B-318 Performance of a LC-MS/MS assay for the multiplexed detection of cannabinoid metabolites

Abstract Background Cannabis and its derived products are increasing in prevalence across the US, driven by the legalization of hemp under the 2018 Farm bill. This legalization facilitated easier access to cannabinoids, such as cannabidiol (CBD). Furthermore, there is great interest in utilizing cannabinoids, including CBD, as a therapeutic agent for a wide variety of diseases. Precise and accurate quantification of cannabinoid and cannabinoid metabolites are critical for characterizing pharmacokinetics and establishing therapeutic efficacy. The aim of this study was to develop and validate an LC-MS/MS assay for the detection of 9 exogenous cannabinoids and the endogenous cannabinoid AEA for ongoing clinical trials investigating CBD as a therapeutic. Methods A quantitative multiplexed LC-MS/MS assay was developed to identify CBN, CBD, THC, 11-OH-THC, THC-COOH, CBG, THC-V, 7-OH-CBD, 7-COOH-CBD, and AEA. Isotope dilution was performed using deuterated Internal standards, followed by protein precipitation in 0.1% formic acid in acetonitrile. After centrifugation, the supernatant was transferred to a Waters Oasis HLB 30 mg solid phase extraction plate and washed using 25% methanol in water. Samples were eluted using 90% acetonitrile, 10% methanol and evaporated under a stream of nitrogen. All analysis was performed using a Waters ACQUITY I-class UPLC System coupled to a Waters Xevo TQS-Micro mass spectrometer. Chromatographic separation was facilitated with a 2.1 x 50 mm ACQUITY 1.7 µm UPLC BEH C18 column with an ACQUITY UPLC BEH C18 VanGuard Pre-column, 1.7 µm, 2.1 mm x 5 mm maintained at 40°C. The linear gradient begins at 50% mobile phase B and ends at 90% of mobile phase B across 4 minutes at a flow rate of 0.4 mL/min (Mobile phase A: 5 mM ammonium formate and 0.1% formic acid in water, Mobile phase B: 0.1% formic acid in acetonitrile). Calibrators and quality controls were prepared in Mass Spec Gold plasma (Golden West BioSolutions), and method validation was conducted in accordance to CLSI C62-A. Assay reproducibility and robustness was characterized using clinical specimens (n=100) that originated from a clinical study evaluating the therapeutic effects of CBD. Results The analytical measurement range (AMR) extended from 0.5 ng/mL to 3000 ng/mL for exogenous cannabinoids and from 0.05 – 50 ng/mL for AEA. External calibration curves for each cannabinoid were linear (R2>0.995). Within run imprecision across the AMR was <15%. Blinded clinical samples with 3 or greater repetitions (n=88) had an AEA mean CV of 6.3% (Range: 0.43-13.3%), 7-COOH-CBD mean CV of 6.8% (Range: 0.02-6.3%), 7-OH-CBD mean CV of 5.4% (Range: 0.42-14.5%), and CBD mean CV of 4.9% (Range: 0.3-12%) Conclusion The validation of a multiplexed LC-MS/MS for exogenous and the endogenous cannabinoid AEA in plasma provides a reliable assay for ongoing clinical trials investigating the therapeutic potential of CBD and other cannabinoids.