Impurity profiling of synthetic cyclic peptides based on orthogonality between hydrophilic-interaction and reversed-phase liquid chromatography.

Owing to their high selectivity and low toxicity, synthetic cyclic peptides hold promise as bioactive therapeutics. The corresponding impurity analysis typically relies on reversed-phase high-performance liquid chromatography (RP-HPLC), which, however, often provides inaccurate results because of the abundance of chemically similar impurities. To bridge this gap, hydrophilic-interaction liquid chromatography (HILIC), a technique orthogonal to RP-HPLC, was herein used with an ultraviolet detector to analyze the impurities of four cyclic peptides. Each of these peptides comprises nine amino acids (both natural and nonnatural) and features neutral, acidic, basic, or zwitterionic residues incorporated at positions 3 and 6. Three polymer-based HILIC columns (acidic, basic and zwitterionic) were newly employed, and seven mobile phases were used to investigate the effects of additives and pH. Derringer's desirability functions based on five criteria (purity, impurity factor, peak symmetry factor, theoretical plate number, and retention time) were employed to identify optimal screening conditions. Ammonium acetate was identified as an effective additive, and polymeric selectors with vinylpyridine and phosphoryl choline residues were shown to be effective irrespective of the peptide features. Optimized HILIC conditions were established for each peptide, and the corresponding impurity elution profiles were compared with those obtained by RP-HPLC using a column filled with octadecylsilyl-functionalized silica. RP-HPLC and HILIC were revealed to be mutually orthogonal, i.e., most impurities with small RP-HPLC retention times had large HILIC retention times, and vice versa. The results help make the purity evaluation of synthetic peptides more accurate, thus paving the way for their efficient purification.