Profiling of branched chain and straight chain saturated fatty acids by ultra-high performance liquid chromatography-mass spectrometry

As a physiologically important class of molecules and essential building blocks, fatty acids (FAs) play an important role in energy storage, membrane structure, and various signaling pathways [1]. FAs can be classified into straight chain (SCFAs) and branched chain fatty acids (BCFAs) which have their unique functions in nature. Generally, de novo SCFAs synthesis in microorganisms and mammals starts from acetyl-CoA and malonyl-CoA that leads to the synthesis of even-chain FAs (see Fig. 1a). For odd-chain FAs, however, propionyl-CoA is required as precursor instead of acetyl-CoA in order to synthesize odd-chain FAs (see Fig. 1b) [2]. BCFAs are commonly saturated FAs substituted with one (mono-) or more (di-/oligo-) methyl-branch(es) (e.g. phytanic and pristanic acid) on the carbon chain. The monomethyl BCFAs have often either an iso structure where the branching point is on the penultimate carbon atom (i.e., one from the end) or an anteiso structure where the branching point is located on the antepenultimate carbon atom (i.e., two from the end). These monomethyl BCFAs are typically derived from branched chain amino acids (BCAAs) including valine, leucine, and isoleucine [3]. In the common biosynthetic pathways, the BCAAs are firstly transaminated to α-ketoacids and then decarboxylated into branched short-chain carboxylic acids and finally the products are elongated by BCFA synthetase, with malonyl-CoA as the chain extender, to form the iso- and anteiso-BCFAs [3,4]. The iso-BCFAs with even chain length (total carbon number), for example iso-16:0 and iso-18:0, are derived from valine (see Fig. 1c) while those with odd chain length (iso-15:0, iso-17:0) from leucine (see Fig. 1d). In contrast, anteiso-BCFAs are derived from isoleucine (see Fig. 1e). In addition to these most common iso and anteiso methyl-positions, some other methyl-positions were also found in nature, for example 10-methyl-hexadecanoic acid (FA 16:0;10Me), 11-methyl-octadecanoic acid (FA 18:0;11Me) in the sponge Verongia aerophoba [5]. Furthermore, 2-, 3-, 4-, and 6-monomethyl-FAs from C7-C12 were the main BCFAs components in the preen gland of the fulmar [6]. The biosynthesis of these kinds of BCFAs remain to a large extent unknown, but most probably the structures were produced by methylmalonyl-CoA instead of malonyl-CoA for the elongation of the fatty acyl chain resulting in the insertion of a methyl branch in the chain.

BCFAs are the common constituents of the microbial lipids present in abundant quantities. In bacteria, BCFAs in membrane are utilized to increase the fluidity and lower the phase transition temperature of the lipid components. Besides, they can also be found in animals including mammals although in much lower amount. In humans, BCFAs have been detected in various tissues (like adipose tissue [7] and biofilm covering the skin of the fetus [8]) and biofluids (milk [9] and serum [10]) which are proven to be associated with energy homeostasis and insulin sensitivity in human body. In general, the analysis of BCFAs is of importance and is attracting more and more attention of researchers in different research areas including microbiology, food chemistry and clinical perspectives due to their biological effects and potential pro-health benefits thus belonging to the group of bioactive FAs. Traditionally, they have been analyzed as fatty acid methyl esters or other derivatives (e.g. picolinyl esters) by gas chromatography (GC) coupled with mass spectrometry (GC–MS) or flame ionization detection (GC-FID), which has become a routine procedure with broad application to biochemical, biomedical and industrial research [11], [12], [13], [14], [15], [16], [17]. These GC methods focus on detailed fatty acid profiling. They are not intended for general lipidomics profiling, because many lipid classes are not volatile enough even after derivatization. Eventually, liquid chromatography coupled to mass spectrometry (LC-MS) has been also applied for FAs analysis especially in lipidomics, which usually does not involve derivatization procedures and covers a wide range of lipid classes [18], [19], [20]. In most of the cases, FAs profiling by LC-MS does not distinguish between straight chain and branched chain FAs. No efforts on the improvement of chromatographic resolution of different FAs isomers including BCFAs (methyl-BCFAs at different branching positions, anteiso-, iso-BCFAs and so on) and SFAs are usually undertaken. For this purpose, a systematic study on the chromatographic resolution of isomeric FAs on different columns including the silica-based polysaccharide chiral stationary phases (CSPs) [21,22] along with the commonly used Acquity UPLC charged surface hybrid (CSH) C18 column (RP C18) was carried out. CSPs have recently shown great potential in uncommon applications like isomer separations in lipidomics [18,[23], [24], [25]] and were also proven to have enantioselectivity for BCFAs in many investigations [26], [27], [28], [29], [30]. On the other hand, RP C18 column was frequently used for lipid profiling including FAs and also Triacylglycerols (TG) isomers with BCFAs [31].

In this study, after the evaluation of different columns and optimization of LC conditions, we report an UHPLC-MS/MS method based on RP C18 column with optimized LC conditions which allows the isomeric analysis of BCFAs with different monomethyl positions including anteiso, iso and other positions (2Me, 3Me, 4Me and so on). Further, the method was applied for the profiling of FAs including BCFAs in Staphylococcus aureus samples. As mammalian samples human platelets and human plasma pools were analyzed as well for evaluating assay specificity when unsaturated FAs are present. The method was proven to be capable for BCFAs profiling in bacterial samples with good selectivity for isomeric FAs especially for saturated monomethyl-BCFAs.

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