Manipulation of ion-pairing reagents for reversed-phase high-performance liquid chromatographic separation of phosphorylated opioid peptides from their non-phosphorylated analogues.

The use of reversed-phase high-performance liquid chromatography for the separation of a mixture of 14 phosphorylated and non-phosphorylated enkephalins is described. The influence of two homologous series of hydrophobic ion-pairing reagents, consisting of perfluorinated carboxylic (trifluoroacetic, pentafluoropropionic and hexafluorobutyric) acids and sodium salts of sulfonic (butane-, hexane- and heptane-) acids, on the retention of enkephalin peptides was investigated. The incorporation of the phosphate group reduces retention time in proportion with the resulting change in hydrophobicity of the peptide. All peptides exhibit increase in retention time with increase in the counter ion hydrophobicity. The increase is proportional to the number of positively charged groups present in a peptide. Phosphopeptides show small increases in retention times than their corresponding non-phospho derivatives. The near-neighbor effect of the Tyr-O-phosphate group is responsible for suppression of the ion-pairing interaction of the mobile phase counter ions with the positively charged terminal amino group of enkephalins.