A non-derivatized high-performance liquid chromatography method for simultaneous quantification of hydroxyl acids and amino acids in ammonolysis reactions

Abstract

A non-derivatized high-performance liquid chromatographic (HPLC) method was developed for the simultaneous quantification of hydroxyl acids and their amination products in ammonolysis reaction mixtures. By optimizing the mobile phase composition and pH (0.04 M KH₂PO₄–5% methanol, pH = 2.7), complete separation of hydroxyl acids and their amination products was achieved, using lactic acid as a model substrate. Lactic acid exhibited excellent linearity within the range of 0.02 to 25 mM (R² = 0.99968), with a relative standard deviation (RSD, n = 6) of 3.51% and a recovery ratio between 96.10% and 102.23%. Similarly, alanine exhibited good linearity from 0.02 to 50 mM (R² = 0.99999) with an RSD of 4.04% and recovery ratios between 97.30% and 100.03%. Both analytes demonstrated good intra-day precision, with RSDs of 4.23% and 1.18%, respectively. The substrate universality tests confirmed the method's ability to rapidly and effectively separate other hydroxyl acids and their corresponding amino acids in ammonolysis reactions. The results of quantitative comparison with AQC, PITC, and OPA derivatization also proved the method to be a reliable approach for the individual or simultaneous quantification of hydroxyl acids and amino acids, offering essential support for monitoring the conversion ratio and selectivity in hydroxyl acid amination processes. Moreover, it provides valuable guidance for optimizing the amination reaction of hydroxyl groups, potentially extending catalytic advantages to other related reactions.