Capillary electrophoresis analysis of hydrolysis, isomerization and enantiornerization of aspartyl model tripeptides in acidic and alkaline solution

In order to investigate the degradation of two aspartyl tripeptides, Gly-Asp-PheNH(2) and Phe-Asp-GlyNH(2) in solution capillary, electrophoresis methods were developed and validated. Separation of most degradation products including those arising from isomerization and enantiomerization of the Asp residues was achieved in a 50 mM sodium phosphate buffer, pH 3.0. Resolution of comigrating compounds could be achieved by addition of cyclodextrins to the background electrolyte. For tripeptide derivatives the assays were linear in the range of 0.015-3.0 mmol/l. Some dipeptides and amino acids exhibited a narrower linear range due to low UV absorbance. The limits of detection were in the range of 0.005-0.1 mmol/l. Incubation of the model peptides was carried out at pH 2 and 10. At pH 2, degradation of the peptides proceeded via C-terminal deamidation and peptide backbone hydrolysis. In contrast, isomerization and enantiomerization were observed in combination with deamidation at pH 10. Generally, degradation of Phe-Asp-GlyNH(2) Proceeded faster compared to Gly-Asp-PheNH2 due to steric hindrance by the phenyl side chain. (c) 2006 Elsevier B.V. All rights reserved.