Optimisation of a gas chromatography-mass spectrometry method for the simultaneous determination of tetrahydrocannabinol and its metabolites in rat urine.
In order to evaluate the effect of irinotecan (IRI) on urinary elimination of delta-9-tetrahydrocannabinol (THC) in a rat experimental model, we developed an analytical method for the determination of the mass concentration of THC and its metabolites 11-hydroxy-delta-9-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in the urine of rats treated only with THC and treated simultaneously with THC and irinotecan. For this purpose, hydrolysis and solid phase extraction conditions of the investigated analytes were optimised and a gas chromatography-mass spectrometry (GC-MS) method was developed to determine all three analytes in rat urine. The most effective hydrolysis method for THC, THC-OH, and THC-COOH conjugates was so-called tandem hydrolysis by the β-glucuronidase enzyme from Escherichia coli at 50 °C for 2 hours and followed by alkaline hydrolysis. The proposed method was then applied for determining concentrations of analytes in 24-hour rat urine. THC was not detected in either sample, THC-OH was detected in 50 % of samples, and THC-COOH in all of the samples. Enhanced urinary THC-COOH excretion was noted in rats administered combined treatment compared to single THC treatment. The method described herein was suitable for determining the mass concentration of THC metabolites in the rat urine due to its sensitivity (detection limits 0.8-1.0 μgL), accuracy (>96 %), and precision (RSD <6 %).