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Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile contaminants and naturally occurring toxins in foods. However, polar compounds, such as hydrophilic polypeptides and quaternary ammonium salts, are often not satisfactorily separated by RPLC and present a challenge for analytical scientists. In this study, hydrophilic interaction liquid chromatography (HILIC), on an amide-based stationary phase in combination with electrospray ionization (ESI) tandem mass spectrometry (MS-MS), is successfully employed to simultaneously separate polar mushroom toxins, including amanitins and phallotoxins, which are cyclic oligopeptides and muscarine, a quaternary ammonium compound, in mushrooms. The sensitivity of different ionization modes is studied, and the positive ionization mode is found to provide a more sensitive and effective tool for the unambiguous identification of the concerned polar toxins because of their characteristic fragmentation patterns. The properties of the mobile phase are also found to have significant impacts on the separation. At a high acetonitrile (ACN) concentration, hydrophilic interaction dominates, and all analytes under study demonstrate a much higher affinity with the stationary phase. The addition of methanol (MeOH) as a modifier could further enhance the HILIC separation for amanitins, phallotoxins, and muscarine. Valley-tovalley separation is achieved upon the optimatization of the mobile phase (comprising of ACN, MeOH, and ammonium formate buffer at pH ~ 3.5) and the solvent gradient. HILIC coupled with ESIMS-MS is demonstrated to be a novel technique for the simultaneous separation and confirmatory analysis of the concerned polar toxins by providing an environment of solubility and retention that could not be achieved through the use of RPLC.

Separation of Polar Mushroom Toxins by Mixed-Mode Hydrophilic and Ionic Interaction Liquid Chromatography—Electrospray Ionization-Mass Spectrometry