Analysis of synthetic cannabinoids in urine, plasma, and edibles utilizing multidimensional liquid chromatography tandem mass spectrometry
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Synthetic cannabinoids (SCs), present a multitude of problems in terms of maintaining up-to-date, reliable, specific, and sensitive methods of detection. Synthetic cannabinoids are novel psychoactive substances originally synthesized for medical use and research purposes. Abuse of these compounds, however, has demonstrated a variety of effects ranging from euphoria to aggressive behavior and loss of consciousness. The most dangerous reported result of synthetic cannabinoids use has been death. The number of synthetic cannabinoid compounds detected drastically increased from two to over 80 compounds within six years. The marketing of these compounds, similar naming, and described pharmacological interactions, create the dangerous and very false perception that SCs are similar to, or the same as, tetrahydrocannabinol in cannabis products. This research focused on the development of a method to detect and quantify seven synthetic cannabinoids in urine, plasma, and gummy bears. The seven synthetic cannabinoids studied include XLR-11, AB-PINACA 5-pentanoic acid metabolite, UR-144 5-pentanoic acid metabolite, 5F-PB-22, AM-2201 4-hydroxypentyl metabolite, JWH-018, and JWH-018 5-hydroxypentyl metabolite. Sample preparation methods and a two dimensional liquid chromatography tandem mass spectrometry method were optimized and developed for analysis of the seven SCs in each matrix. The method was successfully applied to 17 authentic urine case samples previously screened positive for synthetic cannabinoids and a calibration curve for each matrix was generated from spiked samples at varying concentrations. Utilizing two-dimensional (2D) chromatography for the analysis of synthetic cannabinoids allowed for a novel approach to be employed. With this method, 100% organic samples were analyzed with improved resolution and increased sensitivity. The sample preparation method for the urine and plasma samples included a protein precipitation technique with acid followed by solid phase extraction (SPE) on a mixed-mode reversed phase strong anion exchange sorbent. The spiked gummy bear samples were prepared in 50% methanol in water, dissolved by heating, and extracted with SPE on the same sorbent used for the urine and plasma samples. A 200µL injection of the 100% MeOH extracts was injected into 2D-LC-MS/MS for analysis with a loading and diluting solvent consisting of water and 2% ammonium hydroxide and elution solvents containing water or methanol with 0.5% formic acid. These conditions were optimized with an automated method development protocol assessing various conditions such as mobile phase solvents, pH additives, and trap column chemistries. The final chromatography method utilized an ACQUITY ultra performance liquid chromatography (UPLC) ethylene bridged hybrid (BEH) C8 2.1 x 30mm, 10µm trap column and an ACQUITY UPLC high strength silica with tri-functional C18 bonding (HSS T3) analytical column 2.1 x 150mm, 1.7µm. The urine calibration curve produced had a linear dynamic range (LDR) of 0.05-2.5ng/mL for UR-144 5-COOH and AB-PINACA 5-COOH and 0.05-5ng/mL for the other five synthetic cannabinoids. R2 values included 0.992 and 0.993 for UR-144 5-COOH and AB-PINACA 5-COOH, respectively and 0.995 or above for the other five compounds. Synthetic cannabinoids were detected at varying concentrations in all 17 urine case samples. Analysis of plasma and gummy bear samples was also successfully carried out. Plasma calibration curves had a LDR 0.05-10ng/mL with all R2 values above 0.995. Gummy bear calibration curves produced a LDR of 0.05-10ng/mL or 0.05-2.5ng/mL with R2 values over 0.995. All extraction recovery values were greater than 80% with the exception of 63% recovery for AB-PINACA 5-COOH in the gummy bear matrix. Suppression effects of 8%, 19%, and 6.6% were observed for urine, plasma, and gummy bears, respectively. Relatively low recovery values, reduced linear dynamic ranges, and suppression matrix effects for the carboxylic acid analytes assessed in this research suggested an alternative approach may be more successful for the analysis of these particular compound types in all three matrices. Overall, a sensitive, specific, and reliable method was developed with low limits of detection and quantification for efficient and rapid analysis of compounds at trace levels utilizing 2D-LC-MS/MS.