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-c arboxamidomethylpyridinyl)-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 ml 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 < or = 7.9% for I, < or = 9.0%, for II and < or = 9.5% for III across the standard curve range. This method exhibits good linearity and reproducibility for the three analytes.
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA. kerry_riffel@merck.com
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D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D011719:Pyrazines / Q000097:blood*; D011725:Pyridines / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors*
C411402:3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylenecarboxamidomethylpyridinyl)pyrazinone; D011719:Pyrazines; D011725:Pyridines; D013917:Thrombin