Enhancing the Accuracy of Identification and Relative Quantification of Unsaturated Fatty Acids in Serum via a Stable Isotope-Labeled Double Derivatization Strategy

Accurate identification and quantification of fatty acids are critical for investigating their biological function in disease models. Although several derivatization methods have been proposed for identifying the positions of C═C bonds in unsaturated fatty acids, poor ionization efficiency of the carboxyl group leads to lower intensity of molecular ion peaks, making their identification difficult and interfering with the accuracy of quantification based on peak areas of characteristic ion pairs. In this study, a strategy of stable isotope-labeled carboxyl derivatization combined with C═C derivatization was employed for simultaneously the identification and quantification of fatty acids using d₀/d₉-5-amino-N,N,N-trimethylpentane-1-ammonium iodide (d₀/d₉-ATPAI) to label the carboxyl group and m-chloroperoxybenzoic acid to label C═C bonds. The stable isotope-labeled quaternary amine groups in the novel carboxyl derivatization reagent d₀/d₉-ATPAI can enhance the accuracy of the recognition of characteristic ion pairs to facilitate the structural elucidation of various fatty acids. The heavy isotope-labeled fatty acids can be served as internal standards to achieve accurate relative quantification of the C═C position isomers of individual unsaturated fatty acids among samples based on the peak area ratio of the characteristic ion pairs. Unsaturated fatty acid C═C positional isomers were quantified using aldehyde or alkenyl diagnostic ions. In addition, saturated fatty acids were quantified using the m/z 86.09679 cyclamine characteristic ion. This approach enhanced the detection sensitivity of fatty acids by 60,000 times, allowing for the characterization of 70 fatty acids in rat serum, including 26 unsaturated fatty acid C═C positional isomers. Pseudotargeted metabolomics analysis of serum fatty acids revealed alterations in the fatty acid metabolic pathway during diabetic cognitive dysfunction. Overall, the proposed method, with high sensitivity and wide coverage, could provide accurate identification and relative quantification of various fatty acids in complex matrices.