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.

Automated summary

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) in urine. The developed method showed good sensitivity and accuracy for the detection of the target analytes. The method was successfully applied to the analysis of urine samples, and the target metabolites were detected at various concentrations.