title: LC/MS/MS plasma assay for the peptidomimetic VLA4 antagonist I and its major active metabolite II: for treatment of asthma by inhalation.
abstract: In vitro and in animals, I is a potent and specific peptidomimetic for the potential treatment of airway inflammation in the pathogenesis of asthma. Preclinical studies indicated extensive conversion of I to an active metabolite II, and thus, a very sensitive assay for I and II was needed to support an inhalation ascending-dose study in man. The LC/MS/MS plasma/urine assay method (1.0 ml of sample) involves the following: liquid-liquid extraction of acidified plasma into pentane-ethyl acetate (90:10 v/v); evaporation of the organic extract, reconstitution into methanol; addition of water to the methanolic extract and freezing. After thawing, the extract is centrifuged and the clear supernatant injected for chromatography. Extract is chromatographed on a YMC ODS-AM column (50 x 2.0 mm). For detection, a Sciex 365 LC/MS/MS with an electrospray inlet and used in the positive ion, multiple reaction monitoring mode was used to monitor precursor-->fragment ions of m/z 709-->594 for I and m/z 513-->380 for II. The plasma assay was linear over the concentration range of 0.1-100 ng/ml in plasma for I and II. Accuracy and precision for I ranged from 97.9 to 102.1% of nominal with a 0.84-10.65% CV; similarly for II, 98.0-101.7% and 1.39-9.28% CV, respectively. Extraction recovery averaged 63.7% for I and 64.9% for II. This general assay methodology may be applied to assay small acidic peptides and peptidomimetics from biological fluids by LC/MS/MS.
authors.affiliation: WP75A-303, Merck Research Laboratories, West Point, PA 19486, USA. alison_fisher@merck.com
authors.forename: Alison L
authors.initials: AL
authors.lastname: Fisher
authors.forename: Elizabeth
authors.initials: E
authors.lastname: DePuy
authors.forename: Andrew
authors.initials: A
authors.lastname: Jayaraj
authors.forename: Conrad
authors.initials: C
authors.lastname: Raab
authors.forename: Matt
authors.initials: M
authors.lastname: Braun
authors.forename: Michel
authors.initials: M
authors.lastname: Ellis-Hutchings
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: John D
authors.initials: JD
authors.lastname: Rogers
authors.forename: Donald G
authors.initials: DG
authors.lastname: Musson
mesh_terms: D000280:Administration, Inhalation; D000328:Adult; D018927:Anti-Asthmatic Agents / Q000097:blood* / Q000493:pharmacokinetics / Q000652:urine; D001249:Asthma / Q000097:blood* / Q000188:drug therapy / Q000652:urine; D002853:Chromatography, Liquid; D006801:Humans; D039041:Integrin alpha4beta1; D016023:Integrins / Q000037:antagonists & inhibitors*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D015394:Molecular Structure; D016201:Receptors, Lymphocyte Homing / Q000037:antagonists & inhibitors*; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D018927:Anti-Asthmatic Agents; D039041:Integrin alpha4beta1; D016023:Integrins; D016201:Receptors, Lymphocyte Homing