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 Δ9-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 (0.25mL oral fluid and 0.75mL 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 0.5-50ng/mL for THC, 11-OH-THC, CBD and 1-50ng/mL for CBN. The linear dynamic range for THCCOOH was 7.5-500pg/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.

Cannabis withdrawal in chronic, frequent cannabis smokers during sustained abstinence within a closed residential environment

“…D 9 -tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) in plasma and THC, CBD, CBN, and THCCOOH in OF were quantified according to previously published, validated two-dimensional gas chromatography mass spectrometry methods. 25,26 Limits of quantification (LOQ) in plasma were .125 ng/ml (THC and THCCOOH) and .25 ng/ml (11-OH-THC); OF LOQs were .5 ng/ml (THC and CBD), 1 ng/ml (CBN), and 7.5 pg/ml (THCCOOH).…”

Oral Fluid Cannabinoids in Chronic, Daily Cannabis Smokers during Sustained, Monitored Abstinence

“…OF samples were analyzed for THC, CBD, CBN, and THCCOOH by use of a previously published 2-dimensional GC-MS (2D-GC-MS) method that uses 2 analytical systems with different ionization techniques (12 ). Briefly, deuterated internal standards were added to 1-mL calibrators and QC samples [0.25 mL blank authentic OF, 0.75 mL Quantisal buffer mixture (Immunalysis Corporation), and 1 mL participant samples (0.25 mL authentic OF mixed with 0.75 mL Quantisal buffer], followed by 1 mL cold acetonitrile.…”

Techniques and Technologies for The Bioanalysis of Sativex ® , Metabolites and Related Compounds

“…A number of methods to simultaneously analyze multiple cannabinoids including both THC and CBD in oral fluid have recently been published. These methods utilize GC-MS [66][67][68], 2D-GC-MS [69] and LC-MS/MS [37,68,[70][71][72]. SPE is the most common form of sample preparation, however modified versions including solid-phase microextraction (SPME) [68] and micro-extraction by packed sorbent (MEPS) [72] have also been used, as well as LLE [37,71].…”