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.
Riffel
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Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA. kerry_riffel@merck.com
Song
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Gu
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Yan
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Lo
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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