Sensitive Analysis of Underivatized Amino Acids and Peptides Using UHPLC with Charged Aerosol Detection

The quantification of derivatized amino acids by (U)HPLC with UV detection is one of the most widely used methods to date. Unfortunately, separation and detection of underivatized amino acids is difficult as many of them are similar in structure, and few possess adequate chromophores. Derivatizing agents, either pre- or post-column, may overcome some of these issues, but can add time and complexity to the method. Presented here is a simple UHPLC gradient method using a new RSLC 2.2 µm 2.1 x 250 mm column with charged aerosol detection for the measurement of 20 underivatized amino acids. Retention of hydrophilic amino acids was achieved by the addition of a volatile ion-pairing agent heptafluorobutyric acid (HFBA) to the mobile phase. All analytes were resolved in less than 10 minutes with a limit of detection of low nanograms on column, making this approach ideal for the analysis of amino acids standards and starting materials in quality control. The work was expanded with a comparison of charged aerosol detection with inverse gradient and UV detection for monitoring digestion of bovine serum albumin by Trypsin. This technique offers a complementary approach to the traditional UV @ 214 nm with potential for more uniform response across both large and small peptide chains and the ability to measure free amino acids which may be missed because of insufficient chromophores. These fast UHPLC methods provide a simple alternative to the more time consuming or less sensitive amino acid methods typically used to measure analytes in a simple matrix.