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.
Kanazawa
H
H
Kyoritsu College of Pharmacy, Tokyo, Japan.
Nagatsuka
T
T
Miyazaki
M
M
Matsushima
Y
Y
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D000097:Acetonitriles; D000595:Amino Acid Sequence; D000818:Animals; D001920:Bradykinin / Q000097:blood; D002540:Cerebral Cortex / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003342:Corpus Striatum / Q000737:chemistry; D004743:Enkephalin, Leucine / Q000032:analysis; D004744:Enkephalin, Methionine / Q000032:analysis; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007031:Hypothalamus / Q000737:chemistry; D008297:Male; D010455:Peptides / Q000032:analysis*; D051381:Rats; D013050:Spectrometry, Fluorescence
D000097:Acetonitriles; D005456:Fluorescent Dyes; D010455:Peptides; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D004744:Enkephalin, Methionine; D004743:Enkephalin, Leucine; D009327:4-Chloro-7-nitrobenzofurazan; D001920:Bradykinin; C032159:acetonitrile