounds, including more than 100 distinct cannabinoids and other non-cannabinoids.2 Of the known cannabinoids, tetrahydrocannabinol (THC) and cannabidiol (CBD) are the predominant and most well defined compounds for medicinal and recreational purposes. Acting as a partial agonist, THC exerts its pharmacological effects by activating the human cannabinoid receptors CB1 and CB2.3 In contrast, CBD has proved to counteract psychoactive effects from THC and even possess multiple therapeutic properties, including antiepileptic, anti-anxiety, anti- inflammatory, and neuroprotective effects.4-6
The potency of cannabis—defined by the ratio of THC/CBD—varies across different cannabis strains in which higher ratio correlates to a higher risk of psychotic effects of the drug.7 The ratio of THC/CBD is also the most obvious difference between recreational and medicinal cannabis use. Typical THC concentration is never above 10 percent in medical cannabis. However, recreational marijuana may contain THC in the range of 15-30 percent, with the highest concentration being 37 percent.8
In addition to the heterogeneity of the cannabis composition, the current landscape of legalization status of cannabis also adds to its complexity. In the U.S., federal and state laws regarding medical use of cannabis and cannabinoids are in conflict and have led to confusion among patients, caregivers, and healthcare providers. According to Control Act of 1970, the Federal Drug Enforcement Agency lists cannabis as a Schedule 1 substance.9 Under this provision marijuana is considered unacceptable for medical use, has a high potential for abuse, and is believed to be unsafe for use even under medical supervision. Despite the prohibition of medical cannabis use under federal law, some states stand on the side of favoring marijuana use for its medicinal effects. Since California passed the first state law allowing medical marijuana in 1996, today 33 states and the District of Columbia have followed suit, among which 10 allow recreational marijuana consumption as well.10
With the advent of medical or legalized marijuana, patterns of cannabis use have changed dramatically. The questions physicians and legal professionals are often interested in knowing include estimating the time the drug was used last and the concentration in a biological sample that correlates to impairment. These questions are challenging to answer given the non-linear pharmacokinetics (PK) of THC, various routes of consumption (smoking, vaporization, dabbing, oral, dermal), development of tolerance in chronic users, and titration of dose to achieve desired affects by users.11 Thus, a clear limit for impairment has not been established in any biological matrix. Furthermore, due to the complex PK of THC, the presence of THC and/or its metabolites in biological fluids is not sufficient to determine recent use. But it is an acceptable surrogate as evidence for impairment. THC is detectable in blood within seconds after inhalation, and peaks in about three to 10 minutes.11,12 In contrast, orally ingested cannabis peaks at one to two hours after ingestion, demonstrating the PK variability with different modes of consumption. Being highly lipophilic, THC in circulation will quickly distribute to adipose tissue, liver, lung, and spleen, resulting in a rapid drop in levels in blood, albeit with continuing drug effects.11 After consumption by a variety of routes, THC is further metabolized to the psychoactive compound 11-OH-THC, inactivated to THC-COOH, and excreted in feces and urine. As a result of tissue deposition and slow redistribution back to systemic circulation, the half-life of THC and its major metabolites varies from days to months. While occasional users have THC/metabolites detectable for a few days, the detection window in chronic users who usually have large tissue burden, can last for months, confounding the question pertaining to recent use.
Nevertheless, a number of controlled trials have aided in our understanding of THC PK in various biological fluids and identified cannabinoids that could predict recent use. The most common matrices include urine, blood, and oral fluid (OF). Most laboratories offer either an immunoassay-based screening test or a more specific and sensitive test by liquid chromatography (LC) or gas chromatography mass spectrometry (GC-MS), which has the ability to detect individual metabolites. Testing in each of these matrices, although imperfect, elucidates some history of marijuana use and it is important to understand these inadequacies to accurately interpret laboratory results.
Testing in urine
In medical settings, clinicians often test for THC in urine to monitor abstinence in patients enrolled in a transplant program, drug abuse treatment program, or pain management program.11 The most commonly detected marker in urine is THC-COOH. Other THC metabolites are either absent, present at very low concentrations in urine, or commercial standards are unavailable precluding their identification.13 Presence of these metabolites in urine is only indicative of cannabis exposure and cannot be used to predict when it was used last or to glean impairment.
In occasional smokers, the detection window in urine can be several days. In contrast, chronic users can have detectable THC-COOH for months following abstinence.11 As such, mathematical models have been developed to identify new cannabis use from residual excretion in both groups. The premise of the models comprises of comparing creatinine-normalized THC-COOH ratios from two different urine collections.14 In occasional users, a ratio greater than the model’s prediction for the time interval between urine collections is indicative of new use.14 In frequent users, rules that account for residual excretion are incorporated in ratio comparison for a more accurate prediction of residual excretion vs. new use.15 In this model, based on the amount of creatinine normalized THC-COOH excreted in the first urine sample, the algorithm prescribes obtaining a second sample either 48 hours later or on the fifth day. Subsequent comparison of the ratio either predicts residual use, new use, or recommends additional sample collection for further comparison. Thus, urine testing is generally informative in determining continued abstinence or detecting renewed use.
Testing in blood
Testing cannabis metabolites in blood/plasma is indicated for evaluation of possible impairment in cognitive or psychomotor functioning. Although cannabis-impairing effects are directly associated with THC concentration in brain tissue and evidence is lacking regarding the correlation of blood concentration to brain concentration, blood still remains the best-studied and most representative available biological matrix.16 As such, blood THC concentrations have been applied to several mathematical models in an attempt to estimate time of last consumption. The predictive models entail using either blood THC concentration (model I), ratio of THC to THC-COOH (model II), or a combination of both models I and II.17 When these models were validated under controlled smoking conditions, the predictive accuracy of all three were >90 percent but the combined model performed the best with accuracy up to 99.1 percent.17 However, a later study found that the predictive models were not reliable in chronic heavy users.18 Chronic users have a high body burden of THC and metabolites that are slowly released back into blood prolonging the half-life of these biomarkers. Similar to urine, residual THC and its metabolites can be detectable for over a month after sustained abstinence confounding interpretation. Furthermore, the PK of THC is affected by the mode of consumption, wherein smoked and vaporized THC PK are similar but PK following oral consumption differs significantly irrespective of the frequency of use.19 Studies have also identified minor cannabinoids present in the THC plant and other THC metabolites that are typically present for short term in all types of users. These include CBD and cannabinol (CBN) and THC-glucuronide, respectively, which are detectable up to four to five hours. However, absence of these analytes cannot be used to rule out recent use.20
Since the frequency of cannabis intake produces complication in result interpretation, identifying frequent vs. occasional users may add value to clinicians and in legal settings. Chronic users have a high baseline of THC-COOH blood concentrations. Based on a controlled cannabis smoking study, a group from Switzerland has recommended using THC-COOH blood concentrations to help distinguish occasional users (≤ 3 μg/L) from frequent users (≥ 40 μg/L).21 Another criterion was raised with combined THC whole blood concentration ≥ 5 μg/L and THC-COOH/11-OH-THC ratio <20 for identification of recent use in frequent users. These cut-offs demonstrated a detection window < 8 hours for all consumption routes. Notably, using these same criteria, occasional smokers were negative 1.5 hours after inhalation and 12 hours following oral use.19
Overall, in addition to the complex PK of THC, determining recent use from blood testing is affected by the following: consumption route, interpersonal variability in metabolism, frequency of use, dose, and timing of blood collection. All of these factors make it difficult to predict the time of last use but can be achievable to a certain extent with multiple complementary criteria.19
Testing in OF
Oral fluid is increasingly gaining traction in the legal arena in cases of driving under the influence of drugs, and workplace drug testing. Oral fluid sample collection is non-invasive, less likely to be adulterated, and the sample can be collected at the time cannabis use is suspected. Regulatory organizations like Substance Abuse and Mental health Services Administration and Driving Under the Influence of Drugs, Alcohol and Medicines advise testing for THC in OF using a cut-off of 1 and 2 μg/L, respectively.22,23 Positive THC at these concentrations is accepted to indicate recent marijuana use. However, interpretation using these cut-offs requires caution because OF THC PK also varies between chronic and occasional users. THC levels peak to very high levels immediately after smoking due to oral mucosa contamination and drop rapidly. But like blood, chronic users have a high baseline THC level in OF that remains well above these thresholds for longer periods of time. In contrast, recent use in intermittent users can be difficult to capture because the levels may not reach these thresholds or drop rapidly barring detection. Thus, testing for THC alone in OF is of limited value.
PK profile of 11-OH-THC, THC-COOH, and minor cannabinoids found in the THC plant—tetrahydrocannabivarin (THCV), CBD and cannabigerol (CBG)—have been characterized to determine their potential in identifying recent use. Following three different routes of administration in a study by Swortwood et. al.24 11-OH-THC was detectable for up to 1.5 hours but not necessarily consistently in occasional users. THCV was detected up to 8 hours and 12 hours in occasional and frequent users, respectively. However, similar to 11-OH-THC, THCV may be absent in occasional users. CBG was no longer detectable after 32 hours in occasional users and interestingly, after 26 hours in frequent users. CBD was another promising analyte that was detectable for up to 20 hours but further research with high potency THC/CBD formulation is needed to determine its usefulness. THC-COOH is found at very low concentrations in OF, therefore a sensitive assay would be needed to determine its presence. Its presence in OF is a result of THC metabolism and partitioning of THC-COOH from blood to OF. Therefore, detectable THC-COOH in OF can be useful in ruling out passive exposure. Evidently, passive exposure to marijuana smoke can result in OF THC as high as 2 μg/L.25 Oral fluid THC-COOH detection window is highly variable depending on dose, mode of consumption, and frequency of use, with longest documented detection limit of 96 hours.26 Overall, the minor cannabinoids in OF appear to be promising biomarkers for determining recent use but further studies are needed to solidify their usefulness.
Cannabis use in the U.S. continues to experience growth. It remains to be determined which analytes and at what cut-off in a given biologic fluid would be useful in predicting impairment. Despite the caveats, detecting THC and its metabolites in biological samples is currently the best means to identify recent marijuana use. Testing in urine cannot predict time of last use but is useful in identifying resumed use from residual excretion. A stronger correlation exists in blood concentrations in identifying recent exposure, but back-extrapolation of last cannabis usage predicted from blood concentrations is compromised by factors such as frequency of usage and interpersonal PK. Quantitation of a number of cannabinoids and use of certain criteria could aid in interpreting results accurately. Undoubtedly OF has a lot of potential in replacing blood testing but proper interpretation still necessitates development of models to extrapolate last cannabis use and correlation of OF level with impairment. In conclusion, it is important to be aware of the limitations of testing in each of these sample types and understand what is extractable from lab data to draw accurate conclusions about cannabis use.