Optimization of bacterial fatty acid methyl esters separation by gas chromatography - mass spectrometry

Analysis of bacterial fatty acids as their methyl esters (FAMEs) has been extensively used for rapid diagnosis of anaerobic infections, bacterial classification and taxonomy. This makes bacterial FAMEs and especially hydroxy FAMEs analyses of primary importance for clinical and taxonomical interests. However, selection of the appropriate column and other chromatographic parameters makes it an intricate issue for the simultaneous separation of various classes of compounds. For the resolution of these complex mixtures, we have compared four bonded capillary columns OV1, HP-INNOwax, DB5, and VF5-ms of the same length, internal diameter and coated film thickness. Experimental conditions such as gas flow rate and oven temperature programming were optimized to achieve the best resolution on these columns. The standard FAMEs used in this study contained 6 hydroxy fatty acids, two geometric isomers, two positional isomers in branch chain, a cyclopropane and short and long chain components. We have found that OV1 capillary column of low polarity could give best separation of all classes of bacterial FAMEs present in a mixture. Identification of the FAMEs was carried out from their mass spectral fragmentation.