Detection of medroxyprogesterone acetate residues in animal muscle tissues by using enzyme-linked immunosorbent assay and liquid chromatography tandem mass spectrometry

Abstract

Medroxyprogesterone acetate (MPA) has a relative molecular mass of only 344.5 and no immunoreactivity. Methods using carbodiimides and mixed anhydrides were adapted to couple MPA with bovine serum albumin, a 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 according to SDS-PAGE. New Zealand White rabbits were immunized with the conjugate obtained with a coupling rate of 14, and blood was collected after five periods of immunities. Indirect ELISA showed the titer of antiserum to be 2.6×10 5. Based on the purified antibody, a competitive indirect ELISA was developed. It provided a limit of detection of 0.096 ng/mL, recoveries (edible tissues) between 72 and 91% and a working range of 0.1-8.1 ng/mL. Preliminary evaluation of assay performance in terms of specificity, sensitivity, precision, and accuracy showed that this ELISA can be applied to practical detection of MPA in tissues. Moreover, it was compared with high performance liquid chromatography tandem mass spectrometry. The ion pair for quantification of MPA was 345.2/123.1, and linear equation of MPA was Y = 6.68 × 103 X+6.63 × 102. The two analytical methods can be applied to monitor MPA and other anabolic steroid residues in foods.