Protamines are a group of highly basic peptides which is sometimes added to insulin formulations to prolong the pharmacological action. In this study, different methods were investigated in order to identify protamine in insulin formulations. Capillary electrophoresis in aqueous and non-aqueous media was tested to separate these peptides with very close amino acid sequences. Different buffers (phosphate or formate, both acidified) and various additives (principally negatively charged and neutral surfactants) were investigated in order to optimize peptide separation. Finally, a micellar electrokinetic capillary chromatography method using a capillary of 120 cm effective length and an aqueous background electrolyte made up of 100 mM phosphate buffer (pH 2) and 50 mM Thesit gave the best results, providing the separation of the four major protamine peptides within 25 min.

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