Determination of Anabolic Steroid Residues (Medroxyprogesterone Acetate) in Pork by ELISA and Comparison with Liquid Chromatography Tandem Mass Spectrometry



Medroxyprogesterone acetate (MPA) has a relative molecular mass of only 344.5 and it has no immunogenicity. The analytical methods for the carbodiimides and the mixed anhydride were both adopted to couple MPA with bovine serum albumin, the carrier protein. The coupling rates of conjugate using the above methods were estimated to be 14 and 20 by ultraviolet spectrophotometry. The coupling was successful and verified according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. New Zealand White rabbits were immunized with the conjugate, the coupling rate of which was 14, and blood was collected after 5 periods of immunities. Then the titer of antiserum was tested to be 2.6 X 105 by indirect enzyme-linked immunosorbent assay (ELISA). Based on the purified antibody, a competitive indirect ELISA was developed. ELISAs provided a limit of detection of 0.096 ng/g, recoveries (in the edible tissues) between 72% and 91%, and a working range of 0.1 to 8.1 ng/g. Preliminary evaluation of assay performance through specificity, sensitivity, precision, and accuracy showed that this ELISA method can be applied to the practical detection of MPA in tissue samples. Moreover, it was compared with liquid chromatography tandem mass spectrometry (LC/MS/MS). The ion pair for quantification of MPA was 345.2/123.1, and the linear equation of MPA was Y = 6.68 X 103 X + 6.63 X 102. The 2 analytical methods can be applied to monitor MPA and other steroid residues in edible foods.