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

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 (0.2 mL) was first alkalified with 100 μL of sodium hydroxide (3 M) and then extracted with 1 mL of n-hexane containing 1% isopropanol (v/v) and 10% dichloromethane (v/v) by vortex-mixer for 3 min. The mixture was centrifuged at 14,000 rpm for 5 min. 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 (150 mm × 2.1 mm i.d., 5 μm). Mobile phase consisted of methanol–acetonitrile–20 mM 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 1000 ng/mL and the lowest limit of quantification was 3.9 ng/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 12 h, 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 100 mg amantadine.