Fatty acids (FAs) play important roles in several physiological and pathophysiological processes, functioning as both nonesterified free FAs (FFAs) and components of other lipid classes. Although many lipid classes are readily measured using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), the measurement of FFAs by this method is not straightforward because of inconsistent fragmentation behaviours. In this study, we describe a strategy to measure FFAs using conventional reverse-phase LC-ESI-MS/MS, without derivatization. The strategy combines three key methods: (i) an isocratic LC separation with a high organic solvent ratio, (ii) postcolumn base addition, and (iii) pseudo-multiple reaction monitoring. The method facilitates the measurement of ultra-long-chain FAs, the accumulation of which is a common biochemical abnormality in peroxisomal disorders. This study delivers a broad strategy that measures a wide spectrum of FFA species in complex biological samples.
Keywords: LC-MS/MS; ammonium hydroxide; free fatty acid; isocratic LC; pseudo-MRM.
© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.