<p>Reliable determination of long and very long chain fatty acids, which are abundant in marine foods, remains a challenge. In this work, 1-(2-aminoethyl)pyridin-1-ium bromide hydrobromide (AEPy) is proposed for isotope-coded derivatization of fatty acids released from oils through saponification and extraction (esterified and free FAs). Pyridine deuteration was accomplished, and three AEPy isotopologues were synthesized (AEPy-<i>d</i>_<i>n</i>, <i>n</i> = 0,3,5). The introduction of permanent positive charge during derivatization facilitated electrospray ionization process in positive mode. Once the molecular structures were confirmed by high-resolution mass spectrometry and NMR, the main emphasis was placed on setting a liquid chromatography-ion trap mass spectrometry procedure allowing the detection/annotation and quantification of fatty acids (FAs) in real-world samples, although without distinguishing their positional isomers. In an untargeted approach, isotope cluster analysis of data obtained for a mixture of two differentially labeled sample portions allowed annotation of fatty acids, revealing unexpected presence of C24:1 in cod liver oil and C28:8 in menhaden oil. In quantitative analysis based on selected ion monitoring, light AEPy was used to derivatize the calibrator (cod liver oil CRM) and real-world samples, while heavy AEPy derivatives served as internal standards in external calibration and were applied for bracketing isotope dilution. For FAs certified in cod liver oil, the method LOQs were in the range 0.03–0.33&#xa0;mg&#xa0;g⁻¹. Three oil dietary supplements were analyzed, confirming high relative abundance of C20:5, C22:6, C18:1, C16:0 in krill; C18:2, C18:1, C20:5, C22:6 in salmon and C20:5, C22:6, C22:5 in anchovy/sardine products. The two quantification methods provided consistent results; however, bracketing isotope dilution offers better protection against matrix interferences.</p> Graphical Abstract <p></p>