Measurement Uncertainty and Validation for Quantification of Marijuana Metabolite: (−)-11-nor-9-Carboxy-Δ9-THC in Human Urine by GC-MS/MS

Abstract

Background: Since the International Olympic Committee (IOC) was established, sports doping control analyses have revealed a high rate of positive cases for cannabinoids. Cannabinoids were banned in all sports where they were used in competition as per the Prohibited List published annually. Further, it was also included in the threshold drug category. Consequently, developing a reliable method for urine Cannabinoids metabolite quantification plays a pivotal role in sports dope testing. Objective: This work aimed to develop and validate a reliable, cost-effective, robust gas chromatography-tandem mass spectrometry method for detecting (−)-11-nor-9-Carboxy-Δ9- THC component in human urine samples, in compliance with ICH and WADA guidelines. Method: The sample preparation was done by enzymatic hydrolysis for deconjugation, further proceeded with solid phase extraction (SPE), liquid-liquid extraction (LLE), and using an XAD2 column, and N-methyl trimethylsilyl trifluoroacetamide (MSTFA) for derivatization. Results: The linearity was obtained in a range of 50–300 ng/mL, and the correlation coefficient was found to be higher than 0.99. Throughout the entire validation study, the difference in Retention Time (RT) for the analyte, including the Internal Standard (IS), was shown to be less than 1.0%. The quantification limit (LOQ) was calculated at a level of 50 ng/mL in human urine samples for the 3 most abundant ion transitions. The detection limit (LOD) was established at 4 ng/mL. Conclusion: The accuracy, precision, linearity, recovery, quantification limit, and selectivity by GC-MS/MS technique were found acceptable and well satisfactory while following the ICH guidelines. The developed method has been proven to be fit for purpose in accordance with the enforced Guidelines of WADA.