Determination of simvastatin-derived HMG-CoA reductase inhibitors in biomatrices using an automated enzyme inhibition assay with radioactivity detection

Abstract

A robust, automated enzyme inhibition assay method was developed and validated for the determination of HMG-CoA reductase inhibitory activities in plasma and urine samples following simvastatin (SV) administration. The assay was performed on Tecan Genesis 150 and 200 systems equipped with 8-probe and 96-well plates. Plasma samples containing HMG-CoA reductase inhibitors were treated with acetonitrile for protein precipitation before being incubated with HMG-CoA reductase, [ 14C]-HMG-CoA, and NADPH for a fixed length of time at a fixed temperature. The product, [ 14C]-mevalonic acid, was lactonized and separated from excess substrate via a small ion exchange resin column, and radioactivity was counted on a scintillation counter. HMG-CoA reductase inhibitors were measured before and after base hydrolysis. The two values obtained for each sample are referred to as ‘active’ and ‘total’ HMG-CoA reductase inhibitor concentrations. Simvastatin acid (SVA), the β-hydroxy acid of SV, was used as a standard to generate a calibration curve of HMG-CoA reductase activity versus SVA concentration (ng/ml). Three calibration ranges, 0.4–20, 2–50, and 5–100 ng/ml, in human and animal plasma and urine were validated. The assay precision was less than 8.5% CV in plasma and less than 10.4% in urine. The assay accuracy was 93.6–103.0 and 98.1–103.9% for the 0.4–20 and 2–50 ng/ml calibration ranges, respectively, in human plasma, and was 97.3–105.1, 94.4–105.2, and 90.2–95.7% for calibration range 5–100 ng/ml in rat plasma, dog plasma and human urine, respectively.