Quantitative analysis of exogenous peptides in plasma using immobilized enzyme cleavage and gas chromatography-mass spectrometry with negative ion chemical ionization

Cristina D. Márquez, Mu Lan Lee, Susan T. Weintraub, Philip C. Smith

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

A method is presented for the analysis of peptides in plasma at picomole to femtomole levels. Peptides are isolated from plasma by solid-phase extraction, the peptide of interest is purified by reversed-phase high- performance liquid chromatography (HPLC) and selectively digested using immobilized trypsin or chymotrypsin to yield specific di- or tripeptides. These di- and tripeptides are esterified using heptafluorobutyric anhydride, alkylated with pentafluorobenzyl bromide, then quantified by gas chromatography-mass spectrometry with negative ion chemical ionization. This method has been evaluated for a model synthetic heptapeptide, using a deuterium labeled analog as an internal standard. The half-life of the heptapeptide in human plasma was found to be 2 min. Extraction efficiencies of a tritiated peptide of similar size to the heptapeptide, [3H]DSLET, from plasma using either C18 or strong cation-exchange columns were 85±3 and 70±2%, respectively. Quantitation of fragments from the heptapeptide indicated that the analysis was linear from 1-50 ng of the heptapeptide per ml of plasma. This method was subsequently employed for pharmacokinetic studies of the biologically active peptide Met-enkephalin-Arg-Gly-Leu, where linearity was obtained from 50 to 1000 ng/ml in rat plasma. This method demonstrated negligible side reaction by-products due to autolysis, and has potential for extensive use given the wide availability of gas chromatography-mass spectrometry.

Original languageEnglish (US)
Pages (from-to)9-21
Number of pages13
JournalJournal of Chromatography B: Biomedical Applications
Volume700
Issue number1-2
DOIs
StatePublished - Oct 24 1997

Keywords

  • Enzymes
  • Peptides

ASJC Scopus subject areas

  • Chemistry(all)

Fingerprint Dive into the research topics of 'Quantitative analysis of exogenous peptides in plasma using immobilized enzyme cleavage and gas chromatography-mass spectrometry with negative ion chemical ionization'. Together they form a unique fingerprint.

  • Cite this