Stepwise derivatization GC-MS/MS of 39 FFAs in spleen-deficiency rat liver

Free fatty acids (FFAs) are important targets in metabolic diseases. However, accurate quantification of a wide range of C2-C24 FFAs in the liver is challenging due to their structural diversity and large variations in abundance. To address this, a stepwise derivatization pretreatment was employed in this study: after liver homogenization, short-chain fatty acids were first extracted with diethyl ether-water (1:1, V/V). The ether layer was dehydrated with anhydrous Na₂SO₄ and then derivatized with BSTFA. The remaining pellet was extracted with chloroform-methanol (1:2, V/V), and after nitrogen evaporation, the extract was subjected to methyl esterification. A quantitative method for 39 FFAs was established on a Trace1600-TSQ9610 platform (TG-SQC column, MRM mode). Methodological validation showed that for all FFAs, the linear correlation coefficients R² were ≥ 0.991, intra- and inter-day accuracies ranged from 85.94% to 115.28%, precision RSD ≤ 13.16%, stability RSD ≤ 14.91%, and the LLOQ ranged from 0.03 to 10 μg/mL. When applied to the livers of spleen-deficiency SD rats, the method revealed significant decreases in C14:0, C15:0, and C18:0, and significant increases in C9:0, C18:1n9c, and C20:4n6 (VIP > 1, |Log₂FC| > 1, P < 0.05). This method provides a reliable tool for hepatic FFA profiling and the discovery of preliminary candidate biomarkers. Limitations of this study include a small sample size (n = 3 per group), which may affect the robustness of the supervised OPLS-DA model and the interpretation of the differential findings, and the extraction recovery was not independently evaluated.