Accurate quantification of endogenous N-acylethanolamides by chemical isotope labeling coupled with liquid chromatography-tandem mass spectrometry

Abstract

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-d0 (ACC-d0) and acetyl chloride-d3 (ACC-d3), 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.