Determination of urinary metabolites of cannabidiol, ?8-tetrahydrocannabinol, and ?9-tetrahydrocannabinol by automated online ?SPE-LC-MS/MS method

In this study, an automated online micro-solid-phase extraction (mu SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection of metabolites of cannabidiol (CBD), A8-tetrahydrocannabinol (A8-THC), and A9-tetrahydrocannabinol (A9-THC), particularly 7-carboxy-cannabidiol (7-COOH-CBD), 11-nor-9-carboxy-A8-tetrahydrocannabinol (A8-THCCOOH), 11-nor-9-carboxy-A9- tetrahydrocannabinol (A9-THCCOOH), and 11-nor-9-carboxy-A9- tetrahydrocannabinol-glucuronide (A9- THCCOOH-glu) in urine. An instrument top sample preparation (ITSP) cartridge was introduced to increase the sensitivity toward analytes and decrease the matrix effect of the urine. LC-MS/MS analysis was performed in the multiple-reaction monitoring mode, and the analytes were separated using an Acquity UPLC HSS T3 (2.1 x 100 mm, 1.8 mu m) column and gradient elution with water containing 0.05 % acetic acid and methanol as the mobile phase. The calibration range was 0.5-200 ng/mL for all the analytes, with a correlation coefficient (r) of >= 0.996 and a weighting factor of 1/x2. The limits of detection for 7-COOH-CBD, A8-THCCOOH, A9-THCCOOH, and A9- THCCOOH-glu were 0.06, 0.02, 0.03, and 0.1 ng/mL, respectively. The intra-and inter-day accuracy ranged from-8.0 to 6.2 % and-7.3 to 7.8 % with a precision of <= 7.2 % and <= 6.2 %, respectively. The method was also validated for selectivity, recovery, matrix effect, stability, and dilution integrity. The developed method was successfully applied to the analysis of 78 urine samples, and 7-COOH-CBD, A8-THCCOOH, A9-THCCOOH, and A9-THCCOOH-glu were detected in 54 urine samples at normalized concentrations of 1.1, 0.6-939.1, 0.9-2595.0, and 1.3-527.6 ng/mg creatinine, respectively.