Δ9-tetrahydrocannabinol (THC) is the primary target in oral fluid (OF) for detecting cannabis intake. However, additional biomarkers are needed to solve interpretation issues, such as the possibility of passive inhalation by identifying 11-nor-9-carboxy-THC (THCCOOH), and determining recent cannabis smoking by identifying cannabidiol (CBD) and/or cannabinol (CBN). We developed and comprehensively validated a microflow liquid chromatography (LC)–high resolution mass spectrometry method for simultaneous quantification of THC, THCCOOH, CBD and CBN in OF collected with the Oral-Eze® and Quantisal™ devices. One mL OF-buffer solution (0.25mL OF and 0.5mL of Oral-Eze buffer,1:3 dilution, or 0.75mL Quantisal buffer, 1:4 dilution) had proteins precipitated, and the supernatant subjected to CEREX™ Polycrom™ THC solid-phase extraction (SPE). Microflow LC reverse-phase separation was achieved with a gradient mobile phase of 10mM ammonium acetate pH 6 and acetonitrile over 10 min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by targeted-MSMS experiments. The assay was linear 0.5–50 ng/mL for THC, CBD and CBN, and 15–500 pg/mL for THCCOOH. Intra- and inter-day and total imprecision were <10.8%CV and bias 86.5–104.9%. Extraction efficiency was 52.4–109.2%, process efficiency 12.2– 88.9% and matrix effect ranged from −86 to −6.9%. All analytes were stable for 24h at 5°C on the autosampler. The method was applied to authentic OF specimens collected with Quantisal and Oral-Eze devices. This method provides a rapid simultaneous quantification of THCCOOH and THC, CBD, CBN, with good selectivity and sensitivity, providing the opportunity to improve interpretation of cannabinoid OF results by eliminating the possibility of passive inhalation and providing markers of recent cannabis smoking.
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