Quantitative determination of amantadine in human plasma by liquid chromatography–mass spectrometry and the application in a bioequivalence study

Abstract: A sensitive liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) method is developed and validated for rapid determination of amantadine in human plasma. Desloratadine was used as the internal standard (I.S.). Human plasma (0.2mL) was first alkalified with 100μL of sodium hydroxide (3M) and then extracted with 1mL of n-hexane containing 1% isopropanol (v/v) and 10% dichloromethane (v/v) by vortex-mixer for 3min. The mixture was centrifuged at 14,000rpm for 5min. The supernatant was evaporated to dryness and the residue was dissolved in mobile phase. Samples were separated using a Thermo Hypersil-HyPURITYC18 reversed-phase column (150mm×2.1mm i.d., 5μm). Mobile phase consisted of methanol–acetonitrile–20mM ammonium acetate (45:10:45, v/v/v) containing 1% acetic acid with pH 4.0. Amantadine and I.S. were measured by electrospray ion source in positive selective ion monitoring mode. The good linearity ranged from 3.9 to 1000ng/mL and the lowest limit of quantification was 3.9ng/mL. The extraction efficiencies were approximately 70% and recoveries of method ranged from 98.53 to 103.24%. The intra-day relative standard deviations (R.S.D.) were less than 8.43% and inter-day R.S.D. below 10.59%. The quality control samples were stable when kept at room temperature for 12h, at −20°C for 30 days and after four freeze/thaw cycles. The method has been successfully used to evaluation of the pharmacokinetics and bioequivalence of amantadine in 20 healthy volunteers after an oral dose of 100mg amantadine. [Copyright &y& Elsevier]