Nano Liquid Chromatography Directly Coupled to Electron Ionization Mass Spectrometry for Free Fatty Acid Elucidation in Mussel

Abstract

Recently the miniaturization of liquid chromatography (LC) systems and progresses in mass spectrometry instrumentation have enabled direct introduction of the effluent coming from a nanoLC column into the high vacuum region of an electron ionization source. In the present research, a nanoLC system was directly coupled to an electron ionization quadrupole mass spectrometer (EI-MS) without any interface or modification of the ion source. The clear advantage with respect to the atmospheric pressure ionization techniques, normally coupled with LC, is major identification power because of a characteristic, more extensive and reproducible fragmentation pattern, without any matrix effect or mobile phase interferences. In particular, a nanoLC-EI-MS method was developed for the elucidation of the free fatty acid profile in mussel samples, avoiding previous derivatization step, as required when gas chromatographic analysis is involved. A total of 20 fatty acids were reliably identified through the comparison with commercially available libraries. A quantitative determination was also carried out by using the response factors approach along with the internal standard method, allowing for the quantification of 14 fatty acids. Among them, palmitic acid resulted the most abundant, followed by the ω6 arachidonic acid. The quantitative data were compared with those obtained by a well-established technique, such as gas chromatography with flame ionization detection (GC-FID). Both nanoLC-EI-MS and GC-FID methods were validated and comparable similar results were obtained in terms of limit of detection and, limit of quantification, resulting in the picomole range, and sensitivity as well was not significantly different, as demonstrated by comparing the slope values of the calibration curves (p<0.05, performing a t-test).