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.

中文翻译：

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DUID 病例血液中六氢大麻酚 (HHC) 和代谢物的定量

六氢大麻酚（HHC）于2022年5月在欧盟首次报道。HHC具有三个手性碳原子，但只有（6aR,9R,10aR）-HHC（9R-HHC）和（6aR,9S,10aR）-HHC（9S- HHC）已在 HHC 产品中遇到过。本研究的目标是开发和验证一种定量分析 9R-HHC、9S-HHC、11-OH-9R-HHC、9R-HHC-COOH、9S-HHC-COOH 和 8-OH- 的方法。 9R-HHC。此外，一个目标是研究免疫化学交叉反应性。使用 ELISA 对 DUID 病例的血液样本进行大麻筛查呈阳性，并确认四氢大麻酚 (THC)、11-羟基-THC 和 THC-COOH 呈阴性，并使用新验证的 HHC 方法重新分析，以调查 HHC 和代谢物的存在。 LC-MS/MS 方法经过了基质效应、定量下限 (LLOQ)、校准模型、精度、偏差和自动进样器稳定性的验证。分别研究浓度范围为 5-200 ng/mL 的 9R-HHC-COOH 和 9S-HHC-COOH 的 ELISA 方法交叉反应性。发现 9R-HHC-COOH 的交叉反应率为 120%，9S-HHC-COOH 的交叉反应率为 48%。在 LCMSMS 方法中，9R-HHC-COOH、9S-HHC-COOH 和 11-OH-9R-HHC 在两种浓度下均显示出小于 25% 的基质效应，而 8-OH-9R-HHC、9R-HHC 和 9S- HHC 基质效应在两种浓度下均超过 25%，但在进一步验证中显示出良好的精度（日间和日间均＜10%）和低偏差（＜6%）。除羧化代谢物的 LLOQ 为 2.0 ng/mL 外，所有分析物的 LLOQ 均经过研究并确定为 0.2 ng/mL。定量上限分别为20和200 ng/ml。对病例 (N=145) 的重新分析证实了 32 例 (22%) 病例中存在 HHC 和代谢物。经确定，施用 HHC 后血液中的主要代谢物是 9R-HHC-COOH，其次是 11-OH-9R-HHC，并且通过常规 ELISA 大麻筛查发现了推定阳性病例。