Institute of Pharmacology and Toxicology, Rostock University Medical Center, Rostock, Germany.
Institute of Pharmacology and Toxicology, Rostock University Medical Center, Rostock, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Dec 15;1161:122371. doi: 10.1016/j.jchromb.2020.122371. Epub 2020 Sep 6.
The arachidonic acid derivatives N-arachidonoylethanolamine (anandamide; AEA), 2-arachidonoylglycerol (2-AG), N-arachidonoyldopamine (NADA), 2-arachidonoylglycerol ether (noladin ether; 2-AGE) and O-arachidonoylethanolamine (virodhamine; VA) were identified as physiological components of the endocannabinoid (EC) system. In order to gain further profound knowledge about the different EC-induced physiological and pathophysiological effects, appropriate analytical methods are required. The method described here uses liquid chromatography in combination with positive electrospray ionization mass spectrometry (LC-MS/MS) to quantify the concentrations of the above-mentioned EC compounds in cells. Sample preparation prior to LC-MS/MS analysis was performed by means of two liquid extractions with ethyl acetate. The method has been validated according to the bioanalytical guidelines of the Food and Drug Administration (FDA). The lower limits of quantification were 0.03 ng/mL for AEA, 2 ng/mL for 2-AG, 0.03 ng/mL for NADA, 0.3 ng/mL for 2-AGE and 0.15 ng/mL for VA. Linearity was demonstrated up to 10 ng/mL (AEA, NADA, 2-AGE and VA) and 50 ng/mL (2-AG). The values for intra- and inter-day precision and accuracy were within the guideline recommended acceptance criteria for assay validation. Low matrix effects and good recovery were found for AEA, 2-AG and 2-AGE, while a higher matrix effect was observed for NADA and VA. Extraction yields were lowest for VA. The method was used for EC measurement in different cell lines and in mouse brains.
花生四烯酸衍生物 N-花生四烯酰乙醇胺（大麻素；AEA）、2-花生四烯酰甘油（2-AG）、N-花生四烯酰多巴胺（NADA）、2-花生四烯酰甘油醚（noladin 醚；2-AGE）和 O-花生四烯酰乙醇胺（virodhamine；VA）被鉴定为内源性大麻素（EC）系统的生理成分。为了更深入地了解不同 EC 诱导的生理和病理生理效应，需要适当的分析方法。这里描述的方法使用液相色谱法结合正电喷雾电离质谱（LC-MS/MS）来定量细胞中上述 EC 化合物的浓度。LC-MS/MS 分析前的样品制备通过用乙酸乙酯进行两次液相萃取来完成。该方法已根据食品和药物管理局（FDA）的生物分析指南进行了验证。AEA 的定量下限为 0.03ng/mL，2-AG 的定量下限为 2ng/mL，NADA 的定量下限为 0.03ng/mL，2-AGE 的定量下限为 0.3ng/mL，VA 的定量下限为 0.15ng/mL。AEA、NADA、2-AGE 和 VA 的线性度高达 10ng/mL，2-AG 的线性度高达 50ng/mL。日内和日间精密度和准确度的值符合分析验证的指南推荐的接受标准。AEA、2-AG 和 2-AGE 的基质效应较低，回收率较好，而 NADA 和 VA 的基质效应较高。VA 的提取产率最低。该方法用于不同细胞系和小鼠大脑中的 EC 测量。
