Optimization of recombinant β‐glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry

Abstract

Prolonged urinary cannabinoid excretion in chronic frequent cannabis users confounds identification of recent cannabis intake that may be important in treatment, workplace, clinical, and forensic testing programs. In addition, differentiation of synthetic Δ9-tetrahydrocannabinol (THC) intake from cannabis plant products might be an important interpretive issue. THC, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) urine concentrations were evaluated during previous controlled cannabis administration studies following tandem alkaline/E. coli β-glucuronidase hydrolysis. We optimized recombinant β-glucuronidase enzymatic urinary hydrolysis before simultaneous liquid chromatography tandem mass spectrometry (LC-MS/MS) quantification of THC, 11-OH-THC, THCCOOH, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocannabivarin (THCV) and 11-nor-9-carboxy-THCV (THCVCOOH) in urine. Enzyme amount, incubation time and temperature, buffer molarity and pH were optimized using pooled urine samples collected during a National Institute on Drug Abuse, Institutional Review Board-approved clinical study. Optimized cannabinoid hydrolysis with recombinant β-glucuronidase was achieved with 2000 IU enzyme, 2 M pH 6.8 sodium phosphate buffer, and 0.2 mL urine at 37°C for 16 h. The LC-MS/MS quantification method for hydrolyzed urinary cannabinoids was validated per the Scientific Working Group on Toxicology guidelines. Linear ranges were 1-250 μg/L for THC and CBG, 2-250 μg/L for 11-OH-THC, CBD, CBN, THCV and THCVCOOH, and 1-500 μg/L for THCCOOH. Inter-batch analytical bias was 92.4-112.4%, imprecision 4.4-9.3% CV (n = 25), extraction efficiency 44.3-97.1% and matrix effect -29.6 to 1.8% (n = 10). The method was utilized to analyze urine specimens collected during our controlled smoked, vaporized, and edible cannabis administration study to improve interpretation of urine cannabinoid test results.

Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use

“…Additional cannabinoids, for example, CBD, CBN, CBG, THCV, and THCVA were suggested for inclusion into such evaluations in order to improve clinical and forensic result interpretations in blood, plasma, and oral fluid. 18,19,26 The detection of these analytes and other minor cannabinoids as well as their urinary metabolites in in-competition doping control urine samples would constitute an AAF. The herein presented pilot study focused on seven intact minor cannabinoids (i.e., CBG, CBN, CBC, CBDV, CBGA, THCA, and CBDA) without corresponding predicted or established metabolic products.…”

“…Cannabinoids are excreted unconjugated and conjugated (primarily as glucuronidated phase-II metabolites). 18,20,23,24 In order to detect both, free and conjugated urinary cannabinoid metabolites, as well as to increase the analytical sensitivity, a hydrolysis step during the sample preparation is frequently recommended and employed.…”

“…Multiple publications are available for the analysis of cannabis intake, 17–19 with analytical methods being largely based on gas chromatography–mass spectrometry (GC–MS) 20,21 and/or liquid chromatography‐mass spectrometry (LC–MS) 18,19,22 . In order to clarify whether the permissible use of different CBD products can lead to unintentional violations of the antidoping regulations, similar analytical strategies were pursued in this study to assess the risks associated with CBD consumption by athletes during routine doping controls.…”

“…13 However, CBD products are frequently sold as nutritional supplements, novel foods, or cosmetics and can be purchased in pharmacies, organic shops, drugstores, supermarkets, and via the Internet, and a growing receptivity amongst elite athletes is conceivable. [14][15][16] Multiple publications are available for the analysis of cannabis intake, [17][18][19] with analytical methods being largely based on gas chromatography-mass spectrometry (GC-MS) 20,21 and/or liquid chromatography-mass spectrometry (LC-MS). 18,19,22 In order to clarify whether the permissible use of different CBD products can lead to unintentional violations of the antidoping regulations, similar analytical strategies were pursued in this study to assess the risks associated with CBD consumption by athletes during routine doping controls.…”

“…[14][15][16] Multiple publications are available for the analysis of cannabis intake, [17][18][19] with analytical methods being largely based on gas chromatography-mass spectrometry (GC-MS) 20,21 and/or liquid chromatography-mass spectrometry (LC-MS). 18,19,22 In order to clarify whether the permissible use of different CBD products can lead to unintentional violations of the antidoping regulations, similar analytical strategies were pursued in this study to assess the risks associated with CBD consumption by athletes during routine doping controls. Therefore, a GC-MS-based detection method for various cannabinoids in human urine was developed and validated, and 15different commercially available CBD products (oils, pastes, capsules, crystals) were purchased (Table 1).…”