MSACL 2022 Abstract

Introduction
(-)-trans-delta9-Tetrahydrocannabinol ((-)-Δ9-THC), the main psychoactive constituent of cannabis sativa L., is currently used medicinally and recreationally. The plant constituent and bioactive THC form is a specific optically active (-)-trans isomer with stereochemistry (6aR,10aR). Metabolic glucuronidation results in diastereomers with a combination of the beta anomers at the glucuronic linkage, as well as chiral centers at the 6 and 10 positions on the THC molecule. In response to the growing need to test for cannabis use in clinical and forensic settings, cannabinoid Certified Reference Materials (CRMs) and testing methods have been developed for the major metabolite of (-)-Δ9-THC, (-)-11-nor-9-Carboxy-Δ9-THC glucuronide. The first CRM offered for this testing application was (+)-11-nor-9-Carboxy-Δ9-THC glucuronide which is a diastereomer of stereochemistry (6aS,10aS) of what is expected to be the naturally occurring metabolite. Studies have demonstrated that the major glucuronide metabolite identified in cannabis users differs from available reference material. Here we discuss the synthesis, certification, and confirmation of the first reference material for (-)-trans-11-nor-9-Carboxy-Δ9-THC beta-D-glucuronide which matches the major glucuronide metabolite observed in individuals following the use of cannabis.

Methods
(-)-trans-11-nor-9-Carboxy-Δ9-THC beta-D-glucuronide was synthesized via a multi-step sequence from chirally pure (-)-trans-THC intermediates. Multiple reaction monitoring (MRM) liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to evaluate the chromatographic retention of (-)-11-nor-9-Carboxy-Δ9-THC glucuronide in whole blood specimens obtained from individuals which recently smoked cannabis and the synthetic reference.

Results
Synthesized (-)-trans-11-nor-9-Carboxy-Δ9-THC beta-D-glucuronide was certified using an orthogonal testing approach that results in a mass balance purity factor that accounts for chromatographic purity, water, residual solvent, inorganic content, etc. These tests are used in conjunction with structure identification techniques like NMR and LC-MS. MRM LC-MS/MS was then utilized to compare in vivo samples with the newly synthesized (-)-trans metabolite and available CRM, Cerilliant Cat # T-038 (+)-11-nor-9-Carboxy-Δ9-THC glucuronide. This comparison confirmed a difference in the observed retention times of T-038 with the new (-)-trans metabolite and the in vivo metabolite, while the latter two were observed to have matching retention times. Hydrolysis of both the in vivo metabolite and reference materials (T-038 and newly synthesized (-)-trans metabolite) resulted in a non-glucuronidated metabolite with matching retention times. This was an expected outcome as the hydrolyzed product of T-038 is the enantiomer of the synthesized (-)-trans and in vivo metabolite, and would not separate under achiral chromatographic conditions.

Conclusion
(-)-trans-11-nor-9-Carboxy-Δ9-THC beta-D-glucuronide was synthesized and manufactured as a CRM according to ISO 17034 for use in forensic and clinical LC-MS/MS applications. This is the first report that positively identifies the major glucuronidated form of THC as (-)-trans-11-nor-9-Carboxy-Δ9-THC beta-D-glucuronide in humans. The synthesized metabolite was compared against patient samples and chromatographically matched the in vivo metabolite observed in whole blood specimens. (-)-trans-11-nor-9-Carboxy-Δ9-THC beta-D-glucuronide is currently being developed into a solution standard under Cerilliant Cat # T-148-1ML and will be available in conjunction with the existing CRM, T-038. The availability of these reference materials provides the clinical and forensic fields with authentic reference materials for LC-MS/MS assays for the detection of (-)-11-nor-9-Carboxy-Δ9-THC glucuronide in clinical and forensic specimens.