Quantification of14C-Labeled Amino Acids by Reverse-Phase High Performance Liquid Chromatography

This study provided the first evidence indicating that o-phthalaldehyde-amino acid-derivatives are unstable during high performance liquid chromatographic separation on a reverse-phase C18 column. Instability of these derivatives on the column resulted in the formation of 14C-labeled degradation products, and caused excessive 14C-fronting of amino acid peaks. The column halflives of glutamate, arginine, and ornithine were 16, 40, and 54 minutes, respectively. Derivative break-down during derivatization also gave rise to these degradation products in the 14C-distribution profile. Thus, if OPA-derivatization is used to quantify the 14C-distribution in an unknown mixture of amino acids it is necessary to determine correction factors for each amino acid of interest. A method which uses stable phenylthiocarbamate-derivatives did not exhibit these problems and is therefore more suitable for the quantification of 14C-amino acids.