title: Quantitative analysis of exogenous peptides in plasma using immobilized enzyme cleavage and gas chromatography-mass spectrometry with negative ion chemical ionization.
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.
authors.affiliation: College of Pharmacy, University of Texas at Austin, 78712, USA.
authors.forename: C D
authors.initials: CD
authors.lastname: Márquez
authors.forename: M L
authors.initials: ML
authors.lastname: Lee
authors.forename: S T
authors.initials: ST
authors.lastname: Weintraub
authors.forename: P C
authors.initials: PC
authors.lastname: Smith
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D004355:Drug Stability; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives / Q000097:blood; D004800:Enzymes, Immobilized; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D009842:Oligopeptides / Q000097:blood; D010446:Peptide Fragments / Q000097:blood; D010455:Peptides / Q000097:blood*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014357:Trypsin
chemical_list: D004800:Enzymes, Immobilized; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D004744:Enkephalin, Methionine; C033453:enkephalin-Met, Arg(6)-Gly(7)-Leu(8)-; D002918:Chymotrypsin; D014357:Trypsin