title: Sensitive determination of the peptide AP301--a motif of TNF-α--from human plasma using HPLC-MS/MS.
abstract: The AP301 peptide mimics the lectin-like domain of TNF-α. The synthetic peptide AP301 (molecular weight 1923.1amu) is composed of 17 amino acids and contains an intramolecular disulfide bond between the N-terminal and the C-terminal cysteine. AP301 interacts with the endothelial sodium channel (ENaC) and activates pulmonary liquid clearance both in vitro and in animal studies. Currently, AP301 is subject to clinical investigations for the treatment of pulmonary oedema. With HPLC-MS/MS on reversed phase chromatography a determination limit of 1ng AP301/mL human plasma can be achieved. The MS-ionisation was done with ESI positive. 50μL of human plasma was mixed with the internal standard (a stable isotope labelled AP301, with a total of 6 carbon 13) in acetonitrile for protein precipitation. After centrifugation a part of the clear supernatant was injected into HPLC-MS/MS. Validation was performed according to FDA-guideline in three batches [U.S. Department of Health and Human Services, Food and Drug Administration (FDA): Guidance for Industry, Bioanalytical Method Validation, May 2001]. By using a 6xC13 isotopically labelled internal standard good precision, accuracy and linearity can be gained. The inter-batch precision (CV) of the quality control samples in human plasma (conc. 2.50/20/240ng/mL) ranged from 5.54 to 10.15%. The inter-batch accuracy (with reference to the mean value) of the quality control samples in plasma ranged from 96.1% to 99.9%. The analyte was stable in human plasma over three freeze/thaw cycles, or for 4h at room temperature, or for at least 20weeks when stored at below -20°C. This method was used for quantifying AP301 after inhalative application in a phase I-study.
authors.affiliation: pharm-analyt Lab., Baden, Austria. daniel.mascher@pharm-analyt.at
authors.forename: Daniel
authors.initials: D
authors.lastname: Mascher
authors.forename: Werner
authors.initials: W
authors.lastname: Tscherwenka
authors.forename: Hermann
authors.initials: H
authors.lastname: Mascher
authors.forename: Bernhard
authors.initials: B
authors.lastname: Fischer
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017321:Clinical Trials, Phase I as Topic / Q000379:methods; D004355:Drug Stability; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000097:blood* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry*
chemical_list: D010455:Peptides; D014409:Tumor Necrosis Factor-alpha