R E S E A R CH A R T I C L E Human metabolism of the semi-synthetic cannabinoids hexahydrocannabinol, hexahydrocannabiphorol and their acetates using hepatocytes and urine samples Karin Lindbom1 | Caitlyn Norman1 | Steven Baginski2 | Lucas Krebs3 | Darta Stalberga1 | Tobias Rautio4 | Xiongyu Wu4 | Robert Kronstrand1,5 | Henrik Gréen1,5 1Division of Clinical Chemistry and Pharmacology, Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden 2Leverhulme Research Centre for Forensic Science, School of Science and Engineering, University of Dundee, Dundee, UK 3Institute for Chemistry and Bioanalytics, School of Life Sciences, University of Applied Sciences Northwestern Switzerland, Muttenz, Switzerland 4Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden 5Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden Correspondence Henrik Gréen, Division of Clinical Chemistry and Pharmacology, Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden. Email: henrik.green@liu.se Funding information European Commission, Grant/Award Number: E! 113377; European Union's Horizon 2020; Sweden's Innovation Agency, Grant/Award Number: 2019-03566; Strategic Research Area in Forensic Sciences (Strategiområdet forensiska vetenskaper) at Linköping University; Leverhulme Trust, Grant/Award Number: RC-2015-011 Abstract Hexahydrocannabinol (HHC), hexahydrocannabiphorol (HHCP) and their acetates, HHC-O and HHCP-O, respectively, are emerging in Europe as alternatives to tetrahy- drocannabinol (THC). This study aimed to elucidate the metabolic pathways of the semi-synthetic cannabinoids HHC, HHCP, HHC-O and HHCP-O from incubation with human hepatocytes. The metabolites of HHC were also identified in authentic urine samples. HHC, HHCP, HHC-O and HHCP-O were incubated with primary human hepatocytes for 1, 3 and 5 h. Authentic urine samples from cases screened positive for cannabis in blood using ELISA but confirmed negative were analysed both non-hydrolysed and hydrolysed for HHC metabolites. Potential metabolites were identified using ultra-high performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight mass spectrometer (QToF-MS). HHC and HHCP were primarily metabolised through monohydroxylation (monoOH), followed by oxidation to a carboxylic acid metabolite. HHC-O and HHCP-O were rapidly metabolised to HHC and HHCP, respectively. In authentic urine samples, 18 different metabolites were identified, and 99.3% of hydroxylated metabolites were glucuroni- dated. 11-OH-HHC, 50OH-HHC and another metabolite with a monoOH on the side chain were the only metabolites present in all 16 urine samples. The metabolism of HHC and HHCP were similar, although the longer alkyl side chain of HHCP (heptyl) led to greater hydroxylation on the side chain than HHC (pentyl). The use of HHC and HHCP can be differentiated from the use of THC and other phytocannabinoids, but the use of the acetate analogues may not be differentiable from their non- acetate analogues. K E YWORD S hexahydrocannabinol, hexahydrocannabiphorol, HHC acetate, HHCP acetate, metabolism Karin Lindbom and Caitlyn Norman contributed equally to this work. Received: 21 March 2024 Revised: 3 May 2024 Accepted: 15 May 2024 DOI: 10.1002/dta.3740 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2024 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd. Drug Test Anal. 2024;1–15. wileyonlinelibrary.com/journal/dta 1 https://orcid.org/0000-0003-2322-0367 https://orcid.org/0000-0001-5615-1199 https://orcid.org/0009-0005-7679-4403 https://orcid.org/0000-0001-6712-652X https://orcid.org/0000-0002-0857-6261 https://orcid.org/0000-0001-6756-2276 https://orcid.org/0000-0002-4222-9597 https://orcid.org/0000-0002-8015-5728 mailto:henrik.green@liu.se https://doi.org/10.1002/dta.3740 http://creativecommons.org/licenses/by-nc/4.0/ http://wileyonlinelibrary.com/journal/dta http://crossmark.crossref.org/dialog/?doi=10.1002%2Fdta.3740&domain=pdf&date_stamp=2024-05-28 1 | INTRODUCTION Hexahydrocannabinol (HHC), hexahydrocannabiphorol (HHCP) and their corresponding acetates, HHC-O and HHCP-O, respectively (see Figure 1 for chemical structures), are emerging as analogues to tetra- hydrocannabinol (THC) and are marketed as new cannabis alterna- tives. These compounds have been sold in a variety of forms, for example, gummies, bubble gums, vapes or crude material.1 HHC was first identified in Europe in May 20222 and has now been identified in a qualified majority of the EU Member States.1 HHC-O has also emerged on the illicit market in Europe as an alternative to HHC with the first seizures reported in August 2022.3 HHCP was discovered in November 2022,4 and although no detections have been confirmed to date, HHCP-O has been reported to be available for sale on the illicit market.5 Chemically, HHC and its analogues are categorised as tricyclic terpenoid derivatives with a benzopyran ring (or hexahydrobenzochromenes). Although HHC and HHCP are found in small amounts in Cannabis sativa,6,7 the HHC and HHCP detected on the market are synthesised from cannabidiol (CBD) and Δ9-tetrahydrocannabiphorol (THCP), respectively; therefore, they are classed as semi-synthetic cannabinoids.8,9 The synthetic route of HHC-O and HHCP-O has not yet been reported but is likely synthesised using a similar method used for the synthesis of THC acetates.1 HHC has three stereocentres and eight possible stereoisomers8; however, it is typically only found as a mixture of two epimers, (9R) and (9S).8 In HHC- and HHC-O-containing products, the (9R) epimer was found to be about twice as abundant as the (9S).8–11 The (9R) epi- mer has also been found to be about twice as abundant as the (9S) in plasma and serum samples.12 Although there is no pharmacodynamic activity data available for HHCP, HHC-O or HHCP-O, in vivo and in vitro studies of (9R)- and (9S)-HHC found both epimers were partial agonists of the human cannabinoid 1 and 2 (CB1 and CB2) receptors. However, (9R)-HHC had much stronger binding affinity, potency and cannabimimetic effects,8,13–15 demonstrating that the intensity of bio- logical effects can vary based on the epimeric mixtures, which is likely also true for HHC-O. There is also limited information available on the pharmacokinet- ics and metabolism of HHC in humans and animals. Harvey and Brown (1991) examined the in vitro metabolism of (9R)-HHC using GC–MS in hepatic/liver microsomal preparations from five mamma- lian species. This study found only hydroxylated metabolites, where hydroxylation at the 11 position (11-OH-HHC) and 8 position (8-OH-HHC) were dominant. However, hydroxylation at all five posi- tions on the n-pentyl side chain and the 4 carbon position on the aromatic ring was also found.16 In a recent study reporting metabo- lite identification in a human urine sample after ingestion of 20 mg HHC, many of the identified metabolites were glucuronidated and the tentatively identified 40OH-HHC was found to be the major metabolite, followed by 11-OH-HHC and 8-OH-HHC.15 Another study found (9R)-11-COOH-HHC to be the major metabolite in two urine samples after inhalation of 25 mg HHC, followed by (9S)- 11-COOH-HHC, (9R)-11-OH-HHC and 9α-OH-HHC.17 Another study found 11-OH-HHC to be the major hydroxylated metabolite in a human blood sample and following pooled human liver S9 fraction incubations. No glucuronidated metabolites or 8-OH-HHC were found, but a carboxy metabolite was found in the human blood sam- ple.18 Although HHC-O and HHCP-O are believed to go through rapid metabolism and conversion to HHC and HHCP, respectively, in vivo, as seen for other esters, this metabolism has not yet been shown. This study aimed to identify the main metabolites of HHC, HHCP and their corresponding acetates following incubation with human hepatocytes to get a more complete understanding of the human in vitro metabolism of these emerging semi-synthetic cannabi- noids. In addition, the metabolites of HHC in authentic urine samples were identified, and the epimer composition of the (S)- and (R)- hydroxylated and carboxylic acid metabolites in these samples were chromatographically identified. F IGURE 1 Chemical structures with carbon numbers of semi-synthetic cannabinoids analysed in this study: HHC, HHC-O, HHCP and HHCP-O. * indicates the position of the chiral centre for the main (9R) and (9S) epimers. 2 LINDBOM ET AL. 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 2 | METHODS 2.1 | Materials The semi-synthetic cannabinoids (9R)- and (9S)-HHC, (9R)-HHCP, (9R)-HHC-O, (9R)-HHCP-O, 11-hydroxy-(9R)- and 11-hydroxy-(9S)- HHC, (8R)- and (8S)-hydroxy-(9R)-HHC, (8R)- and (8S)-hydroxy-(9S)- HHC and 11-carboxy-(9R)- and 11-carboxy-(9S)-HHC (purity ≥98%) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). The 50OH-HHC reference standard was synthesised in-house, and details of the synthesis and characterisation data can be found in the Sup- porting Information. Acetonitrile (LC–MS grade), formic acid, metha- nol (LC–MS grade) and the reagents for the hepatocyte incubations, Williams E medium, L-glutamine and HEPES buffer were obtained from Thermo Fisher Scientific (Gothenburg, Sweden). Ethanol was from Kemetyl AB (Jordbro, Sweden). Cryopreserved primary human hepatocytes using the 20-donor pool and thawing medium (InVitroGro HT) were purchased from Bioreclamation IVT (Brussels, Belgium). The internal standard solution for the urine samples (0.4 μg/mL each of 11-nor-9-carboxy-Δ9-THC-D9, Δ9-THC-D3, 11-hydroxy-Δ9- THC-D3, cannabidiol-D3 and Δ9-tetrahydrocannabinolic acid A-D3) was prepared from reference material obtained from Cerilliant (Round Rock, TX, USA). Urine sample preparation used high-purity water made on site using a MilliQ Gradient production unit (Millipore, Billerica, MA, USA). Finden B-One β-glucuronidase was purchased from Kura Biotech, Puerto Varas, Chile. 2.2 | Hepatocyte incubations Hepatocyte incubations and metabolite identification studies were performed as previously described19,20 with slight modifications. In short, the 9R epimers of the cannabinoids were diluted to a working concentration of 10 μM in Williams E medium, supplemented with HEPES buffer and L-glutamine. Pooled human hepatocytes (HHeps) were thawed to 37�C and added to 48 mL of InVitroGro HT medium. This solution was centrifuged at 100 � g for 5 min at room temperature, following which the supernatant was removed, and the pellet re-suspended in 50 mL of supplemented Williams E medium. The re-suspended pellet was centrifuged at 100 � g for 5 min at room temperature, the supernatant removed and the final pellet was re-suspended in Williams E medium with a cell concentra- tion of 2 � 106 cells/mL. The HHeps were then incubated in an IncuLine® IL-10 digital incubator (VWR, Stockholm, Sweden) with the drug solutions (at a final concentration of 5 μM) in duplicates for 1, 3 and 5 h at 37�C. One hundred microlitres of ice-cold acetonitrile was added to stop the reactions. The samples were centrifuged at 1100 � g for 15 min at 4�C, and the supernatants were transferred to the injection plate for LC-QToF-MS analysis. Degradation controls (drug without HHeps) and negative controls (HHeps without drug) were also incubated for 5 h. 2.3 | Authentic urine samples Cases sent to the National Board of Forensic Medicine between January and May 2023 were included in agreement with ethical approval from the Swedish Ethical Review Authority (2018/186:31). Urine samples were selected from cases that screened positive for cannabis in blood using an enzyme-linked immunosorbent assay (ELISA) but confirmed negative for THC, 11-OH-THC and 11-carboxy-THC and positive for HHC in blood.21 Urine samples were prepared as both non-hydrolysed and hydro- lysed. The non-hydrolysed urine samples were prepared by combining 50 μL urine with 50 μL MilliQ water, 25 μL methanol and 25 μL inter- nal standard in methanol (corresponding to 200 ng/mL of each com- pound). The hydrolysed urine samples were prepared by mixing 50 μL urine with 50 μL Kura B-One β-glucuronidase (room temperature) and incubating at room temperature for 2 h. Then, 25 μL methanol and 25 μL internal standard in methanol were added. Negative non- hydrolysed and hydrolysed urine samples and a mixture of B-One β-glucuronidase, MilliQ water and methanol were analysed as nega- tive controls. 2.4 | Instrumental analysis The analytical workflow was based on an established standardised protocol19,20 to ensure comparability between substances and runs. Further optimisation with regards to collision energy, gradient and retention times were established using reference standards of the substances prior to the analysis. Optimisation was performed with the goal of producing molecular fragments of appropriate sizes (approximately 80–350 m/z) and to ensure the parent compound eluted between 10 and 13 min. As the resulting metabolites are gen- erally more polar in nature than the parent, they usually elute prior to the parent compound. Due to the racemic composition of the sub- stances in the urine samples, the analysis was also performed using methanol as a mobile phase to achieve a greater separation of the hydroxy and carboxy epimers. The HHeps incubated and urine samples were analysed with a LC-QToF-MS system comprised of a 1290 Infinity UHPLC system (Agilent Technologies) coupled to a 6550 iFunnel QToF MS (Agilent Technologies) with a Dual Agilent Jet Stream electrospray ionisation source. Separation was achieved by injecting 10 μL of the sample onto an Acquity HSS T3 column (150 mm � 2.1 mm, 1.8 μm; Waters, Sollentuna, Sweden) fitted with an Acquity VanGuard precolumn (Waters). Mobile phase (A) consisted of water and (B) of acetonitrile both with the addition of 0.1% formic acid. For separation, the flow rate was 0.5 mL/min and the following gradient used: 10% B (0–0.6 min); 10% to 50% B (0.6–2 min); 50% to 90% B (2–13 min); 90% to 95% B (13–15 min); 95% B (15–18 min); 95% to 10% B (18–18.1 min); 10% B (18.1–19 min). The column temperature was 60�C. MS data were acquired using positive ionisation and an auto MS/MS acquisition with the following settings: scan range 100–950 m/z (MS) and LINDBOM ET AL. 3 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense T A B L E 1 (9 R )- H H C ,( 9 R )- H H C -O ,( 9 R )- H H C P an d (9 R )- H H C P -O m et ab o lit es w it h bi o tr an sf o rm at io n; m o le cu la r fo rm ul as ;m ea n re te nt io n ti m es (R T ); ac cu ra te (c al cu la te d )m as se s o f th e pr o to na te d m o le cu le s; m as s er ro rs fr o m al ls am pl es ;p ea k ar ea s af te r 1 ,3 an d 5 h in cu ba ti o ns fo r tw o re pl ic at e sa m pl es ;a nd m aj o r fr ag m en t io ns (a ls o in d ic at iv e o f b io tr an sf o rm at io n ). M et # B io tr an sf o rm at io n Fo rm ul a M ea n R T (m in ) A cc ur at e m as s [M + H ]+ (m /z ) M as s er ro r (p pm ) # 1 pe ak ar ea (� 1 0 3 ) # 2 p ea k ar ea (� 1 0 3 ) M aj o r fr ag m en t io n s M in M ax 1 h 3 h 5 h 1 h 3 h 5 h (9 R )- H H C C 2 1 H 3 2 O 2 1 1 .9 0 3 1 7 .2 4 7 4 �2 .3 1 4 .0 4 3 9 1 0 4 4 5 0 2 4 4 6 4 2 8 1 .0 6 9 9 ,1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 , 2 3 1 .1 3 8 0 H 1 A M o no O H + G LU C C 2 7 H 4 0 O 9 4 .8 9 5 0 9 .2 7 4 7 �1 .0 1 0 .9 8 4 8 9 1 0 5 4 8 2 5 4 5 2 8 6 8 7 2 8 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 5 9 .1 7 0 0 , 3 3 3 .2 4 2 4 H 1 B M o no O H + G LU C C 2 7 H 4 0 O 9 5 .3 9 5 0 9 .2 7 4 8 �1 .0 9 1 .9 8 4 9 0 9 6 2 5 6 3 3 6 5 7 2 3 4 7 3 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 5 9 .1 7 0 0 , 3 3 3 .2 4 2 4 H 2 C ar bo xy lic ac id (9 R ) C 2 1 H 3 0 O 4 7 .3 3 3 4 7 .2 2 1 8 �1 .0 3 1 .1 1 3 0 7 5 1 5 4 1 2 1 8 3 4 0 0 3 3 1 1 2 1 .1 0 1 2 ,1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 , 2 4 5 .1 5 3 6 H 3 A D iO H + G LU C C 2 7 H 4 0 O 1 0 3 .2 1 5 2 5 .2 6 9 2 �1 .8 5 1 .3 2 1 1 2 2 4 4 2 7 6 1 0 9 2 2 5 2 7 9 1 3 5 .1 1 6 8 ,1 9 1 .1 0 6 7 ,2 0 9 .1 1 7 2 , 2 7 5 .1 6 1 7 H 4 D eh yd + di O H + G LU C C 2 7 H 3 8 O 1 0 5 .2 9 5 2 3 .2 5 3 4 �1 .2 7 0 .0 9 7 4 3 1 9 1 1 8 9 5 0 1 5 7 1 7 6 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 1 9 .1 3 8 0 , 3 0 1 .2 1 4 2 H 3 B D iO H + G LU C C 2 7 H 4 0 O 1 0 3 .8 3 5 2 5 .2 6 9 2 �1 .3 3 0 .5 6 9 6 1 7 4 1 4 8 9 1 1 5 2 1 3 7 1 3 5 .1 1 6 8 ,2 0 9 .1 1 7 2 ,2 2 3 .1 3 2 9 H 3 C D iO H + G LU C C 2 7 H 4 0 O 1 0 3 .4 2 5 2 5 .2 6 9 4 �2 .2 3 1 .3 4 5 9 1 1 3 1 0 7 5 8 1 0 3 1 0 5 1 9 1 .1 0 6 7 ,2 0 9 .1 1 7 2 ,2 5 7 .1 5 3 6 H 1 C M o no O H + G LU C C 2 7 H 4 0 O 9 4 .3 9 5 0 9 .2 7 3 9 �2 .1 6 0 .7 3 3 0 5 1 3 6 2 4 4 6 2 9 1 9 3 .1 2 2 3 ,2 1 9 .1 3 8 0 (9 R )- H H C -O C 2 3 H 3 4 O 3 1 4 .0 0 3 5 9 .2 5 7 3 �3 .7 1 �0 .9 4 n. d. n. d. n. d. 3 6 n .d . n .d . 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,3 1 7 .2 4 7 5 H O 1 A A ce ta te lo ss + m o no O H + G LU C C 2 7 H 4 0 O 9 4 .9 0 5 0 9 .2 7 3 2 �4 .8 8 �1 .7 0 1 7 9 6 1 9 7 8 3 1 8 7 5 3 8 6 1 8 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 5 9 .1 7 0 0 , 3 3 3 .2 4 2 4 H O 1 B A ce ta te lo ss + m o no O H + G LU C C 2 7 H 4 0 O 9 5 .4 0 5 0 9 .2 7 3 0 �4 .5 2 �2 .5 3 5 8 3 1 7 4 2 7 7 9 2 8 5 2 8 9 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 1 9 .1 3 8 0 , 3 3 3 .2 4 2 4 H O 2 A ce ta te lo ss + ca rb o xy lic ac id (9 R ) C 2 1 H 3 0 O 4 7 .3 3 3 4 7 .2 2 0 9 �3 .5 �1 .2 9 7 4 2 9 6 3 6 7 8 1 3 0 8 2 1 8 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 4 5 .1 5 3 6 , 3 0 1 .2 1 6 2 H O 3 A A ce ta te lo ss + di O H + G LU C C 2 7 H 4 0 O 1 0 3 .2 1 5 2 5 .2 6 7 8 �4 .0 5 �2 .3 1 4 4 1 2 8 1 8 4 5 9 1 1 4 1 7 8 1 9 1 .1 0 6 7 ,2 0 9 .1 1 7 2 ,2 7 5 .1 6 1 7 , 3 3 1 .2 2 6 8 H O 4 A ce ta te lo ss ((9 R )- H H C ) C 2 1 H 3 2 O 2 1 1 .9 0 3 1 7 .2 4 7 4 �3 .7 6 �2 .5 6 5 5 8 4 6 3 1 1 7 1 1 7 9 6 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 3 3 .1 5 3 6 H O 5 A ce ta te lo ss + de hy d + di O H + G LU C C 2 7 H 3 8 O 1 0 5 .2 9 5 2 3 .2 5 2 1 �3 .9 2 �1 .9 9 n. d. 9 3 1 4 5 n .d . 9 2 1 0 2 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 4 5 .1 5 3 6 , 3 2 9 .2 1 1 1 H O 3 B A ce ta te lo ss + di O H + G LU C C 2 7 H 4 0 O 1 0 3 .8 3 5 2 5 .2 6 7 5 �5 .8 6 �2 .3 5 2 8 8 3 9 9 2 6 7 8 8 0 2 0 9 .1 1 7 2 ,2 2 3 .1 3 2 9 H O 3 C A ce ta te lo ss + di O H + G LU C C 2 7 H 4 0 O 1 0 3 .4 5 5 2 5 .2 6 7 7 �6 .8 6 �2 .0 9 2 4 6 4 7 7 n .d . 5 2 6 8 1 9 1 .1 0 6 7 ,2 5 7 .1 5 3 6 (9 R )- H H C P C 2 3 H 3 6 O 2 1 3 .5 8 3 4 5 .2 7 9 2 �4 .1 4 8 .1 7 5 3 4 0 5 4 6 3 2 3 5 1 3 8 4 5 7 8 4 8 5 6 8 1 .0 6 9 9 ,1 2 3 .0 4 4 1 ,2 2 1 .1 5 3 6 , 2 3 5 .1 6 9 2 P 1 A M o no O H + G LU C C 2 9 H 4 4 O 9 6 .4 3 5 3 7 .3 0 9 0 4 .3 1 7 .2 7 3 2 5 1 2 0 9 2 1 1 9 4 5 4 9 7 0 1 5 7 2 2 2 1 .1 5 3 6 ,2 3 5 .1 6 9 2 ,2 8 7 .2 0 0 6 P 2 A D iO H + G LU C C 2 9 H 4 4 O 1 0 3 .7 2 5 5 3 .3 0 3 9 3 .6 3 7 .0 5 1 7 3 6 3 5 8 9 7 1 8 5 4 5 2 8 9 6 1 6 3 .0 7 5 4 ,2 1 9 .1 3 8 0 ,2 3 7 .1 4 8 5 , 3 4 1 .2 4 7 5 4 LINDBOM ET AL. 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 50–950 m/z (MS/MS); precursor intensity threshold of 5000 counts; precursor number per cycle, 5; fragmentor voltage, 380 V; CE, 3 eV at 0 m/z ramped up by 8 eV per 100 m/z; gas temperature, 150�C; gas flow, 18 L/min; nebuliser gas pressure, 345 kPa; sheath gas tempera- ture, 375�C; and sheath gas flow, 11 L/min. Hydrolysed urine samples were also analysed using mobile phases (A) water and (B) methanol, both supplemented with 0.1% formic acid. A longer gradient was used with a total run time of 26 min and the following gradient: 10% B (0–0.6 min); 10% to 50% B (0.6–2 min); 50% to 90% B (2–20 min); 90% to 95% B (20–23 min); 95% B (23– 25 min); 95% to 10% B (25–25.1 min); 10% B (25.1–26 min). All other instrumental parameters were the same as described above. 2.5 | Data and statistical analysis The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology.22 Agilent Mas- sHunter Qualitative Analysis software (version B.07.00) was used for data analysis. The criteria for metabolite identification has been described previously,23 but in brief, the data were searched for all the molecular formulas corresponding to potential modification of the parent compound by known biotransformations and any combina- tions thereof (up to three modifications). Each potential metabolite identification required mass errors <5 ppm for protonated molecules (values >5 ppm accepted for saturated or very small peaks, where the mass accuracy could deviate), a consistent isotopic pattern, a product ion spectrum consistent with the proposed structure and related to the parent compound, a retention time plausible for the proposed structure and the absence of identical peaks with the same mass spec- trum in negative and degradation controls. For the HHeps incubations, the total peak area for each metabo- lite was calculated by summing the peak areas for both replicates at all incubation times, which were then summed to calculate the total peak area for each parent compound. The total peak area of the par- ent compounds was not included in these calculations. For the urine samples, the total peak area for each metabolite was calculated by summing the peak areas for all samples, which were then summed to calculate the total peak area for all urine samples where HHC and/or its metabolites were identified. The total peak area of the parent com- pounds were included in these calculations. The percentage total peak areas were calculated using these values for different metabolites or biotransformations. 3 | RESULTS AND DISCUSSION 3.1 | Hepatocyte incubations Following incubation with HHeps, eight metabolites were identified for (9R)-HHC, eight for (9R)-HHC-O, six for (9R)-HHCP and three for (9R)-HHCP-O. The identified metabolites are listed in Table 1. The metabolites are numbered according to their total peak area, fromT A B L E 1 (C o nt in ue d) M et # B io tr an sf o rm at io n Fo rm ul a M ea n R T (m in ) A cc ur at e m as s [M + H ]+ (m /z ) M as s er ro r (p pm ) # 1 pe ak ar ea (� 1 0 3 ) # 2 p ea k ar ea (� 1 0 3 ) M aj o r fr ag m en t io n s M in M ax 1 h 3 h 5 h 1 h 3 h 5 h P 3 D eh yd + di O H C 2 3 H 3 4 O 4 9 .2 7 3 7 5 .2 5 4 5 2 .3 9 6 .2 9 4 6 1 2 9 2 8 8 3 3 1 5 2 2 6 7 1 6 3 .0 7 5 4 ,2 2 1 .1 5 3 6 ,2 3 5 .1 6 9 2 , 3 2 9 .2 4 7 5 P 1 B M o no O H + G LU C C 2 9 H 4 4 O 9 7 .0 7 5 3 7 .3 0 8 4 3 .0 9 6 .9 9 7 7 1 4 4 1 3 2 1 8 6 1 6 3 1 8 4 2 2 1 .1 5 3 6 ,2 4 7 .1 6 9 3 ,2 8 7 .2 0 0 6 , 3 6 1 .1 2 7 4 P 4 D eh yd + tr iO H + G LU C C 2 9 H 4 2 O 1 1 3 .5 8 5 6 7 .2 8 2 3 �0 .0 4 6 .6 8 1 9 7 6 1 1 2 1 2 5 0 1 4 9 1 2 1 .1 0 1 2 ,2 3 3 .1 1 7 2 ,2 7 5 .1 2 7 8 , 3 5 5 .2 2 6 8 P 2 B D iO H + G LU C C 2 9 H 4 4 O 1 0 4 .8 6 5 5 3 .3 0 3 6 2 .4 6 7 .7 1 1 4 6 0 1 0 8 1 9 4 2 7 5 2 3 7 .1 4 8 5 ,2 5 1 .1 6 1 7 ,3 0 3 .1 9 5 5 (9 R )- H H C P -O C 2 5 H 3 8 O 3 1 5 .4 8 3 8 7 .2 8 9 4 �3 .0 7 2 .1 4 9 1 4 1 0 3 8 2 9 4 6 2 2 1 .1 5 3 6 ,3 4 5 .2 7 8 8 P O 1 A ce ta te lo ss + m o no O H + G LU C C 2 9 H 4 4 O 9 6 .4 4 5 3 7 .3 0 7 0 0 .3 0 3 .2 4 6 1 4 4 7 6 0 0 5 8 4 0 4 5 6 7 2 2 1 .1 5 3 6 ,2 8 7 .2 0 0 6 ,3 6 1 .2 7 3 7 P O 2 A ce ta te lo ss + di O H + G LU C C 2 9 H 4 4 O 1 0 3 .7 6 5 5 3 .3 0 1 3 �3 .4 0 2 .7 4 1 8 1 8 9 3 3 2 2 7 1 9 7 3 3 4 1 7 7 .0 9 1 0 ,2 1 9 .1 3 8 0 ,2 3 7 .1 4 8 5 P O 3 A ce ta te lo ss ((9 R )- H H C P ) C 2 3 H 3 6 O 2 1 3 .5 6 3 4 5 .2 7 9 5 �0 .4 2 5 .0 1 3 8 8 4 8 8 8 8 9 2 1 5 5 2 2 1 .1 5 3 6 ,3 4 5 .2 7 8 8 N ot e: M et ab o lit es ar e o rd er ed fr o m m o st to le as t ab un da nt ac ro ss al li nc ub at io ns . A bb re vi at io n: n. d. ,n o t de te ct ed . LINDBOM ET AL. 5 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense highest to lowest. Proposed structures of the metabolites are orga- nised in suggested metabolic pathways in Figures 2 and 3. The mass spectra and proposed fragmentation patterns of the parent com- pounds and all metabolites can be found in the Supporting Information. It should be noted that for (9R)-HHC and (9R)-HHCP, there are some large differences in peak areas between replicates for the parent and some metabolites. Given that the addition of an internal standard did not improve the stability, this variation is likely due to experimen- tal variability, such as the saturation effect in the LC-QToF-MS detec- tor, or the high lipophilicity of the compound, which may have led to some of the compound sticking to pipette tips or plastic incubation plates. For the acetate analogues HHC-O and HHCP-O, all metabolites had acetate loss, resulting in similar metabolites with the same relative abundance as their non-acetate analogues HHC and HHCP, respec- tively. Therefore, the metabolites of HHC-O and HHCP-O will be dis- cussed with the HHC and HHCP metabolites. As expected, this indicates that HHC-O and HHCP-O were first rapidly metabolised to HHC (HO4) and HHCP (PO3), respectively. Therefore, the use of HHC-O and HHCP-O is unlikely to be differentiated from that of HHC and HHCP, respectively, in biological samples, particularly urine. This is also likely to be true for other recently emerged acetate analogues of other phytocannabinoids and semi-synthetic cannabi- noids, including THC acetates and CBD di-acetate.30,31 This may be problematic in jurisdictions where the acetate analogues are not con- trolled. In the future, authentic urine and other biological samples from people who have used phytocannabinoids and semi-synthetic cannabinoids should be examined in comparison with that of their acetate analogues to determine if their use can be differentiated. 3.2 | (9R)-HHC In QToF-MS analysis, (9R)-HHC (m/z 317.2474) was fragmented into three major product ions: m/z 81.0699, representing the cyclohexyl ring; m/z 193.1223, representing the aromatic ring and pentyl side chain; and m/z 231.1380, representing the three-ring core. The observed fragment ions were used as the basis for elucidating the structures of the metabolites. Following incubation with HHeps, eight metabolites were identi- fied for (9R)-HHC and (9R)-HHC-O. The metabolites eluted between 3.21 and 7.33 min with the parent drug eluting at 11.90 min for (9R)-HHC and 14.00 min for (9R)-HHC-O (see Table 1). The observed biotransformations included monohydroxylations (monoOH), dihy- droxylations (diOH), dehydrogenation (dehyd) in combination with F IGURE 2 Proposed metabolic pathways of HHC and HHC-O following duplicate 1, 3 and 5 h incubations with HHeps and analysis of authentic urine samples for HHC. Markush bonds represent the probable location of the group. * indicates the position of the chiral centre for the main (9R) and (9S) epimers. F IGURE 3 Proposed metabolic pathways of (9R)-HHCP and (9R)- HHCP-O following duplicate 1, 3 and 5 h incubations with HHeps. Markush bonds represent the probable location of the group. 6 LINDBOM ET AL. 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense diOH (ketone or carboxylic acid formation) and glucuronidation (GLUC). All metabolites were glucuronidated except the metabolite formed from combined dehydrogenation and diOH (H2; HO2). The glucuronidations were characterised by the addition of m/z 176 to the overall mass of the metabolites. All modifications occurred at either the cyclohexyl ring or pentyl side chain, where all identified metabo- lites had a biotransformation at the cyclohexyl ring and 37.5% of metabolites had a biotransformation at the pentyl side chain. The two most abundant metabolites were produced via monoOH on the cyclohexyl ring and glucuronidation (H1A-B; HO1A-B). These metabolites were characterised by the addition of m/z 16 to the par- ent, which is consistent with the addition of a hydroxy group. The absence of modifications to the fragment ions of the parent demon- strates the hydroxylations occurred on the cyclohexyl ring, although the exact locations of the hydroxy groups could not be determined as indicated by the Markush bonds in Figure 2. Apart from glucuronidation, diOH was the most common bio- transformation (H2–H4; HO2, HO3, HO5). The three metabolites with diOH combined with glucuronidation (H3A-C; HO3A-C) were found to have one hydroxy group added to the pentyl side chain and one added to the cyclohexyl ring. This was characterised by the addi- tion of m/z 32 to the overall mass, consistent with the addition of two hydroxyl groups, and the presence of a fragment ion at m/z 209.1172, which indicates an addition of m/z 16, consistent with one hydroxy group, to the aromatic ring and pentyl side chain mass fragment of the parent (m/z 193.1223) with no further modifications. The remaining two metabolites with diOH were combined with dehydrogenation (H2 and H4; HO2 and HO5), representing the formation of a carbox- ylic acid on the cyclohexyl ring. 3.3 | (9R)-HHCP (9R)-HHCP (m/z 345.2792) was fragmented into three major product ions: m/z 81.0699, representing the cyclohexyl ring; m/z 123.0441, representing the aromatic ring; and m/z 221.1536, representing the aromatic ring and heptyl side chain. The observed fragment ions were used as the basis for elucidating the structures of the metabolites. Following incubation with HHeps, six metabolites were identified for (9R)-HHCP. The metabolites eluted between 3.58 and 9.27 min with the parent drug eluting at 13.58 min (see Table 1). The observed biotransformations included monoOH, diOH, dehydrogenation in combination with diOH (ketone or carboxylic acid formation), dehy- drogenation in combination with trihydroxylation (triOH) and glucuro- nidation. Similar to HHC, all metabolites were glucuronidated, except the metabolite formed from combined dehydrogenation and diOH (P3). All modifications occurred at either the cyclohexyl ring or heptyl side chain, where all identified metabolites had a biotransformation at the cyclohexyl ring and half had a biotransformation at the heptyl side chain. The most abundant metabolite was formed from monoOH and glucuronidation (P1A; PO1). The exact location of the monoOH on metabolites P1A-B and PO1 could not be determined but given no modifications were observed on the major mass fragments of the par- ent, it was determined the hydroxy group was added to the cyclohexyl ring or isopropyl (carbons 12 or 13 on Figure 1). Apart from glucuronidation, diOH was the most common bio- transformation (P2A-B and P3; PO2). Two of the metabolites with diOH (P2A-B; PO2) had one hydroxy group added to the heptyl side chain and the other to the cyclohexyl ring or isopropyl. The other metabolite with diOH was combined with dehydrogenation (P3), representing the formation of a carboxylic acid on the cyclohexyl ring. The metabolite with triOH and dehydrogenation (P4) had a carboxylic acid on the cyclohexyl ring and a hydroxy group on the heptyl side chain. Because HHC and HHCP produced similar metabolites, they likely follow a similar metabolic pathway. All modifications of HHC and HHCP occurred at either the cyclohexyl ring or pentyl side chain or isopropyl for HHCP; however, while all identified metabolites of both HHC and HHCP had a biotransformation at the cyclohexyl ring, HHCP showed greater biotransformation on the side chain, where half of its metabolites had a hydroxy group on the side chain in comparison to 37.5% of metabolites of HHC. This is likely due to the longer alkyl side chain of HHCP (heptyl) than HHC (pentyl). This relationship has been previously observed with phytocannabinoids, where tetrahydrocanna- binolic acid (THCA) had greater metabolism on its pentyl side chain than the propyl side chain of tetrahydrocannabivarin (THCV).27 This has also been identified in structure-metabolism relationships of syn- thetic cannabinoid receptor agonists (SCRAs), where greater metabo- lism on the alkyl chain tails has been observed for SCRAs with longer alkyl chains.28 3.4 | HHC authentic urine samples The analysis of the non-hydrolysed and hydrolysed urine samples in acetonitrile resulted in the identification of 21 and 18 metabolites for HHC, respectively. The glucuronidated parent compound HHC (N1A-B) was identified in the non-hydrolysed samples (5.9% of total peak area) with both epimers being present, although it was not possi- ble to determine which of the metabolites (N1A or N1B) corresponded to each epimer. The parent compounds (9R)- and (9S)-HHC were iden- tified in the hydrolysed samples and confirmed with reference stan- dards, where the (9S) epimer was found to be more abundant (1.8% of total peak area) than the (9R) epimer (1.3% of total peak area). The metabolites identified eluted between 3.20 and 7.63 min in non- hydrolysed urine samples and 4.00 and 9.51 min in the hydrolysed urine samples, with the parent drug HHC eluting at 11.69 (9S) and 11.79 min (9R). The identified metabolites are listed in Table 2 for the non-hydrolysed and Table 3 for the hydrolysed urine samples. Follow- ing the parent compounds, or glucuronidated parent compounds in the case of the non-hydrolysed urine samples, metabolites are num- bered according to their prevalence in the urine samples, from the most to least prevalent, followed by abundance based on total peak areas across all samples. No HHC or metabolites were detected in Urine Sample 7 despite the corresponding blood sample containing LINDBOM ET AL. 7 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense T A B L E 2 H H C m et ab o lit es id en ti fi ed in 1 6 no n- hy dr o ly se d u ri ne sa m pl es w it h bi o tr an sf o rm at io n, m o le cu la r fo rm ul as ,m ea n re te nt io n ti m es (R T ), ac cu ra te (c al cu la te d )m as se s o f th e p ro to n at ed m o le cu le s, m as s er ro rs fr o m al ls am pl es ,m aj o r fr ag m en t io ns (a ls o in di ca ti ve o f bi o tr an sf o rm at io n) an d pe ak ar ea s fo r 1 6 ur in e sa m pl es . M et # B io tr an sf o rm at io n Fo rm ul a M ea n R T (m in ) A cc ur at e m as s [M + H ]+ (m /z ) M as s er ro r (p p m ) M as s fr ag m en ts M in M ax N 1 A H H C + G LU C C 2 7 H 4 0 O 8 6 .6 8 4 9 3 .2 8 0 3 �2 .1 7 3 .9 2 1 9 3 .1 2 2 3 ,3 1 7 .2 4 7 6 N 1 B H H C + G LU C C 2 7 H 4 0 O 8 6 .9 2 4 9 3 .2 8 0 3 �1 .7 2 3 .4 4 1 9 3 .1 2 2 3 ,3 1 7 .2 4 7 6 N 2 M o no O H (s id e ch ai n) + G LU C C 2 7 H 4 0 O 9 4 .0 6 5 0 9 .2 7 5 0 �1 .5 9 3 .7 8 1 9 1 .1 0 6 7 ,2 5 9 .1 6 9 3 ,3 1 5 .2 3 1 9 N 3 A M o no O H (8 )+ G LU C C 2 7 H 4 0 O 9 3 .9 0 5 0 9 .2 7 4 7 �4 .2 6 3 .7 6 1 9 3 .1 2 2 3 ,2 5 9 .1 6 9 3 ,3 1 5 .2 3 1 9 N 3 B M o no O H (1 1 )+ G LU C C 2 7 H 4 0 O 9 4 .8 8 5 0 9 .2 7 5 3 �1 .0 5 4 .3 0 1 9 3 .1 2 2 3 ,2 5 9 .1 6 9 3 ,3 3 3 .2 4 2 4 N 3 C M o no O H (1 1 )+ G LU C C 2 7 H 4 0 O 9 5 .3 5 5 0 9 .2 7 5 0 �3 .1 9 3 .8 9 1 9 3 .1 2 2 3 ,2 5 9 .1 6 9 3 ,3 3 3 .2 4 2 4 N 4 A D iO H + G LU C C 2 7 H 4 0 O 1 0 3 .5 1 5 2 5 .2 7 0 3 �4 .6 6 5 .5 8 1 9 3 .1 2 2 3 ,2 0 1 .0 9 1 0 ,2 2 1 .1 5 3 6 ,2 7 1 .1 6 9 2 N 5 A D eh yd + di O H + G LU C C 2 7 H 3 8 O 1 0 3 .4 4 5 2 3 .2 5 3 5 �7 .2 0 7 .4 0 2 0 7 .1 0 1 6 ,2 7 3 .1 4 8 5 N 3 D M o no O H (8 )+ G LU C C 2 7 H 4 0 O 9 3 .9 8 5 0 9 .2 7 5 2 �5 .2 6 5 .6 6 1 9 3 .1 2 2 3 ,2 3 3 .1 5 3 6 ,2 5 9 .1 6 9 3 ,3 1 5 .2 3 1 9 N 6 D eh yd + di O H (c ar bo xy lic ac id )+ G LU C C 2 7 H 3 8 O 1 0 5 .2 7 5 2 3 .2 5 4 1 �4 .7 0 4 .0 2 1 9 3 .1 2 2 3 ,2 4 5 .1 5 3 6 ,3 2 9 .2 1 1 1 N 5 B D eh yd + di O H + G LU C C 2 7 H 3 8 O 1 0 3 .5 9 5 2 3 .2 5 3 3 �7 .6 8 3 .2 5 2 0 7 .1 0 1 6 ,2 3 3 .1 1 7 7 ,2 7 3 .1 4 8 5 N 4 B D iO H + G LU C C 2 7 H 4 0 O 1 0 3 .4 2 5 2 5 .2 7 0 0 �6 .0 8 4 .7 6 1 9 1 .1 0 6 7 ,2 5 7 .1 5 3 6 N 4 C D iO H + G LU C C 2 7 H 4 0 O 1 0 3 .2 0 5 2 5 .2 7 0 0 �4 .6 2 4 .6 2 1 9 3 .1 2 2 3 ,2 5 7 .1 5 3 6 ,2 1 5 .1 0 6 7 ,3 3 1 .2 2 6 8 N 7 A C ar bo xy lic ac id (9 R ) C 2 1 H 3 0 O 4 7 .2 7 3 4 7 .2 2 2 0 �4 .1 2 3 .9 8 1 9 3 .1 2 2 3 ,2 4 5 .1 5 3 6 N 5 C D eh yd + di O H + G LU C C 2 7 H 3 8 O 1 0 4 .8 6 5 2 3 .2 5 3 3 �5 .2 9 1 .0 0 1 9 3 .1 2 2 3 ,2 0 7 .1 0 1 6 ,2 4 5 .1 5 3 6 ,2 5 9 .1 6 9 3 N 8 A D iO H C 2 1 H 3 2 O 4 4 .0 0 3 4 9 .2 3 8 0 0 .6 1 3 .4 1 1 9 1 .1 0 6 7 ,2 1 5 .1 0 6 7 ,2 5 7 .1 5 3 6 N 9 D eh yd + di O H C 2 1 H 3 0 O 4 4 .5 3 3 4 7 .2 2 2 2 �1 .0 1 3 .1 8 1 8 9 .0 9 1 0 ,2 0 7 .1 0 1 6 N 8 B D iO H C 2 1 H 3 2 O 4 4 .4 0 3 4 9 .2 3 8 4 2 .3 7 3 .8 2 1 9 1 .1 0 6 7 ,2 7 5 .1 6 1 7 N 8 C D iO H C 2 1 H 3 2 O 4 4 .1 3 3 4 9 .2 3 7 8 0 .0 7 2 .1 0 1 9 1 .1 0 6 7 ,2 5 7 .1 5 3 6 ,3 3 1 .2 2 6 8 N 7 B C ar bo xy lic ac id (9 S) C 2 1 H 3 0 O 4 7 .6 0 3 4 7 .2 2 2 3 0 .8 2 3 .8 2 1 9 3 .1 2 2 3 ,2 5 9 .1 6 9 3 ,3 0 1 .2 1 6 2 N 1 0 M o no O H (1 1 ) C 2 1 H 3 2 O 3 7 .6 3 3 3 3 .2 4 3 0 1 .7 2 1 .7 2 1 3 7 .1 3 2 5 ,1 9 3 .1 2 2 3 N ot e: F o llo w in g th e gl uc ur o ni da te d pa re nt co m po un ds ,m et ab o lit es ar e o rd er ed fr o m m o st to le as t pr ev al en t, fo llo w ed by ab un da nc e ba se d o n to ta lp ea k ar ea ac ro ss al ls am p le s. U ri n e Sa m p le 7 w as n o t in cl ud ed in th e ta bl e as no H H C o r m et ab o lit es w er e fo un d, de sp it e be in g po si ti ve fo r H H C in bl o o d. 8 LINDBOM ET AL. 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense T A B L E 2 (C o nt in ue d) M et # P ea k ar ea (� 1 0 3 )i n ur in e sa m pl es 1 2 3 4 5 6 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 N 1 A n. d. 2 1 5 0 n. d. 1 3 8 2 0 n. d. n. d. n. d. 7 0 n. d. n .d . 1 5 0 2 8 3 6 9 6 6 7 8 N 1 B n. d. n. d. 1 0 6 n. d. 9 3 n. d. 2 0 n. d. n. d. 5 0 n. d. n .d . 1 0 4 2 7 1 4 4 3 5 2 1 N 2 8 9 2 2 2 1 0 7 3 5 0 2 2 6 2 5 6 3 9 3 1 8 2 1 3 7 4 7 5 1 7 6 8 0 5 8 1 2 9 5 2 3 1 3 2 0 3 4 N 3 A 1 6 4 1 1 5 4 0 5 2 2 5 2 2 1 3 1 1 3 1 2 6 1 0 8 3 2 3 8 8 4 4 3 1 2 1 3 1 8 5 2 1 1 5 2 N 3 B n. d. 5 1 8 0 6 6 4 3 0 1 2 0 6 2 2 4 1 1 4 1 9 5 4 7 7 1 4 1 2 9 4 5 1 6 9 4 1 8 1 5 2 3 7 0 N 3 C n. d. 5 4 3 2 4 4 8 1 5 4 8 8 1 4 4 9 9 1 3 3 2 5 3 7 0 n .d . 9 6 4 9 4 1 0 1 1 2 1 8 3 N 4 A 9 8 1 1 0 n. d. 3 3 n. d. 2 5 8 2 6 0 8 8 1 2 0 3 1 4 1 0 2 3 5 1 2 7 1 7 4 1 8 3 0 9 2 1 N 5 A n. d. 4 0 2 0 2 3 n. d. 4 4 1 2 0 1 9 9 5 8 1 3 0 3 1 1 1 2 4 8 1 1 8 7 9 3 2 7 3 8 4 1 N 3 D 7 1 9 0 3 5 1 3 2 1 0 6 n. d. 1 4 1 9 4 8 1 2 8 7 n. d. n .d . 1 7 1 2 8 8 7 2 6 1 0 3 7 N 6 n. d. n. d. 4 7 3 n. d. 2 5 2 5 2 8 6 1 0 9 4 2 0 2 5 2 1 4 0 3 2 2 4 1 4 0 1 6 0 9 5 5 N 5 B 3 1 n. d. 3 2 1 2 6 n. d. 2 7 3 2 2 9 5 2 1 8 4 4 8 n .d . 9 9 3 9 1 6 0 2 5 2 N 4 B 5 8 8 9 1 1 9 3 n. d. 5 8 n. d. 2 5 6 n. d. 1 6 0 5 1 9 2 9 7 6 3 2 4 4 3 3 0 n .d . 9 3 8 N 4 C 1 2 2 1 4 1 8 3 1 n. d. n. d. n. d. 3 9 9 n. d. 2 5 0 8 7 2 n. d. 9 4 3 3 3 5 2 7 7 7 1 1 4 4 5 N 7 A n. d. n. d. 2 5 5 n. d. n. d. 7 4 n. d. n. d. 1 0 0 n. d. 1 2 7 n .d . n .d . n .d . 4 1 9 1 4 0 N 5 C n. d. n. d. 3 4 0 2 7 n. d. 2 2 3 n. d. 6 6 n. d. 9 1 n. d. n .d . n .d . n .d . n .d . n .d . N 8 A n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. 1 5 5 n .d . n .d . n .d . 1 3 8 3 n .d . N 9 n. d. n. d. 6 0 n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n .d . n .d . n .d . 1 3 0 9 n .d . N 8 B n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n .d . n .d . n .d . 4 1 1 2 9 N 8 C n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n .d . n .d . n .d . 3 4 6 7 8 N 7 B n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n .d . n .d . n .d . 3 0 3 0 N 1 0 n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n .d . n .d . n .d . 2 0 9 n .d . N ot e: F o llo w in g th e gl uc ur o ni da te d pa re nt co m po un ds ,m et ab o lit es ar e o rd er ed fr o m m o st to le as t pr ev al en t, fo llo w ed by ab un da nc e ba se d o n to ta lp ea k ar ea ac ro ss al ls am p le s. U ri n e Sa m p le 7 w as n o t in cl ud ed in th e ta bl e as no H H C o r m et ab o lit es w er e fo un d, de sp it e be in g po si ti ve fo r H H C in bl o o d. LINDBOM ET AL. 9 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense T A B L E 3 H H C m et ab o lit es id en ti fi ed in 1 6 hy dr o ly se d ur in e sa m pl es w it h bi o tr an sf o rm at io n, m o le cu la r fo rm ul as ,m ea n re te nt io n ti m es (R T ), ac cu ra te m as se s o f th e pr o to na te d m o le cu le s, m as s er ro rs fr o m al ls am pl es ,m aj o r fr ag m en t io ns (a ls o in di ca ti ve o f bi o tr an sf o rm at io n) an d pe ak ar ea s fo r 1 6 ur in e sa m pl es . M et # B io tr an sf o rm at io n Fo rm ul a M ea n R T (m in ) A cc ur at e m as s [M + H ]+ (m /z ) M as s er ro r (p p m ) M as s fr ag m en ts M in M ax H H C (9 S) C 2 1 H 3 2 O 2 1 1 .6 9 3 1 7 .2 4 7 4 �1 .3 5 1 .4 3 9 8 .0 8 4 8 ,1 9 3 .1 2 2 3 H H C (9 R ) C 2 1 H 3 2 O 2 1 1 .7 9 3 1 7 .2 4 7 4 �2 .1 2 3 .7 8 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 M 1 A M o no O H (1 1 ) C 2 1 H 3 2 O 3 7 .6 7 3 3 3 .2 4 2 5 �0 .5 8 4 .1 3 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 5 9 .1 6 9 3 M 2 A M o no O H (5 0 O H ) C 2 1 H 3 2 O 3 7 .0 9 3 3 3 .2 4 2 6 �0 .7 5 4 .9 5 1 9 1 .1 0 6 7 ,2 0 9 .1 1 7 2 ,2 5 9 .1 6 9 3 M 2 B M o no O H (s id e ch ai n) C 2 1 H 3 2 O 3 7 .3 7 3 3 3 .2 4 2 7 �3 .3 7 2 .6 9 1 9 1 .1 0 6 7 ,2 5 9 .1 6 9 3 ,3 1 5 .2 3 1 9 M 3 A D eh yd + di O H C 2 1 H 3 0 O 4 4 .5 3 3 4 7 .2 2 2 2 �3 .3 6 4 .3 7 1 8 9 .0 9 1 0 ,2 0 7 .1 0 1 6 ,2 3 1 .1 0 1 6 ,2 5 7 .1 5 3 6 M 4 A D iO H C 2 1 H 3 2 O 4 4 .8 9 3 4 9 .2 3 7 4 �3 .5 1 4 .6 2 1 9 3 .1 2 2 3 ,2 0 1 .0 9 1 0 ,2 5 7 .1 5 3 6 ,2 7 1 .1 6 9 2 M 4 B D iO H C 2 1 H 3 2 O 4 4 .0 0 3 4 9 .2 3 7 9 �2 .7 3 .7 7 1 9 1 .1 0 6 7 ,2 7 5 .1 6 1 7 M 4 C D iO H C 2 1 H 3 2 O 4 4 .1 3 3 4 9 .2 3 7 9 �3 .2 2 4 .8 6 1 9 1 .1 0 6 7 ,2 5 7 .1 5 3 6 ,2 7 5 .1 6 1 7 M 3 B D eh yd + di O H C 2 1 H 3 0 O 4 4 .7 8 3 4 7 .2 2 2 4 �2 .9 2 5 .6 2 1 7 7 .0 9 1 0 ,2 0 7 .1 0 1 6 ,2 7 3 .1 4 8 5 M 3 C D eh yd + di O H C 2 1 H 3 0 O 4 4 .2 6 3 4 7 .2 2 2 2 �1 .0 3 4 .0 1 1 8 9 .0 9 1 0 ,2 0 7 .1 0 1 6 ,2 7 3 .1 4 8 5 M 5 A C ar bo xy lic ac id (R ) C 2 1 H 3 0 O 4 7 .2 7 3 4 7 .2 2 2 3 �0 .9 1 5 .9 5 1 9 3 .1 2 2 3 ,2 0 7 .1 3 8 0 ,2 4 5 .1 5 3 6 ,3 0 1 .2 1 6 2 M 5 B C ar bo xy lic ac id (S ) C 2 1 H 3 0 O 4 7 .5 8 3 4 7 .2 2 1 9 �7 .1 5 3 .3 3 1 9 3 .1 2 2 3 ,2 3 1 .1 3 8 0 ,2 4 5 .1 5 3 6 ,3 0 1 .2 1 6 2 M 4 D D iO H C 2 1 H 3 2 O 4 5 .4 0 3 4 9 .2 3 7 5 �1 .7 5 3 .2 9 1 8 9 .0 9 1 0 ,2 0 7 .1 0 1 6 ,2 5 9 .1 6 9 3 M 1 B M o no O H (8 ) C 2 1 H 3 2 O 3 6 .8 1 3 3 3 .2 4 2 4 �2 .6 3 1 .7 8 N o sp ec M 2 C M o no O H (s id e ch ai n) C 2 1 H 3 2 O 3 5 .8 0 3 3 3 .2 4 2 4 �1 .8 2 3 .5 5 1 9 1 .1 0 6 7 ,2 0 9 .1 1 7 2 ,2 5 9 .1 6 9 3 M 4 E D iO H C 2 1 H 3 2 O 4 4 .4 0 3 4 9 .2 3 8 0 0 .8 2 3 .5 1 1 9 1 .1 0 6 7 ,2 5 7 .1 5 3 6 M 1 C M o no O H C 2 1 H 3 2 O 3 9 .5 1 3 3 3 .2 4 2 4 �1 .6 3 1 .7 2 1 9 3 .1 2 2 3 ,2 2 1 .1 5 3 6 ,2 5 9 .1 6 9 3 ,3 1 5 .2 3 1 9 M 1 D M o no O H (8 ) C 2 1 H 3 2 O 3 7 .0 4 3 3 3 .2 4 2 6 �0 .7 5 0 .6 1 1 9 3 .1 2 2 3 ,2 3 3 .1 1 7 2 ,2 5 9 .1 6 9 3 ,3 1 5 .2 3 1 9 M 1 E M o no O H (8 ) C 2 1 H 3 2 O 3 6 .6 5 3 3 3 .2 4 2 3 �0 .6 1 0 .4 2 1 9 3 .1 2 2 3 ,2 3 3 .1 5 3 6 ,2 5 9 .1 6 9 3 ,3 1 5 .2 3 1 9 N ot e: M et ab o lit es ar e o rd er ed fr o m m o st to le as t pr ev al en t, fo llo w ed by ab un da nc e ba se d o n to ta lp ea k ar ea ac ro ss al ls am pl es .U ri ne Sa m pl e 7 w as n o t in cl u d ed in th e ta b le as n o H H C o r m et ab o lit es w er e fo un d, de sp it e be in g po si ti ve fo r H H C in bl o o d. A bb re vi at io n: n. d. ,n o t de te ct ed . 10 LINDBOM ET AL. 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense T A B L E 3 (C o nt in ue d) M et # P ea k ar ea (� 1 0 3 )i n ur in e sa m pl es 1 2 3 4 5 6 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 n. d. n. d. 3 4 n. d. 9 5 n. d. n. d. n. d. n. d. 4 5 n. d. n .d . 9 6 1 8 7 4 7 0 4 1 9 n. d. n. d. 7 2 n. d. 6 5 n. d. n. d. n. d. n. d. 2 5 n. d. n .d . 6 0 1 3 3 2 8 4 3 2 0 M 1 A 7 4 1 0 8 1 1 8 2 1 1 9 5 0 3 2 5 1 3 4 7 2 2 0 3 0 4 6 5 1 2 0 8 5 5 5 4 4 1 2 7 1 3 6 6 2 5 1 9 0 M 2 A 6 7 1 6 6 3 5 5 4 5 2 2 1 1 7 6 2 8 4 1 6 3 8 7 3 7 1 1 0 5 4 7 5 7 6 3 1 3 2 1 6 8 1 9 0 4 M 2 B 3 9 8 1 7 4 9 2 1 1 0 2 1 0 4 1 9 9 5 7 6 3 1 9 2 8 5 4 3 1 6 1 1 7 6 1 2 4 8 7 0 1 M 3 A 6 5 7 4 3 5 6 4 n. d. 2 8 1 2 0 5 2 8 4 9 1 5 9 3 8 1 3 8 0 1 0 2 3 2 4 2 2 5 1 9 6 5 1 7 6 2 M 4 A 7 5 1 3 4 5 2 7 1 2 7 5 2 8 8 4 0 6 1 9 8 1 1 6 6 2 3 1 7 8 n .d . 7 6 3 4 7 1 4 2 8 9 7 4 M 4 B 5 6 n. d. 1 2 1 7 n. d. 1 1 4 1 7 2 4 0 6 1 1 5 1 9 6 5 3 0 3 8 3 9 4 2 7 1 5 4 4 2 7 3 9 1 6 0 3 M 4 C 1 1 0 n. d. 1 0 5 8 n. d. 1 2 8 1 2 9 2 9 8 5 2 1 1 2 2 5 5 2 0 5 7 5 1 3 0 2 4 3 7 2 9 6 9 9 M 3 B 4 7 4 2 7 0 5 n. d. n. d. 2 2 1 7 7 8 2 1 1 0 3 1 8 1 4 0 4 9 2 1 4 2 2 9 7 5 7 7 7 5 M 3 C n. d. 2 4 6 8 8 n. d. n. d. n. d. 2 7 9 n. d. 1 0 4 3 3 7 1 8 8 n .d . 1 0 0 1 5 3 1 3 6 2 1 1 5 6 M 5 A n. d. n. d. 2 7 1 n. d. n. d. 7 0 5 1 2 5 2 3 9 2 8 1 7 7 n .d . n .d . 1 2 1 4 6 1 7 1 1 M 5 B n. d. n. d. 5 8 8 2 n. d. n. d. 2 2 n. d. 2 8 3 1 n. d. n .d . n .d . 4 1 7 4 2 2 2 M 4 D n. d. 9 8 4 9 6 n. d. n. d. n. d. 3 3 8 n. d. 2 9 2 0 4 6 3 n .d . n .d . n .d . 8 6 8 2 2 9 M 1 B 2 2 n. d. 7 0 n. d. n. d. n. d. 2 4 n. d. n. d. 5 1 n. d. n .d . 4 9 n .d . 1 1 0 1 3 1 M 2 C n. d. n. d. 3 1 5 1 1 9 9 9 8 8 8 5 n. d. n. d. n. d. 2 3 n .d . n .d . n .d . n .d . n .d . M 4 E n. d. n. d. 4 4 5 n. d. n. d. n. d. n. d. n. d. n. d. 4 0 1 n. d. n .d . 2 4 3 n .d . 1 2 5 0 1 3 2 6 M 1 C n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. 8 9 n. d. n .d . 8 2 4 1 3 9 6 7 4 M 1 D n. d. n. d. 1 0 8 n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n .d . n .d . n .d . 3 7 4 4 3 0 M 1 E n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n .d . n .d . 2 8 1 0 4 1 4 2 N ot e: M et ab o lit es ar e o rd er ed fr o m m o st to le as t pr ev al en t, fo llo w ed by ab un da nc e ba se d o n to ta lp ea k ar ea ac ro ss al ls am pl es .U ri ne Sa m pl e 7 w as n o t in cl u d ed in th e ta b le as n o H H C o r m et ab o lit es w er e fo un d, de sp it e be in g po si ti ve fo r H H C in bl o o d. A bb re vi at io n: n. d. ,n o t de te ct ed . LINDBOM ET AL. 11 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense HHC, so this sample is not included in Table 2 or 3. The proposed metabolic pathways for HHC from analysis of non-hydrolysed and hydrolysed urine samples are shown in Figure 2. Apart from glucuronidation, the same biotransformations were found in both the non-hydrolysed and hydrolysed urine samples, which consisted of monoOH, diOH and diOH in combination with dehydrogenation (ketone or carboxylic acid formation). The metabo- lites of HHC identified in authentic urine samples were also similar to the metabolites of HHC identified after incubation with HHeps, although there were more than double the number of metabolites in the urine samples. This is likely due to only the (9R)-HHC being incu- bated with HHeps in this study, whereas the urine samples contained metabolites of both epimers, as shown by the identification of both epimers of the parent compound (glucuronidated in non-hydrolysed) and multiple stereoisomers of metabolites, such as three stereoiso- mers of 8-OH-HHC, which were not identified in the samples from HHeps incubations. This is in agreement with previous studies that found HHC-containing products contain a mixture of both the (9R) and (9S) epimers.1,8,10–12 Similar to the metabolites from incubation with HHeps, 99.3% of hydroxylated metabolites were glucuronidated in the non-hydrolysed urine samples. Metabolites with monoOH in combination with glucur- onidation were the most prevalent, accounting for 46.4% of the total peak area of the metabolites. A metabolite with a monoOH on the pentyl side chain (N2; 13.6% of total peak area) and a metabolite with a monoOH on the cyclohexyl ring, 8-OH-HHC (N3A; 6.7% of total peak area) were the only metabolites found in all 16 urine samples. Two 11-OH-HHC with glucuronide metabolites were the next most prevalent metabolites, found in 15 (N3B; 12.5% of total peak area) and 14 urine samples (N3C; 8.1% of total peak area). These are likely the (9R) and (9S) epimers of 11-OH-HHC, although it was not possible to determine which metabolite corresponded to which epimer. There were also two additional metabolites with a monoOH that were less prevalent and abundant, an 8-OH-HHC with glucuronide metabolite (N3D; 5.5% of total peak area) and a non-glucuronidated 11-OH-HHC metabolite (N10; 0.3% of total peak area). The remaining 13 metabolites had a diOH, where 83.5% (based on total peak area) were glucuronidated. Three of these metabolites had only diOH (N8A-C), three had diOH with a glucuronide (N4A-C), three had diOH in combination with dehydrogenation (N7A-B, N9) and four had diOH in combination with dehydrogenation and glucuronida- tion (N5A-C, N6). Two of the metabolites with diOH and dehydrogena- tion were confirmed by comparison with reference standards to be the (9R)- and (9S)-carboxylic acid metabolites (N7A and N7B, respec- tively). The remaining metabolites with diOH combined with dehydro- genation could not be confirmed but are presumed to be ketone formations. Extensive glucuronidation was found in the non-hydrolysed urine samples and following HHeps incubation, which is similar to phyto- cannabinoids like THC.24–26 In addition, Schirmer et al. (2023) found extensive glucuronidation of HHC metabolites in a urine sample col- lected 2 h after ingestion of HHC.15 Unfortunately, glucuronidated parent compounds and metabolites are not available as certified refer- ence standards. Because confirmation of identifications require com- parison of the samples with certified reference standards, hydrolysing toxicological samples where use of these or other semi-synthetic can- nabinoids is suspected is necessary. The urine samples in this study were hydrolysed and an overlaid chromatogram from hydrolysed Urine Sample 17 is provided in Figure 4 to demonstrate the relative abundance and retention times of the identified metabolites and parent compound. No glucuroni- dated metabolites were identified in the hydrolysed urine samples, indicating complete hydrolysis. However, complete cleavage of glucu- ronides by β-glucuronidase was only achieved after using longer incu- bation times (2 h) than in the standard manufacturer recommended method of 15 min (data from standard incubation time not shown). The need for longer incubation times was also observed by Schirmer et al. (2023) where after hydrolysis of the urine sample, half of the metabolites (4 of 8) were still glucuronidated.15 The parent compound, (9R)- and (9S)-HHC, only accounted for 3.1% of the total peak area, demonstrating the extensive metabolism of HHC. Similar to the non-hydrolysed samples, metabolites with a monoOH were the most prevalent and abundant in the hydrolysed samples, accounting for 38.1% of the total peak area of identified metabolites. The 11-OH-HHC metabolite (M1A; 19.4% of total peak area) and two metabolites with a monoOH on the pentyl side chain F IGURE 4 The overlaid chromatogram of metabolites of HHC found in Urine Sample 17, demonstrating the abundance and retention times of all the metabolites. 12 LINDBOM ET AL. 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense (M2A and M2B; 9.3 and 5.3% of total peak area, respectively) were the only metabolites found in all 16 urine samples. There was a third metabolite with a monoOH on the pentyl side chain (M2C), but it was less abundant (1.0% of total peak area). In addition, although the 8-OH-HHC with glucuronide metabolite was the most prevalent and abundant in the non-hydrolysed urine samples, the three 8-OH-HHC metabolites identified in the hydrolysed urine samples (M1B, M1D and M1E) only accounted for 2.17% of the total peak area. There was also one additional monoOH metabolite (M1C) where the exact location of the hydroxy group could not be determined. To confirm the locations and epimeric structure of the monoOH, two hydrolysed urine samples (16 and 17) were analysed alongside the reference standards using methanol and a longer gradient on the LC-QToF-MS to clarify the conformation. An overlaid chromatogram of Urine Sample 17 from the analysis with methanol is provided in Figure 5. As shown in Figure 5, both the (9R)- and (9S)-11-OH-HHC were confirmed to be present in the urine samples when run in meth- anol with a longer gradient, which improved separation of the epi- mers, whereas due to co-elution of the epimers, as can be seen in Figure 4, only one 11-OH-HHC metabolite (M1A) was identified when analysed using acetonitrile. Therefore, within the hydrolysed urine samples, the most prevalent and abundant metabolite for HHC was confirmed to be 11-OH-HHC (M1A). These results are consistent with prior studies of HHC metabolism. Manier et al. (2023) found a metab- olite with monoOH in human plasma and following pooled human liver S9 fraction incubation of HHC, although the exact location of the monoOH was not confirmed.18 In addition, Schirmer et al. (2023) identified both (9R)- and (9S)-11-OH-HHC as primary metabolites in urine15 and Kobidze et al. (2024) identified (9R)-11-OH-HHC in urine.17 It is also consistent with metabolism results of THC and CBD, where 11-OH-THC and 11-OH-CBD, respectively, are their principal metabolites. 11-OH-THC is an active metabolite,24,26,29 so future work should examine if 11-OH-HHC also displays activity at the can- nabinoid receptors. Three stereoisomers of 8-OH-HHC, (8R, 9S), (8S, 9S) and (8R, 9R), were also confirmed to be present in varying amounts in the urine samples. (8S)-OH-(9R)-HHC is also believed to present, although the reference standard was not available for confirmation. The (8R, 9R) and presumed (8S, 9R) epimers were found to be the most abundant of the 8-OH-HHC epimers. In comparison, Schirmer et al. (2023) only identified the (8R,9R)-8-OH-HHC epimer,15 and Kobidze et al. (2023) identified no 8-OH-HHC metabolites but found 9α-OH-HHC.17 As can be seen in Figure 5, the epimers of the 8-OH-HHC metabolites predominantly correspond to (9S)-HHC, while the epimers of the 11-OH-HHC metabolites predominantly correspond to (9R)-HHC. It should be noted that similar to the 11-OH-HHC epimers, the 8-OH- HHC epimers could only be confirmed when run in methanol with a longer gradient. Further research might be conducted to improve the separation of the epimers, such as by using a chiral column and pro- vide an opportunity for quantitative measurement of the epimers when all reference standards become available. 50OH-HHC was also confirmed to be the most abundant metabo- lite with a monoOH on the pentyl side chain (M2A) following compari- son with an in-house synthesised reference standard. Unfortunately, reference standards of the other isomers were not available for com- parison, so the locations on the side chain of the hydroxylations for the other metabolites (M2B and M2C) could not be confirmed. In com- parison, Schirmer et al. (2023) identified one metabolite with a monoOH on the pentyl side chain, which they tentatively identified to be at carbon four (40OH-HHC).15 There were five metabolites with diOH (M4A-E) in the hydrolysed urine samples, which accounted for 31.7% of the total peak area, but the exact locations of the hydroxy groups were unable to be deter- mined. There were also five metabolites with diOH in combination with dehydrogenation (M3A-C and M5A-B). Two of these metabolites were confirmed by comparison with reference standards to be the (9R)- and (9S)-carboxylic acid metabolites (M5A and M5B, respec- tively), where the (9R) epimer was found to be more abundant (2.9% of total peak area) than the (9S) epimer (0.7% of total peak area). The remaining metabolites with diOH combined with dehydrogenation (M3A-C; 23.52% of total peak area) could not be confirmed but are presumed to be ketone formations. F IGURE 5 The overlaid chromatogram and chemical structures of the epimers of HHC, 8-OH-HHC, 11-OH-HHC and carboxy-OH-HHC from running Urine Sample 17 in methanol with a longer gradient. *This is presumed to be the (8S, 9R) epimer of 8-OH-HHC, although a reference standard was not available for confirmation. LINDBOM ET AL. 13 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense The (R)- and (S)-carboxylic acid metabolites of HHC (M5A and M5B, respectively) were only found in about half of the urine samples in this study (10 and eight urine samples, respectively) with relatively low abundance, whereas one other study found (9R)- and (9S)- 11-COOH-HHC to be the most abundant metabolites in two urine samples.17 Given the carboxylic acid metabolite of THC and CBD are their primary metabolites,24–26 this indicates HHC may follow some of the same metabolic pathways as phytocannabinoids but that there are also some important differences. It should also be noted that none of the metabolites of HHC or HHCP were the same as those of THC or CBD, so the use of these semi-synthetic cannabinoids, which are currently not controlled in many jurisdictions, can be differentiated from the use of controlled phytocannabinoids. 4 | CONCLUSIONS In this study, glucuronidation, hydroxylation and dehydrogenation were the only biotransformations identified for HHC and HHCP fol- lowing incubation with HHeps and in authentic urine samples for HHC. HHC and HHCP were found to be extensively glucuroni- dated, where 99.3% of hydroxylated metabolites were glucuroni- dated in the non-hydrolysed urine samples. Metabolites with a monoOH were the most prevalent and abundant, accounting for 38.1% of the total peak area of identified metabolites in the hydro- lysed urine samples. The 11-OH-HHC metabolite (M1A), 50OH-HHC (M2A) and another metabolite with a monoOH on the pentyl side chain (M2B) were the only metabolites found in all 16 hydrolysed urine samples. Given that 11-OH-HHC (M1A), 50OH-HHC (M2A) and another metabolite with a monoOH on the pentyl side chain (M2B) were the only metabolites detected in all 16 urine samples where metabolites were identified, these metabolites along with the parent drug are sug- gested as suitable urinary markers to identify consumption of HHC and HHC-O. Following incubation with HHeps, the metabolites with monoOH + GLUC (P1A) and diOH + GLUC (P2A) were found to be the most abundant for HHCP and HHCP-O; therefore, these metabo- lites or their hydrolysed equivalents are suggested as suitable urinary markers to identify consumption of HHCP and HHCP-O. It is recom- mended that clinical and forensic toxicologists add these metabolites and characteristic ions to their targeted and semi-targeted analytical methods. AUTHOR CONTRIBUTIONS Conceptualisation: Robert Kronstrand and Henrik Gréen. Methodology: Karin Lindbom, Steven Baginski, Lucas Krebs, Darta Stalberga, Tobias Rautio, Xiongyu Wu, Robert Kronstrand and Henrik Gréen. Data cura- tion: Karin Lindbom, Caitlyn Norman, Tobias Rautio and Robert Kronstrand. Data analysis: Karin Lindbom, Caitlyn Norman, Steven Baginski, Lucas Krebs, Darta Stalberga, Tobias Rautio, Robert Kronstrand and Henrik Gréen. Writing—original draft: Karin Lindbom, Caitlyn Norman and Henrik Gréen. Writing—review and editing: All. Supervision: Robert Kronstrand and Henrik Gréen. ACKNOWLEDGEMENTS The authors acknowledge Goodness Ogechi Akubuiro for her assis- tance in the synthesis of 50OH-HHC. This study received funding from the Eurostars-2 Joint Program (European Commission, E! 113377 [Eurostars-2], NPS-REFORM) with co-funding from the European Union's Horizon 2020 research and innovation programme, Sweden's Innovation Agency (Grant 2019-03566) and the Strategic Research Area in Forensic Sciences (Strategiområdet forensiska vetenskaper) at Linköping University. The Leverhulme Research Centre for Forensic Science is funded by the Leverhulme Trust (Grant RC-2015-011). CONFLICT OF INTEREST STATEMENT The authors do not report any conflicts of interest. ORCID Caitlyn Norman https://orcid.org/0000-0003-2322-0367 Steven Baginski https://orcid.org/0000-0001-5615-1199 Lucas Krebs https://orcid.org/0009-0005-7679-4403 Darta Stalberga https://orcid.org/0000-0001-6712-652X Tobias Rautio https://orcid.org/0000-0002-0857-6261 Xiongyu Wu https://orcid.org/0000-0001-6756-2276 Robert Kronstrand https://orcid.org/0000-0002-4222-9597 Henrik Gréen https://orcid.org/0000-0002-8015-5728 REFERENCES 1. Ujváry I, Evans-Brown M, Gallegos A, et al. Hexahydrocannabinol (HHC) and Related Substances. Publications Office of the European Union; 2023. 2. European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). EU Early Warning System Formal Notification. [Notifica- tion of 6a,7,8,9,10,10a-hexahydro-6,6,9-trimethyl-3-pentyl-6H[1] dibenzo[b,d]pyran-1-ol (hexahydrocannabinol; HHC) in Europe.] EU- EWS-RCS-FN-2022-0031. 2022. 3. European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). EU Early Warning System Formal Notification. 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Cannabis. 2023;6:13-21. doi: 10.26828/cannabis/2023/000164 SUPPORTING INFORMATION Additional supporting information can be found online in the Support- ing Information section at the end of this article. How to cite this article: Lindbom K, Norman C, Baginski S, et al. Human metabolism of the semi-synthetic cannabinoids hexahydrocannabinol, hexahydrocannabiphorol and their acetates using hepatocytes and urine samples. Drug Test Anal. 2024;1‐15. doi:10.1002/dta.3740 LINDBOM ET AL. 15 19427611, 0, D ow nloaded from https://analyticalsciencejournals.onlinelibrary.w iley.com /doi/10.1002/dta.3740 by F H N ordw estschw eiz, W iley O nline L ibrary on [06/03/2025]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense info:doi/10.1007/s11419-023-00667-9 info:doi/10.1007/s11419-023-00667-9 info:doi/10.1089/can.2022.0253 info:doi/10.1002/dta.3570 info:doi/10.1111/j.1749-6632.1971.tb13985.x info:doi/10.1021/acschembio.3c00254 info:doi/10.1177/14690667231200139 info:doi/10.1177/14690667231200139 info:doi/10.1016/0091-3057(91)90359-A info:doi/10.1016/0091-3057(91)90359-A info:doi/10.1016/j.jpba.2023.115918 info:doi/10.1016/j.jpba.2023.115918 info:doi/10.1093/jat/bkad079 info:doi/10.1002/dta.2888 info:doi/10.1208/s12248-019-0381-3 info:doi/10.1208/s12248-019-0381-3 info:doi/10.1093/jat/bkae030 info:doi/10.1111/bph.14153 info:doi/10.1111/bph.14153 info:doi/10.1002/dta.3203 info:doi/10.1002/dta.3203 info:doi/10.7812/TPP/19.200 info:doi/10.1002/cbdv.200790152 info:doi/10.1080/00498254.2023.2194981 info:doi/10.1093/jat/bkad065 info:doi/10.1093/jat/bkad065 info:doi/10.1172/JCI107431 info:doi/10.1093/jat/bkac036 info:doi/10.26828/cannabis/2023/000164 info:doi/10.1002/dta.3740 Human metabolism of the semi-synthetic cannabinoids hexahydrocannabinol, hexahydrocannabiphorol and their acetates using he... 1 INTRODUCTION 2 METHODS 2.1 Materials 2.2 Hepatocyte incubations 2.3 Authentic urine samples 2.4 Instrumental analysis 2.5 Data and statistical analysis 3 RESULTS AND DISCUSSION 3.1 Hepatocyte incubations 3.2 (9R)-HHC 3.3 (9R)-HHCP 3.4 HHC authentic urine samples 4 CONCLUSIONS AUTHOR CONTRIBUTIONS ACKNOWLEDGEMENTS CONFLICT OF INTEREST STATEMENT REFERENCES