Simultaneous quantification of major cannabinoids and metabolites in human urine and plasma by HPLC‐MS/MS and enzyme‐alkaline hydrolysis

A high performance liquid chromatography coupled to tandem mass spectrometry (HPLC‐MS/MS) method for simultaneous quantification of Δ9‐tetrahydrocannabinol (THC), its two metabolites 11‐hydroxy‐Δ9‐tetrahydrocannabinol (11‐OH‐THC) and 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinol (THC‐COOH), and four additional cannabinoids (cannabidiol (CBD), cannabigerol (CBG), tetrahydrocannabivarin (THCV), and cannabinol (CBN)) in 1 mL of human urine and plasma was developed and validated. The hydrolysis process was studied to ensure complete hydrolysis of glucuronide conjugates and the extraction of a total amount of analytes. Initially, urine and plasma blank samples were spiked with THC‐COOH‐glucuronide and THC‐glucuronide, and four different pretreatment methods were compared: hydrolysis‐free method, enzymatic hydrolysis with Escherichia Coli β‐glucuronidase, alkaline hydrolysis with 10 M NaOH, and enzyme‐alkaline tandem hydrolysis. The last approach assured the maximum efficiencies (close to 100%) for both urine and plasma matrices. Regarding the figures of merit, the limits of detection were below 1 ng/mL for all analytes, the accuracy ranged from 84% to 115%, and both within‐day and between‐day precision were lower than 12%. Finally, the method was successfully applied to real urine and plasma samples from cannabis users. Copyright © 2016 John Wiley & Sons, Ltd.