FAA Office of Aerospace Medicine
Civil Aerospace Medical Institute

Report No: DOT/FAA/AM-15/6

Title and Subtitle: Comparison of Species-Specific &#223-Glucuronidase Hydrolysis of Cannabinoids in Human Urine

Report Date: March 2015

Authors: Kemp PM, Cliburn KD

Abstract: Toxicological investigations of fatal aviation accidents include testing for Delta-9-tetrahydrocannabinol (THC), the active component of marijuana, and its major metabolite, 11-nor-9-carboxy-Delta-9-THC (THCCOOH) in postmortem fluids and tissues from deceased pilots. These cannabinoids, as well as others, exist in human urine as their glucuronide conjugates, formed during Phase II metabolism to increase their solubility for excretion. Prior to the analysis of cannabinoids in urine by gas chromatography/mass spectrometry (GC-MS), it is desirable to cleave (hydrolyze) the glucuronic acid to enhance the detection of the unconjugated compounds. In the current study, &#223-glucuronidases from 3 mollusk species (Helix pomatia, Patella vulgate, and Haliotis rufescens), 1 bacteria (Escherichia coli), and bovine liver were compared for their quantitative efficiency in hydrolyzing the glucuronide bond in spiked, blank urine.

THC and THCCOOH were selected for this project to study the hydrolysis efficiency of the various enzymes on the ester-linked THCCOOH-glucuronide and the ether-bonded THC-glucuronide. An unconjugated metabolite, 11-hydroxy-THC (11-OH-THC), was included to evaluate the effect of the various hydrolysis conditions on its recovery. The hydrolysis variables evaluated were pH, time, and temperature of incubation. Peak area ratio of analyte/deuterated internal standard was used to quantify cannabinoid recovery. All of the enzymes were able to hydrolyze both the THC and THCCOOH glucuronides, but there were significant species-specific differences. The &#223-glucuronidases from Haliotis rufescens, Escherichia coli, and Patella vulgata produced the greatest recovery of THC and THCOOH during the pH experiments.

While recovery of THCCOOH was acceptable using Helix pomatia and bovine liver enzymes, these glucuronidases did not hydrolyze the ether-bonded THC conjugate as efficiently as the other three. Further recovery experiments with Haliotis rufescens, Escherichia coli, and Patella vulgata investigated the effect of incubation time and temperature on the cannabinoid recoveries. Experimental incubation times (hours) were 0.25, 1.0, 4.0, 8.0, and 16 at optimum pH for each enzyme and 37^oC incubation temperature. Experimental temperatures (^oC) were 25, 37, 50, 60, and 90 at optimum pH for each enzyme and a 16 h incubation time. The results demonstrated that &#223-glucuronidases from Haliotis rufescens, Escherichia coli, and Patella vulgata would provide the best recovery of both ester-linked and ether-linked cannabinoid metabolites with GC-MS analysis. Further research will now analyze actual postmortem urine from THC-positive pilots involved in aviation accidents to identify a more complete metabolic profile of cannabinoids.

Key Words: Forensic Science, Toxicology, Hydrolysis, Glucuronidase, Gas Chromatography/Mass Spectrometry, Cannabinoids, Tetrahydrocannabinol, Aviation

No. of Pages: 15