Iridium Isotope Tag-Assisted LC-MS Method for Global Profiling and Quantification of Nonvolatile Serum Fatty Acids in Nonalcoholic Fatty Liver Mice

Highly accurate and sensitive measurements of fatty acids (FAs) in biological samples are essential for advancing our understanding of their diverse biofunctions. In this work, based on the characteristic isotope pattern of iridium (^191/193Ir), we employed an iridium-encoded amine (Ir-NH₂) as the derivatization reagent to establish a selective and sensitive liquid chromatography–mass spectrometry (LC-MS) method for rapid identification and accurate quantification of FAs in biological samples. Upon derivatization, nonvolatile FAs were transformed into amide derivatives tagged with a charged iridium tag, exhibiting improved sensitivity and selectivity in the electrospray ionization (ESI) positive ion mode. By leveraging the unique 2.002 Da mass shift and the 3:5 peak intensity ratio from the natural ¹⁹¹Ir and ¹⁹³Ir isotopes, we can rapidly and efficiently screen the potential carboxyl-containing metabolites from biological samples. Compared to other existing methods, our technique offers higher sensitivity, better signal-to-noise ratio, lower detection limit (1.2–8.4 pg/mL), and easier quantification due to the clear identification of iridium-tagged derivatives. With this method, a total of 58 FAs, including both saturated and unsaturated types, were detected in mice serum lipid extracts, with carbon chain lengths varying from C9 to C24. More importantly, this method was successfully employed for global profiling of nonvolatile serum FAs from mice with nonalcoholic fatty liver disease (NAFLD), providing a novel means for detecting them and offering new avenues for exploring their functional roles and disease associations.