Disclosing chronic use of cannabis has always been a difficult task due to the fact that hair testing of THC alone is not conclusive of use and hair testing of THC-COOH require specific and sensitive instrumentation and laboratory skill, not available for many analytical laboratories. We hypothesized the presence of THC-COOH glucuronide in hair as possible alternative biomarker of repeated consumption of cannabis products.

We developed and validated an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to identify and quantify THC-COOH glucuronide in hair after applying one hour digestion of 25mg keratin matrix with M3 (Comedical Spa, Italy) reagent at 100°C, using deuterated THC-COOH glucuronide as internal standard. An amount of 10μl was injected in the ULPC-MS/MS system. Chromatographic separation was carried out on a Acquity UPLC HSS C18 column (2,1mm × 150mm, 1.8μm) using a linear gradient elution with two solvents:0.1% formic acid in acetonitrile (solvent A) and 5mM ammonium formate pH 3 (solvent B). The flow rate was kept constant at 0.40mL/min during the analysis. The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). The applied ESI conditions were the following: capillary voltage 1,3kV, desolvation temperature 600°C, source temperature 150°C, cone gas flow rate 20L/h, desolvation gas flow rate 1000L/h and collision gas flow rate 0.13mL/min, cone voltage of 65V. Following transitions were considered: 521.2→345.03 (Collision energy:16), 521.2 →327.07 (Collision energy: 22),521.2 → 299.09 (Collision energy: 32), with the first one used for quantification.

Linear calibration curves were obtained for THC-CCOH glucuronide with correlation coefficients (r²) of 0.99 and a LOQ of 0.1pg/mg hair. Analytical recovery was between 70.9 and 100.7%. Intra and inter-assay imprecision and inaccuracy were always lower than 10%. No additional peaks due to endogenous substances which could have interfered with the detection of the analytes under investigation were observed in drug-free hair samples. No psychoactive drugs other than the compounds under investigation interfered with the assay. Blank hair samples injected after the highest point of the calibration curve did not present any traces of carryover. The matrix effect in quality control samples ranged from 80 to 101%. Preliminary analysis on 9 different hair samples of consumers disclosed the presence of THC-CCOH glucuronide in the range of 0.30–1.22pg/mg with by median value of 0.58pg/mg hair.

Simple extraction, identification and quantification of THC-COOH glucuronide in hair by UPLC-MS/MS was developed, validated and tested for its feasibility in clinical samples and provided a good start to consider THC-CCOH glucuronide as alternative hair biomarker of repeated cannabis consumption.