Integrated LC-MS metabolomics with dual derivatization for quantification of FFAs in fecal samples of hepatocellular carcinoma patients

Free fatty acids (FFAs) display pleiotropic functions in human diseases. Short-, medium-, and long-chain fatty acids (SCFAs, MCFAs, and LCFAs) are derived from different origins, and precise quantification of these FFAs is critical for revealing their roles in biological processes. However, accessing stable isotope-labeled internal standards (SIL-IS) is difficult, and different chain lengths of FFAs challenge the chromatographic coverage. Here, we developed a metabolomics strategy to analyze FFAs based on isotope-free liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) integrated with dual derivatization. Samples and dual derivatization internal standards (DD-ISs) were synthesized using 2-dimethylaminoethylamine (DMED) or dansylhydrazine (Dns-Hz) as a "light" label under mild and efficient reaction conditions, and N, N-diethyl ethylene diamine (DEEA) or N, N-diethyldansulfonyl hydrazide (Dens-HZ) as a "heavy" label. General MRM parameters were designed to analyze these FFAs. The limit of detection (LOD) of SCFAs varied from 0.5 to 3 nM. Furthermore, we show this approach exhibits good linearity (R2=0.99374 to 0.99929), there is no serious substrate interference, and no quench steps are required, confirming the feasibility and reliability of the method. Using this method, we successfully quantified 15 types of SCFAs in fecal samples from hepatocellular carcinoma (HCC) patients and healthy individuals; among these, propionate, butyrate, isobutyrate, and 2-methylbutyrate were significantly decreased in the HCC group compared to the healthy control group. These results indicate that the integrated LC-MS metabolomics with isotope-free and dual derivatization is an efficient approach for quantifying FFAs, and may be useful for identifying lipid biomarkers of cancer.