Two "ghost peaks" appeared in the process of developing an impurities analytical method for an electron-rich aniline compound by RP-HPLC. Surprisingly, the "ghost peaks" disappeared when the mobile phase was re-prepared. The "ghost peak" slowly began to reappear and gradually increased after increasing the injection volume or changing different instruments and columns. In this paper, the structures of two "ghost peaks" were analyzed by LC-MS/MS, which were identified as the dimer of the main peak and the oxidation products of the dimer. The mechanism was confirmed by studying the "ghost peaks" appearance phenomenon in the mobile phase with different pH values, different types of buffer salts, and different brand chromatographic columns. The potential chemical behavior of this type of compound on the column was clearly revealed in the process of RP-HPLC separation. The coupling reaction of electron-rich aniline compounds can be induced by partial or complete dissociation of the silanol group to Si-O-in the stationary phase of a reversed-phase chromatographic column under neutral and basic conditions of the mobile phase. Adding inorganic salts to the mobile phase can inhibit the reaction, and the higher salt concentration, the stronger the effect.
Keywords: Electron-rich aniline compound; Ghost peak; In reversed-phase liquid chromatography; Mechanism and solution.
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