Towards enantioselective ultrahigh performance liquid chromatography–mass spectrometry‐based metabolomics of branched‐chain fatty acids and anteiso‐fatty acids under reversed‐phase conditions using sub‐2‐μm amylose‐ and cellulose‐derived chiral stationary phases

Abstract

Branched‐chain fatty acids (BCFAs) are mostly saturated fatty acids with one or more methyl, seldom ethyl, branches in the alkyl chain. They are derived from branched‐chain amino acids, ruminant‐derived food, or biosynthetic side products of acetyl‐CoA carboxylase. They possess iso‐ (branching at penultimate carbon) and anteiso‐fatty acid structure (branching at antepenultimate carbon) or are branched at any other position of the carbon chain. Except for iso‐fatty acids, BCFAs are chiral. They are commonly analyzed by GC‐MS, while there is a lack of enantioselective LC‐MS methods. In this work, we present a methodology for targeted enantioselective UHPLC‐ESI‐MS/MS metabolomics of BCFAs. It makes use of precolumn derivatization with 1‐naphthylamine and reversed‐phase elution conditions. A homologous series of short BCFA analytes with distinct chain lengths (having up to eight carbon atoms), branching type (methyl or ethyl), and position of branching (2, 3, and 4, anteiso and iso) has been systematically studied on six commercially available polysaccharide UHPLC columns. Chiralpak IB‐U exhibited the highest and broadest enantioselectivity while IH‐U maintained enantioselectivity also for BCFAs with chirality distant from the carboxylic function (i.e., with other branching than in 2‐position). The method was used to assign the absolute configuration of a 4‐methylhexanoic acid side chain of a natural product from Streptomyces sp. SHP 22‐7. The potential of the corresponding UHPLC‐ESI‐QTOF‐MS/MS assay for analyzing stereoselectively BCFAs and other short organic acids by untargeted analysis in human urine was further elucidated in a preliminary proof‐of‐principle test.