Validation of a microsampling-compatible liquid–liquid extraction method for cannabinoid quantitation in 50 µL of whole blood using liquid chromatography–mass spectrometry

Recently developed dried blood analysis methods for cannabinoid quantitation utilize small blood volumes, making them microsampling-compatible, but are limited by 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), cannabinol (CBN), and cannabigerol (CBG), 0.20 ng/mL for cannabidiol (CBD), 0.50 ng/mL for 11-hydroxy-THC (11-OH-THC), and 1.0 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 one of the first validated LLE approaches for cannabinoid quantitation in less than 100 µL of liquid whole blood. Further, it achieves sub-ng/mL sensitivity, exceeding the LLOQs of most published methods which require ≥100 µL of whole blood. We anticipate particular utility for our method in obtaining evidence from suspected impaired drivers at the roadside when paired with capillary microsampling, such as via finger prick. This approach enables measurement of THC levels at the time of driving, thereby overcoming current limitations, including the decrease in THC levels that occurs with delayed blood sampling, requirement for larger sample volumes (≥100 µL), and dependence on trained phlebotomists for venipuncture.