Liquid chromatographic determination of free and total fatty acids in milk and milk products as their 2-nitrophenylhydrazides.

A high-performance liquid chromatographic (HPLC) method with direct derivatization is described for the determination of both free fatty acids and total fatty acids in milk and milk products. The method is based on the reaction of these acids with 2-nitrophenylhydrazine hydrochloride with and without saponification of the samples, and there are no sample work-up steps. The derivatized fatty acids were separated into two groups, short-chain fatty acids (C2:0-C6:0) and long-chain fatty acids (C8:0-C22:6), as their hydrazides, by a simple solvent extraction. The HPLC of each acid group was performed isocratically with short retention times on a YMC-FA column. The results showed good recovery and reproducibility using 2-ethylbutyric acid and margaric acid as internal standards for the two acid groups. The method is simple, rapid and reliable and has advantages with regard to resolution, analysis time and sensitivity over previous methods.