Schirmer, Willi (2025). Analytical characterization of semi-synthetic cannabinoids in Forensics: Detection, metabolism and synthetic origin. (Thesis). Universität Bern, Bern

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Abstract

In mid 2022, hexahydrocannabinol (HHC) emerged as an unregulated, legal substitute for cannabis. HHC is a hydrogenated derivative of the main psychoactive compound found in cannabis, Δ9-tetrahydrocannabinol (Δ9-THC), which also shows psychoactive effects in human. It was therefore commonly abused and set new challenges for forensic investigators due to its novelty. Prior 2023, only scarce information about the detection and metabolism of HHC were available. After the regulation of HHC in Switzerland in April 2023, other semi-synthetic cannabinoids emerged, which were even more potent, such as hexahydrocannabiphorol (HHCP) or Δ9-tetrahydrocannabiphorol (Δ9-THCP). These potent successors shared the same core structure, but they have a longer side chain. Distributors of these products claim that they are obtained from the cannabis plant or from extracted cannabidiol (CBD). Two distinct products were investigated to get an insight into the synthetic procedures. A product that was declared as pure HHCP wax (Publication I) and a vape pen, which contained 10% Δ9-THCP according to the labeling (Publication II). After initial GC-MS analysis of the HHCP wax, it was found that this recreational product contained several compounds with mass spectra similar to or indistinguishable from HHCP. Six compounds were isolated from the wax using flash column chromatography and their structures were elucidated by NMR. Besides the main compound (9R)-HHCP, the side products iso-HHCP, cis-HHCP, abn-HHCP, and two α,β-unsaturated ketone intermediates were identified. The isolated products were derivatized using R- and S-mosher acid chloride and analyzed by GC-MS. Besides iso-HHCP, the isolated compounds were enantiopure. Furthermore, a fraction was collected that contained bisalkylated cannabinoids, which are common side products in total synthetic approaches for the synthesis of phytocannabinoids. The presence of the intermediates indicated that a derived route from Tietze et al. for the synthesis of HHC has been used. The vape pen, which contained Δ9-THCP was analyzed by GC-MS and it was found that this product contained several compounds with mass spectra similar to or indistinguishable from Δ9-THCP. Besides Δ9-THCP, the vape pen liquid contained cannabiphorol (CBP), cannabidiphorol (CBDP), Δ8-THCP, and 5-heptylresorcinol, which were confirmed by comparison with reference standards. Additionally, four other compounds were tentatively identified by comparison with mass spectra from THC analogs, these were 8-oxo-Δ9-THCP, cis-Δ9-THCP, 7,8-dihydrocannabidiphorol, and 6a,10a-dihydrocannabiphorol. Furthermore, the vape pen liquid also contained a fraction with bisalkylated cannabinoids, which are commonly found in total synthetic approaches of phytocannabinoids. The compounds Δ9-THCP and 5-heptylresorcinol were isolated from the vape pen by flash column chromatography. The isolated Δ9-THCP was derivatized with mosher acid chloride and it was found that this compound was enantiopure, too. The presence of 5-heptylresorcinol, a homolog of olivetol, indicates that this product was synthesized by using a total synthetic approach as olivetol is a known precursor for the total synthesis of Δ9-THC. It is however unclear which other starting material has been used as several terpenes can be used for the total synthesis. This vape pen liquid contained various unspecified terpenes, presumably for olfactory and sensory reasons. In order to prove the abuse of new emerging substances, analytical targets have to be identified, which are the abundant and characteristic metabolites of these new compounds. As another topic, this thesis describes the identification of Phase I and II metabolites of the semi-synthetic cannabinoids HHC (Publication III), HHCP (Publication IV), and tetrahydrocannabidiol (H4CBD, Publication V) in human urine. These studies involved one or two volunteers who ingested a small amount of the semi-synthetic cannabinoids and collected urine over the course of up to three days. The Phase I and II metabolites were identified using LC-QqTOF. The Phase I metabolites were also analyzed after trimethylsilylation with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and sensitive detection methods for the Phase I metabolites were developed using LC-QqLIT. For HHC, it was found that the most abundant Phase I metabolites were side chain hydroxylated metabolites. The main metabolite was tentatively identified as 4’OH-HHC. Besides 4’OH-HHC, both epimers of 11-OH-HHC have been found to be abundant metabolites and therefore good analytical targets. The epimers of 11-nor-9-carboxy-HHC (HHC-COOH) and (8R,9R)-8-OH-HHC were identified as minor metabolites. Further metabolites with hydroxylation positions on the alicyclic moiety or on the side chain were detected but their structures were not elucidated. The detected Phase II metabolites were glucuronides of HHC and its hydroxylated structures. In the case of HHCP, the identified Phase I metabolites were mainly mono- and bishydroxylated metabolites. Hydroxylation was observed on the alicyclic moiety and/or the side chain. The bishydroxylated Phase I metabolites were more abundant than the monohydroxylated metabolites, indicating that cannabinoids with a longer side chain undergo further metabolisation. The most abundant Phase I metabolites of HHCP were two bishydroxylated metabolites, which showed a hydroxylation position on the side chain and another one on the alicyclic moiety of the molecule. Phase II metabolites were glucuronides of HHCP, which were formed to a lesser extent, and the glucuronides of Phase I metabolites. After HHCP ingestion, the urine samples were tested negative with homogeneous enzyme immunoassay tests designed for Δ9-THC. This indicates that positive cases for HHCP might remain undetected in routine case work. After the ingestion of H4CBD, deglucuronidated urine samples revealed several hydroxylated Phase I metabolites in high abundance, and bishydroxylated and carboxylated Phase I metabolites in low abundance. The carboxylated metabolites 7-COOH-H4CBD and 5”COOH-H4CBD, as well as the hydroxylated metabolites (1R,6R)-6-OH-H4CBD, (1R,6S)-6-OH-H4CBD, 2”OH-H4CBD and both epimers of 7-OH-H4CBD were tentatively identified. The Phase II metabolites were glucuronides of H4CBD and of its Phase I metabolites. ESI+ product ion mass spectra of H4CBD and its side chain hydroxylated Phase I metabolites showed very uncommon fragmentation patterns. In some cases, loss of both oxygens from the resorcinol unit was observed. The corresponding Phase II metabolites showed degradation of the glucuronide moiety to form furanyl radical cations. This emphasises the necessity that mass spectra of newly emerging compounds and their metabolites have to be investigated carefully as they might not just show shifted signals in respect to a well-known derivative. With the emergence of new semi-synthetic cannabinoids, detection methods have to keep up to date. Publication VI describes the development and validation of a qualitative screening method for the detection of semi-synthetic cannabinoids in human blood by LC-QqTOF. This method covers 24 different phyto- and semi-synthetic cannabinoids with recovery rates ranging from 87-118%, matrix effects ranging from 24-93% and limits of detection ranging from 0.8-16 ng/mL. During the development of this method, it was observed that more hydrophobic analytes showed non-specific binding to glass vials, which led to diminished or even complete loss of detection signals. The initially used glass vials did not show this behavior for the analysis of phytocannabinoids and their metabolites, and finally switching to polypropylene vials solved this issue. Further preanalytically investigations should be carried out, as this issue can occur when physiological samples are stored in unsuitable containers with the consequence of analyte loss remaining unnoticed.

Item Type:	Thesis
Dissertation Type:	Cumulative
Date of Defense:	7 November 2025
Subjects:	300 Social sciences, sociology & anthropology > 360 Social problems & social services 500 Science > 540 Chemistry 600 Technology > 610 Medicine & health
Institute / Center:	08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP)
Depositing User:	Hammer Igor
Date Deposited:	20 Jan 2026 17:52
Last Modified:	20 Jan 2026 17:52
URI:	https://boristheses.unibe.ch/id/eprint/7067

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