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 devel oped for plasma, which usually requires maximum sensitivity. The HPLC-W met hod is for urine. In both methods, analytes are extracted using liquid-liqu id extraction, and analyzed by reversed-phase high-performance liquid chrom atography. 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. Th e urine method has an LOQ of 8.12 ng/ml (20 nM) and the linear dynamic rang e is 8.12-8120 ng/ml (20-20 000 nM). Both methods are fast, specific and se nsitive. Various validation procedures have proven that both methods are ru gged, robust and reproducible. The research also suggested that, while LC-M S-MS provides superior sensitivity and selectivity for the determination of drugs and their metabolites at very low concentrations, HPLC with a conven tional detector such as UV is still useful in the analysis when the sensiti vity requirement is not crucial. (C) 2000 Elsevier Science B.V. All rights reserved.