Evaluation of the dissociation behavior of hydrophobic ion pairs via HPLC analysis

The objective of this study was to investigate the stability of hydrophobic ion pairs (HIP) of tuftsin and perfluorooctanoic acid in physiological media via high performance liquid chromatography (HPLC).

HIP was formed between the model peptide tuftsin and perfluorooctanoic acid (PFOA). Precipitation efficiency and logP of HIP was determined via HPLC. HIP was characterized via mass spectrometry (MS). Furthermore, the impact of monovalent salts (NaCl, KCl), divalent salts (CaCl₂, MgCl₂), phosphate containing media (pure phosphate, PBS), bile salts (sodium taurocholate, sodium deoxycholate) and fatty acid (sodium myristate) as well as the impact of pH on dissociation of HIP was analyzed via HPLC.

HIP formed in a charge ratio of 1:1 (tuftsin: PFOA) showed a 2512-fold increase in lipophilicity compared to tuftsin. The formation of HIP 1:1 was confirmed by mass spectrometry, showing a peak at the expected mass of 1327 m/z. Pure tuftsin eluted from a C18 reversed phase column after 1.8 min, while the peak of HIP eluted at 6.6 min. The stability of HIP in presence of physiological relevant compounds decreased in the following rank order: sodium deoxycholate > sodium myristate > magnesium chloride = calcium chloride > phosphate > sodium taurocholate > sodium chloride = potassium chloride. The lower the pH, the lower was the stability of HIP.

HPLC analysis offers a valuable method to study the dissociation of hydrophobic ion pairs in physiological relevant media.