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Abstract INTRODUCTION: The testing of whole blood samples for tetrahydrocannabinol (Δ-9-THC) consumption is routine and has been around for many decades. Δ-9-THC is metabolized into 11-Hydroxy-Δ9-tetrahydrocannabinol (11-OH-Δ-9-THC) and further into 11-nor-9-carboxy-Δ-9-THC (Δ-9-THC-COOH). It is important to test for the parent and both metabolites to properly monitor for THC usage. As more isomers of Δ-9-THC become available on the market, testing becomes more complicated and novel methods are needed to achieve isomeric resolution. One such isomer, Δ-8-THC, is federally unregulated in the United States and readily available for purchase in many stores. This compound forms its own hydroxylated and carboxylated metabolites, (11-OH-Δ-8-THC and Δ-8-THC-COOH), that must be chromatographically resolved from their isomeric metabolites. The resolution of isomeric metabolites is key in reporting accurate specimen findings and poor resolution, especially when one isomer is in much greater abundance than the other, can result in quantitation issues and invalid data. In this study, an LC-MS/MS method was developed to adequately resolve all three isomeric pairs of compounds in whole blood.
OBJECTIVES: The primary objective of this study was to develop an LC-MS/MS method to adequately resolve isomeric pairs Δ-8-THC and Δ-9-THC, 11-OH-Δ-8-THC and 11-OH-Δ-9-THC, and Δ-8-THC-COOH and Δ-9-THC-COOH in a whole blood matrix.
METHODS: An LC-MS/MS method was developed on a Raptor FluoroPhenyl 100 x 3.0 mm, 2.7 µm column using a mobile phase A of water and a mobile phase B of methanol, both acidified with 0.1% formic acid. The flow rate was 0.8 mL/min and the column temperature was 40°C. The gradient started at 64% B and held for 6.50 minutes, ramped up to 68% B at 6.60 minutes, and held at 68% B until 13.00 minutes. At 13.10 minutes the gradient ramped up to 100% B until 14.00 minutes, before returning to start conditions at 14.10 minutes and held until 16.00 minutes for ample re-equilibration. Whole blood samples were prepared using a liquid-liquid extraction. Extracts were dried down and reconstituted in 50:50 MPA:MPB. Δ-8/9-THC and 11-OH-Δ-8/9-THC were collected in ESI+ mode, while Δ-8/9-THC-COOH was collected in ESI- mode.
RESULTS: All three isomer pairs were adequately resolved using the developed method. Calibration curves were tested ranging from 0.5-100 ng/mL for 11-OH-Δ-8/9-THC and Δ-8/9-THC, and 2.5-500 ng/mL for Δ-8/9-THC-COOH. Linearity was demonstrated using a 1/x2 weighted linear regression, and all analytes showed acceptable R2 values. The method showed acceptable inter-day and intra-day precision and accuracy. No matrix or cross-analyte interferences were observed.
CONCLUSION: An LC-MS/MS method was successfully developed for reliable and accurate testing of Δ-8/9-THC isomers and isomer metabolites in whole blood. The method was determined to be quick, rugged, and sensitive enough to meet reporting guidelines for clinical and forensic toxicology laboratories.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
