Determination the concentration of phenytoin in healthy human plasma and study on its bioequivalence Article Abstract
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Published: May 28, 2006
Objective To establish a sensitive and selective LC/MS/MS method for the determination of phenytoin in healthy human plasma to study on the bioequivalence of different formulations containing phenytoin. Methods Phenytoin and the internal standard osalmid were extracted from plasma with liquid - liquid extraction, then separated on a Zorbax SB -C18 column. The mobile phase consisted of methanol -water -formic acid (90: 10: 0. 2) , at a flow - rate of 0. 6 mL · min-1. A Finnigan TSQ quantum ultra tandem mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source was used as the detector and operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor to product ion combinations of m/z 253 → m/z 182 and m/z 230→ m/z 121 was performed to detect phenytoin and the internal standard, respectively. The method was used to evaluate the bioequiavailability of phenytoin in compound preparation. Results The linear calibration curves were obtained in the concentration range of 2.5 ~ 3000 ng · mL-1 for phenytoin. The lower limit of quantification was 2. 5 ng · mL-1. The intra - and inter - day RSD over the entire concentration range were less than 7.0%. Accuracy determined at three concentrations (2. 5, 60. 0 and 2 400. 0 ng · mL-1 for phenytoin) ranged from -0.5% to 2. 3%. The bioavailability of two formulations was (107.5±18.9) %. Conclusion The method was proved to be ro - bust, convenient and suitable for bioequivalence evaluation of different formulations containing phenytoin.