Study of the impact of histidine configuration and position on analytical separation of tripeptides

Therapeutic peptides have gained prominence in the pharmaceutical market, but among other reasons, their development was hindered by the risk of amino acid epimerization, particularly histidine (His), which is prone to alter its chiral configuration (L or D) during synthesis. Although strategies like solid phase peptide synthesis (SPPS) and protective groups have helped limit epimerization, they did not eliminate the issue. Currently, a combination of analytical techniques such as Marfey's method, liquid chromatography (LC), and mass spectrometry (MS) is used to assess peptide optical quality. Whereas the former only provides information at the amino acid level after peptide hydrolysis, the latter deals with intact sequences. While effective, these methods are nonetheless time-consuming (multi-steps) and can be challenging, especially for peptides with similar sequences differing only in amino acid position and configuration. The aim of this study was to develop a new analytical method to address this challenge and enable the rapid determination of peptide optical quality in routine analysis. Using duplicate sets of 8 synthetic tripeptides where only the absolute configuration of His was changed (L-His (H) or d-His (h)) within the peptide composed of alanine (Ala, A) and phenylalanine (Phe, F) i.e. H/hAF, H/hFA, AH/hF and AFH/h, we examined how His configuration and position within the model sequence influenced analytical separation by LC and capillary zone electrophoresis (CZE). The use of both separation techniques under different conditions (pH, concentration of organic solvents, gradient slope) allowed to understand how to optimize the parameters to differentiate these peptides. The optimized chromatographic conditions enabled the separation of the 8 tripeptides based not only on the configuration of His but also on its position within the sequence. In addition, these studies showed the impact of the position of the His on the rate of epimerization during synthesis. These results highlight the importance of the configuration and position of amino acids within peptide sequences, especially in the case of His. Furthermore, the LC method demonstrated high sensitivity and reliability, with detection (LOD) and quantification (LOQ) limits of 0.9 µg·L⁻¹ and 2.7 µg·L⁻¹, respectively, suitable for identifying epimerization at the 0.1 % threshold. The method exhibited excellent repeatability on retention time and peak area (coefficient of variation ≤ 0.3 %).