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

17beta-19-nortestosterone (17beta-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 17beta-NT in antifatigue functional foods. A polyclonal antibody against 17beta-NT was produced from rabbits immunized with the 17beta-NT-BSA conjugate, and a competitive direct enzyme-linked immunosorbent assay was developed for the rapid detection of 17beta-NT. The concentration causing 50% inhibition (IC(50)) 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. C(18) 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 17beta-NT in various antifatigue functional food samples.