Detection and quantitation of cannabidiol and delta(9) tetrahydrocannabinol in oral fluid of a therapeutic-use cannabidiol donor using the QSight 220 CR LC-MS/MS
Gardner, Jenna Elizabeth
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Cannabidiol (CBD) is one of over 80 active cannabinoids found in Cannabis Sativa and is the second most abundant cannabinoid derived from the plant following d(9)-Tetrahydrocannabinol (THC). As opposed to THC, CBD does not appear to have any psychotropic effects, rather CBD is often utilized for its therapeutic properties, which include effects such as antinociception, anti-convulsion, and anti-inflammation. During the extraction of CBD from plant material, THC may be co-extracted. Therefore, screening and quantitating potential THC levels in individuals using CBD products is important in instances where the legality of use of THC does not match that of CBD. In recent years, oral fluid has gained recognition as a non-invasive and expedient matrix for both drug testing and forensic casework. Due to its relevance, oral fluid was selected for analysis. This project evaluated the detection and quantitation of CBD, THC, and two primary metabolites in oral fluid samples of a therapeutic-use cannabidiol donor using Biotage Supported Liquid Extraction (SLE) and subsequent testing by PerkinElmer QSight 220 CR LC-MS/MS in positive ionization mode All calibrators and quality controls were prepared by fortifying synthetic oral fluid with certified reference standards. Standards and samples were prepared in a 1:3 dilution with extraction buffer. Calibrators were prepared at 0.25, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, and 500 ng/mL, with quality controls analyzed at 0.75, 70, and 425 ng/mL. Internal standard was added to the appropriate samples to account for any variation produced by sample preparation. SLE was performed using ISOLUTE SLE+ 1 mL columns with elution in hexane:ethyl acetate:methyl tert-butyl ether (80:10:10), followed by evaporation using an Organomation Multivap Nitrogen Evaporator (Berlin, Massachusetts). All samples were reconstituted in 100 uL of 0.1% formic acid in deionized water:0.1% formic acid in acetonitrile (70:30). Validation parameters were assessed using Academy Standards Board (ASB) Standard 036-Standard Practices for Method Validation in Forensic Toxicology, including linear dynamic range (LDR), limit of detection (LOD), limit of quantitation (LOQ), analyte recovery, ion suppression/enhancement, and carryover. Following reconstitution, samples were placed onto the autosampler for injection and subsequent chromatographic separation using a PerkinElmer Brownlee C18 2.1x50 millimeter (mm), 2.7 micrometer (um) column. Analysis of the samples by mass spectrometry was performed in positive mode with multiple reaction monitoring (MRM). Total run time including equilibration was 10.5 minutes. All compounds were quantified using linear calibration models with 1/X weighting (1/concentration) and measured values were normalized by their respective internal standards. The LDR was determined to be 0.25 to 500 ng/mL. For all analytes, LOD was assessed and determined to be 0.25 ng/mL with an LOQ of 1 ng/mL. Carryover was assessed by running a double blank following a sample spiked at 500 ng/mL with no analytes observed. The donor samples were collected at several timepoints around the oral administration of an 8 mg dose of CBD. These timepoints included: prior to administration, at the time of administration, 30 minutes post-administration, 45 minutes post-administration, 60 minutes post-administration, 90 minutes post-administration, and 120 minutes post-administration. CBD was quantified within the diluted oral fluid samples from below LOQ to 325.75 ng/mL. THC was detected above LOD but below LOQ, concentrations which lie below typical cut-offs used in both workplace drug testing and forensic casework. The two metabolites were not detected above the LOD. As such, the CBD product can be concluded to be of reasonable purity as it relates to legal implications. Overall, the use of laminar flow mass spectrometry was effective in detecting various cannabinoids in oral fluid samples following SLE sample extraction.