Double derivatization-enhanced LC-MS/MS for isomer-resolved identification and quantification of fatty acids

Background Fatty acids (FAs) are essential metabolites involved in energy storage, signaling, and inflammation regulation, and their biological functions are closely linked to the position and geometric configuration of carbon–carbon double bonds (C C). However, the isomer-resolved characterization and highly sensitive detection of FAs in complex biological matrices remain challenging due to their co-elution, similar fragmentation, and low ionization efficiency. Therefore, there remains a critical need for an efficient approach that can simultaneously enhance chromatographic separation, enable unambiguous C C characterization, and improve detection sensitivity for FA isomers in biological samples. Results In this study, we developed a double derivatization strategy combining magnesium monoperoxyphthalate hexahydrate (MMPP) epoxidation with N,N-diethyl-1,2-ethanediamine (DEEA) amidation, coupled with LC–MS/MS, for isomer-resolved FA analysis. The double derivatization enhanced the chromatographic resolution of positional and cis/trans isomers, enabled reliable localization of C C positions via Δ16 Da diagnostic ion pairs, and improved detection sensitivity by 16–32 fold relative to epoxidation alone. Cis -trans configurations were further supported by parallel linear relationships between retention time and C C position under identical LC conditions, providing a standards-sparing criterion for structural assignment. Applied to mouse plasma, the strategy identified 69 FAs, including 55 unsaturated species, representing an increase of 46 over the underivatized approach. Quantitative analyses and differential analyses showed most FAs were elevated in hepatitis B virus (HBV) mice, with altered isomer ratios pointing to disrupted desaturase activity and oxidative stress. Significance In conclusion, this double derivatization LC-MS/MS platform provided improved structural resolution, higher detection sensitivity, and more reliable configurational assignment of FAs without requiring extensive standards. Its successful application to HBV mouse plasma demonstrated its suitability for complex biological matrices and highlighted its potential for biomarker discovery and mechanistic studies of lipid-related metabolic diseases.