Determination of a novel thrombin inhibitor in human plasma and urine utilizing liquid chromatography with tandem mass spectrometric and ultraviolet detection.
An LC-MS-MS method and an HPLC-UV method have been developed for the assay of a novel thrombin inhibitor in human fluids. The LC-MS-MS method is developed for plasma, which usually requires maximum sensitivity. The HPLC-UV method is for urine. In both methods, analytes are extracted using liquid-liquid extraction, and analyzed by reversed-phase high-performance liquid chromatography. A tandem mass spectrometer in the multiple reaction monitoring (MRM) mode is used for detection of the analytes in the plasma method. UV is the detector for the urine method. The plasma method has a lower limit of quantitation (LOQ) of 0.1 ng/ml with a linearity range of 0.1-100 ng/ml. The urine method has an LOQ of 8.12 ng/ml (20 nM) and the linear dynamic range is 8.12-8120 ng/ml (20-20000 nM). Both methods are fast, specific and sensitive. Various validation procedures have proven that both methods are rugged, robust and reproducible. The research also suggested that, while LC-MS-MS provides superior sensitivity and selectivity for the determination of drugs and their metabolites at very low concentrations, HPLC with a conventional detector such as UV is still useful in the analysis when the sensitivity requirement is not crucial.
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Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA. henry_song@merck.com
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D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011719:Pyrazines / Q000097:blood* / Q000652:urine*; D011725:Pyridines / Q000097:blood* / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012997:Solvents; D013917:Thrombin / Q000037:antagonists & inhibitors*
C411402:3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylenecarboxamidomethylpyridinyl)pyrazinone; D011719:Pyrazines; D011725:Pyridines; D012997:Solvents; D013917:Thrombin