Despite efforts to control synthetic cannabinoids, clandestine manufacturers continue to modify their structures to avoid legal consequences, creating an ever-changing analytical target for forensic laboratories (1). Forensic toxicology laboratories often lack the needed resources or do not have the capabilities to test for these compounds and metabolites, requiring specimens to be submitted to reference laboratories (2). Drug stability can be affected by long storage times, temperature and preservatives (3). Although these factors can be controlled, systematic research is necessary to identify their impacts on the stability of these new synthetic cannabinoids that are continually emerging.
The purpose of this research is to assess the stability of 17 synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in human urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) over thirty-five weeks. The analysis methods were validated in accordance to the Academy Standards Board (ASB) method validation guidelines for quantitative analysis and stability evaluation of the following analytes in blood: 4-cyano CUMYL-BUTINACA, ADB-PINACA, EMB-FUBINACA, JWH-250, MO-CHMINACA, 5-fluoro-3,5-ABPFUPPYCA, 5-fluoro ADB-PINACA, APP-PICA, CUMYL-THPINACA, PB-22, XLR11, 5-fluoro PY-PINACA, MDMB-FUBICA, MEP-CHMICA, NM2201, RCS-8, and UR144. The stability analysis in urine includes the following metabolites: 5-fluoro MDMB-PICA metabolite 7, 5-fluoro PB-22 3-carboxyindole, AB-FUBINACA metabolite 3, ADB-PINACA N-(4-hydroxypentyl), ADB-PINACA pentanoic acid, UR-144 Degradant N-pentanoic acid, PB-22 N-(5-hydroxypentyl), MDMB-FUBICA metabolite 3, UR-144 N-(5-hydroxypentyl), and JWH-250 N-pentanoic acid.
Research samples were prepared by spiking with certified reference standards (Cayman Chemical, Ann Arbor, MI, USA) of each select synthetic cannabinoid in certified drug-free human whole blood (Boston Medical Center, Boston, MA, USA; Biological Specialty Corporation, Colmar, PA) and drug-free urine that was received as donations following the approved Institutional Review Board guidelines (Boston University School of Medicine, Boston, MA, USA). Blood samples were aliquoted into 6 mL BD Vacutainer Plastic Collection Tubes (Fisher Scientific, Waltham, MA, USA) and urine samples were stored in 15 mL Falcon Conical Centrifuge Tubes (Fisher Scientific, Waltham, MA, USA). Stability under room temperature (20ºC), refrigerator (4ºC), and freezer (-20ºC) at low and high concentrations were evaluated at select time points. A 5% solution of potassium oxalate and sodium fluoride or ethylenediaminetetraacetic acid (EDTA) was added to the preserved blood samples by the manufacturer prior to storage. The anticoagulant, potassium oxalate, was only added in solution to the preserved samples whereas none was added to the nonpreserved samples. Short-term urine samples were preserved with 1% of sodium fluoride prior to storage. Extraction of analytes was conducted using supported-liquid extraction (SLE) ISOLUTE 1 mL cartridges (Biotage, Charlotte, NC, USA) and reconstituted in 100 μL of 50:50 mixture of 0.1% formic acid in millipore deionized water and 0.1% formic acid in acetonitrile (Fisher Scientific, Waltham, MA, USA).
Analysis was performed in triplicate using a reverse-phase C18 column (Waters XBridge C18 3.5 μM, 2.1 x 50 mm, Milford, MA, USA) on the Shimadzu Prominence Ultra-Fast Liquid Chromatography (UFLC, Kyoto, Japan) with SCIEX 4000 Q-Trap Electrospray Ionization Tandem Mass Spectrometry (ESI/MS/MS, Waltham, MA, USA) in positive ionization mode. The total run time was 8 minutes with a flow rate of 0.6 mL/min and injection volume of 10 μL. Linear calibration curves for each analyte with the exception of a quadratic regression for PB-22, all had acceptable R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 0.5 – 25 ng/mL was used for all analytes in blood except for NM2201 and APP-PICA with a limit of quantitation (LOQ) of 0.1 ng/mL and MO-CHMINACA with a working range of 0.5 – 15 ng/mL. A linear working range of 5 – 40 ng/mL was utilized for all metabolites in urine. No signs of carryover were observed. In general, analytes were considered stable if the average area ratio between the analyte and internal standard at the time point was within ± 20% of the average area ratio response at time point zero. In some cases, it was necessary to evaluate the complete picture of the stability data by reviewing analyte area, concentration, and overall stability data trend between timepoints at the low and high concentrations. In certain situations, an analyte was considered stable even if specific timepoints for a concentration were outside the ±20% range. For example, in cases where one concentration at a timepoint was within the ±20% range and the other concentration fell within ±30% range the analyte was considered stable overall.
Long-term stability results revealed that all synthetic cannabinoids were stable at 21 to 35 weeks in frozen blood preserved with sodium fluoride except for APP-PICA. The preservatives are recommended to be added to blood to reduce the possibility of matrix inferences and minimize detrimental impacts on the stability of synthetic cannabinoids. Analytes experienced lower degradation in the order of samples that were kept frozen, refrigerated, and then at room temperature. Blood analytes that were stable up to 35 weeks in freezer generally had a core structure of a carbonyl substituent on a pyrazole or pyrrole with surrounding nonpolar groups; whereas compounds with two polar carbonyl functional groups present were found to experience degradation much earlier at 1 week or less in room temperature and refrigerator storage conditions. 5-fluoropentyl analogs, like XLR11 and 5-fluoro ADB-PINACA, in comparison to their counterpart analyte, UR144 and ADB-PINACA, were unstable at earlier time points under all storage conditions. Instability in the majority of the urine metabolites was not observed until after 9 weeks and was generally consistent across all storage conditions.
The validated methods demonstrate a sensitive and reliable way to positively identify 17 different synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in urine for rapid time stability analysis at various storage conditions. The use of SLE improved sample preparation efficiency by decreasing the extraction time from 1 hour to 30 minutes compared to traditional extraction methods, such as solid-phase extraction (SPE) and liquid-liquid extraction (LLE). Further studies into additional matrices, such as oral fluid, longer storage times, and other emerging synthetic cannabinoid analytes would expand the scope of this research.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/38697 |
| Date | 11 October 2019 |
| Creators | Phung, Erika Dang |
| Contributors | Botch-Jones, Sabra |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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