Optimization of Ninhydrin Test for Amino Acids by Multiple Reaction Monitoring Analysis

The ninhydrin test is a well-known amino acid identification test used in official specification monographs worldwide. Ruhemann's purple (RP) is considered a key product; however, its formation and the color used for identification can be affected by reaction conditions. Since the quality of various reagents used in the ninhydrin test has varied over time, the reaction efficiency can also be affected. In this study, ninhydrin tests for amino acids and related compounds, as specified in Japan's specifications and standards for food additives (JSFA), were performed using high-purity reagents. Results suggested that the colors of the reaction mixtures for several amino acids, including L-asparagine, L-cystine, and L-lysine salts, were not well-suited for their identification tests, potentially leading to misjudgments. To improve the accuracy of the judgment, optimal reaction conditions were investigated using an RP monitoring method via UHPLC/MS/MS. An analysis of the reaction mixtures of L-cystine by HPLC-PDA revealed that sufficient production of RP, relative to other brown-colored by-products, is important for achieving the desired purple coloration. Based on these findings, improved ninhydrin test conditions that can be adopted in JSFA in the future were established. This study provides valuable insights into the ninhydrin reaction based on theoretical and scientific results and contributes to the improvement of identification tests using the ninhydrin reaction specified by the JSFA.