Development of liquid chromatography–ion mobility mass spectrometry method for profiling novel N-acyl amides in fecal samples

Abstract

Lipids are vital in multiple physiological functions, such as cell signaling and structure. N-acyl amides (NAAs), comprising an acyl tail and an amine group, are a large and diverse class of lipids and are hypothesized to facilitate various physiological functions. Methods for analyzing lipids typically employ high-resolution mass spectrometry coupled with liquid-chromatogram (LC) separation. Ion mobility spectrometry (IMS) provides an additional separation dimension to the data, improving the signal-to-noise of the method. This study aimed to develop and optimize a qualitative LC-IMS mass spectrometry method to detect novel N-acyl amides in biological matrices, specifically studying their dynamics in in-vitro colon simulation chyme samples. This study used synthetic standard mixtures of 1,426 NAAs provided by our collaborators. Method development started with constructing a preliminary in-house library of the NAAs, chromatographic gradient and peak shape optimization, and optimization of MS settings. Fecal samples from in-vitro colon simulation vessels, mimicking the physiology of the human colon, were analyzed to understand these compounds' dynamics and the effect of fecal microbes on the NAA profile. This study resulted in an in-house library comprising retention times, ion mobility values and fragmentation spectra of 910 NAAs. The library can be used to screen novel metabolites in biological samples. A total of 52 NAAs were detected in colon simulation samples, with four showing statistically significant associations in the vessels. The results of this study are encouraging and the method is nearly ready to be implemented in larger sample sets.