title: Simultaneous quantification of cannabinoids and metabolites in oral fluid by two-dimensional gas chromatography mass spectrometry.
abstract: Development and validation of a method for simultaneous identification and quantification of Delta9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), and metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in oral fluid. Simultaneous analysis was problematic due to different physicochemical characteristics and concentration ranges. Neutral analytes, such as THC and CBD, are present in ng/mL, rather than pg/mL concentrations, as observed for the acidic THCCOOH biomarker in oral fluid. THCCOOH is not present in cannabis smoke, definitively differentiating cannabis use from passive smoke exposure. THC, 11-OH-THC, THCCOOH, CBD, and CBN quantification was achieved in a single oral fluid specimen collected with the Quantisal device. One mL oral fluid/buffer solution (0.25 mL oral fluid and 0.75 mL buffer) was applied to conditioned CEREX Polycrom THC solid-phase extraction (SPE) columns. After washing, THC, 11-OH-THC, CBD, and CBN were eluted with hexane/acetone/ethyl acetate (60:30:20, v/v/v), derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide and quantified by two-dimensional gas chromatography electron ionization mass spectrometry (2D-GCMS) with cold trapping. Acidic THCCOOH was separately eluted with hexane/ethyl acetate/acetic acid (75:25:2.5, v/v/v), derivatized with trifluoroacetic anhydride and hexafluoroisopropanol, and quantified by the more sensitive 2D-GCMS-electron capture negative chemical ionization (NCI-MS). Linearity was 0.5-50 ng/mL for THC, 11-OH-THC, CBD and 1-50 ng/mL for CBN. The linear dynamic range for THCCOOH was 7.5-500 pg/mL. Intra- and inter-assay imprecision as percent RSD at three concentrations across the linear dynamic range were 0.3-6.6%. Analytical recovery was within 13.8% of target. This new SPE 2D-GCMS assay achieved efficient quantification of five cannabinoids in oral fluid, including pg/mL concentrations of THCCOOH by combining differential elution, 2D-GCMS with electron ionization and negative chemical ionization. This method will be applied to quantification of cannabinoids in oral fluid specimens from individuals participating in controlled cannabis and Sativex (50% THC and 50% CBD) administration studies, and during cannabis withdrawal.
authors.affiliation: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Blvd., Baltimore, MD 21224, USA.
authors.forename: Garry
authors.initials: G
authors.lastname: Milman
authors.forename: Allan J
authors.initials: AJ
authors.lastname: Barnes
authors.forename: Ross H
authors.initials: RH
authors.lastname: Lowe
authors.forename: Marilyn A
authors.initials: MA
authors.lastname: Huestis
mesh_terms: D002138:Calibration; D002186:Cannabinoids / Q000032:analysis*; D013759:Dronabinol / Q000031:analogs & derivatives / Q000032:analysis; D005410:Flame Ionization; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D012015:Reference Standards; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry*
chemical_list: D002186:Cannabinoids; C016780:11-nor-delta(9)-tetrahydrocannabinol-9-carboxylic acid; D013759:Dronabinol; C025351:delta(9)-tetrahydrocannabinolic acid