Quantitation of hexahydrocannabinol (HHC) and metabolites in blood from DUID-cases

Hexahydrocannabinol (HHC) was first reported in the EU in May 2022. HHC has three chiral carbon atoms, but only (6aR,9R,10aR)-HHC (9R-HHC) and (6aR,9S,10aR)-HHC (9S-HHC) have been encountered in HHC products. The goal of this study was to develop and validate a method for the quantitative analysis of 9R-HHC, 9S-HHC, 11-OH-9R-HHC, 9R-HHC-COOH, 9S-HHC-COOH, and 8-OH-9R-HHC. In addition, an objective was to investigate the immunochemical cross reactivity. Blood samples from DUID-cases screened positive for cannabis using ELISA and confirmed negative for tetrahydrocannabinol (THC), 11-hydroxy-THC, and THC-COOH were reanalyzed with a newly validated HHC method to investigate the presence of HHC and metabolites. The LC-MS/MS method was validated for matrix effects, lower limit of quantification (LLOQ), calibration model, precision, bias, and autosampler stability. Cross reactivity on an ELISA method was investigated separately for 9R-HHC-COOH and 9S-HHC-COOH at a concentration range between 5-200 ng/mL. The cross reactivity was found to be 120% for 9R-HHC-COOH and 48% for 9S-HHC-COOH. In the LCMSMS method 9R-HHC-COOH, 9S-HHC-COOH, and 11-OH-9R-HHC showed matrix effects less than 25% at both concentrations while 8-OH-9R-HHC, 9R-HHC, and 9S-HHC matrix effects exceeded 25% at both concentrations but showed good precision (<10% for both inter and between day) and low bias (<6%) in the further validation. The LLOQ was investigated and established at 0.2 ng/mL for all analytes except the carboxylated metabolites that had an LLOQ of 2.0 ng/mL. The upper limit of quantification was 20 and 200 ng/ml respectively. Reanalysis of cases (N=145) confirmed HHC and metabolites in 32 cases (22%). It was determined that the major metabolite in blood after administration of HHC was 9R-HHC-COOH followed by 11-OH-9R-HHC and that presumptive positive cases are caught by the routine ELISA screening for cannabis.