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

Hideko Kanazawa, Tomomi Nagatsuka, Michiko Miyazaki, Yoshikazu Matsushima

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26 Citations (Scopus)

Abstract

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 berate buffer (pH 8.0) at 40°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 (100x2 mm I.D., 2 μm) 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.

Original languageEnglish
Pages (from-to)23-29
Number of pages7
JournalJournal of Chromatography A
Volume763
Issue number1-2
DOIs
Publication statusPublished - 1997 Feb 28

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Keywords

  • Bradykinin
  • Derivatization
  • Enkephalins
  • LC
  • Peptides

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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