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Optimization of recombinant β-glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry.
Drug Test Anal. 2018 Mar; 10(3):518-529.DT

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.

Authors+Show Affiliations

Chemistry and Drug Metabolism Section, IRP, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, 21224, USA.Chemistry and Drug Metabolism Section, IRP, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, 21224, USA.Chemistry and Drug Metabolism Section, IRP, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, 21224, USA.Chemistry and Drug Metabolism Section, IRP, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, 21224, USA. University of Maryland School of Medicine, Baltimore, MD, 21224, USA.

Pub Type(s)

Journal Article
Validation Study

Language

eng

PubMed ID

28815938

Citation

Sempio, Cristina, et al. "Optimization of Recombinant Β-glucuronidase Hydrolysis and Quantification of Eight Urinary Cannabinoids and Metabolites By Liquid Chromatography Tandem Mass Spectrometry." Drug Testing and Analysis, vol. 10, no. 3, 2018, pp. 518-529.
Sempio C, Scheidweiler KB, Barnes AJ, et al. Optimization of recombinant β-glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry. Drug Test Anal. 2018;10(3):518-529.
Sempio, C., Scheidweiler, K. B., Barnes, A. J., & Huestis, M. A. (2018). Optimization of recombinant β-glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry. Drug Testing and Analysis, 10(3), 518-529. https://doi.org/10.1002/dta.2230
Sempio C, et al. Optimization of Recombinant Β-glucuronidase Hydrolysis and Quantification of Eight Urinary Cannabinoids and Metabolites By Liquid Chromatography Tandem Mass Spectrometry. Drug Test Anal. 2018;10(3):518-529. PubMed PMID: 28815938.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Optimization of recombinant β-glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry. AU - Sempio,Cristina, AU - Scheidweiler,Karl B, AU - Barnes,Allan J, AU - Huestis,Marilyn A, Y1 - 2017/08/16/ PY - 2017/03/22/received PY - 2017/05/21/revised PY - 2017/06/12/accepted PY - 2017/8/18/pubmed PY - 2018/10/3/medline PY - 2017/8/18/entrez KW - LC-MS/MS KW - cannabinoids KW - urine KW - β-glucuronidase SP - 518 EP - 529 JF - Drug testing and analysis JO - Drug Test Anal VL - 10 IS - 3 N2 - 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. SN - 1942-7611 UR - https://www.unboundmedicine.com/medline/citation/28815938/Optimization_of_recombinant_β_glucuronidase_hydrolysis_and_quantification_of_eight_urinary_cannabinoids_and_metabolites_by_liquid_chromatography_tandem_mass_spectrometry_ L2 - https://doi.org/10.1002/dta.2230 DB - PRIME DP - Unbound Medicine ER -