Rapid LC–QTOF–MS screening method for semi-synthetic cannabinoids in whole blood
Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern, Bern 3008, Switzerland
Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern 3012, Switzerland
Center for Forensic Science Research and Education, Fredric Rieders Family Foundation, Horsham, PA 19044, United States
Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern, Bern 3008, Switzerland
Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern 3012, Switzerland
Center for Forensic Science Research and Education, Fredric Rieders Family Foundation, Horsham, PA 19044, United States
Toxicology, NMS Labs, Horsham, PA 19044, United States
Center for Forensic Science Research and Education, Fredric Rieders Family Foundation, Horsham, PA 19044, United States
Abstract
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.
Article notes
Untitled section
Collection date 2026 Feb.
Introduction
The emergence of Δ8-tetrahydrocannabinol (Δ8-THC) and hexahydrocannabinol (HHC) on the recreational cannabinoid market has led to a new class of new psychoactive substances (NPSs) with cannabimimetic effects, commonly referred to as semi-synthetic cannabinoids [1]. Semi-synthetic cannabinoids are structurally related to Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive substance in Cannabis sativa L. (marijuana). Currently, the most prevalent semi-synthetic cannabinoids worldwide are HHC and Δ8-THC [1]. They both occur naturally in small amounts in the cannabis plant [2, 3]. However, it is unclear if HHC is naturally formed or only as a degradation product from the disproportionation of Δ9-THC to cannabinol (CBN) and HHC [4]. The first description of HHC was in 1940 after the hydrogenation of cannabis extracts [5]. Even though their natural occurrence in cannabis, the low content of Δ8-THC and HHC in the cannabis plant makes it cost prohibited for extraction. Both can be synthesized from cannabidiol (CBD), which is legal in many countries, including the United States through the enactment of the 2018 “Farm Bill” [6], and are then sprayed on hemp, or used as an ingredient in vaping liquids or edibles [1]. Acidic catalyzed ring-closure of CBD yields Δ9-THC and Δ8-iso-THC, respectively. Further acid catalyzed isomerization converts the products to the thermodynamically more stable Δ8-THC and Δ4(8)-iso-THC, respectively (see Fig. 1). The product composition depends on the reaction conditions (e.g. choice of acid, solvent, temperature, and time) [7]. After hydrogenation of the THC mixture, HHC is obtained as a mixture of two epimers: (9R)-HHC and (9S)-HHC [8, 9]. While hydrogenation of Δ9-THC delivers an excess of (9S)-HHC, the hydrogenation of Δ8-THC yields an excess of (9R)-HHC [9]. The iso-THCs are undesired side-products commonly found in recreational products containing Δ8-THC and have not been identified in Cannabis sativa L. [10–12]. Their hydrogenation delivers iso-HHC, which is a synthetic impurity in recreational HHC products [13]. Like Δ9-THC, Δ8-THC is a partial agonist on the CB1 cannabinoid receptor but it is commonly accepted to be less potent than Δ9-THC [14]. Of the two HHC epimers, (9R)-HHC shows a greater cannabimimetic potency [9, 15] and a greater binding affinity towards CB1 [16–18]. The iso-THCs potentially lack any cannabimimetic properties, and their pharmacology has not been studied yet due to suggested lower significance and importance.
Even though Δ9-THC and all its double-bonded isomers are listed in Schedule I of the United Nations (UN) Convention on Psychotropic Substances of 1971 [19], Δ8-THC is not legally regulated in the United States due to an ambiguity in the Agriculture Improvement Act of 2018 (“Farm Bill”). Hemp derived products are in a legal gray area in the United States as long as the product does not contain more than 0.3% Δ9-THC by dry weight [20]. In 2021, HHC entered the recreational drug market and spread rapidly as a legal substitute for Cannabis [1]. The regulation of HHC in many European countries has led to other unregulated semi-synthetic cannabinoids being available on the recreational drug market [21, 22]. Some semi-synthetic cannabinoids on the recreational market, like 11-OH-Δ9-THC and 8-OH-Δ9-THC, are known active metabolites of Δ9-THC [23–25]. Their synthesis is not as simple as for Δ8-THC or HHC [26, 27]. Other semi-synthetic cannabinoids like Δ9-THCP or Δ9-tetrahydrocannabihexol (Δ9-THCH) have been previously discovered as natural trace compounds in Cannabis sativa L. A content of 29 µg/g was reported for Δ9-THCP and 7 µg/g for Δ9-THCH, respectively [28, 29], indicating that they cannot be cost efficiently be extracted from the cannabis plant. Instead, these semi-synthetic cannabinoids that do not share the pentyl moiety of Δ8-THC or HHC are synthesized from 5-alkylresorcinols or 5-alkylcyclohexane-1,3-diones [30–32]. Suppliers claim that these semi-synthetic cannabinoids are made from CBD; however, this seems unlikely because of the alteration of the cannabinoid pentyl substituent. A general structure for semi-synthetic cannabinoids is depicted in Fig. 2. Semi-synthetic cannabinoids are derivatives of Δ9-THC and share the dibenzopyran moiety with it. The differences to Δ9-THC are the position or presence of the double bond. Acylation or alkylation of the phenolic hydroxy group, or homologation of the pentyl group is encountered as well. Other derivatives are hydroxylated at the positions C8, C10, or C11. Recently, three novel THC analogs were identified bearing an allyl or a propen-2-yl group at position C2. These derivatives are likely products after a Claisen rearrangement of THC allyl ethers [33]. The herein presented method expands an already existing screening method to include semi-synthetic cannabinoids [34]. Recently, a targeted method for the determination of THC isomers, analogs, homologs, and metabolites in blood and urine was published [35].
Adsorption of Δ9-THC on different surfaces is a well-known phenomenon [36]. Compounds with a large octanol-water partition coefficient (log Pow), like semi-synthetic cannabinoids, may adsorb to hydrophobic surfaces, such as polymers or siloxane groups of glass [37]. For THC analogues, it is known that their lipophilicity (log Pow) increases by a factor of approximatively three for each methylene group added to the side chain [38]. This might lead to preanalytical challenges when such hydrophobic compounds are analyzed. Storage and workup of samples might lead to analyte loss due to non-specific binding of the highly hydrophobic substances on storage containers, test tubes or vials prior analysis. Adsorption of Δ9-THC has been described on polystyrene, glass, and polypropylene (PP) [36, 39, 40].
Materials and methods
Chemicals and reagents
Δ9-Tetrahydrocannabiorcol (Δ9-THCO), Δ9-THC-ethyl (Δ9-THCE), Δ9-THC-butyl (Δ9-THCB), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabihexol (Δ9-THCH), Δ9-tetrahydrocannabiphorol (Δ9-THCP), Δ9-THC-octyl (Δ9-THC-C8), Δ9-tetrahydrocannabinol methyl ether (Δ9-THC-OMe), Δ9-tetrahydrocannabinol acetate (Δ9-THC-O), cannabinol (CBN), cannabidiol (CBD), cannabigerol (CBG), (9R)-hexahydrocannabinol ((9R)-HHC), (9S)-hexahydrocannabinol ((9S)-HHC), (9R)-hexahydrocannabihexol ((9R)-HHCH), (9R)-hexahydrocannabiphorol ((9R)-HHCP), (9R)-hexahydrocannabinol acetate ((9R)-HHC-O), Δ8-tetrahydrocannabinol (Δ8-THC), (±)-9α-hydroxy hexahydrocannabinol (9α-OH-HHC), (±)-9β-hydroxy hexahydrocannabinol (9β-OH-HHC), (−)-11-hydroxy-Δ8-tetrahydrocannabinol (11-OH-Δ8-THC), (±)-hydroxy-Δ9-tetrahydrocannabinol (11-OH-Δ9-THC), (±)-11-nor-9-carboxy-Δ9-tetrahydrocannabinol (11-COOH-Δ9-THC), and 11-nor-(9R)-carboxy-hexahydrocannabinol (11-COOH-(9R)-HHC) at concentrations of 1.0 mg/mL were purchased from Cayman Chemical (Ann Arbor, MI, United States). Cannabidiol-D3 (CBD-D3) and Δ9-tetrahydrocannabinol-D3 (THC-D3) at concentrations of 100 µg/mL were purchased from Cerilliant (Round Rock, TX, United States). Bond Elut C18 Solid Phase Extraction cartridges (500 mg, 3 mL) were purchased from Agilent (Santa Clara, CA, United States). Hexanes (95% n-hexane), ethyl acetate (EtOAc), tert-butyl methyl ether (TBME), and LC-MS grade solvents (e.g., methanol (MeOH), acetonitrile (MeCN), and water) were purchased from Honeywell Chemicals (Charlotte, NC, United States), formic acid ampules (1 mL) were purchased from Thermo Fisher Scientific (Waltham, MA, United States), ammonium formate was purchased from Millipore Sigma (St Louis, MO, United States), phosphoric acid was purchased from VWR (Radnor, PA, United States). A solution of MeOH in water (90%, V%) with formic acid (0.1%, V%) was used to reconstitute samples prior to analysis. The internal standard (ISTD) solution consisted of CBD-D3 and THC-D3 (γ = 500 ng/mL) in reconstitution solution. A solution of n-hexane, TBME and EtOAc (8/1/1; V/V/V) was used for the liquid extraction from blood. Drug-free human blood preserved with sodium fluoride and potassium oxalate was purchased from BioIVT (Westbury, NY, United States). Drug-free status was determined through comprehensive toxicology screening onsite after basic and acidic liquid-liquid-extraction described previously [34, 41].
Library semi-synthetic cannabinoids
Reference mass spectra from the cannabinoid standards were implemented in the library by measuring diluted cannabinoid standards (40 ng injected from each analyte). Standard solutions (2 µL, γ = 1 mg/mL) were diluted with 1.0 mL mobile phase B (γ = 2 µg/mL). From these solutions, 20 µL were injected on a Sciex Exion liquid chromatograph coupled to a Sciex X500R quadrupole time-of-flight mass spectrometer (LC–QTOF–MS) using SWATH-acquisition.
Reference mixtures 1 and 2
For the determination of limits of detection (LOD), recovery rates and matrix effects, two different mixtures were prepared. Reference mixture 1 was prepared by mixing 2 µL of 13 different standards (γ = 1 mg/mL) and diluting the mixture with 974 µL of mobile phase B (γ = 2 µg/mL). The standards were Δ9-THCO, 9α-OH-HHC, 11-OH-Δ9-THC, Δ9-THCE, 11-COOH-Δ9-THC, Δ8-THC, CBG, Δ9-THCB, CBN, Δ9-THCH, Δ9-THCP, (9S)-HHC, and Δ9-THC-C8.
Reference mixture 2 was prepared by mixing 2 µL of 11 different standards (γ = 1 mg/mL) and diluting the mixture with 978 µL of mobile phase B (γ = 2 µg/mL). The standards were 11-OH-Δ8-THC, 11-COOH-(9R)-HHC, 9β-OH-HHC, CBD, Δ9-THC, (9R)-HHCH, Δ9-THC-O, (9R)-HHCP, (9R)-HHC, (9R)-HHC-O, and Δ9-THC-OMe.
Blood samples were fortified with the Reference mixtures 1 and 2 prior extraction for the determination of the recovery rates and LOD, and after the extraction for the determination for the matrix effects.
LC–QTOF–MS method
A previously published method with adjustments to the electrospray voltage was used for the measurements [34]. Five-hundred microliters of blood was mixed with 8 µL ISTD working solution in a test tube. To this solution, 1 mL H3PO4 (5%, m%) and 3 mL extraction mixture were added. The mixture was rotated for 15 min and then centrifuged for 5 min (3398×g). To facilitate the phase separation, the test tubes were put in the freezer for 15 min at −80°C. The organic phase was decanted into a clean test tube and evaporated to dryness. The residue was dissolved in 200 µL reconstitution solution. Instrumental analysis was performed as described above using an Exion LC coupled to a X500R mass spectrometer (Sciex, Framingham, MA, United States) equipped with an ESI twinspray electrode assembly. The mass spectra were measured in positive ionization mode utilizing SWATH acquisition. The mobile phase A was an aqueous ammonium formate solution (10 mM, pH 3) containing formic acid, and mobile phase B was a solution of MeOH and MeCN (1/1, V/V) containing formic acid (0.1%, V%). The injection volume was 20 µL. Chromatography was performed at 30 °C on a Kinetex C18 column, 50 × 3 mm, 2.6 µm, 100 Å (Phenomenex, Torrance, CA, United States). A gradient method was used for chromatographic separation with the following conditions: 0–1 min: 5% B, 1–10 min: 5%–95% B, 10–13 min: 95% B, 13–13.1 min: 95%–5% B, 13.1–15.5 min: 5%. The curtain gas was set to 45 psi, ion source gas 1 to 40 psi, ion source gas 2 to 75 psi, the electrospray voltage was set to 4000 V and the source temperature was 600°C. Precursor ions from 100 to 510 Da were selected. Fragmentation was induced by using a collision energy spread of 35 ± 15 V [42]. The fragment scan range was 40–510 Da. The declustering potential was 80 V. Data were acquired on SciexOS (version 3.3.143) and processed with Peak View (version 2.2.0.11391) and MasterView (version 1.1.1944.0) (Fig. 3).
Method validation
A laboratory validation plan was developed and evaluated according to a previous screening validation performed by Krotulski et al. [34]. Validation experiments included recovery, matrix effects, limit of detection, carryover and inter- and intra-day reproducibility (mass-accuracy, retention times and analyte area ratio to CBD-D3). The validation was evaluated for suitability for the method’s intended use: qualitative broad-based semi-synthetic cannabinoid identification.
For the inter-day reproducibility, the blood samples were fortified on each day with the reference mixtures 1 and 2, respectively. A five-fold determination was performed on three different days. Intra-day reproducibility was calculated by comparing the retention times, mass accuracy, and area ratios of the analyte to CBD-D3 over a five-fold determination.
Recovery and matrix effects
Analyte recovery rates and matrix effects were evaluated for a liquid–liquid extraction (LLE) and a solid-phase extraction (SPE) protocol. The method was validated using the LLE protocol described below; however, both extraction protocols were evaluated for optimal recovery and matrix effects. Recovery rates and matrix effects rates were determined in triplicate.
Liquid–liquid extraction
In a test tube 0.5 mL blood, 1 mL H3PO4 (5%, m%), and 8 µL of the ISTD solution (γ = 20 ng/mL) were added. For the determination of the recovery rates, 2.5 µL reference mixture 1 or mixture 2 (γ = 25 ng/mL) were added, additionally. The solution was mixed, and 3 mL of the extraction solvent mixture was added. The test tubes were rotated for 15 min and centrifuged for 5 min (3398×g). Afterwards, the test tubes were put in the freezer at −80°C for 15 min, the organic phase was transferred to a new test tube and the solvent was evaporated in a TurboVap at 40°C for 30 min. The residue was dissolved in 200 µL reconstitution solution, transferred to a vial and analyzed on the X500R QTOF-MS.
For the determination of the matrix effects 0.5 mL blood, 1 mL H3PO4 (5%, m%) and 8 µL of the ISTD solution (γ = 20 ng/mL) were added into a test tube. The mixture was extracted and separated as mentioned for the determination of the recovery rates. After separation of the organic phase, 2.5 µL reference mixture 1 or mixture 2 (γ = 25 ng/mL) was added and the solvent was evaporated. The residue was dissolved in 200 µL reconstitution solution, transferred to a vial and analyzed on the X500R QTOF-MS.
Solid phase extraction
A validated method for the extraction of Δ9-THC and its metabolites from serum was used with the slight modification that the cartridges were additionally eluted with EtOAc [43]. C18 Bond Elut cartridges were conditioned with 2 mL MeOH and 2 mL AcOH (0.1 M). Five-hundred microliter blood was mixed with 8 µL ISTD (γ = 20 ng/mL). For the determination of the recovery rates, 2.5 µL reference mixture 1 or mixture 2 (γ = 25 ng/mL) were added. The samples were briefly vortexed and loaded on the cartridges. After loading, the cartridges were washed with 1 mL AcOH (0.1 M) and 1 mL MeCN (40%, V%). The samples were eluted using 1.5 mL MeCN and then with 1.5 mL EtOAc. The solutions were evaporated to dryness, dissolved in 200 µL reconstitution solution, transferred to a PP vial and analyzed on the X500R QTOF–MS.
The determination of the matrix effects was determined similar with the difference that the 2.5 µL reference mixture 1 or mixture 2 (γ = 25 ng/mL) were added to the organic phase after the extraction step. The eluted solutions were evaporated to dryness and dissolved in 200 µL reconstitution solution, transferred to a PP vial and analyzed on the X500R QTOF–MS.
Limit of detection
Limits of detection (LOD) were determined from fortified blood samples at different concentrations (γ = 0.8, 2, 4, 8, and 16 ng/mL) after LLE. The given LOD show a signal-to-noise ratio greater than 3 (S/N > 3). The noise near the analyte signal was used for the determination of the S/N. The LOD were determined from one measurement per level.
Inter- and intra-day reproducibility
For investigation of the reproducibility inter- and intra-day measurements were performed, and mass accuracy, relative standard deviation of retention time (RSD(Rt)) and signal area (RSD(A)) were calculated. For inter- and intra-day reproducibility blank blood was fortified to a concentration of γ = 10 ng/mL with reference mixture 1 and 2, respectively. For the inter-day reproducibility, a five-fold determination on three different days were prepared. The intra-day reproducibility was determined from a five-fold determination.
Carryover
For the investigation of carryover effects, blank solutions were injected after reference solutions (γ = 2000 ng/mL).
Results
Validation
Validation of the LC–QTOF–MS screening method was performed successfully. The method was validated for the LLE protocol using PP vials. The recovery rates ranged from 87% to 118% and the matrix effects ranged from 24% to 93% (see Table 1 and Supplementary Material S2). LOD ranged from 0.8 to 16 ng/mL, they are summarized in Table 1. For the inter-day reproducibility the mass errors of the analytes ranged from −2.1 to 1.3 ppm. The relative standard deviations of the retention times were RSD(Rt) ≤ 0.32%, and the relative standard deviations of the peak area were RSD(A) ≤ 48.2%. The values are summarized in Table 1. For the intra-day reproducibility, the mass errors of the analytes ranged from -3.7 to 0.2 ppm. The relative standard deviations of the retention times were RSD(Rt) ≤ 0.11%, and the relative standard deviations of the peak area were RSD(A) ≤ 22.5%. The values are summarized in Table 1. No carryover was observed.
| Analyte | Mass [M + H+]+ | LOD/ng*mL−1 | Recovery/% | M.E./% | Rt/min | Interday reproducibility | Intraday reproducibility | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Area RSD (%) | Mass accuracy RSD (ppm) | Rt RSD (%) | Area RSD (%) | Mass accuracy RSD (ppm) | Rt RSD (%) | ||||||
| Δ9-THCO | 259.1693 | 2 | 108 ± 6.9 | 70 | 9.47 | 29.2 | 0.2 | 0.30 | 12.9 | −0.1 | 0.09 |
| 9β-OH-HHC | 333.2424 | 4 | 97 ± 5.7 | 43 | 9.73 | 17.9 | −0.1 | 0.31 | 5.9 | −0.2 | 0.07 |
| 11-OH-Δ9-THC | 331.2268 | 2 | 104 ± 9.9 | 32 | 9.75 | 33.1 | −0.5 | 0.29 | 13.2 | −1.4 | 0.08 |
| 11-OH-Δ8-THC | 331.2268 | 4 | 95 ± 5.2 | 46 | 9.78 | 15.3 | 0.1 | 0.32 | 9.0 | −0.5 | 0.09 |
| Δ9-THCE | 273.1849 | 0.8 | 100 ± 8.1 | 30 | 9.82 | 18.5 | −0.4 | 0.30 | 5.3 | −0.5 | 0.05 |
| 11-COOH-(9R)-HHC | 347.2217 | 8 | 88 ± 12.4 | 63 | 9.85 | 11.8 | −0.9 | 0.31 | 5.1 | −1.0 | 0.11 |
| 11-COOH-Δ9-THC | 345.2060 | 0.8 | 100 ± 6.2 | 59 | 9.93 | 14.9 | −2.0 | 0.28 | 6.4 | −3.1 | 0.05 |
| 9α-OH-HHC | 333.2424 | 2 | 101 ± 6.4 | 41 | 10.09 | 26.7 | −0.9 | 0.28 | 4.8 | −1.9 | 0.10 |
| CBD | 315.2319 | 2 | 101 ± 4.8 | 66 | 10.13 | 13.6 | 0.1 | 0.27 | 10.8 | −0.6 | 0.11 |
| CBG | 317.2475 | 2 | 99 ± 9.6 | 61 | 10.17 | 20.2 | −0.2 | 0.23 | 6.3 | −0.6 | 0.05 |
| Δ9-THCB | 301.2162 | 2 | 102 ± 8.2 | 62 | 10.53 | 20.3 | −2.1 | 0.27 | 5.0 | −3.0 | 0.08 |
| CBN | 311.2006 | 2 | 106 ± 8.2 | 50 | 10.62 | 19.8 | −1.9 | 0.27 | 9.5 | −2.6 | 0.05 |
| Δ9-THC | 315.2319 | 2 | 100 ± 5.2 | 24 | 10.85 | 19.6 | −0.8 | 0.26 | 10.8 | −1.6 | 0.07 |
| Δ8-THC | 315.2319 | 2 | 111 ± 8.1 | 24 | 10.92 | 20.9 | −1.0 | 0.26 | 6.7 | −1.9 | 0.09 |
| (9S)-HHC | 317.2475 | 2 | 101 ± 10.2 | 67 | 11.01 | 32.9 | −1.5 | 0.25 | 10.4 | −2.1 | 0.09 |
| (9R)-HHC | 317.2475 | 2 | 102 ± 5.6 | 44 | 11.06 | 25.9 | −0.8 | 0.26 | 10.5 | −1.7 | 0.08 |
| Δ9-THCH | 329.2475 | 8 | 106 ± 4.6 | 93 | 11.13 | 18.8 | −0.1 | 0.25 | 6.1 | −0.7 | 0.05 |
| (9R)-HHCH | 331.2632 | 4 | 118 ± 4.4 | 24 | 11.34 | 21.3 | 1.3 | 0.16 | 22.5 | 0.5 | 0.06 |
| Δ9-THCP | 343.2632 | 4 | 97 ± 5.4 | 53 | 11.40 | 17.0 | −0.8 | 0.24 | 11.0 | −1.8 | 0.10 |
| Δ9-THC-O | 357.2424 | 2 | 92 ± 10.7 | 80 | 11.49 | 48.2 | −2.4 | 0.25 | 19.6 | −3.7 | 0.06 |
| (9R)-HHCP | 345.2788 | 16 | 87 ± 10.0 | 25 | 11.58 | 16.7 | 0.5 | 0.25 | 11.0 | −0.1 | 0.07 |
| (9R)-HHC-O | 359.2581 | 2 | 94 ± 11.6 | 42 | 11.60 | 16.9 | −0.1 | 0.22 | 18.4 | −0.6 | 0.08 |
| Δ9-THC-C8 | 357.2788 | 8 | 90 ± 7.0 | 35 | 11.66 | 22.8 | 0.0 | 0.24 | 2.6 | −0.6 | 0.06 |
The recovery rates from the LLE using non-silanized glass vials ranged from 0% to 113% (see Supplementary Material S1 and S3). The matrix effects ranged from 11% to 76% (see Supplementary Material S1). For the SPE protocol the recovery rates ranged from 28% to 90% (see Supplementary Material S1 and S4), and the matrix effects ranged from 33% to 171% (see Supplementary Material S1).
Application to authentic samples
The validated LC–QTOF–MS method described above was applied to authentic driving under the influence of drugs (DUID) samples where the consumption of cannabinoids other than or in addition to Δ9-THC was suspected. Twenty-three deidentified human blood specimens collected between December 2023 and February 2024 were provided by NMS Labs (Horsham, PA, United States) and were subjected to the optimized workflow described in this manuscript. The semi-synthetic cannabinoid HHC and its metabolite HHC-COOH were identified in one blood specimen (1 of 23). THC and THC-COOH were identified in eighteen samples (18 of 23). Four samples (4 of 23) did not contain any cannabinoids described in the method above the listed LOD (Table 1).
For the confirmation of the results of the qualitative QTOF screening method on THC isomers, the described samples were re-analyzed using an in-house research method (LC-QqQ). This method enables the chromatographic separation of the THC isomers: Δ8-THC and Δ9-THC were both qualitatively identified in the majority of the samples (18 of 23), three were tested positive for only Δ9-THC (3 of 23), one sample was only positive for Δ8-THC (1 of 23) and one sample did not contain any THC isomers (1 of 23).
Discussion
Adsorption
During the recovery experiments, it was found that the larger semi-synthetic cannabinoids (by mass) showed a low recovery rate. (9R)-HHCH, Δ9-THCP, Δ9-THCP-O, Δ9-THC-C8, and Δ9-THC-OMe were initially not detected after extraction. It was found that this problem occurred when analysis was performed from non-silanized glass vials with inserts. Vials made from PP did not show this behavior and were therefore used for validation and further analyses, even though adsorption of phytocannabinoids has been described on glass and on PP [39, 40]. It is assumed that the adsorption of the more hydrophobic analytes emerged from the interactions with the hydrophobic siloxane groups on the glass surface. These groups would not be affected through silanization, only the hydrophilic silanol groups would be desactivated.
The analyte recovery rates from the tested SPE protocol dropped noticeably with the lipophilicity of the analytes. It is assumed that the more lipophilic analytes experienced such a strong retention that elution did not occur completely, even additional elution with EtOAc showed no improved recovery rates. The SPE protocol, which was tested for this method was from a validated method for the determination of Δ9-THC and its metabolites from serum [43]. For the extraction of semi-synthetic cannabinoids from blood another SPE protocol should be developed.
Limitations
Missing forensic markers and sensitivity
For most analytes described in this method, no established forensic markers (e.g. metabolites) are known due to scarce research on the metabolism of semi-synthetic cannabinoids. It is unclear to what extent the analytes are metabolized and if the consumption of such substances leads to a detectable concentration of the unmetabolized consumed drug in blood, particularly if the consumption was days prior. Work on HHCP has shown that semi-synthetic cannabinoids with a longer side-chain might metabolize excessive to mono- and bishydroxylated phase-I metabolites [44, 45]. Semi-synthetic cannabinoids with a longer side-chain are more potent than Δ9-THC, lower LODs should therefore be achieved for these compounds [17, 18]. This might however not be possible due to their poor ionizability. Currently unknown phase-I or phase-II metabolites are better targets and should be included after their respective discovery and availability as reference standard.
Resolution of THC isomers
Initially, the panel included more THC isomers, which could be synthesized from CBD, namely Δ8-THC, Δ9-THC, Δ8-iso-THC, Δ8-THC, (9S)-Δ6a,10a-THC, (9S)-Δ7-THC, exo-THC and (6aR, 9R)-Δ10-THC. Only Δ8- and Δ9-THC were then included due to poor separation of the THC isomers using the method described. From the possible isomers with natural configuration, only Δ8-THC has a significant prevalence besides the main cannabinoid Δ9-THC. The isomers (6aR, 9S)-Δ10-THC, (9R, 10aR)-Δ6a-THC, (9S, 10aR)-Δ6a-THC, (9R)-Δ7-THC and the enantiomer of the initially included cannabinoid (9R)-Δ6a,10a-THC were not measured. These THC isomers could also be prepared from CBD. To the authors’ knowledge no method exists which separates all THC isomers in a single chromatographic method, which is somewhat inhibited by the introduction of new isomers to the recreational market yearly. Our LC–QTOF–MS qualitative screening method did not fully resolve Δ9-THC from Δ8-THC and 11-OH-Δ9-THC from 11-OH-Δ8-THC. The HHC epimers (9S)-HHC and (9R)-HHC are coeluting within this screening method (see Supplementary Material S5–S8).
Interferences from regio- and stereo-chemical synthetic impurities
Previous work on HHCP and THCP has shown that these substances possess isomeric impurities such as abnormal cannabinoids or cis-cannabinoids [31, 32]. These unnatural regio- and stereo-isomers show similar chromatographic properties and very similar fragmentation patterns which might interfere with the detection of the semi-synthetic cannabinoids. These impurities were not included in this screening method and might interfere with the detection of semi-synthetic cannabinoids in authentic specimens.
Conclusion
Due to the emergence of semi-synthetic cannabinoids on the recreational market and their potent cannabimimetic effects, existing screening methods should be updated to include these novel substances in their screening panel. There is limited information in the literature about comprehensive screening for semi-synthetic cannabinoids and our LC–QTOF–MS demonstrates the feasibility for application to authentic forensic biological specimens. The lipophilicity of these cannabinoid substances might lead to adsorption on material used for sample preparation or storage and should be taken into account if an analytical method should cover these analytes.
Supplementary Material
Contributor Information
Willi Schirmer, Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern, Bern 3008, Switzerland; Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern 3012, Switzerland.
Sara E Walton, Center for Forensic Science Research and Education, Fredric Rieders Family Foundation, Horsham, PA 19044, United States.
Wolfgang Weinmann, Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern, Bern 3008, Switzerland.
Stefan Schürch, Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern 3012, Switzerland.
Barry K Logan, Center for Forensic Science Research and Education, Fredric Rieders Family Foundation, Horsham, PA 19044, United States; Toxicology, NMS Labs, Horsham, PA 19044, United States.
Alex J Krotulski, Center for Forensic Science Research and Education, Fredric Rieders Family Foundation, Horsham, PA 19044, United States.
Supplementary data
Supplementary data is available at Journal of Analytical Toxicology online.
Funding
The University of Bern is acknowledged for funding the research stay of W.S. at the CFSRE through their funding program “UniBE Short Travel Grants for (Post)Docs”. The Center for Forensic Research and Education (CFSRE) is funded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice (Award Number 15PNIJ-22-GG-04434-MUMU, “Implementation of NPS Discovery – An Early Warning System for Novel Drug Intelligence, Surveillance, Monitoring, Response, and Forecasting using Drug Materials and Toxicology Populations in the United States”).
Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.
Acknowledgments
The authors would like to acknowledge the staff and scientists at the CFSRE for their assistance. Ayako Chan-Hosokawa from NMS Labs is acknowledged for providing insights and the DUID samples.
References
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References
- 1. Ujváry I. Hexahydrocannabinol and closely related semi-synthetic cannabinoids: A comprehensive review. Drug Test Anal 2024;16:127–61. 10.1002/dta.3519
- 2. Hively RL, Mosher WA, Hoffmann FW. Isolation of trans-Δ6-tetrahydrocannabinol from marijuana. J Am Chem Soc 1966;88:1832–3. 10.1021/ja00960a056
- 3. Basas-Jaumandreu J, de las Heras FXC. GC-MS metabolite profile and identification of unusual homologous cannabinoids in high potency Cannabis sativa. Planta Med 2020;86:338–47. 10.1055/a-1110-1045
- 4. Garrett ER, Gouyette AJ, Roseboom H. Stability of tetrahydrocannabinols II. J Pharm Sci 1978;67:27–32. 10.1002/jps.2600670108
- 5. Adams R, Pease DC, Cain CK et al. Structure of cannabidiol. VI. Isomerization of cannabidiol to tetrahydrocannabinol, a physiologically active product. Conversion of cannabidiol to cannabinol. J Am Chem Soc 1940;62:2402–5. 10.1021/ja01866a040
- 6. United States Department of Agriculture (USDA), Agricultural Marketing Service, https://www.ams.usda.gov/rules-regulations/hemp/enforcement
- 7. Marzullo P, Foschi F, Coppini DA et al. Cannabidiol as the substrate in acid-catalyzed intramolecular cyclization. J Nat Prod 2020;83:2894–901. 10.1021/acs.jnatprod.0c00436
- 8. Collins A, Ramirez G, Tesfatsion T et al. Synthesis and characterization of the diastereomers of HHC and H4CBD. Nat Prod Commun 2023;18:1934578X231158910. 10.1177/1934578X231158910
- 9. Russo F, Vandelli MA, Biagini G et al. Synthesis and pharmacological activity of the epimers of hexahydrocannabinol (HHC). Sci Rep 2023;13:11061. 10.1038/s41598-023-38188-5
- 10. Radwan MM, Wanas AS, Gul W et al. Isolation and characterization of impurities in commercially marketed Δ8-THC products. J Nat Prod 2023;86:822–9. 10.1021/acs.jnatprod.2c01008
- 11. Gul W, Shahzadi I, Sarma N et al. Development and validation of a GC-FID method for the quantitation of Δ8-tetrahydrocannabinol and impurities found in synthetic Δ8-tetrahydrocannabinol and vaping products. Planta Med 2024;90:316–32. 10.1055/a-2249-7824
- 12. Geci M, Scialdone MA-O, Tishler J. The Dark side of cannabidiol: the unanticipated social and clinical implications of synthetic Δ8-THC. Cannabis Cannabinoid Res 2023;8:270–82. 10.1089/can.2022.0126
- 13. Tanaka R, Kikura-Hanajiri R. Identification of hexahydrocannabinol (HHC), dihydro-iso-tetrahydrocannabinol (dihydro-iso-THC) and hexahydrocannabiphorol (HHCP) in electronic cigarette cartridge products. Forensic Toxicol 2024;42:71–81. 10.1007/s11419-023-00667-9
- 14. Hollister LE, Gillespie HK. Delta-8- and delta-9-tetrahydrocannabinol; Comparison in man by oral and intravenous administration. Clin Pharma Ther 1973;14:353–7. 10.1002/cpt1973143353
- 15. Reggio PH, Greer KV, Cox SM. The importance of the orientation of the C9 substituent to cannabinoid activity. J Med Chem 1989;32:1630–5. 10.1021/jm00127a038
- 16. Nasrallah DJ, Garg NK. Studies pertaining to the emerging cannabinoid hexahydrocannabinol (HHC). ACS Chem Biol 2023;18:2023–9. 10.1021/acschembio.3c00254
- 17. Janssens LK, Van Uytfanghe K, Williams JB et al. Investigation of the intrinsic cannabinoid activity of hemp-derived and semisynthetic cannabinoids with β-arrestin2 recruitment assays—and how this matters for the harm potential of seized drugs. Arch Toxicol 2024;98:2619–30; 10.1007/s00204-024-03769-4
- 18. Persson M, Kronstrand R, Evans-Brown M et al. In vitro activation of the CB1 receptor by the semi-synthetic cannabinoids hexahydrocannabinol (HHC), hexahydrocannabinol acetate (HHC-O) and hexahydrocannabiphorol (HHC-P). Drug Test Anal 2025;17: 487–93. 10.1002/dta.3750
- 19. United Nations Convention on Psychotropic Substances 1971, https://www.unodc.org/pdf/convention_1971_en.pdf
- 20. Congress. Agriculture Improvement Act of 2018, Government. U.S. Government Publishing Office, November 17, 2023. https://www.govinfo.gov/app/details/COMPS-15214/
- 21. European Union Drugs Agency EUDA, EU Drug Market: New psychoactive substances—Distribution and supply in Europe: Semi-synthetic cannabinoids, June 2024
- 22. United Nations Office on Drugs and Crime UNODC, SMART Forensics Update, Beyond plants: semi-synthetic diversify the cannabis market, Vol. 01, May 2024
- 23. Gasse A, Pfeiffer H, Köhler H et al. 8β-OH-THC and 8β,11-diOH-THC—minor metabolites with major informative value? Int J Legal Med 2018;132:157–64. 10.1007/s00414-017-1692-5
- 24. Radwan MM, ElSohly MA, El-Alfy AT et al. Isolation and pharmacological evaluation of minor cannabinoids from high-potency Cannabis sativa. J Nat Prod 2015;78:1271–6. 10.1021/acs.jnatprod.5b00065
- 25. Huestis MA. Pharmacokinetics and metabolism of the plant cannabinoids, Δ9-tetrahydrocannibinol, cannabidiol and cannabinol. In: Pertwee RG (ed.) Cannabinoids. Berlin Heidelberg: Springer, 2005, 657–90.
- 26. Pitt CG, Fowler MS, Sathe S et al. Synthesis of metabolites of Δ9-tetrahydrocannabinol. J Am Chem Soc 1975;97:3798–802. 10.1021/ja00846a040
- 27. Linciano P, Citti C, Russo F, et al. Cannabis extracts and uses thereof. US Patent US2022064090A1, filed 9 July 2021, and issued 3 March 2022.
- 28. Citti C, Linciano P, Russo F et al. A novel phytocannabinoid isolated from Cannabis sativa L. with an in vivo cannabimimetic activity higher than Δ9-tetrahydrocannabinol: Δ9-Tetrahydrocannabiphorol. Sci Rep 2019;9:20335. 10.1038/s41598-019-56785-1
- 29. Linciano P, Citti C, Russo F et al. Identification of a new cannabidiol n-hexyl homolog in a medicinal cannabis variety with an antinociceptive activity in mice: cannabidihexol. Sci Rep 2020;10:22019. 10.1038/s41598-020-79042-2
- 30. Caprari C, Ferri E, Vandelli MA et al. An emerging trend in novel psychoactive substances (NPSs): designer THC. J Cannabis Res 2024;6:21. 10.1186/s42238-024-00226-y
- 31. Schirmer W, Gjuroski I, Vermathen M et al. Isolation and characterization of synthesis intermediates and side products in hexahydrocannabiphorol. Drug Test Anal 2025;17:531–43. 10.1002/dta.3759
- 32. Schirmer W, Schürch S, Weinmann W. The identification of synthetic impurities in a vape pen containing Δ9-tetrahydrocannabiphorol using gas chromatography coupled with mass spectrometry. Psychoactives 2024;3:491–500. 10.3390/psychoactives3040030
- 33. Dadiotis E, Mpakaoukas S, Mitsis V et al. Identification of three novel tetrahydrocannabinol analogs in the European market. Drug Test Anal 2025;17:1594–600. 10.1002/dta.3866
- 34. Krotulski AJ, Varnum SJ, Logan BK. Sample mining and data mining: combined real-time and retrospective approaches for the identification of emerging novel psychoactive substances. J Forensic Sci 2020;65:550–62. 10.1111/1556-4029.14184
- 35. Muir LS, Doumit SE, Seither JZ et al. Comprehensive LC-MS/MS analysis of THC isomers, analogs, homologs and metabolites in blood and urine. J Anal Toxicol 2025;49:322–31. 10.1093/jat/bkaf023
- 36. Christophersen AS. Tetrahydrocannabinol stability in whole blood: plastic versus glass containers. J Anal Toxicol 1986;10:129–31. 10.1093/jat/10.4.129
- 37. Murakoshi M, Fukuzawa K, Sato Y et al. Adsorption phenomenon and development of low adsorption vials for LC and LC/MS. Shimadzu Poster 2017.
- 38. Thomas BF, Compton DR, Martin BR. Characterization of the lipophilicity of natural and synthetic analogs of delta 9-tetrahydrocannabinol and its relationship to pharmacological potency. J Pharmacol Exp Ther 1990;255:624–30. 10.1016/S0022-3565(25)23000-2
- 39. Stephenson JB, Carter DN, Richardson M. Surface adsorption of cannabinoids in LC–MS/MS applications. Forensic Toxicol 2020;38:292–6. 10.1007/s11419-019-00505-x
- 40. Molnar A, Lewis J, Fu S. Recovery of spiked Δ9-tetrahydrocannabinol in oral fluid from polypropylene containers. Forensic Sci Int 2013;227:69–73. 10.1016/j.forsciint.2012.11.006
- 41. Krotulski AJ, Mohr ALA, Friscia M et al. Field detection of drugs of abuse in oral fluid using the Alere™ DDS®2 mobile test system with confirmation by liquid chromatography tandem mass spectrometry (LC–MS/MS). J Anal Toxicol 2018;42:170–6. 10.1093/jat/bkx105
- 42. Dresen S, Gergov M, Politi L et al. ESI-MS/MS library of 1,253 compounds for application in forensic and clinical toxicology. Anal Bioanal Chem 2009;395:2521–6. 10.1007/s00216-009-3084-2
- 43. Stumptner D, Kempf J, Weinmann W. Increase of extraction efficiency for THC in serum by larger SPE cartridges. XIII. GTFCh-Symposium, Ausgewählte Aspekte der Forensischen Toxikologie: Verlag Dr Dieter Helm, Heppenheim, 2004, 374–78.
- 44. 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 2025;17:372–86. 10.1002/dta.3740
- 45. Schirmer W, Schürch S, Weinmann W. Identification of hexahydrocannabiphorol metabolites in human urine. Drug Test Anal 2025;17:1681–93. 10.1002/dta.3871
Associated Data
Supplementary Materials
Data Availability Statement
The data underlying this article will be shared on reasonable request to the corresponding author.