Separation of positional and geometrical fatty acid isomers as 2-nitrophenylhydrazide derivatives by high-performance liquid chromatography

Previously, following derivatization to their 2-nitrophenylhydrazide (2-NPH) derivatives, fatty acid (FA) abundances have been evaluated using high-performance liquid chromatography (HPLC). Although the method was sensitive, resolution was insufficient for many of the biologically important FAs. We have developed an enhanced separation of 24 FAs by use of different column temperature, stationary phase, and mobile phase gradient conditions. We applied this method to analysis of mouse skeletal muscle phospholipid and triglyceride. This further development of the chromatographic separation of 2-NPH FAs may lead to greater utility of this HPLC approach.