Hexahydrocannabinol (HHC) is a cannabinoid that has been known since 1940 but has only recently found its way into recreational use as a psychoactive drug. HHC has been used as a legal alternative to tetrahydrocannabinol (THC) in many countries, but first countries already placed it under their narcotic substances law. Our aim was to evaluate a reliable analytical method for the proof of HHC consumption by LC-MS/MS and GC-MS. We identified the two epimers of HHC and metabolites after HHC consumption by two volunteers (inhalation by use of a vaporizer and oral intake). LC-HR-MS/MS, LC-MS/MS and GC-MS with literature data (EI-MS spectra of derivatives) and reference compounds - as far as commercially available - were used for metabolite identification. Phase-II-metabolites (glucuronides) of HHC and OH-HHC were found in urine samples with LC-HR-MS/MS and LC-MS/MS. The main metabolite was tentatively identified with GC-MS as 4'OH-HHC (stereochemistry on C9 and C4' unknown). Another major side-chain hydroxylated metabolite found by LC-MS/MS could not be unambiguously identified. Both epimers of 11-OH-HHC were found in considerable amounts in urine. (8 R, 9 R)-8-OH-HHC was identified as a minor metabolite with GC-MS and LC-MS/MS. While (9 S)-HHC was found in urine after oral intake and inhalation of HHC, the more psychoactive epimer (9 R)-HHC was only found in urine after inhalation. Several other minor metabolites were detected but not structurally identified. We found that after oral or inhalative consumption the urinary main metabolites of a diastereomeric mixture of HHC are different from the respective, major Δ⁹-THC metabolites (11-OH-Δ⁹-THC and 11-nor-9-carboxy-Δ⁹-THC). Although a sensitive LC-MS/MS and GC-SIM-MS method were set-up for the reference compounds (9 R)-11-nor-9-carboxy-HHC and (9 S)-11-nor-9-carboxy-HHC, these oxidation products were not detected in urine with these techniques. To further increase sensitivity, a GC-MS/MS method was developed, and the 11-nor-9-carboxy metabolites of HHC were confirmed to be present as minor metabolites.

After the Swiss ban of hexahydrocannabinol (HHC) in March 2023, other semisynthetic dibenzopyran cannabinoids emerged on the Swiss gray market. Hexahydrocannabiphorol (HHCP) was the most prominent of them due to its potent cannabimimetic effects, as anecdotal reports from recreational users suggest. In October 2023, a class wide ban of dibenzopyran cannabinoids was introduced in Switzerland to prevent new similar substances from entering the drug market. Various vendors in online shops claim that HHCP is made from CBD, even though they possess different alkyl chain lengths. An HHCP sample was analyzed by gas chromatography coupled to mass spectrometry (GC‐MS), showing that a mixture of molecules with the same or a similar molecular mass as HHCP was present. Six different substances could be isolated from this sample using column chromatography. Four phenols ((9R)‐HHCP, iso‐HHCP, cis‐HHCP, and abn‐HHCP) and two ketones (possible intermediates to (9R)‐HHCP and abn‐HHCP) were identified by various nuclear magnetic resonance spectroscopy (NMR) techniques. (9S)‐HHCP was obtained in an impure fraction. In addition, a fraction was obtained that showed characteristic molecular and fragment ions consistent with bisalkylated products from the synthesis of similar compounds. The presence of abnormal cannabinoids (abn‐HHCP) and bisalkylated cannabinoids is a confirmation that this sample was produced purely synthetically as initially suspected, as these compounds have not been reported in Cannabis. Chiral derivatization of the phenols with Mosher acid chlorides showed that only iso‐HHCP was present as a scalemic mixture, indicating a good stereocontrol of this synthetic procedure.

Δ⁹-Tetrahydrocannabiphorol (Δ⁹-THCP, THCP) a psychoactive cannabinoid recently found in Cannabis sativa L., is widely used as a legal marijuana substitute. THCP is encountered in sprayed Cannabis, edibles, and vape liquids. The distributors of such products claim that the THCP in use originates from a natural source. The legal status of this substance varies from country to country. THCP and similar cannabinoids with a dibenzoyprane structure have been banned in Switzerland since October 2023. A vape liquid, which contains 90% THCP and 10% terpenes according to the distributor, was analyzed by gas chromatography coupled with mass spectrometry (GC-MS). Besides CBP, CBDP, Δ⁹-THCP and Δ⁸-THCP and some terpenes, other compounds were found which probably result from a synthetic procedure. This sample contained 5-heptylresorcinol, the heptyl homologue of olivetol, a common precursor for the synthesis of tetrahydrocannabinol (THC). Bisalkylated compounds (m/z 476) were found as a result of the reaction of one equivalent of 5-heptylresorcinol with two equivalents of (+)-p-mentha-1,8-dien-4-ol or another precursor. Similar bisalkylated compounds are known as undesired side products of the synthesis of THC. The sample contained unidentified isomers of Δ⁹-THCP, presumably abnormal cannabinoids (abn-Δ⁹-THCP; abn-Δ⁸-THCP) and iso-cannabinoids (iso-THCP). Chiral derivatization with Mosher acid chlorides revealed that the Δ⁹-THCP in the sample was enantiopure.

Hexahydrocannabiphorol (HHCP) is an emerging semisynthetic cannabinoid, which has been known since 1942 from research of tetrahydrocannabinol (THC) analogs and homologs. After the ban of hexahydrocannabinol (HHC) in many European Countries HHCP emerged as a replacement among other similar compounds. First countries already placed HHCP under their narcotic substance law. The aim of this research was to identify human Phase I and II metabolites in urine after oral HHCP consumption. Enzymatic immunoassays of urine samples were tested negative for cannabinoids after a single oral consumption of 4‐mg HHCP from a Δ⁹‐THC abstinent volunteer. The HHCP sample consumed in the self‐experiment was purchased from an online store and analyzed beforehand using GC–MS. LC–HR‐MS/MS and GC–MS after derivatization were used for the identification of metabolites. Hydroxylated metabolites were found with hydroxylation on the side chain or on the alicyclic part of the molecule. Bishydroxylated HHCP metabolites were found in similar abundance as the monohydroxy metabolites. All of the bishydroxylated metabolites besides a minor metabolite had a hydroxyl group on the side chain and another hydroxyl group on the alicyclic part of the molecule. In addition, the corresponding glucuronides were identified by LC–HR‐MS/MS. The exact positions and stereochemistry of the hydroxylation sites could not be determined. Due to the extensive metabolism of HHCP and the lacking cross‐reactivity of urine samples after consumption in Δ⁹‐THC specific immunoassays, it is recommended to include HHCP metabolites in routine screening methods. Monohydroxylated and bishydroxylated metabolites of HHCP and their respective glucuronides are suggested as forensic targets.

Semi-synthetic cannabinoids are a class of new psychoactive substances (NPSs) with structural similarities to the main psychoactive phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) found in Cannabis sativa L. The first semi-synthetic cannabinoids, which were used as legal substitutes for marijuana, were Δ8-tetrahydrocannabinol (Δ8-THC) and hexahydrocannabinol (HHC). Δ8-THC emerged around 2019 on the recreational drug market in the United States after it became legal due to an ambiguity in the Agricultural Improvement Act 2018 (Farm Bill 2018). It was never legal outside the United States as the isomers of THC are regulated in the United Single Convention on Narcotic Drugs from 1971. HHC, a hydrogenated derivative of THC, followed as a legal substitute on the European recreational drug market. Many countries already placed HHC in their narcotic substance law, which led to the emergence of other structurally related derivatives of THC. An existing rapid screening method for the qualitative analysis of various new psychoactive substances was expanded for semi-synthetic cannabinoids in whole blood using a LC–QTOF–MS system. This method was validated for 24 different phytocannabinoids and semi-synthetic cannabinoids in blood. Recovery rates of the analytes from a liquid–liquid-extraction ranged from 87% to 118%, matrix effects ranged from 24% to 93%, and limits of detection (LOD) ranged from 0.8 to 16 ng/mL.

The pharmacokinetics of the semisynthetic cannabinoid hexahydrocannabinol (HHC) and its 11-nor-carboxy- as well as 11-hydroxy-metabolites has already been the subject of research in various recent publications. However, further metabolism studies showed the presence of other, especially (side-chain-)hydroxylated metabolites in urine and serum. This study aimed to investigate the pharmacokinetics of these metabolites after oral (25 mg fruit gum) and inhalative (three puffs from vape) consumption, using serum and urine samples from a previous study. Serum (up to 48 h) and urine (up to five days) samples of six participants (three per consumption group) were collected at different time points and analyzed by LC-QqTOF and LC-QqLIT. Three metabolites (M1: side-chain-hydroxylated; M2: hydroxylated on alicyclic moiety; M3: 8-OH-HHC) were further investigated. The peak area ratios of analytes and internal standards were plotted against the time after consumption to obtain pharmacokinetic information. Overall, this study proved the presence of these metabolites in biological samples and provided pharmacokinetic insights for the first time. Differences between serum and urine, oral and inhalative consumption as well as inter-individual differences were observed. The investigated metabolites could partially serve as unambiguous consumption markers for HHC, as they are unlikely to be formed in vivo from corresponding Δ ⁹ -tetrahydrocannabinol (THC) metabolites.