Considerations about the in situ derivatization and fractionation of EFA and NEFA in biological and food samples

Despite their important role in tissues, fluids and foods, the analysis of non-esterified fatty acids (NEFA) as methyl esters (NEFAME) is performed using expensive, cumbersome and time-consuming procedures that needs of isolation, fractionation and derivatization steps. However, Yi et al. [1] • Increment of the transesterification performance by adding hexane to the reaction mixture, decreasing the time for the derivatization of acylglycerols from 10 min to 3–4 min and stopping the reaction with sulfuric acid. • Avoid cross-contamination of the NEFAME extract by adding 500 μL of water after collection of EFA methyl esters (EFAME). • Samples are spiked with three internal standards: a triacylglycerol (to calculate the concentration of EFA), a free fatty acid (to calculate NEFA) and a FAME (to control isolation of FAME and cross-contamination).