Simvastatin is a lactonic prodrug which has a large first pass metabolism in the liver. Its most important metabolite is simvastatin acid, an active form of simvastatin that is HMG-CoA reductase inhibitor and that causes decrease in cholesterol synthesis in liver. Because of the simvastatin’s low aqueous solubility and its large first pass metabolism simvastatin has extremely low bioavailability.
The aim of our work was to develop a sensitive, accurate and precise method for determination of simvastatin and simvastatin acid in small aliquots of rat plasma (100 µL). The developed method should serve as analytical support the pharmacokinetic preclinical study, where a novel simvastatin prodrug and various simvastatin formulations were compared in terms of their pharmacokinetic properties To select the optimal sample preparation technique, three methods were tested: protein precipitation, solid phase extraction using different cartridges and liquid extraction using different organic solvents. The samples were analysed by liquid chromatography coupled to tandem mass spectrometry. Additionally two different columns and their influence on analyte response were tested and compared. The method development was performed using human plasma as a surrogate, while the final method which utilized the liquid-liquid extraction by terc-buthyl methyl ether was accomplished on rat serum.
The selectivity, accuracy and precision were tested in concentration range between 0.5 µg/L and 50 µg/L. We concluded that simvastatin is a problematic analyte due to high variability during the sample preparation and poor stability. Better precision was achieved with simvastatin acid. We proved that the chosen method does not suffer from matrix effect, and the process efficiency is around 15% for simvastatin and around 30% for simvastatin acid. Regardless of the low process efficiency, the method has been successfully applied to a large number of study samples.
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