title: In vitro degradation of the antimicrobial human peptide HEM-gamma 130-146 in plasma analyzed by a validated quantitative LC-MS/MS procedure.
abstract: In stability studies during preclinical drug development, the human antimicrobial peptide hHEM-gamma 130-146 shows progressive N-terminal degradation in plasma. To determine this effect, we developed and validated a selective and quantitative muHPLC-MS/MS procedure for this compound. Following deproteinization by precipitation, reversed-phase separation is performed with a time-saving two-column design online coupled to an ion trap mass spectrometer for electrospray ionization MS detection. Using a linear calibration curve obtained with synthetic external standards ranging nearly two orders of magnitude, we achieved good precision (repeatability and reproducibility: 5-15%), accuracy (-3 to 15%), and ruggedness with a lower limit of quantification at 0.29 microg/ml plasma (0.15 microM). Because of good linearity (r2>0.999), the recovery (84+/-3%) and ion suppression (86+/-4% remaining intensity) were calculated from specifically prepared calibration curves. The developed procedure was applied to human and animal plasma samples. Incubations in the presence and absence of proteinase inhibitors revealed at least an aminopeptidase M activity for the initial N-terminal truncation of tryptophan (W130) and a putative glutaminyl-peptide cyclotransferase activity for the resulting intermediate starting with the bared glutamine residue (Q131). The calculated periods of half-change demonstrated exceeding interspecies variations, whereas the intraspecies variations were only between 20 and 30%. The current procedure is valuable as a generic method for pharmaceutical purposes, and data give important information for further development toward a potential natural drug candidate.
authors.affiliation: IPF PharmaCeuticals GmbH, D-30625 Hannover, Germany. h.john@ipf-pharmaceuticals.de
authors.forename: Harald
authors.initials: H
authors.lastname: John
authors.forename: Kham Dieu
authors.initials: KD
authors.lastname: Huynh
authors.forename: Christiane
authors.initials: C
authors.lastname: Hedtmann
authors.forename: Michael
authors.initials: M
authors.lastname: Walden
authors.forename: Axel
authors.initials: A
authors.lastname: Schulz
authors.forename: Friedrich Birger
authors.initials: FB
authors.lastname: Anspach
authors.forename: Wolf-Georg
authors.initials: WG
authors.lastname: Forssmann
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000097:blood* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D006801:Humans; D008565:Membrane Proteins / Q000097:blood* / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods* / Q000592:standards
chemical_list: D023181:Antimicrobial Cationic Peptides; D008565:Membrane Proteins; D010446:Peptide Fragments; C095128:NCKAP1L protein, human