Spotlight on Endocannabinoids in Healthy Individuals Using Volumetric Absorptive Microsampling Combined with LC-MS/MS Analysis

Endocannabinoids (eCBs) are crucial to regulating physiological function in the human body. This study investigates the applicability of volumetric absorptive microsampling (VAMS) for eCB analysis in blood samples. First, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify a series of primary eCBs and eCB-like lipid mediators (i.e., AEA, OEA, SEA, PEA, LEA, 2-AG, and 2-OG). We then evaluated VAMS, considering several key parameters such as temperature, humidity, and hematocrit levels. This research systematically compared, for the first time, eCB levels in whole blood and plasma samples. We demonstrated an accuracy between 72.5% and 98.9%, with an interday and intraday precision below 8%, for all eCBs and eCB-like lipid mediators. The method exhibited minimal matrix effects, ranging from -15.0% to +9.0%. Most eCBs were stable under varying temperatures and storage conditions. However, drying time significantly affected the detected levels of 2-AG and 2-OG, suggesting that <i>ex vivo</i> biosynthesis may occur during drying. Interestingly, substantial differences in eCB levels were found between whole blood and plasma samples, emphasizing the importance of cellular components in eCB distribution. For example, 2-AG levels in whole blood ranged from 219-1,143 ng mL^-1; plasma samples exhibited lower levels, ranging from 1.73-8.44 ng mL^-1. These results highlight the need for standardized sampling methods to ensure accurate and consistent measurements of eCBs in both blood and plasma samples. While this work has significant implications for clinical eCB research, further investigation is necessary to understand the underlying mechanisms and clinical impact of eCB and eCB-like lipid mediators research.