Optimisation and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for cannabis and...

The two most abundant phytocannabinoids are Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD). Their major metabolites include 7-hydroxy-cannabidiol (CBD-OH), 7-carboxy-cannabidiol (CBD-COOH), 11-hydroxy-Δ⁹-tetrahydrocannabinol (THC-OH) and 11-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH). As THC and CBD metabolites are metabolised to their glucuronide conjugates, it is essential to account for these via enzyme de-conjunction during analysis of urine. The new generation of recombinant β-glucuronide enzymes have the potential to improve processing speed and hydrolysis efficiency for quantification of phytocannabinoids and their metabolites. This study aimed to optimise the de-glucuronidation and preparation of urine samples for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of phytocannabinoids and their metabolites, and use the method to quantify cannabinoids in authentic urine samples. Two β-glucuronidase enzymes (B-One and BGTurbo) were compared for their ability to hydrolyse the glucuronide forms of CBD-OH, CBD-COOH, THC-OH and THC-COOH. Enzyme volume was varied to optimise hydrolysis efficiency. Subsequent sample treatment using protein precipitation, filtration or a combination of both were compared. Hydrolysis efficiency was calculated, and validation parameters were measured. Treating a volume of 200 μL of urine with 200 μL of B-One enzyme, followed by protein precipitation using acetonitrile and filtration with a regenerated cellulose syringe filter, was optimal in terms of 100% hydrolysis efficiency and 93% cannabinoid recovery. The lower limit of quantification (LLOQ) was 0.2 ng/mL for CBD and THC, 1 ng/mL for CBD-OH and THC-OH, and 0.5 ng/mL for CBD-COOH and THC-COOH. Calibration curves extending to 100 ng/mL were linear with correlation coefficients exceeding 0.998; as the method involved a 6-fold dilution, this method can be applied to measurement of up to 600 ng/mL without additional dilution. Three authentic urine samples ranged in concentration of THC and CBD metabolites, from non-detectable up to a maximum of 385 ng/mL for THC-COOH. Single-step hydrolysis using B-One enzyme and combined precipitation with filtration for clean sample preparation allows for rapid cleavage of cannabinoid glucuronides with high recovery.