Detection of bioactive oligopeptides after microbore HPLC with electrochemical detection of their Cu(II) complexes: effect of operating parameters on sensitivity and selectivity.

We used a microbore reversed phase column for acetonitrile/0.1% aqueous TFA gradient elution separation of peptides with the detection of their copper complexes by electrochemical detection. The copper complexes are formed in a short (1 or 1.5 min) postcolumn reactor following mixing of the eluent with the postcolumn reaction phase. Detection can be at an upstream anode or a downstream cathode of a dual-electrode electrochemical detector. The following parameters have been investigated for their effect on the sensitivity and the selectivity of the procedure: postcolumn pH, buffer type, temperature, reaction time, and anode potential. Of the 23 bioactive peptides used, there are several that fall into classes according to their chemical and electrochemical behavior with copper(II): those with a blocked terminal amine, those with aspartate in the third position, those that have an electroactive amino acid, and those that have a cyclic structure formed by the amide backbone through a Cys-Cys disulfide bridge. Depending on these attributes, the operating parameters have an influence on the sensitivity of the determination. Among the more well-defined results are the following. Uncomplicated peptides with a free amine terminus react rapidly in the postcolumn reactor and give signals in the range predicted by theory. There is evidence that longer peptides, and those with a blocked amine terminus, have a sensitivity limited by kinetic factors. The oxidations of tyrosine and tryptophan in peptides are dramatically influenced by buffer type at pH 9.8. At pH 8.0, there is no signal from several peptides in phosphate buffer, while in borate there is a signal.