Endocannabinoids and endocannabinoid-related compounds (ERCs) are involved in many physiological processes. They are released on demand from phosphoinositide and N-acylphosphatidyl ethanolamine (NAPE) precursors and comprise 2-monoacylglycerols (2-MGs) and FA ethanolamides (FEAs). Despite the abundance of advanced quantitative methods, however, their determined concentrations in blood plasma are inconsistent because 2-MGs and FEAs undergo artifactual de novo formation, chemical isomerization, and degradation during sample collection and storage. For a comprehensive survey of these compounds in blood and plasma, we have developed and validated an ultra-HPLC-MS/MS method to quantify 24 endocannabinoids, ERCs, and their phospholipid precursors. Immediate acidification of EDTA-blood to pH 5.8 blocked artifactual FEA formation for at least 4 h on ice. The 2-MGs were stabilized after plasma harvest with 0.5 M potassium thiocyanate at pH 4.7. FEA and MG plasma concentrations in six healthy volunteers ranged between 0.04-3.48 and 0.63-6.18 ng/ml, respectively. Interestingly, only 1-5% of circulating FEAs were present in their free form, while the majority was bound to NAPEs. Similarly, 97% of 2-arachidonoylglycerol (2-AG) was bound to a potential phosphoinositide pool. The herein-described stabilization and extraction methods may now be used to reliably and comprehensively quantify endocannabinoids, ERCs, and their phospholipid precursors in clinical studies.

中文翻译：

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通过 UHPLC-MS/MS 对人血浆中 24 种循环内源性大麻素、内源性大麻素相关化合物及其磷脂前体进行定量。

内源性大麻素和内源性大麻素相关化合物（ERC）参与许多生理过程。它们根据需要从磷酸肌醇和N-酰基磷脂酰乙醇胺 (NAPE) 前体中释放，并包含 2-单酰基甘油 (2-MG) 和 FA 乙醇酰胺 (FEA)。然而，尽管有大量先进的定量方法，但它们在血浆中测定的浓度并不一致，因为 2-MG 和 FEA 在样品收集和储存过程中会人为地从头形成、化学异构化和降解。为了对血液和血浆中的这些化合物进行全面调查，我们开发并验证了一种超 HPLC-MS/MS 方法来量化 24 种内源性大麻素、ERC 及其磷脂前体。立即将 EDTA 血液酸化至 pH 5.8，在冰上阻止人工 FEA 形成至少 4 小时。收获血浆后，用 0.5 M 硫氰酸钾（pH 4.7）稳定 2-MG。六名健康志愿者的 FEA 和 MG 血浆浓度分别为 0.04-3.48 和 0.63-6.18 ng/ml。有趣的是，只有 1-5% 的流通 FEA 以其游离形式存在，而大多数与 NAPE 结合。同样，97% 的 2-花生四烯酰甘油 (2-AG) 与潜在的磷酸肌醇库结合。本文描述的稳定和提取方法现在可用于在临床研究中可靠且全面地量化内源性大麻素、ERC 及其磷脂前体。