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Development of Enzyme-Linked Immunosorbent Assay for Determination of Boldenone in Dietary Supplements

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Abstract

There is an increasing interest in the investigation and implementation of methods for the analysis of anabolic steroids in dietary supplements. In this study, a competitive indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of boldenone. For this purpose, an antiserum against boldenone was raised in a rabbit using boldenone-17-hemisuccinate-bovine serum albumin as an immunization conjugate. Based on the ELISA standard curve, the detection limit was as low as 0.014 ± 0.007 ng mL−1 with the IC50 value of 0.293 ± 0.084 ng mL−1 and linear working range of 0.065–1.529 ng mL−1. The intra- and inter-assay variations were found to be satisfactory. Relative standard deviations were calculated in the range of 3.8–10.5 and 7.3–12.9 %. The developed ELISA was applied in the analysis of extracts obtained from spiked samples of dietary supplements. Ethanol extracts were applied into the immunoassay without a cleanup procedure (only diluted) to minimize the effect of the matrix. Recoveries from spiked samples were from 86.0 to 115.7 %. An excellent correlation between ELISA and UHPLC-MS/MS was obtained with the linear equation of y = 1.0256 x − 0.7772 (R 2 = 0.9999). This ELISA as proposed here could be successfully applied for the simple monitoring of boldenone in dietary supplements.

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Acknowledgments

This work was supported by the Grant of the Ministry of the Interior of the Czech Republic (VG20112015045).

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Authors and Affiliations

  1. Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technicka 3, 166 28, Prague 6, Czech Republic

    Lucie Fojtíková, Ladislav Fukal, Martina Blažková, Sandra Sýkorová & Barbora Holubová

  2. Forensic Laboratory of Biologically Active Substances, University of Chemistry and Technology, Prague, Technicka 3, 166 28, Prague 6, Czech Republic

    Martin Kuchař & Petra Mikšátková

  3. Department of Chemistry of Natural Compounds, University of Chemistry and Technology, Prague, Technicka 3, 166 28, Prague 6, Czech Republic

    Martin Kuchař, Petra Mikšátková & Oldřich Lapčík

Authors
  1. Lucie Fojtíková
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  2. Ladislav Fukal
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  3. Martina Blažková
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  4. Sandra Sýkorová
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  5. Martin Kuchař
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  6. Petra Mikšátková
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  7. Oldřich Lapčík
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  8. Barbora Holubová
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Corresponding author

Correspondence to Barbora Holubová.

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Funding

This study was funded by Ministry of the Interior of the Czech Republic (grant number VG20112015045).

Conflict of Interest

Lucie Fojtíková has no conflict of interest. Ladislav Fukal has no conflict of interest. Martina Blažková has no conflict of interest. Sandra Sýkorová has no conflict of interest. Martin Kuchař has no conflict of interest. Petra Mikšátková has no conflict of interest. Oldřich Lapčík has no conflict of interest. Barbora Holubová has no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of laboratory animals were followed.

Informed Consent

Informed consent was obtained from all the individual participants involved in the study.

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Fojtíková, L., Fukal, L., Blažková, M. et al. Development of Enzyme-Linked Immunosorbent Assay for Determination of Boldenone in Dietary Supplements. Food Anal. Methods 9, 3179–3186 (2016). https://doi.org/10.1007/s12161-016-0511-9

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  • DOI: https://doi.org/10.1007/s12161-016-0511-9

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