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

Article Details

Citation

Liu L, Zhang R, Zhao JJ, Rogers JD, Hsieh JY, Fang W, Matuszewski BK, Dobrinska MR

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

J Pharm Biomed Anal. 2003 Apr 24;32(1):107-23.

PubMed ID
12852453 [ View in PubMed
]
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 beta-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 50, 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.

DrugBank Data that Cites this Article

Drug Targets
DrugTargetKindOrganismPharmacological ActionActions
Mevastatin3-hydroxy-3-methylglutaryl-coenzyme A reductaseProteinHumans
Yes
Inhibitor
Details
Simvastatin3-hydroxy-3-methylglutaryl-coenzyme A reductaseProteinHumans
Yes
Inhibitor
Details