Validated enzyme-linked immunosorbent assay for determination of rosuvastatin in plasma at picogram level

Authors


Correspondence to: Prof. Ibrahim A. Darwish, Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia. E-mail: idarwish@ksu.edu.sa

Abstract

In this study, a highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed and validated for the determination of rosuvastatin (ROS) in plasma samples at picogram level. The assay employed a polyclonal antibody that specifically recognizes ROS with high affinity, and ROS conjugate of bovine serum albumin (ROS–BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between ROS, in plasma sample, and the immobilized ROS-BSA for the binding sites on a limited amount of the anti-ROS antibody. The bound anti-ROS antibody was quantified with horseradish peroxidase-labelled second anti-rabbit IgG antibody (HRP-IgG) and 3,3`,5,5`-tetramethylbenzidine (TMB) as a substrate for the peroxidase enzyme. The concentration of ROS in the sample was quantified by its ability to inhibit the binding of the anti-ROS antibody to the immobilized ROS-BSA and subsequently the colour intensity in the assay wells. The assay limit of detection was 25 pg ml–1 and the effective working range at relative standard deviations (RSD) of ≤ 5% was 40–2000 pg ml–1. Analytical recovery of ROS from spiked plasma was 96.2 – 104.8 ± 2.12 – 5.42%. The precision of the assay was satisfactory; RSD was 2.47 – 4.46 and 3.24 – 5.27% for the intra- and inter-assay precision, respectively. The analytical procedure is convenient, and one can analyze ~ 200 samples per working day, facilitating the processing of large-number batch of samples. The proposed ELISA has a great value in routine analysis of ROS for its pharmacokinetic studies. Copyright © 2011 John Wiley & Sons, Ltd.

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