High-performance liquid chromatographic determination of free fatty acids and esterified fatty acids in biological materials as their 2-nitrophenylhydrazides

High-performance liquid chromatography (HPLC) in conjunction with direct derivatization is described for the determinations of both free fatty acids (FFAs) and esterified fatty acids (EFAs) in biological materials. The method is based on the reaction of these acids with acidic 2-nitrophenylhydrazine hydrochloride (2-NPH·HCl) with and without saponification of the samples, and there are no sample work-up steps. The derivatized FAs were extracted into n-hexane and separated isocratically with short retention times. These acids were consisted saturated and mono- and polyunsaturated FAs including cis–trans isomers and double-bond positional isomers. The analytical results showed good recovery and reproducibility using an internal standard. The method is simple, rapid and reliable and has several advantages with regard to resolution, analysis time, selectivity and sensitivity over previous methods. Thus, the present method can serve as a useful tool for routine determinations of FFAs and EFAs in various fields.