Enhanced Chromatographic Separation for Quantitative Accuracy of Palytoxin

Palytoxin (PlTX) is one of the most toxic non-protein molecules extracted from soft corals, posing significant risks within the food chain. To mitigate these risks, regulatory agencies prioritize developing reliable analytical techniques for monitoring and risk management of PlTX in marine organisms. The analysis of PlTX using liquid chromatography-tandem mass spectrometry (LC/MS/MS) has become a preferred method due to its high sensitivity and specificity. However, previous studies have reported challenges in achieving sharp peak shapes and adequate resolution in PlTX detection, which can compromise the accuracy and reliability of quantification. Therefore, PlTX monitoring is actively performed using an emerging and enhanced LC/MS/MS method. To ensure accurate quantification of PlTX, which is present in trace amounts in marine organisms, it is essential to optimize chromatographic separation and ionization. In this study, we optimized the separation conditions to improve PlTX quantification through the adjustment of a buffer system, specifically increasing the concentration of ammonium formate using reverse-phase liquid chromatography coupled with multiple reaction-monitoring mass spectrometry (RPLC/MRM-MS). This optimization significantly improved peak sharpness and resolution, enhancing the overall analytical performance of LC–MS/MS for PlTX detection in marine samples. Our findings suggest that the optimized method can serve as a reliable reference for developing standardized protocols aimed at improving PlTX analysis.