After the method’s viability was tested, the method was tested against the fatty acid metabolisms of Syrian golden hamsters.
Chinese researchers, led by Liu Yang of the Chinese Academy of Medical Sciences & Peking Union Medical College in Beijing, China, used stable isotope derivatization coupled with liquid chromatography–triple quadrupole mass spectrometry (ID-LC-QQQ-MS) to better analyze fatty acids in biological samples. Their findings were published in the Journal of Chromatography B (1).
Fatty acids (FAs) are generally comprised of 12–26 carbon atoms, with the majority of them falling within the 16–20 carbon range. FAs containing 16 and 18 carbon atom free FAs (FFAs) are the most prevalent in various organisms, whereas FAs with carbon chain lengths shorter than 12 or exceeding 22 carbon atoms are less frequently encountered. Despite their relatively simple molecular structure, FAs play crucial roles in biological regulation: 1) they are vital constituents of cell membrane lipids and are essential for membrane-mediated cellular activities; and 2) they not only serve as an energy source for cells and regulate energy metabolism but also contribute to systemic immune responses.
Abnormal FAs are linked to various health issues, including cardiovascular diseases, insulin resistance, and Alzheimer’s disease. Consequently, profiling FAs using innovative analytical techniques is critical for elucidating their functions in physiological and pathological contexts.
FFAs, which are characterized by a hydrocarbon chain terminated at one end by a carboxylic acid group (COOH), can fulfill diverse roles in biological processes, including cellular structure integrity, energy storage, and signaling (2). Various methods have proven useful for (FFAs. Gas chromatography–mass spectrometry (GC–MS) is commonly used for FFA analysis; however, it is most effective for thermally stable and readily volatile analytes. FFAs can also be directly quantified using liquid chromatography–MS (LC–MS) in negative-ion mode. Regardless, the negative ion mode of ESI-MS/MS lacks the sensitivity needed for detecting low-abundance FFAs.
In this experiment, the scientists used a method combining stable isotope derivatization coupled with liquid chromatography–triple quadrupole mass spectrometry (ID-LC-QQQ-MS) to thoroughly identify and relatively quantify FAs in serum samples. This method involves the derivatization of FFAs’ carboxyl groups using a pair of isotope reagents; this process results in the formation of trimethylaminoethyl ester (FA-TMAE-h3/d3). This FA can yield two distinct neutral fragments with masses of 59 and 62 Da during collision-induced dissociation (CID). A quadruple neutral loss scan (QNLS) approach was utilized for the non-targeted profiling of FFAs in serum samples. The derivative peak pairs displaying matching retention times and distinct mass differences were extracted from the two QNLS spectra, after which they were recognized as potential FFAs.
Following these efforts, a multiple reaction monitoring (MRM) detection protocol was established for the relative quantification of FA in the serum of Syrian golden hamsters. These hamsters, which were observed under diverse conditions, including a standard chow diet, a high-fat diet (HFD), and fenofibrate treatment, were subjected to various treatments, utilizing a pooled sample labeled with a heavy isotope as an internal standard. When partial least squares discriminant analysis (PLS-DA) was used, notable variations were found in these 23 FAs across the four groups.
Overall, the approach created in this study was deemed promising for identifying and quantifying FFAs in real samples.
(1) Yang, L.; Wang, Q; Chen, W.; Yang, J.; Lin, Y.; Lubman, D. M. Trimethylaminoethyl Ester Derivatization and Stable Isotope Derivatization for Enhanced Analysis of Fatty Acids in Biological Samples by Electrospray Ionization Tandem Mass Spectrometry. J. Chromatogr. B 2025, 1264, 124733. DOI:
(2) What are Free Fatty Acids: Properties, Sources, and Significance. MetwareBio 2025.
