Capillary electrophoresis analysis of the degradation of the aspartyl tripeptide Phe-Asp-GlyOH at pH 2.0 and 7.4 under forced conditions.

The degradation of the tripeptide l-Phe-alpha-l-Asp-GlyOH was studied at 80 degrees C and pH 2.0 and 7.4 by capillary electrophoresis. Separation of most known as well as unknown degradation products was achieved in a 50mM sodium phosphate buffer, pH 3.0. The diastereomers l-Phe-alpha-l-Asp-GlyOH/l-Phe-alpha-d-Asp-GlyOH could only be separated upon addition of 16mg/ml carboxymethyl-beta-cyclodextrin and 5% acetonitrile to the background electrolyte. Compound identification was performed by capillary electrophoresis-electrospray ionization-mass spectrometry. In addition to Asp isomerization and epimerization products as well as hydrolysis products four diketopiperazine derivatives were identified. Moreover, two degradation products were observed containing the amino acids Asp, Gly and Phe but the unequivocal assignment could not be accomplished based on the mass spectra. Following validation with regard to linearity, range, limit of detection, limit of quantitation and precision the assay was applied to the analysis of the incubation solutions. While peptide backbone hydrolysis dominated at pH 2.0, isomerization and enantiomerization yielding beta-Asp and d-Asp peptides as well as cyclization to diketopiperazine derivatives were observed at pH 7.4. The diketopiperazines were the dominant reaction products amounting to about 85% of the compounds detected after the maximal incubation time of 240h. A kinetic model was used to fit the concentration versus time data.