4.7. LC-MS Analysis
SEPARATION, LENS
Measurements were performed on the UHPLC-MS/MS (Ultimate 3000RS equipped with TSQ Quantis MS/MS detector, Thermo Fischer Scientific, Waltham, MA, USA). Heated electrospray ion source (H-ESI) was set to a positive ion mode with spray voltage of 4000 V, while Sheat, Aux, and Sweep gasses were set to 50, 25, and 2 arbitrary units (Arb), respectively. Ion transfer tube was set to 325 °C and Vaporizer temperature was set to 280 °C. Collision-induced dissociation (CID) gas pressure was constant at 1.5 mTorr. Sample injection volume was 1 μL. Separation was performed on Accucore C18 column (150 mm × 2.1 mm, 2.6 µm, Thermo Fischer Scientific, Waltham, MA, USA) equipped with Accucore C18 guard column (10 mm × 2.1 mm, 2.6 µm) using gradient elution with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Solvent gradient was programmed as follows: 0 min 60% B, 8 min 68% B, 8.1 min 95% B, 9.1 min 95% B, 9.3 min 60% B, and 11 min 60% B. Total run time was 11 min. Column flow was 0.5 mL/min. Column temperature was held at 40 °C, while autosampler temperature was held at 15 °C. Needle was washed in solvent mixture comprised of acetonitrile/methanol/water/formic acid = 40:40:20:2 (Quantitative analysis of CBD, 7-OH-CBD, and 7-COOH-CBD was performed using external calibration curves for each analyte (ranging from 0 ng/mL to 2000 ng/mL). Each compound was introduced to the MS/MS system separately to optimize fragment transitions, collision energies, and ion focusing lens voltages, which were all performed using Chromeleon MS tuning console v. 7.2.10 (23925) and are shown in the following Limit of detection (LOD) and limit of quantification (LOQ) were estimated using Blank Samples approach. Ten (10) blank samples were analyzed and LOD was calculated as 3.9*(s