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A highly sensitive and specific polyclonal antibody-based enzyme immunoassay for therapeutic monitoring and pharmacokinetic studies of atorvastatin

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Abstract

This study describes the development and validation of a highly sensitive and specific enzyme immunoassay (EIA) for therapeutic monitoring and pharmacokinetic studies of atorvastatin (ATR). The assay employs a polyclonal antibody that recognizes ATR with high specificity and affinity, and ATR conjugated to bovine serum albumin (ATR-BSA) immobilized onto microwell plates as a solid phase. The assay involved a competitive binding reaction between ATR and the immobilized ATR-BSA for the binding sites on a limiting amount of the anti-ATR antibody. The bound anti-ATR antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin secondary antibody and 3,3′,5,5′-tetramethylbenzidine as a substrate for the peroxidase enzyme. The concentration of ATR in the sample was quantified by its ability to inhibit the binding of the anti-ATR antibody to the immobilized ATR-BSA and subsequent color development in the assay wells. The conditions for the EIA were investigated and optimized for the determination of ATR in plasma samples. The limit of detection was 0.04 ng mL−1 and the effective working range at relative standard deviations (RSD) of ≤5% was 0.1–10 ng mL−1. Mean analytical recovery of ATR from spiked plasma was 99.3 ± 2.8%. The precision of the assay was satisfactory; RSD were 2.7–4.6 and 3.3–5.7% for intra- and inter-assay precision, respectively. The reliability of the EIA was confirmed by HPLC. The EIA is convenient, and one can analyze ∼ 200 samples per working day, facilitating the processing of large-number of samples of ATR.

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Acknowledgment

The authors thank King Abdulaziz City for Science and Technology for funding the work (KACS-16-98AT).

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia

    Ibrahim A. Darwish, Abdul-Rahman M. Al-Obaid & Hamoud A. Al-Malaq

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  1. Ibrahim A. Darwish
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  2. Abdul-Rahman M. Al-Obaid
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  3. Hamoud A. Al-Malaq
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Correspondence to Ibrahim A. Darwish.

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Darwish, I.A., Al-Obaid, AR.M. & Al-Malaq, H.A. A highly sensitive and specific polyclonal antibody-based enzyme immunoassay for therapeutic monitoring and pharmacokinetic studies of atorvastatin. Microchim Acta 170, 67–74 (2010). https://doi.org/10.1007/s00604-010-0390-5

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  • DOI: https://doi.org/10.1007/s00604-010-0390-5

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