A Disposable Pipette Extraction-UHPLC-MS/MS Method Based on Removal of Phospholipids to Determine Anandamide, 2-Arachidonoylglycerol, Cannabidiol, and Δ9-Tetrahydrocannabidiol in Plasma Samples

Cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC), the main components of Cannabis sativa plants, can interact with specific cell receptors known as cannabinoid receptors (CBs). The endogenous compounds anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are CB agonists, and, alongside enzymes, they constitute the endocannabinoid system (ECS) and take part in neuromodulation. Several LC-MS/MS methods have been developed to quantify these compounds in biological matrixes, but a fast and simple method that can determine these analytes in plasma samples simultaneously is not available. Here, we propose a disposable pipette extraction technique containing a zirconia-based sorbent (DPX(Zr)) combined with UHPLC-MS/MS analysis to determine CBD, THC, AEA, and 2-AG in plasma samples, simultaneously. The method combines simple protein precipitation (PPT) with a one-step DPX procedure to remove phospholipids, one of the most common endogenous interferents in biological samples. Optimization of the combined PPT-DPX sample preparation method reduced the matrix effect and improved the sensitivity of the analytical method. The validated DPX(Zr)-UHPLC-MS/MS method reported LLOQs of 0.1 ng mL⁻¹ for AEA and 2-AG and 1 ng mL⁻¹ for CBD and THC. The method demonstrated intra- and interassay accuracy and precision of less than 20% for the LLOQ, and less than 15% for the other calibration points. Additionally, no carryover or significant matrix effect was observed. We applied this method to determine AEA, 2-AG, and CBD in plasma samples obtained from obsessive-compulsive disorder patients treated with CBD.