Detection and quantitation of fluorescamine-labeled bradykinin, its analogues and metabolites using high-performance liquid chromatography

A sensitive technique is described for detecting and quantitating fluorescamine-labeled kinins and their usual metabolic products using reversed-phase high-performance liquid chromatography (HPLC) linked with a fluorescence detector. Kinins and their enzymatic products were labeled with fluorescamine, subjected to HPLC, and scanned for the fluorescence signal with excitation at 390 nm and emission at 476 nm. The fluorescence signal was linear with bradykinin, Lys-bradykinin and Met-Lys-bradykinin in amounts upward from 2.5 ng. Separation of the fluorescamine-labeled kinins using HPLC was carried out with a solvent system of methanol-triethylammonium formate buffer. Labeled kinins were eluted in the following order: bradykinin, Lys-bradykinin, and Met-Lys-bradykinin. When native (unlabeled) kinins were subjected to HPLC using a solvent system of acetonitrile-triethylammonium formate buffer, the minimum amount of native kinin detected at 210 nm was 1 microgram. All three kinins showed linearity at 210 nm in amounts upward from 1 microgram. Kinins were eluted in the following order: Lys-bradykinin, bradykinin and Met-Lys-bradykinin. The different elution patterns of kinins by means of these two separation techniques provide a useful method for identification of purified kinins. The fluorescamine label provides a 400-fold more sensitive detection technique than ultraviolet absorbance of the native kinins and may be used to identify the metabolic products of kinins.