Highly sensitive determination of polyunsaturated fatty acids by permanently charged derivatization-liquid...

Polyunsaturated fatty acids (PUFAs) are essential dietary components. However, they must be obtained from food because the human body cannot synthesize them in sufficient quantities. Furthermore, PUFA content can vary slightly depending on the type of food and storage conditions, making it necessary to establish accurate and highly sensitive methods for their quantification. However, their sensitive determination remains challenging because of the poor ionization efficiency of conventional liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-MS analyses. In this study, we developed a highly sensitive LC with tandem mass spectrometry (LC-MS/MS) method for PUFA analysis based on permanently charged derivatization using TPP-AEP. This derivatization reagent has a quaternary ammonium structure, which is required for efficient ionization under mild electrospray ionization (ESI)-MS conditions. Consequently, the [M]⁺ ions of PUFAs derivatized with TPP-AEP were detected with high sensitivity. Solid-phase extraction (SPE) was used for sample preparation to remove impurities. Furthermore, a C18 column was used for LC separation with an acidic mobile phase to achieve good peak shapes and stable detection sensitivity in PUFA analysis. The developed method exhibited excellent linearity (r² ≥ 0.9983) and achieved a low limit of quantification in the range of 2.80-3.29 ng/mL for the examined PUFAs (5,8,11,14,17-eicosapentaenoic acid, 4,7,10,13,16,19-docosahexaenoic acid, arachidonic acid, and linoleic acid). Application of the method to PUFAs extracted from muscle tissues of four marine fish species demonstrated that the combination of lipid extraction, alkaline hydrolysis, derivatization, SPE, and LC-MS/MS allowed for the quantification of both free and total PUFA content with high sensitivity.