Qualitative Study of Free Fatty Acids Based on Two-step Derivatization-HPLC-MS Method

OBJECTIVE To establish a two-step derivatization and high performance liquid chromatography-mass spectrometry method, and to qualitatively study the free fatty acids in biological samples, with a focus on distinguishing double bond isomers.

METHODS Six representative free fatty acids with different saturation were subjected to two-step derivatization using m-chloroperoxybenzoic acid and N,N-diethylethylenediamine. High performance liquid chromatography-mass spectrometry was used for analysis and detection.

RESULTS Verified by 6 free fatty acid standards, the analysis approach of mass spectrometry fragmentation data backtracking structure was feasible and reliable. A total of 38 kinds of free fatty acids were identified from Beagle plasma, including 13 monounsaturated, 17 polyunsaturated and 8 saturated. It could distinguish 10 kinds of double bond isomers and accurately analyze the double bond positions of low abundance docosahexadilute acids.

CONCLUSION The established two-step derivatization and high performance liquid chromatography mass spectrometry method provides a new idea for the qualitative analysis of free fatty acids in vivo, especially for the differentiation of double bond isomerism, and also provides methodological support for related metabolomics and lipidomics studies.