Determination of peptides by high-performance liquid chromatography with laser-induced fluorescence detection

Peptides were determined by high-performance liquid chromatography (HPLC) with laser-induced fluorescence detection. Detection was based on pre-column fluorescence derivatization of peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in acetonitrile (MeCN)-0.1 M borate buffer (pH 8.0) at 40 degrees C for 10 min. The peptide derivatives were separated on a reversed-phase column with trifluoroacetic acid-MeCN and determined fluorometrically at 530 nm with excitation at 470 nm. The method was applied to the determination of enkephalins in rat brain and to a degradation study of bradykinin in human plasma. Optimization of the reaction conditions and the use of a semi-micro-column (100 x 2 mm I.D., 2 microns) made the detection limit of the peptides as low as 5-10 fmol. The detection limits of enkephalin and bradykinin were 20 and 5 fmol, respectively, using HPLC with laser-induced fluorescence detection. The method was sensitive enough to permit the quantitative determination of opioid peptides and bradykinin in tissue and plasma samples.