A simple LC-MS/MS method for the simultaneous determination of 18 free fatty acids in human serum

Free fatty acids (FFAs) hold significant clinical importance, not only aiding in the assessment of lipid metabolism status but also being closely associated with various diseases such as metabolic disorders and cardiovascular diseases. Consequently, the detection of FFAs is crucial for clinical research and disease diagnosis. Previous methods for FFA analysis predominantly relied on GC-MS, which often required complex derivatization procedures. This study developed a targeted LC-MS/MS method for the simultaneous quantification of 18 FFAs in human serum and validated its applicability for clinical sample analysis. Stable isotope-labeled internal standards were used for all analytes to correct for variability from matrix effects and ionization efficiency. Samples were prepared using a straightforward protein precipitation procedure. Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm), with quantification achieved through multiple reaction monitoring (MRM) in negative ion mode. Through optimization of chromatographic separation conditions, mass spectrometry parameters, and sample preparation protocols, coupled with systematic methodological validation, efficient separation and precise quantification of 18 FFAs were achieved. This method was ultimately applied to the analysis of serum samples from healthy individuals, and reference intervals were established based on the test results, providing a benchmark for future studies on FFA concentrations in vivo. The method offers advantages of simplicity, high sensitivity, and strong reproducibility, providing reliable technical support for mechanism research, early diagnosis, and therapeutic evaluation of diseases associated with FFAs.