Detection of peptides by precolumn derivatization with biuret reagent and preconcentration on capillary liquid chromatography columns with electrochemical detection

The separation and detection of biuret complexes of neuropeptides by capill ary liquid chromatography with electrochemical detection was explored. Capi llaries of 25-mu m inner diameter packed with base-resistant, polymer-based reversed-phase particles were used for separation, and C-fiber electrodes were used for detection. Detection at the C-fiber electrode was found to ha ve some differences in relative sensitivity for peptides compared to glassy carbon electrodes used previously. On-column preconcentration of preformed complexes allowed up to 1-mu L samples to be injected with minimal band br oadening resulting in a 100-fold improvement in concentration detection lim it with no effect on mass detection limit. Concentration detection limits r anged from 5 to 59 pM, depending upon the peptide, corresponding to 5-59 am ol injected. The low concentration detection limit was possible because of minimal baseline disturbances, minimal formation of unwanted products, and high efficiency of complex formation associated with biuret derivatization. The method was applied to determination of vasopressin and bradykinin in d ialysates collected with Ei-min sampling frequency from the rat supraoptic nucleus.