Enantiomeric separation of TAPP, H–Tyr–(d)Ala–Phe–Phe–NH2, by capillary electrophoresis using 18-crown-6-tetracarboxylic acid as a chiral selector

A capillary electrophoresis method for the enantiomeric separation of the tetrapeptide H–Tyr–(d)Ala–Phe–Phe–NH2 (TAPP), has been developed and validated. The preferred background electrolyte (BGE) consisted of 0.1 M aqueous phosphoric acid adjusted to pH 3.0 with triethanolamine. The chiral selectors 18-crown-6-tetracarboxylic acid (18C6H4) and heptakis(2,6-di-O-methyl)-β-cyclodextrin (2,6-DM-β-CD) were compared and the crown ether 18C6H4 was found to be superior. The separation of TAPP and its enantiomer was accomplished within 30 min with a resolution greater than 3.5. The method was then investigated with respect to selectivity, linearity, accuracy, range, precision, detection limit (DL), quantitation limit (QL) and robustness, essentially following International Conference of Harmonisation (ICH) guidelines for the validation of analytical methods. The DL and QL for the TAPP enantiomer were found to be 0.3 and 0.8%, respectively, at the target TAPP concentration of 1 mg/ml. Robustness was tested using a full factorial design for the following four experimental variables varied at two levels: pH of the BGE, chiral selector concentration in the BGE, phosphoric acid concentration in the BGE, and temperature. The method showed good performance with respect to all of the validation parameters, and proved to be robust to changes in the experimental parameters within the tested domain.