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

Hideko Kanazawa, Tomomi Nagatsuka, Michiko Miyazaki, Yoshikazu Matsushima

Research output: Contribution to journalArticle

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

Fingerprint

High performance liquid chromatography
Bradykinin
Lasers
Fluorescence
High Pressure Liquid Chromatography
Peptides
Enkephalins
Limit of Detection
Plasma (human)
Trifluoroacetic Acid
Opioid Peptides
Rats
Brain
Buffers
Tissue
Derivatives
Plasmas
Degradation

Keywords

  • Bradykinin
  • Derivatization
  • Enkephalins
  • LC
  • Peptides

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Determination of peptides by high-performance liquid chromatography with laser-induced fluorescence detection. / Kanazawa, Hideko; Nagatsuka, Tomomi; Miyazaki, Michiko; Matsushima, Yoshikazu.

In: Journal of Chromatography A, Vol. 763, No. 1-2, 28.02.1997, p. 23-29.

Research output: Contribution to journalArticle

Kanazawa, Hideko ; Nagatsuka, Tomomi ; Miyazaki, Michiko ; Matsushima, Yoshikazu. / Determination of peptides by high-performance liquid chromatography with laser-induced fluorescence detection. In: Journal of Chromatography A. 1997 ; Vol. 763, No. 1-2. pp. 23-29.
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