title: Development and validation of a highly sensitive and robust LC-MS/MS with electrospray ionization method for simultaneous quantitation of itraconazole and hydroxyitraconazole in human plasma: application to a bioequivalence study.
abstract: A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of itraconazole (ITZ) and hydroxyitraconazole (OH-ITZ) with 500 microL of human plasma using fluconazole as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Solid phase extraction process was used to extract ITZ, OH-ITZ and IS from human plasma. The total run time was 3.0 min and the elution of ITZ, OH-ITZ and IS occurred at 2.08 min, 1.85 min and 1.29 min, respectively; this was achieved with a mobile phase consisting of 0.2% (v/v) ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPurity C(18) (50 mm x 4.6 mm, 5 microm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.50 ng/mL for both ITZ and OH-ITZ. A linear response function was established for the range of concentrations 0.5-263 ng/mL (r>0.998) for both ITZ and OH-ITZ. The intra- and inter-day precision values for ITZ and OH-ITZ met the acceptance as per FDA guidelines. ITZ and OH-ITZ were stable in the battery of stability studies, viz., bench-top, auto-sampler, dry extract and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.
authors.affiliation: Department of Chemistry, Jawaharlal Nehru Technological University Kukatpally, Hyderabad, India.
authors.forename: D Vijaya
authors.initials: DV
authors.lastname: Bharathi
authors.forename: Kishore Kumar
authors.initials: KK
authors.lastname: Hotha
authors.forename: P V Vidya
authors.initials: PV
authors.lastname: Sagar
authors.forename: Sanagapati Sirish
authors.initials: SS
authors.lastname: Kumar
authors.forename: Pandu Ranga
authors.initials: PR
authors.lastname: Reddy
authors.forename: A
authors.initials: A
authors.lastname: Naidu
authors.forename: Ramesh
authors.initials: R
authors.lastname: Mullangi
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D017964:Itraconazole / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013810:Therapeutic Equivalency
chemical_list: C084794:hydroxyitraconazole; D017964:Itraconazole