Simultaneous Quantification of Commonly Used Counter Ions in Peptides and Active Pharmaceutical Ingredients by Mixed Mode Chromatography and Evaporative Light Scattering Detection

Abstract

In academia and industry, the analysis of counter ions in small molecules and synthetic peptides represents a great challenge. Due to the frequent use of salt forms and the application of a wider range of counter ions in pharmaceutically used substances, simple and generic methods for quantification are required. Especially, the analysis of trifluoracetic acid (TFA) in synthetic peptides is of high interest. Quantification of TFA is needed to assess the content and safety of synthetic peptides and for the interpretation of functional assay results, respectively. In here, a full quantitative mixed mode high performance liquid chromatography based method coupled to evaporative light scattering detection is presented. Finally, 14 positively and negatively charged counter ions were simultaneously quantified within 30 minutes. The method was validated in terms of specificity, accuracy, precision, limit of quantification, sample stability and carry over as proposed by the International Council of Harmonization. In order to prove the applicability of the procedure, small molecules reference substances and synthetic peptides were analyzed, respectively. The obtained results indicated a successful determination of counter ions in small molecules and differences to expected concentrations of prepared peptide solutions. Furthermore, an unexpectedly high content of sodium was observed for synthetic peptides.

Keywords: Counter ion analysis; Evaporative light scattering detection; Method development; Mixed-mode chromatography; Solid phase peptide synthesis; Trifluoracetic acid.