Validation of a Microsampling-Compatible LC-MS/MS Method for Cannabinoid Quantitation in Whole Blood

Recently developed dried blood sampling methods for cannabinoid quantitation require small blood volumes, making them microsampling-compatible, but have notable limitations including hematocrit-related bias for dried blood spots (DBS) and higher consumable costs for volumetric absorptive microsampling (VAMS®). To address these issues, we developed a highly sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method capable of quantifying cannabinoids in 50 µL of liquid whole blood, providing a practical microsampling alternative to dried blood approaches. Using liquid–liquid extraction (LLE) with sodium hydroxide alkalinization and acetonitrile precipitation, followed by quantitative analysis on an Agilent 6495 liquid chromatography-triple quadrupole (LC-TQ) mass spectrometer, we achieved lower limits of quantitation (LLOQs) of 0.10 ng/mL for Δ9-tetrahydrocannabinol (THC) and cannabinol (CBN), 0.20 ng/mL for cannabigerol (CBG), 0.30 ng/mL for cannabidiol (CBD), 0.50 ng/mL for 11-hydroxy-THC (11-OH-THC), and 5.0 ng/mL for 11-nor-9-carboxy-THC (THC-COOH). Calibration was linear from the LLOQ to 300 ng/mL for all analytes. To our knowledge, this is the first validated approach for cannabinoid quantitation in less than 100 µL of liquid whole blood with an LLOQ for THC comparable to that with the most sensitive LC-MS/MS methods using standard blood volumes. The achieved LLOQs for other cannabinoids are also suitable for forensic toxicology applications. We anticipate particular utility for obtaining evidence from suspected impaired drivers at the roadside when paired with finger-prick sampling and liquid blood microcollection tubes.This approach enables measurement of THC levels at the time of driving and thereby overcoming current limitations, including the decrease in THC levels that occurs if blood sampling is delayed, the requirement for larger sample volumes (≥100 µL), and dependence on trained phlebotomists for venipuncture.