title: Simultaneous analysis of oxybutynin and its active metabolite N-desethyl oxybutynin in human plasma by stable isotope dilution LC-MS/MS to support a bioequivalence study.
abstract: An isotope dilution high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determination of oxybutynin and its pharmacologically active metabolite N-desethyl oxybutynin in human plasma. Extraction of oxybutynin, its metabolite and their deuterated analogs as internal standards (ISs) from 300 μL human plasma was carried out by liquid-liquid extraction with methyl tert-butyl ether-ethyl acetate solvent mixture. Chromatographic separation of analytes was performed on Cosmosil C18 (150 mm × 4.6 mm, 5 μm) column under isocratic conditions with acetonitrile-1.0mM ammonium acetate (90:10, v/v) as the mobile phase. Six endogenous plasma phospholipids (496.3/184.0, 524.3/184.0, 758.5/184.0, 786.5/184.0, 806.5/184.0 and 810.5/184.0) were monitored to determine the extraction efficiency under different extraction conditions. The precursor→product ion transition for both the analytes and ISs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ionization mode. The method was validated over a concentration range of 0.050-10.0 ng/mL for oxybutynin and 0.500-100 ng/mL for N-desethyl oxybutynin. The mean extraction recovery for analytes (80.4%) and ISs (76.9%) was consistent across five QC levels. Bench top, wet and dry extract, freeze-thaw and long term stability was evaluated for both the analytes. The method was applied to support a bioequivalence study of 5mg tablet formulation in 74 healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 344 incurred samples.
authors.affiliation: Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad 380009, India.
authors.forename: Primal
authors.initials: P
authors.lastname: Sharma
authors.forename: Daxesh P
authors.initials: DP
authors.lastname: Patel
authors.forename: Mallika
authors.initials: M
authors.lastname: Sanyal
authors.forename: Hiren
authors.initials: H
authors.lastname: Berawala
authors.forename: Swati
authors.initials: S
authors.lastname: Guttikar
authors.forename: Pranav S
authors.initials: PS
authors.lastname: Shrivastav
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D007201:Indicator Dilution Techniques; D059625:Liquid-Liquid Extraction / Q000379:methods; D008333:Mandelic Acids / Q000097:blood* / Q000737:chemistry*; D013607:Tablets / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013810:Therapeutic Equivalency
chemical_list: D008333:Mandelic Acids; D013607:Tablets; C032681:desethyloxybutynin; C005419:oxybutynin
keywords: Bioequivalence study; LC–MS/MS; N-desethyl oxybutynin; Oxybutynin; Phospholipids; Sensitive