Reversed-phase liquid chromatography/mass spectrometry approach for (un)targeted analysis of polar to mid-polar metabolites.

Herein, we aim to establish a straightforward and versatile reversed-phase liquid chromatography/mass spectrometry (RP-LC/MS) methodology for analyzing a wide range of polar and mid-polar metabolites utilizing a single instrument, column, and mobile phase. We present a comprehensive evaluation of three C18 columns compatible with aqueous solutions using 19 mobile phases in terms of the number of detected metabolites, chromatographic performance, and MS response. The RP-LC/MS platform utilizes the HSS T3 column with a mobile phase consisting of 0.2 % formic acid, acetonitrile, and propan-2-ol, effectively separating polar and mid-polar metabolites through various mobile phase gradients. Our developed method outperforms the conventional hydrophilic interaction liquid chromatography metabolomic method, yielding a higher number of detected metabolites and better chromatographic performance. The RP-LC/MS platform demonstrates excellent intrabatch and interbatch retention time repeatability (<0.8 %). Furthermore, the determined concentrations of metabolites show strong agreement with certified and published concentrations of metabolites in the SRM 1950 plasma sample. We successfully annotate 71 polar metabolites, 36 acylcarnitines, 23 endocannabinoids, 42 oxylipins, and 16 fatty acids in plasma, placenta, and brain samples. The developed RP-LC/MS approach represents a robust and adaptable technique for the targeted or untargeted analysis of polar and mid-polar metabolites employing a single chromatographic column and mobile phase. This is achieved through the simple modification of the gradient program and MS conditions. Consequently, this methodology offers a highly valuable tool for conducting comprehensive, large-scale metabolomic investigations on a variety of biological samples.