Due to the strong hydrophilic property, melamine is less retained by the reversed phased column. In our study, we found melamine without derivatization eluted fast on a reversed phase C18 column. It was even difficult to separate it from the reagent peak, no matter how slow gradient elution process was used. Under this condition, peaks caused by reagent or matrix may coelute and interfere with the qualitative and quantitative results. This phenomenon
no longer existed when derivatization was applied. In this study, the hydrophobicity of melamine was increased through derivatizing with hydrophobic MASC. Correspondingly, the interaction between melamine derivative and the reversed phase C18 column was also greatly increased. Therefore, mobile phase simply composed of acetonitrile and water was sufficient enough for the HPLC analysis of melamine. As shown in Fig. 2, melamine eluted at 4.8 min on a C18 column.