title: Determination of Azelastine in Human Plasma by Validated Liquid Chromatography Coupled to Tandom Mass Spectrometry (LC-ESI/MS/MS) for the Clinical Studies.
abstract: A liquid chromatography coupled to tandem mass spectrometry (LC-ESI/MS/MS) was validated to determine azelastine in human plasma. Azelastine and internal standard (IS, clomipramine) were separated using a mobile phase of acetonitrile:(5 mM)-ammonium acetate solution (70:30, v/v, pH=6.4) with flow rate of 0.25 mL/min over YMC C8 column. One mL of plasma was extracted by n-hexane: 2-propanol (97:3, v/v) and then injected into HPLC system after reconstitution by acetonitrile: (5 mM)-ammonium acetate (1:1, v/v) solution. Detection was carried out on API5000 MS system by multiple reactions monitoring mode. The ionization was optimized using ESI (+) and selectivity was achieved at m/z 382.2→112.2 for azelastine and m/z 315.3→228.0 for IS. Total run-time (<2.0 min) and linearity (10 (LLOQ) ~5000 pg/mL) were good. No endogenous compounds were found around the retention time. The inter- and intra-day precision and accuracy were 4.13~17.91% and 87.57~109.70%, respectively. This validated method was successfully applied to a bioequivalence study in 23 healthy Korean male volunteers from the blood samples taken up to 96 h after orally administered 2 tablets of 1 mg of reference and test formulations of azelastine in a double-blind, randomized, cross-over design. The mean peak plasma concentrations (Cmax ± SD) of 1.02 ± 0.37 and 1.10 ± 0.43 ng/mL were reached at 5.9 and 5.6 h for reference and test azelastine, respectively. The mean total area under the curve (AUC0-infinity) were 25.96 ± 10.84 and 28.24 ± 11.09 ng·h/mL for reference and test formulations, respectively. The reference and test azelastine formulations can be considered bioequivalent from the obtained pharmacokinetics by LC-ESI/MS/MS.
authors.affiliation: The Institute of Biomedical Science, Hanyang University, Seoul, Republic of Korea; ; Department of Pharmacology, College of Medicine, Hanyang University, Seoul, Republic of Korea;
authors.forename: Yoo-Sin
authors.initials: YS
authors.lastname: Park
authors.forename: Shin-Hee
authors.initials: SH
authors.lastname: Kim
authors.forename: Young-Jae
authors.initials: YJ
authors.lastname: Kim
authors.forename: Seok-Chul
authors.initials: SC
authors.lastname: Yang
authors.forename: Min-Ho
authors.initials: MH
authors.lastname: Lee
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Shaw
authors.forename: Ju-Seop
authors.initials: JS
authors.lastname: Kang
keywords: LC-ESI/MS/MS; azelastine; bioequivalence; pharmacokinetics