Analysis of non-esterified fatty acids in human samples by solid-phase-extraction and gas chromatography/mass spectrometry

•We developed a method to quanitfy non-esterified fatty acids.•Separation of the NEFA-fraction is achieved through solid-phase-extraction.•Quantitation is achieved using GC/MS.•This allows the comparison of circulating and bound FAs.

The determination of the fatty acid (FA) profile of lipid classes is essential for lipidomic analysis. We recently developed a GC/MS-method for the analysis of the FA profile of total FAs, i.e. the totality of bound and unbound FAs, in any given biological sample (TOFAs). Here, we present a method for the analysis of non-esterified fatty acids (NEFAs) in biological samples, i.e. the fraction that is present as extractable free fatty acids. Lipid extraction is performed according to Dole using 80/20 2-propanol/n-hexane (v/v), with 0.1% H2SO4. The fatty acid-species composition of this NEFA-fraction is determined as FAME after derivatization with our GC/MS-method on a BPX column (Shimadzu). Validation of the NEFA-method presented was performed in human plasma samples. The validated method has been used with human plasma, cells and tissues, as well as mammalian body fluids and tissue samples. The newly developed solid-phase-extraction (SPE)–GC–MS method allows the rapid separation of the NEFA-fraction from a neutral lipid extract of plasma samples. As a major advantage compared to G–FID-methods, GC–MS allows the use of stable isotope labeled fatty acid precursors to monitor fatty acid metabolism.