Capillary liquid chromatography of multiple peptides with on-line capillary electrophoresis immunoassay detection

A competitive immunoassay for neuropeptide Y (NPY) based on capillary elect rophoresis (CE) with laser-induced fluorescence detection was developed uti lizing polyclonal antisera as the immunoreagent and fluorescein-labeled NPY as the tracer. The assay was performed with on-line mixing of reagents, au tomated injections, and a 3 s separation time. The assay had a detection li mit of 850 pm. To detect NPY at lower concentrations, the assay was coupled on-line to reversed-phase capillary liquid chromatography (LC). In this ar rangement, 5 muL samples were preconcentrated by capillary LC and eluted by a gradient of isopropanol-containing mobile phase. The resulting chromatog raphic peaks were monitored by the CE immunoassay. With preconcentration, t he concentration detection limit was improved to 40 pm and NPY could be mea sured in push-pull perfusion samples collected from the paraventricular nuc leus of freely moving rats. The technique was extended to simultaneous dete ction of NPY and glucagon secretion from islets of Langerhans.