State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau.
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau.
Anal Chim Acta. 2021 Sep 22;1179:338839. doi: 10.1016/j.aca.2021.338839. Epub 2021 Jul 7.
N-acylethanolamides (NAEs) are a class of naturally occurring lipid molecules with pleiotropic activities ranging from energy homeostasis to analgesic functioning. However, the comprehensive quantitation of endogenous NAEs is challenged by the sub-trace level (nM) in complex biological samples and the limited availability of stable isotope labeled internal standards (SIL-IS). Herein, a sensitive method was developed to accurately determine 20 NAEs in biological samples by chemical isotope labeling strategy coupled with liquid chromatography - tandem mass spectrometry (LC-MS/MS). A pair of efficient derivatization reagents, acetyl chloride-d (ACC-d) and acetyl chloride-d (ACC-d), were used to label NAEs in biological samples and NAE standard mixture, respectively. The heavily labeled NAE derivatives of the standard substances were used as one-to-one internal standards to minimize the matrix effects and potential ion suppression in MS analysis. Although no chemical moiety with high ionization capability was introduced, the detection sensitivity of the derivatized NAEs were substantially enhanced, as evidenced by 6- to 170-fold increase in LOQs, compared to non-derivatized NAEs. The derivatized NAEs provided the stable and abundant specific product ions in MS/MS spectrum, which were used as the quantitation ions for multiple reaction monitoring (MRM) analysis. The validated LC-MS/MS method was also successfully applied to determine NAEs in serum samples and liver tissues from control and alcohol-fed mice, which shown its practicability in the analysis of endogenous NAE in biological samples. Collectively, the proposed method offers a sensitive and accurate quantification of endogenous NAEs, which may facilitate the understanding of NAE metabolisms and their functions in the physiological and pathological processes.
N-酰基乙醇胺（NAEs）是一类具有多种活性的天然存在的脂质分子，其活性范围从能量平衡到镇痛作用。然而，内源性 NAE 的全面定量分析受到复杂生物样品中痕量水平（nM）和有限的稳定同位素标记内标（SIL-IS）的限制。本文采用化学同位素标记策略结合液相色谱-串联质谱（LC-MS/MS），开发了一种灵敏的方法，能够准确测定生物样品中的 20 种 NAE。使用一对高效的衍生化试剂，乙酰氯-d（ACC-d）和乙酰氯-d（ACC-d），分别对生物样品和 NAE 标准混合物中的 NAE 进行标记。将标准物质的大量标记 NAE 衍生物用作一一对应的内标，以最大程度地减少 MS 分析中的基质效应和潜在的离子抑制。尽管没有引入具有高电离能力的化学基团，但衍生化 NAE 的检测灵敏度得到了显著提高，与非衍生化 NAE 相比，LOQs 提高了 6-170 倍。衍生化 NAE 在 MS/MS 谱中提供了稳定且丰富的特征产物离子，可作为多重反应监测（MRM）分析的定量离子。验证后的 LC-MS/MS 方法还成功应用于测定对照和酒精喂养小鼠血清样品和肝组织中的 NAE，表明其在生物样品中内源性 NAE 分析中的实用性。总之，该方法为内源性 NAE 的灵敏准确定量提供了一种新方法，有助于理解 NAE 代谢及其在生理和病理过程中的功能。
