Determination of simvastatin-derived HMG-CoA reductase inhibitors in biomatrices using an automated enzyme inhibition assay with radioactivity detection.
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.
Liu
Lida
L
Drug Metabolism, Merck Research Laboratories, WP 75A-303, West Point, PA 19486, USA. lida_liu@merck.com
Zhang
Rena
R
Zhao
Jamie J
JJ
Rogers
John D
JD
Hsieh
John Y K
JY
Fang
Wei
W
Matuszewski
Bogdan K
BK
Dobrinska
Michael R
MR
D000818:Animals; D001322:Autoanalysis; D002250:Carbon Radioisotopes; D004285:Dogs; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000032:analysis* / Q000097:blood / Q000652:urine; D007202:Indicators and Reagents; D051381:Rats; D015203:Reproducibility of Results; D012371:Robotics; D012680:Sensitivity and Specificity; D019821:Simvastatin / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000652:urine
D002250:Carbon Radioisotopes; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007202:Indicators and Reagents; D019821:Simvastatin