= Emerging. More than 5 years before clinical availability. (19.79%)
= Expected to be clinically available in 1 to 4 years. (37.97%)
= Clinically available now. (42.25%)
MSACL 2022 : Suhandynata

MSACL 2022 Abstract

Self-Classified Topic Area(s): Tox / TDM / Endocrine > Troubleshooting

Podium Presentation in De Anza 3 on Wednesday at 14:40 (Chair: Karen Phinney / Andrew T Nelson)

Get Your Reference Right!: Synthesis, Certification, and Confirmation of the First Reference Material for (-)-trans-11-nor-9-Carboxy-Δ9-THC beta-d-glucuronide

Suhandynata RT. (1), Koswatta P.(2), Lund K. (1), Menlyadiev M.(1), Sreenivasan U.(2), Lima H.M.(2), Fitzgerald R.L.(1)
(1) University of California, San Diego, CA (2) MilliporeSigma

Raymond Suhandynata (Presenter)
University of California, San Diego

Presenter Bio: Dr. Suhandynata recently completed his Clinical Chemistry fellowship at the UC San Diego Center for Advanced Laboratory Medicine, under the direction of Dr. Robert Fitzgerald. He attended the State University of New York at Stony Brook where he received his Ph.D. in Biochemistry and Structural Biology. His graduate work was performed under the mentorship of Dr. Nancy Hollingsworth, where he applied quantitative phospho-proteomics to identify novel kinase targets by LC-MS/MS. His post-doctoral work was performed at the Ludwig Institute for Cancer Research at the University of California San Diego, and focused on applying quantitative LC-MS/MS to identify sumoylated proteins and the roles they play in chromosome segregation. As a Clinical Chemistry fellow, he led efforts in the development, validation, and implementation of COVID-19 serology assays in the clinical laboratory.

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.


Financial Disclosure

DescriptionY/NSource
Grantsno
SalaryyesUniversity of California San Diego
Board Memberno
Stockno
Expensesno
IP Royaltyno

Planning to mention or discuss specific products or technology of the company(ies) listed above:

yes