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Abstract
A High Performance Liquid Chromatographic (HPLC) method was developed for the analysis of Bay 12-9566 (4-[4-4-(chlorophenyl)Phenyl]-4-oxo-2S-(phenylthiomethyl) butanoic acid), a metalloproteinase inhibitor, and its three metabolites, M1 (sulfoxide), M2 (ρ-hydroxy) and M3 (reduction product) in plasma. Sample preparation involved precipitation of plasma proteins using acidified acetonitrile. A reverse phase chromatography with gradient elution, ultraviolet detection at 290 nm and internal standard were used for separation and quantitation of all analytes. The quantitation range was 0.10 μg/mL to 50 μg/mL for BAY 12-9566, and 0.10 μg/mL to 3.00 μg/mL for all three metabolites. The limit of detection for BAY 12-9566 and for all three metabolites was 0.05 μg/mL. Intra-day accuracy for BAY 12-9566 and the three metabolites ranged from 95.0 to 105.0% and precision (CV) ranged from 0.0 to 4.76%.
Inter-day accuracy and precision were based on quality control samples analyzed concurrently with subject plasma samples during a 15 month period. Accuracy ranged from 95.7 to 104.8% and precision ranged from 1.46 to 7.31%.
ACKNOWLEDGMENTS
The authors are sincerely grateful to Drs. Suzan Bjorge, William Brubaker, and Ms. Lisa Zadjura for the pre-clinical investigation of BAY 12-9566 and its metabolism and to Dr. Harold Kluender for the synthesis of BAY 12-9566 and the three metabolites. The encouragement and support of Drs. A. H. Heller, K. Kumor, and A. Shah during the development and implementation of this HPLC procedure are also gratefully acknowledged.