Development of a sensitive and reproducible method for analysis of 22 underivatized amino acids and application of the method to quantify 17 amino acids in wine. Accurate quantification of proline, using an isotopically labeled internal standard.
The goal of this application is to analyze the non-proteinogenic amino acid carbocisteine and its impurities using HPLC-CAD (Charged Aerosol Detection) and to obtain improved sensitivity for the impurities when compared to the existing method.
In this Application Update, we combine into a single method individual improvements made to the standard AAA-Direct gradient program. These individual improvements are now in the AAA-Direct manual. This update shows that such combined changes to the AAA-Direct method improve the performance of our previously published method. This new method successfully determines amino acids in yeast extract-peptone-dextrose yeast culture medium (YPD Broth), and Dulbecco’s modified Eagle’s (with F-12), M199, L-15 (Leibovitz), and McCoy’s 5A media for mammalian cell culture.
Amino acids present in protein formulations serve as buffers, bulking agents, stabilizers, and antioxidants. Eight specific amino acids are commonly used as excipients in protein therapeutic formulations due to their physicochemical properties. These include Arginine, Aspartic acid, Glutamic acid, Lysine, Proline, Glycine, Histidine, and Methionine. The UHPLC column, an Acclaim PA2 column, provides excellent separation characteristics for these un-labeled amino acids. Gradient conditions are adjusted by selecting appropriate ion-pairing reagents, pH and level of organic solvents.
This application note shows that AAA-Direct can be used to determine peptides with lysine hydroxylation. All common amino acids, including hydroxylysine (Hyl), were separated by AAA-Direct, and the presence or absence of Hyl in the fractions confirmed peak identities. The techniques described in this application note show several ways that peptides can be evaluated for identity and purity, how peptides can be desalted, and how much desalting is needed for successful AAADirect amino acid analysis.
