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

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 (R2 = 0.9999). This ELISA as proposed here could be successfully applied for the simple monitoring of boldenone in dietary supplements.