Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Feb 1;883-884:161-71. doi: 10.1016/j.jchromb.2011.06.025. Epub 2011 Jun 22.
Analysis of the endocannabinoid (EC) system's key molecules 2-arachidonoyl glycerol (2AG) and arachidonoyl ethanolamide (anandamide, AEA) is challenging due to several peculiarities. 2AG isomerizes spontaneously to its biologically inactive analogue 1-arachidonoyl glycerol (1AG) by acyl migration and it is only chromatographically distinguishable from 1AG. Matrix-effects caused primarily by co-extracted phospholipids may further compromise analysis. In addition, 2AG and 1AG are unstable under certain conditions like solvent evaporation or reconstitution of dried extracts. We examined effects of different organic solvents and their mixtures, such as toluene, ethyl acetate, and chloroform-methanol, on 2AG/1AG isomerisation, 2AG/1AG stability, and matrix-effects in the UPLC-MS/MS analysis of 2AG and AEA in human plasma. Toluene prevented, both, 2AG isomerisation to 1AG and degradation of 2AG/1AG during evaporation. Toluene extracts contain only 2% of matrix-effect-causing plasma phospholipids compared to extracts from the traditionally used solvent mixture chloroform-methanol. Toluene and all other tested organic solvents provide comparable 2AG and AEA extraction yields (60-80%). Based on these favourable toluene properties, we developed and validated a UPLC-MS/MS method with positive electrospray ionization (ESI+) that allows for simultaneous accurate and precise measurement of 2AG and AEA in human plasma. The UPLC-MS/MS method was cross-validated with a previously described fully-validated GC-MS/MS method for AEA in human plasma. A close correlation (r(2)=0.821) was observed between the results obtained from UPLC-MS/MS (y) and GC-MS/MS (x) methods (y=0.01+0.85x). The UPLC-MS/MS method is suitable for routine measurement of 2AG and AEA in human plasma samples (1 mL) in clinical settings as shown by quality control plasma samples processed over a period of 100 days. The UPLC-MS/MS method was further extended to human urine. In urine, AEA was not detectable and 2AG was detected in only 3 out of 19 samples from healthy subjects at 160, 180 and 212 pM corresponding to 12.3, 14.5 and 9.9 pmol/mmol creatinine, respectively.
分析内源性大麻素（EC）系统的关键分子 2-花生四烯酰甘油（2AG）和花生四烯酰乙醇胺（大麻素，AEA）具有挑战性，因为存在几个特点。2AG 通过酰基迁移自发异构化为其生物活性较低的类似物 1-花生四烯酰甘油（1AG），并且仅通过色谱法与 1AG 区分开。主要由共提取的磷脂引起的基质效应可能进一步影响分析。此外，2AG 和 1AG 在某些条件下不稳定，例如溶剂蒸发或干燥提取物的重新配制。我们研究了不同有机溶剂及其混合物（如甲苯、乙酸乙酯和氯仿-甲醇）对 UPLC-MS/MS 分析人血浆中 2AG 和 AEA 时 2AG/1AG 异构化、2AG/1AG 稳定性和基质效应的影响。甲苯可防止 2AG 异构化为 1AG 和 2AG/1AG 在蒸发过程中的降解。与传统使用的溶剂混合物氯仿-甲醇相比，甲苯提取物中仅含有 2%引起基质效应的血浆磷脂。甲苯和所有其他测试的有机溶剂提供相当的 2AG 和 AEA 提取产率（60-80%）。基于这些有利的甲苯特性，我们开发并验证了一种带有正电喷雾电离（ESI+）的 UPLC-MS/MS 方法，可同时准确、精密地测量人血浆中的 2AG 和 AEA。UPLC-MS/MS 方法与之前描述的完全验证的人血浆 AEA 的 GC-MS/MS 方法进行了交叉验证。从 UPLC-MS/MS（y）和 GC-MS/MS（x）方法获得的结果之间观察到密切相关（r（2）=0.821）（y=0.01+0.85x）。UPLC-MS/MS 方法适用于临床环境中处理 100 天期间的质控血浆样品的人血浆样品中 2AG 和 AEA 的常规测量。UPLC-MS/MS 方法进一步扩展到人类尿液。在尿液中，AEA 无法检测到，只有 19 个健康受试者的样本中的 3 个样本中检测到 2AG，浓度分别为 160、180 和 212 pM，相当于 12.3、14.5 和 9.9 pmol/mmol 肌酐。
