Determination of urinary metabolites of cannabidiol, Δ8-tetrahydrocannabinol, and Δ9-tetrahydrocannabinol by automated online μSPE–LC–MS/MS method.

• Automated online μSPE–LC–MS/MS developed and validated. • Developed method applied to detect cannabinoid metabolites in urine samples. • Isomeric separation achieved between Δ8-THCCOOH and Δ9-THCCOOH. • Metabolites detected in normalized concentrations in 54 urine samples. • Method useful to determine the legal boundaries for CBD, Δ8-THC, and Δ9-THC. In this study, an automated online micro-solid-phase extraction (μSPE)–liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the detection of metabolites of cannabidiol (CBD), Δ8-tetrahydrocannabinol (Δ8-THC), and Δ9-tetrahydrocannabinol (Δ9-THC), particularly 7-carboxy- cannabidiol (7-COOH-CBD), 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THCCOOH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH), and 11-nor-9-carboxy-Δ9- tetrahydrocannabinol-glucuronide (Δ9-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 × 100 mm, 1.8 µm) 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, Δ8-THCCOOH, Δ9-THCCOOH, and Δ9-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, Δ8-THCCOOH, Δ9-THCCOOH, and Δ9-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. [ABSTRACT FROM AUTHOR]