Automated enzyme inhibition assay method for the determination of atorvastatin-derived HMG-CoA reductase inhibitors in human plasma using radioactivity detection.
BACKGROUND
A Tecan-based enzyme inhibition assay has been developed for the determination of atorvastatin-derived 'active' and 'total' (active inhibitors plus atorvastatin lactone and other potential inhibitors following base hydrolysis) 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor concentrations in human plasma. Atorvastatin is an inhibitor of HMG-CoA reductase, which is a key rate-limiting enzyme in the cholesterol biosynthesis. Previously, atorvastatin-derived HMG-CoA reductase inhibitors were measured via enzyme inhibition assays by manual operation.


METHODS
In this work, an enzyme assay procedure based on 8-tip Tecan robotics and set-up in a 96-well plate format with customized hardware is presented. Following protein precipitation of the plasma sample, an aliquot of the resulting supernatant is mixed with HMG-CoA reductase and (14)C-labeled HMG-CoA prior to incubation. The product, (14)C-mevalonic acid, is lactonized, separated from unreacted (14)C-substrate, and counted in a liquid scintillation counter. Plasma HMG-CoA reductase inhibitor concentrations are measured against atorvastatin as the standard. Tecan Genesis 150 and 200 robotic workstations were used for the protein precipitation, enzyme incubation, and product separation.


RESULTS
The standard calibration range for the assay was 0.4-20 ng eq/mL. Intra-day precision (%CV) data for the calibration standard and quality control (QC) samples (n=5 replicates) were both <or=8%, with an accuracy between 88 and 113% of nominal values. Initial inter-day precision of the QC samples was <or=6%, with an accuracy range of 94-111% of nominal values.


CONCLUSIONS
The assay procedure provides high throughput analysis of clinical samples to support pharmacokinetic studies.
Valesky
Robert J
RJ
Merck Research Laboratories, Department of Drug Metabolism, Sumneytown Pike, WP75B-300, West Point, PA, USA. robert_valesky@merck.com
Liu
Lida
L
Musson
Donald G
DG
Zhao
Jamie J
JJ
D000069059:Atorvastatin; D006538:Heptanoic Acids / Q000032:analysis* / Q000097:blood* / Q000737:chemistry; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000097:blood*; D007202:Indicators and Reagents; D015394:Molecular Structure; D011758:Pyrroles / Q000032:analysis* / Q000097:blood* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007202:Indicators and Reagents; D011758:Pyrroles; D000069059:Atorvastatin; D006903:Hydroxymethylglutaryl CoA Reductases