A liquid chromatography-mass spectrometry method for analysis of intact fatty-acid-based lipids extracted from soil

Fatty-acid-based lipids are considered to be indicators of the structure and function of soil microbial biomass. Identifying and quantifying intact fatty-acid-based lipids in soil extracts is challenging because lipids range from polar (e.g. phospholipids and other membrane lipids) to non-polar (e.g. triacylglycerols involved in energy storage). The aim of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method to identify and quantify intact fatty-acid-based lipids in soil extracts. Reversed-phase LC separated fatty-acid-based lipids ranging from polar to non-polar in one chromatographic analysis. Within classes, individual lipid species were separated predictably from one another on the basis of carbon numbers and degree of unsaturation, such that chromatographic retention time could be used to aid identification. Lipid species were identified based on intact mass of the lipid and by fragmentation into structure-specific fragments that enabled compound classes and fatty acyl chains to be determined. Detection limits in positive and negative mode were better than 10nmoll(-1) for most lipid standards. In an extract of soil, 74 lipids from 10 classes were identified. The main non-polar lipids were 12 diacylglyerol species and 16 triacylglycerol species. Polar lipids included phosphatidylcholines, phosphatidylethanolamines, monomethyl-phosphatidylethanolamines, phosphatidylinositols and three classes of betaine lipid (diacylgycerol-N-trimethylhomoserine; diacylglycerylhydroxymethy-N,N,N-trimethyl--alanine; and diacylglycerylcarboxyhydroxymethylcholine). The LC-MS method developed provides a powerful analytical tool for analysis of fatty-acid-based lipids ranging from polar to non-polar and should be useful for the culture- and gene-independent assay of soil function.