Drug Safety & Pharmacovigilance

There are four established methods for analyzing amino acids: pre-labeled, post-labeled, ion-pairing reversed phase and normal-phase, but each of these methods has disadvantages. The pre-labeled method has problems with derivitization efficiency and cost, while the post-labeled method is usually not compatible with LC-MS due to non-volatile mobile phases. The ion-pairing reversed-phase method has difficulty separating polar amino acids; on the other hand, the normal-phase mode has problems separating all the compounds, especially the Leu and Ile isomers. We have developed a novel amino acid separation column for LC-MS (MS) which can separate all 20 amino acids in protein using a mixed-mode stationary phase structure. We have also estimated separation and detection characteristics using LC-MS instruments. We found two methods to successfully analyze the complete array of 20 amino acids: 1) high throughput separation with Leu/Ile separation in 5min and 2) simple gradient separation. We also found that detection can occur not only in single MS mode but also in triple MS mode. In addition, no derivitization is required and a standard LC-MS (MS) system is sufficient for the analysis. This novel HPLC method will be a powerful tool for amino acid LC-MS(MS) analysis in many different biochemistry applications.