In-vial one-step FAME derivatization in biological samples followed by GC-FID and GC×GC-MS analysis

The characterization of fatty acid (FA) composition in biological matrices requires efficient derivatization strategies capable of converting lipid species into volatile derivatives suitable for gas chromatographic (GC) analysis. In the present study, FAs from biological samples, including human serum and associated lipoproteins and extracellular vesicles, were analyzed to investigate lipid profile associated with metabolic alterations. To enable GC analysis, FAs were derivatized using a simplified in-vial transesterification approach, which allows the direct conversion of lipid-bound fatty acids into FA methyl esters (MEs) without the need for preliminary lipid extraction.
Briefly, biological samples were subjected to acid-catalyzed transesterification using sulfuric acid in anhydrous methanol (1%), promoting the conversion of esterified lipids into their corresponding methyl esters. The reaction was monitored through the addition of two internal standards introduced to assess the efficiency of the transesterification process. Following derivatization, FAMEs were recovered through hexane and subsequently analyzed by GC coupled with flame ionization detection (FID) and comprehensive
multidimensional GC (GC × GC) coupled to mass spectrometry (MS) analysis.
This streamlined derivatization workflow minimizes solvent consumption while ensuring efficient conversion of FAs within complex biological matrices