Cbn d3

We purchased high-purity methanol (≥99.9%), 2-propanol (≥99.9%), formic acid (≥99.5%), and ammonium formate (≥99%) from Fisher Scientific (Fair Lawn, NJ). We purchased liquid chromatography–mass spectrometry (LC–MS) grade water (J.T. Baker, ≥99.9%) and acetonitrile (Burdick & Jackson, ≥99.9%) from VWR (Radnor, PA). We purchased ammonium sulfate, sodium hydroxide (NaOH), ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA-Na₂), and 0.5 M dibutylammonium acetate from Sigma-Aldrich Laboratories, Inc. (St. Louis, MO). We purchased native and isotopically labeled standards, including THC, THC-d₃, CBD, CBD-d₃, CBN, and CBN-d₃ from Cerilliant (Round Rock, TX). We bought the SPE (C18, 100 mg) column and 96-well plate from Phenomenex (Torrance, CA). All chemicals were used without further purification.

Optima-grade ammonium formate, formic acid, and methanol were purchased from Fisher Scientific (Hanover Park, IL, USA). Ultrapure Type 1 water was generated in-house as needed from a Millipore Direct-Q3 system. Certified reference materials for a quality assurance test mix containing amitriptyline, diazepam, fluoxetine, methadone, nicotine, nordiazepam, norfluoxetine, nortriptyline, paroxetine, and trazodone were acquired from Cerilliant (Round Rock, TX, USA), as were CBD, CBD-d3, Δ9-THC, Δ9-THC-d3, cannabigerol (CBG), cannabidivarin (CBDV), cannabichromene (CBC), cannabinol (CBN), and CBN-d3. Δ9-Tetrahydrocannabinolic acid Δ9-THCA-A), Δ8-THC, and cannabidiolic acid (CBDA) were purchased from Cayman Chemical (Ann Arbor, MI, USA).

THC, 11-OH-THC, THCCOOH, CBD, CBN, THC-d₃, 11-OH-THC-d₃, THCCOOH-d₉, CBD-d₃, CBN-d₃, and THCCOOH-glucuronide-d₃ were purchased from Cerilliant (Round Rock, TX, USA). CBG was from Restek (Bellefonte, PA, USA), THCV was from RTI International (Research Triangle Park, NC, USA), and THCVCOOH and THC-glucuronide were acquired from ElSohly Laboratories (Oxford, MS, USA). Ammonium acetate and acetonitrile (LC-MS grade) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Methanol and water (LC-MS grade) and formic acid (ACS-grade) were from Fisher Scientific (Fair Lawn, NY, USA). WAX-S tips (1 mL tip containing 20 mg resin and 40 mg salt) were purchased from DPX Labs (Columbia, SC, USA). Chromatography was performed on a Kinetex® C₁₈ column (Phenomenex® Inc., Torrance CA, USA; 2.1 mm × 50 mm, 2.6 μm) combined with a SecurityGuard™ C₁₈ guard column (4 × 2.0 mm).

Reference standards (Δ9-THC, 11-OH-THC, CBD, and cannabinol [CBN]) and isotopically labeled internal standards (THC-d3, 11-OH-THC-d3, CBD-d3, CBN-d3) were purchased from Cerilliant (Round Rock, TX). BondElut® certify (Varian, Lake Forest, CA) C-18 cartridges were used for solid-phase extraction. HPLC grade water, 0.1% formic acid in acetonitrile (ACN), and methanol (MeOH) were purchased from Fisher Scientific. Reagent grade ammonium formate and pure sodium hydroxide pellets were purchased from Sigma-Aldrich.
We followed the Wei et al saponification-solid-phase extraction method for sample clean up and extraction with minor modifications.^{15 (link)}Chromatographic separation was achieved on reverse-phase C-18 column (MAC-MOD Ace 5 C-18, 15cm · 2.1mm). The mobile phase consisted of 5 mM ammonium formate in 0.05% formic acid (Solvent A) and ACN in 0.1% formic acid (Solvent B). A gradient elution was performed over 15 minutes at a flow rate of 200 μL/min. Retention times for 11-OH-THC, CBD, THC were 9.35, 10.62, and 11.70 minutes in positive ion mode, respectively. Quantitation of THC, 11-OH-THC, and CBD and isotopically labeled internal standards was achieved on a triple quadrupole mass spectrometer (API-4000, AB Sciex) in positive ion mode. CBN and CBN-d3 were detected in negative ion mode.