Development of a Solid-Phase Extraction–Enzyme-Linked Immunosorbent Assay for the Determination of 17β-19-Nortestosterone Levels in Antifatigue Functional Foods.

17β-19-nortestosterone (17β-NT) has been illegally used in antifatigue functional foods to promote muscle growth and improve endurance. A rapid and sensitive solid-phase extraction–enzyme-linked immunosorbent assay (SPE-ELISA) method was developed and successfully applied to analyze the levels of 17β-NT in antifatigue functional foods. A polyclonal antibody against 17β-NT was produced from rabbits immunized with the 17β-NT-BSA conjugate, and a competitive direct enzyme-linked immunosorbent assay was developed for the rapid detection of 17β-NT. The concentration causing 50% inhibition ( IC50) and the limit of detection (LOD) were found to be 0.08 and 0.0055 ng/mL, respectively; this was better than methods previously reported that had a LOD of 2.4 ng/mL. C18 cartridges were investigated for use in removing the effects of matrix in foods, and the sample purification protocol was optimized. Using the developed SPE-ELISA method, recoveries of functional food samples were obtained in the range of 71% to 91.5%. Moreover, 2 kinds of antifatigue functional foods were analyzed using the established ELISA and HPLC methods. The correlation coefficient of the results obtained using the 2 methods was greater than 0.98. Thus, the preliminary evaluation of the SPE-ELISA method proved that it is a specific, sensitive, and precise tool that can be used for the practical detection of 17β-NT in various antifatigue functional food samples. [ABSTRACT FROM AUTHOR]