Cannaboid Testing in Forensic Toxicology in the Wave of Legalized Marijuana

Currently, 26 US states have approved medical marijuana in their legislature, while another 16 have decriminalized possession and 4 states (plus DC) have legalized marijuana use. Marijuana per se driving laws for driving under the influence of drugs (DUID) were enacted in 17 states, making it illegal for someone to operate a vehicle with detectable THC (Δ9-tetrahydrocannabinol) or its major metabolite, THCCOOH (11-nor-9-carboxy-THC), in blood and/or urine. A majority of those states have zero tolerance laws while some of them enforce specific THC driving cutoffs (1-5 ng/mL THC). However, low THC levels can be detected in frequent cannabis smokers’ blood up to 30 days after last use during sustained abstinence, making cannabinoid data interpretation difficult.

In a recent national US survey of adults reporting past month (current) marijuana use, nearly half of respondents reported using bowls, pipes, or joints. Interestingly, 16.1% admitted to using marijuana edibles/drinks and 7.6% used a vaporizer or “other electronic device”. As different modes of marijuana use become more popular, more data are needed to fully understand the impairing effects as well as the impact on THC levels in biological samples.

Recent marijuana research and cutting edge analytical equipment and technology have allowed forensic toxicologists to analyze for cannabinoids in a variety of samples, including blood, urine, oral fluid, and hair. Researchers are currently identifying markers of recent use in order to differentiate those who smoked or used marijuana recently from those who may have used days prior. Some of these state-of-the-art methods incorporate liquid-liquid extraction (LLE), solid-phase extraction (SPE), or disposable pipette extraction (DPX) when coupled with gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Perhaps the most common biological fluid for drug testing is blood, especially for DUID cases. Cannabinoids detected in blood can reflect recent use and acute impairment. However, sensitive analytical techniques are capable of quantifying THC at low levels, particularly in frequent smokers, long after the window of acute impairment. Blood collection requires trained phlebotomists or medical professionals to collect the sample, often occurring up to 4 hours after the time of a crash or traffic stop for DUI investigations. For this reason, researchers are hoping to identify markers of recent use within a 6- to 8-hour detection window after marijuana consumption. Aiming to identify those markers, a comprehensive analytical method for detecting and quantifying THC, minor cannabinoids, and their metabolites was developed using DPX WAX-S and LC-MS/MS1. The method allows for monitoring multiple cannabinoids in an attempt at discriminating between medicinal and recreational marijuana use. However, whole blood is not always available for clinical or forensic toxicology testing, so serum may be used. Routine forensic toxicology laboratories may not yet have access to LC-MS/MS and may still rely on GC-MS techniques. Oftentimes, LLE is still also used in many laboratories. While SPE is routinely automated in order to cut-down on workload, recent technology, Gerstel MultiPurpose Sampler (MPS), has allowed for automating LLE. A recent publication used the Gerstel MPS for extracting cannabinoids in serum, with a reduced sample volume and reduced organic solvent for higher-throughput GC-MS cannabinoid testing2.

While blood is often analyzed for recent drug use, urine is more often analyzed in workplace drug testing, pain management, and drug treatment compliance testing. The main, inactive metabolite of THC, 11-nor-9-carboxy-THC (THC-COOH) is routinely quantified in urine to determine approximately past-month marijuana use. While LLE or SPE are useful sample preparation approaches, DPX is a more efficient and timely technique for extracting cannabinoids from urine, allowing for direct analysis on an LC-MS/MS system. Our recent application note utilizes the Gerstel MPS with DPX Option for analysis of THC-COOH in hydrolyzed urine in a fully-automated procedure that can be easily implemented in routine forensic toxicology testing. However, for those opting for more specific testing and direct analysis of phase II glucuronidated metabolites, a DPX method was recently developed for quantification of THC, minor cannabinoids, and glucuronidated metabolites in a small sample volume of urine using DPX WAX-S tips in combination with LC-MS/MS3.

Oral fluid is an increasingly popular biological fluid for drug testing, particularly in a DUID setting. Oral fluid is advantageous over blood as it reflects recent drug intake without the need for trained medical professionals to collect the sample. Additionally, oral fluid is not easily adulterated and does not require same-sex collectors like with urinalysis. Oral fluid THC levels are primarily a result of oromucosal contamination with peak levels occurring during or immediately after smoking, and then rapidly decrease. THC in oral fluid is a general indication of recent (within 1 day) cannabis use. When workplace oral fluid testing was initially proposed, few analytical methods were sensitive enough to quantify THC levels often detected in oral fluid (low ng/mL). To address the analytical challenge, Gerstel scientists developed a stir bar sorptive extraction (SBSE) using the Gerstel-Twister coupled with GC-MS to reduce sample preparation and achieve analytical sensitivity necessary. However, THC can be detected in oral fluid for around one day, so toxicologists are trying to identify recent use markers in oral fluid, particularly analytes which would be suitable regardless of smoking frequency or mode of consumption. A recent clinical study from the National Institute on Drug Abusestudied oral fluid cannabinoids after smoked, vaporized, and edible marijuana and identified some minor cannabinoids, cannabigerol (CBG) and  tetrahydrocannabivarin (THCV), that may be useful for detecting recent (within 8 hour) marijuana use. The methodology, combining hydrolysis, SPE, and LC-MS/MS could be easily automated for routine, high-throughput oral fluid cannabinoid testing, especially with increased oral fluid regulatory testing guidelines.

While detecting THC and other cannabinoids for identifying recent use is relevant to DUID and workplace testing, some compliance programs are more concerned with identifying drug use across a wider detection window. While urine is a useful biological fluid for this type of testing, it is an invasive procedure requiring same-sex collectors and samples can easily be easily manipulated. Hair THC testing is useful for documenting specific times of use over past weeks or even months, but sample preparation can be tedious and difficult. In order to comply Society of Hair Testing recommended, but challenging, cutoffs, researchers developed a fully-automated protocol using the Gerstel MPS in combination with GC-MS that allowed for decreased sample preparation time and necessary sensitivity4.

As states continue to add medical and recreational marijuana to their ballots in the coming elections, forensic toxicologists need to be aware of the implications for THC testing. While a positive THC test used to be sufficient for punitive action, cannabinoid data interpretation is now more difficult than ever as we have to differentiate recent use from prior use. Sensitive analytical methodologies are now available for quantifying THC and other cannabinoids and metabolites in blood, urine, oral fluid, and hair and can be easily automated and implemented in forensic toxicology laboratories. As more research is published on different markers of recent use that can be implemented regardless of smoking frequency or mode of marijuana use, toxicologists will be responsible for more challenging analytical testing.

1Scheidweiler KB, Newmeyer MN, Barnes AJ, Huestis MA. J Chromatogr A, 1453: 34-42 (2016).

2Purschke K, Heinl S, Lerch O, Erdmann F, Veit F. Anal Bioanal Chem, 408: 4379-7388 (2016).

3Andersson M, Scheidweiler KB, Sempio C, Barnes AJ, Huestis MA. Anal Bioanal Chem, 408: 6461-6471 (2016).

4Heinl S, Lerch O, Erdmann F. J Anal Toxicol, 40:498-503 (2016).


Madeleine J. Swortwood, Ph.D.

Assistant Professor, Forensic Science Department, College of Criminal Justice, Sam Houston State University