Chemicals and reagents
Acetonitrile (LC–MS grade), ammonium formate 10 M (99.995%), ethanol absolute, ethyl acetate p.a., methanol (ChromasolvTM), potassium hydroxide (purris, p.a., ≥ 86% (T) pellets) and superoxide dismutase (SOD) (≥ 3,000 units/mg protein from bovine erythrocytes, 1 mg/mL solution, dissolved in 0.5 M potassium phosphate buffer pH 7.5) were purchased from Sigma-Aldrich (Steinheim, Germany); sodium carbonate and n-hexane (Lichrosolv®) from Merck (Darmstadt, Germany); formic acid (98–100%, p.a.) from Applichem (Darmstadt, Germany); potassium hydrogen phosphate (≥ 99%, p.a.) and sodium hydrogen carbonate from Carl Roth (Karlsruhe, Germany); β-glucuronidase (E. coli K 12) from Roche Diagnostics (Mannheim, Germany); pHLMs (50 donors, 20 mg/mL protein in 250 mM sucrose), NADPH regenerating solutions A/B (reductase activity 0.43 µmol/min × mL), and 0.5 M potassium phosphate buffer (pH 7.5) from Corning (Corning, NY, USA). NADPH regenerating solution A consisted of 26 mM NADP+, 66 mM glucose-6-phosphate, and 66 mM MgCl2 in water. NADPH regenerating solution B consisted of 40 U/mL glucose-6-phosphate dehydrogenase in 5 mM sodium citrate. Deionized water was prepared using a Medica® Pro deionizer from ELGA (Celle, Germany).
Cumyl-PINACA (purity > 98%) was kindly provided by the Slovenian National Forensic Laboratory (Ljubljana, Slovenia). The 5F-Cumyl-PINACA, ADBICA-d9, AKB48-d9, JWH-007-d9, JWH-398-d9, MAM-2201-d5, PB-22-d9, RCS-4d9, UR-144-d5 and XLR-11-d5 were purchased from Cayman Chemical (Ann Arbor, MI, USA); JWH-015-d7, JWH-018-d11, JWH-081-d9, JWH-122-d9, JWH-200-d5, JWH-210-d9 and JWH-250-d5 from LGC Standards (Wesel, Germany); JWH-018 and JWH-073-d9 from Chiron AS (Trondheim, Norway). AB-CHMINACA, AB-FUBINACA, AB-PINACA, AM-2201, EG-018, MDMB-CHMICA and THJ-2201 were bought as ‘research chemical’ via the Internet; identity and purity (≥ 95%) were determined using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS).
The cAMP biosensor assay with CB1 as target was conducted by DiscoveRx (Fremont, CA, USA) with the substances AB-CHMINACA, AB-FUBINACA, AB-PINACA, AM-2201, Cumyl-PINACA, EG-018, JWH-018, MDMB-CHMICA and THJ-2201. Each compound was tested in duplicates; to induce response, 20 µM forskolin was used.
For LC–MS/MS analysis, mobile phase A consisted of 1% acetonitrile, 0.1% formic acid, and 2 mM ammonium formate in water, while mobile phase B consisted of acetonitrile with 0.1% formic acid, and 2 mM ammonium formate.
Instrumentation
GC–MS analysis
For GC–MS analysis, a 6890 series chromatography system combined with a 5973-series mass selective detector, a 7683 B series injector and Chemstation G1701GA version D.03.00.611 (Agilent, Waldbronn, Germany) was used. The method parameters were as described elsewhere [
25]. Briefly they were: injection port temperature 270 °C, carrier gas helium, flow rate 1 mL/min, and oven temperature 100 °C for 3 min, ramped to 310 °C with 30 °C/min and held for 10 min. Electron ionization (EI) mode with 70 eV ionization energy and scan mode from
m/z 40–550 were applied. The obtained mass spectra were compared to commonly used EI–MS spectra libraries (Cayman Chemical, Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG), National Institute of Standards and Technology (NIST), Wiley and Maurer-Pfleger-Weber (MPW)), to the EDND of the European Monitoring Center for Drugs and Drug Addiction (EMCDDA) and to an in-house library of previously identified synthetic cannabinoids.
NMR analysis
NMR spectra of the unknown compound found in three of the e-liquids (called ‘c-liquids’ by some vendors) were recorded in CDCl3 at room temperature with a DRX 400 (Bruker Physik AG, Ettlingen, Germany). Chemical shifts are reported in ppm relative to CHCl3 (1H: d = 7.27) and CDCl3 (13C: d = 77.23) as internal standards. Compounds were fully characterized by 1D-1H as well as 13C NMR at 400 MHz and 100 MHz, respectively. Selective 1D-TOCSY (total correlation spectroscopy), 1D-ROESY (rotation frame nuclear Overhauser effect spectroscopy) as well as 2D-1H/13C HSQC (heteronuclear single quantum coherence), 1H/1H COSY (correlation spectroscopy) and 1H/13C HMBC (heteronuclear multiple-bond correlation spectroscopy) spectra were recorded.
LC–MS/MS analysis of serum samples
For analysis of the serum samples, a routinely used LC–MS/MS screening method, currently covering 94 compounds with at least two ion transitions for each analyte and one ion transition for each internal standard in positive scheduled multiple reaction monitoring (+sMRM) was applied [
26]. Briefly, a QTRAP™ 4000 triple quadrupole linear ion trap instrument (Sciex, Darmstadt, Germany) equipped with a TurbolonSpray
® interface and coupled to a Shimadzu Prominence HPLC system consisting of two LC-20 AD SP isocratic pumps, SIL 20 AC autosampler, CTO-20A controller (Shimadzu, Duisburg, Germany) and Analyst
® software version 1.6.2 were used (Sciex). Chromatographic separation was performed on a Kinetex
® C18 column (2.6 µm, 100 Å, 100 × 2.1 mm; Phenomenex, Aschaffenburg, Germany) applying gradient elution as follows: starting concentration of 20% mobile phase B was held for 1 min, then linearly increased to 60% B for 1.5 min, further increased to 65% B for 1.5 min, held for 1.5 min, further increased to 90% B for 2.5 min, held for 2.0 min, decreased to starting conditions of 20% B for 0.1 min and held for 1.9 min for re-equilibration. The total flow rate was 0.5 mL/min. The autosampler and the column oven temperatures were set to 10 and 40 °C, respectively. The monitored ion transitions used for Cumyl-PINACA were
m/z 350→215 and 350→232.
The LC–MS/MS system used for the analysis of the urine and the pHLM assay samples consisted of a Nexera X2 UHPLC (Shimadzu) coupled to a QTRAP™ 5500 triple quadrupole linear ion trap instrument (SCIEX) equipped with the TurboIonSpray® probe. Chromatographic separation was performed on the Kinetex® C18 column (2.6 µm, 100 Å, 100 × 2.1 mm) applying gradient elution as follows: starting concentration of 5% mobile phase B was held for 0.5 min, then linearly increased to 35% B for 0.5 min, further increased to 50% B for 3.0 min, further increased to 90% B for 4.0 min, held for 2.0 min, decreased to starting conditions of 5% for 0.5 min and held for 2.5 min for re-equilibration. The total flow rate was 0.4 mL/min. The autosampler and the column oven temperatures were set to 10 and 40 °C, respectively.
The MRM ion transitions of the parent compounds were optimized carefully in positive ionization mode (see Supplementary Tables S1 and S2). Enhanced product ion (EPI) scan experiments were performed using hypothetical masses of anticipated phase I metabolites, and the obtained spectra were compared to the EPI spectrum of the respective parent compound. In order to detect unexpected metabolites not covered by the EPI scan approach, precursor ion scan experiments for the characteristic fragment ion at m/z 145 and for the m/z of its modified analogs 161 (monohydroxylation), 177 (dihydroxylation) and 179 (dihydrodiol) were conducted. The relative abundances of the characterized metabolites in the urine samples obtained during the self-administration study were compared using an MRM method comprising the most abundant ion transitions of each metabolite (see Supplementary Fig. S1 and Table S3).
Sample treatments
E-liquids
A 1-mL volume of the e-liquids bought during the monitoring of the online market was extracted by adding 1 mL of methanol and 1 mL n-hexane using an overhead shaker for 5 min at lowest rotation speed (Reax 2; Heidolph, Schwabach, Germany). Subsequently, samples were centrifuged at 2860 × g for 5 min (Heraeus Megafuge 1.0; Thermo Scientific, Schwerte, Germany). Ten microliters of the supernatant were evaporated to dryness at 40 °C under a gentle stream of nitrogen. Prior to analysis by the GC–MS system (injection volume 1 µL), the samples were reconstituted in 100 µL of dry ethyl acetate.
For NMR analysis, 1 mL of an e-liquid was extracted five times using n-hexane/ethyl acetate (99:1, v/v). The combined supernatants were evaporated to dryness and resulted in 4 mg of a raw extract containing 5F-Cumyl-PINACA, which was used for structure elucidation by NMR spectroscopy.
Pooled human liver microsome assay
To investigate the tentative phase I metabolism of Cumyl-PINACA and 5F-Cumyl-PINACA (extracted from e-liquids), in vitro experiments with pHLM were performed in triplicates. A 0.5-µL volume of a substrate solution (1 ng/mL in acetonitrile) was added to 49.5 µL of a reaction mixture consisting of 2.5 µL pHLM, 2.5 µL NADPH regenerating solution A, 0.5 µL NADPH regenerating solution B, 5.0 µL SOD, 10 µL 0.5 M phosphate buffer, and 29 µL deionized water. Incubation was performed for 60 min at 37 °C. The reaction was terminated by the addition of ice-cold acetonitrile, and the supernatant was analyzed after 1:10 dilution with mobile phase A/B (50:50, v/v). Blank pHLM samples were processed in the same way serving as negative controls.
Serum samples
A 1-mL aliquot of a serum sample was spiked with 10 µL of an internal standard solution mixture (25 ng/mL each, ADBICA-d9, AKB48-d9, JWH-007-d9, JWH-015-d7, JWH-018-d11, JWH-073-d9, JWH-081-d9, JWH-122-d9, JWH-200-d5, JWH-210-d9, JWH-250-d5, JWH-398-d9, MAM-2201-d5, PB-22-d9, RCS-4-d9, UR-144-d5, XLR-11-d5). A 0.5-mL volume carbonate buffer (pH 10) and 1.5 mL of extraction mixture 1 (n-hexane/ ethyl acetate, 99:1, v/v) were added. After gentle mixing for 5 min, the sample was centrifuged and 1 mL of the supernatant was transferred to an HPLC vial. Subsequently, 1.5 mL of extraction mixture 2 (n-hexane/ethyl acetate, 80:20, v/v) was added to the residue, and the mixing and centrifugation steps were repeated. A 1-mL volume of the supernatant was added to the same HPLC vial, and after evaporation under a gentle stream of nitrogen at 40 °C and reconstitution in 100 µL of mobile phase (A/B, 80:20, v/v), the sample was analyzed using LC–MS/MS.
Urine samples
In order to cleave conjugates, 0.5 mL of an urine sample was incubated at 45 °C for 1 h after addition of 0.5 mL phosphate buffer (pH 6) and 30 µL β-glucuronidase. In a next step, 1.5 mL acetonitrile and 0.5 mL ammonium formate (10 M) were added. After shaking and centrifugation, 1 mL of the acetonitrile phase was transferred into a glass vial and evaporated to dryness under a gentle nitrogen stream at 40 °C. The residue was reconstituted in 200 µL mobile phase A/B (50:50, v/v) directly before LC–MS/MS analysis. Blank urine samples were prepared serving as negative controls.