Simultaneous determination of a novel thrombin inhibitor and its two metabolites in human plasma by liquid chromatography/tandem mass spectrometry

I, 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylene-carboxamidomethylpyridinyl)-pyrazinone dihydrochloride monohydrate, is a potent, orally active thrombin inhibitor. The compound also has two metabolites which have been shown to have thrombin inhibitory activity: benzylic alcohol M3 metabolite (II) and hydroxymethylpyrazinone M7 metabolite (III). A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of I and its two metabolites in human plasma has successfully been developed. The method consists of treating 0.5 mi plasma with 2 N NaOH and extracting I, II, III and internal standard (IV) with ethyl acetate:methyl-t-butyl ether (1:3,v/v). The analytes were then back-extracted into 2% formic acid. The analytes were chromatographed by high performance liquid chromatography (HPLC) and detected by MS/MS. Positive ionization was used on a heated nebulizer probe monitoring precursor-->product ion combinations in multiple reaction monitoring mode. The linear range is 0.5-1000 nM for I and III and 0.75-1000 nM for II. Recoveries were 88, 74, 78 and 102.1% for I, II and III and the internal standard, respectively in human plasma. Intraday variation using this method was less than or equal to 7.9%; for I, less than or equal to 9.0% for II and less than or equal to 9.5% for III across the standard curve range, This method exhibits good linearity and reproducibility for the three analytes. (C) 2000 Elsevier Science B.V. All rights reserved.