Development of Validated LC-MS/MS Method for Pharmacokinetic and Bioequivalence Studies of Azelastine in Korean Healthy Volunteers

Development of Validated LC-MS/MS Method for Pharmacokinetic and Bioequivalence Studies of Azelastine in Korean Healthy Volunteers

The pharmacokinetic (PK) and bioequivalence (BE) studies of two formulations of azelastine (AZ) was carried out in 18 healthy male Korean volunteers according to a randomized crossover-design and were performed by a new validated liquid chromatography coupled to tandem mass spectrometry (LCMS/MS) method for quantitative analysis of plasma AZ. Subjects were given single dose of 2 tablets of 1 mg AZ of each formulation with 240 ml of water to subjects on 2 treatment days separated by one week washout period. After dosing, serial blood samples were collected for a period of 96 hours. AZ and the clomipramine (IS) were separated using a mobile phase of acetonitrile-5 mM NH4AC (8:2, v/v, pH=6.4) with flow rate of 0.25 ml/min over Luna C18 column. Analysis required 0.2 ml of plasma and involved a solid extraction with an Oasis HLB cartridge by on-line clean-up extraction automation system and then directly injected into HPLC with an API 3000 MS system by multiple reactions monitoring (MRM) mode. Several PK parameters (including AUC0-t, AUC0-infinity, Cmax, Tmax, T1/2 and Ke) were determined from the plasma concentration of AZ of both formulations. The AUC and Cmax was tested for BE after log-transformation of the data. The ionization was optimized using ESI (+) and selectivity was achieved by MS-MS analysis, m/z 112.0 and m/z 86.1 for AZ and IS, respectively. The calibration curves with r2>0.999 were linear over a working range of 20∼2000 pg/ml. No endogenous compounds were found to interfere with the analysis. The inter- and intra-day accuracy was in the ranges of 89.9∼109.3% and precision of inter- and intra-day expressed as relative standard deviation were 2.73∼8.79 %. No significant difference was found based on ANOVA; 90% confidance intervals (91.77∼101.94% for AUC0-t; 90.44∼ 106.27% for AUC0-infinity; 92.22∼108.57% for Cmax) for the test and reference drugs were found within FDA guideline of 80∼125%. Based on these statistical considerations, it was concluded that test drug was bioequivalent to the reference drug and therefore, may be interchangeably in the management of allergic or bronchial asthma and prophylaxis of mucositis due to cancer chemoradiotherapy. This validated method was found to be good performance on PK and BE studies for AZ. (Cancer Prev Res 12, 84-91, 2007).

Author (s)  Details

Professor Ju-Seop Kang,
Department of Pharmacology and Clinical Pharmacology Laboratory, College of Medicine, Hanyang University, Seoul 133-791, Korea.

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