MANIPULATION OF ION-PAIRING REAGENTS FOR REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SEPARATION OF PHOSPHORYLATED OPIOID-PEPTIDES FROM THEIR NONPHOSPHORYLATED ANALOGS

The use of reversed-phase high-performance liquid chromatography for t he separation of a mixture of 14 phosphorylated and non-phosphorylated enkephalins is described. The influence of two homologous series of h ydrophobic ion-pairing reagents, consisting of perfluorinated carboxyl ic (trifluoroacetic, pentafluoropropionic and hexafluorobutyric) acids and sodium salts of sulfonic (butane-, hexane- and heptane-) acids, o n the retention of enkephalin peptides was investigated. The incorpora tion of the phosphate group reduces retention time in proportion with the resulting change in hydrophobicity of the peptide. All peptides ex hibit increase in retention time with increase in the counter ion hydr ophobicity. The increase is proportional to the number of positively c harged groups present in a peptide. Phosphopeptides show small increas es 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 co unter ions with the positively charged terminal amino group of enkepha lins.