Anthony P. DeCaprio* and Meena Swaminathan | Florida International University Abstract: The continuous increase in cannabis use for its perceived medical benefits and as a recreational drug in the United States has created a great demand in forensic toxicology for biomarkers of Cannabis exposure that can reliably indicate medicinal versus recreational Cannabis use, recent versus past exposure use, and occasional versus chronic use. In addition, markers that can be sampled non-invasively and with relative ease are needed. Current markers of Cannabis intake include ∆9-tetrahydrocannabinol (∆9-THC) and its metabolites 11-Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). However, these exhibit complex pharmacokinetics, making it difficult to interpret concentrations to determine the time and frequency of exposure. More importantly, blood or urine levels of THC and metabolites correlate poorly with degree of impairment in driving under the influence scenarios. Cannabis contains ~120 unique chemical entities considered to be “cannabinoids,” some of which are psychoactive and some that can modify the overall activity of the combined product. These include major and minor components, few of which have been assessed as potential exposure biomarkers in conventional or alternative sample matrices. The possibility that profiles or patterns of multiple cannabinoids may provide more reliable correlations with Cannabis exposure or impairment than individual molecules has not been extensively explored. Oral fluid (OF) and exhaled breath condensate (EBC) are alternative, non-invasive sample matrices that hold promise for identification of cannabis exposure biomarkers. OF is currently being explored as a matrix for Cannabis exposure analysis. EBC is an easily collected specimen derived from lung lining fluid that consists of condensed water vapor, dissolved volatiles, and water-soluble polar and non-volatile small molecules, including metabolites. Although licit drugs and metabolites have been measured in EBC, this matrix has not been explored as an alternative sample in forensic toxicology studies, despite its obvious advantages. This presentation describes the development of an analytical method to determine a battery of major and minor cannabinoids and metabolites as potential Cannabis exposure biomarkers in EBC and OF. Liquid chromatographic-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) can detect and quantify 25 cannabinoid analytes, including the difficult to separate analytes Δ8-THC, Δ9-THC, cannabichromene, and cannabinol, which have similar multiple reaction monitoring transitions. Limit of quantitation values for the great majority of target cannabinoids in OF and EBC are in the 1–10 ng/mL range. In addition, the recruitment of a cohort of 240 adult Cannabis users with well-characterized Cannabis use profiles is described. Participants provided samples of EBC, OF, and urine and completed an exposure questionnaire detailing characteristics of exposure and type of Cannabis used. OF was collected using Quantisal™ (Immunalysis™) devices. EBC was obtained using RTube™ devices (Respiratory Research, Inc), which yield ~1 mL of sample with 5–10 minutes of normal breathing. Urine was also collected for initial confirmation of recent or chronic use using dipstick analysis. Initial cannabinoid data analysis will use descriptive statistics, one-way analysis of variance, and correlation analysis to isolate possible trends and correlations. Then, data will be further investigated with multivariate analyses, including principal component analysis, to explore the relationships between individual or groupings of analytes and exposure parameters, including type of cannabis used and the timing and frequency of use.