Twin Derivatization Strategy for High-Coverage Quantification of Free Fatty Acids by Liquid Chromatography–Tandem Mass Spectrometry

Free fatty acids (FFAs) are vitally important components of lipids that modulate biological metabolism in various ways. Although the molecular structures are simple, the analysis of FFAs is still challenging due to their unique properties and wide concentration range. In the present study, a high-coverage liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the quantification of FFAs in serum samples using two structural analogues 5-(dimethylamino)naphthalene-1-sulfonyl piperazine (Dns-PP) and (diethylamino)naphthalene-1-sulfonyl piperazine (Dens-PP) as twin derivatization reagents. The Dns labeling of FFAs could significantly enhance their MS response via the introduction of the easily ionizable moiety of a tertiary amine-containing part and aid fragmentation in the multiple reaction monitoring (MRM) mode. Our results demonstrated that the detection sensitivities of FFAs were increased by 50–1500 fold compared with the nonderivatization method. At the same time, Dens-labeled standards were used as one-to-one internal standards to ensure accurate quantifications. Thirty-eight FFAs, covering short-, medium-, and long-chain, could be quantified in wide dynamic range with the lower limit of quantification (LLOQ) varied from 2 to 20 nM. Using this method, we analyzed serum FFAs in rat models of cisplatin-induced nephrotoxicity and irinotecan-induced gastrointestinal toxicity, respectively. The findings were further compared with those revealed by previous untargeted metabolomics. The results indicate that twin derivatization-based LC-MS provides a more accurate view of global FFA alternation and has great application potential in the fields of targeted metabolomics.