Development and characterisation of an immunoaffinity column for the selective extraction of methandrostenolone from food and feed samples

Methandrostenolone (MA) is used as veterinary medicine for promoting food-producing animal growth. Accumulation of MA in a human body would cause multiple side effects. In this study, an anti-MA monoclonal antibody (mAb)-based immunoaffinity column (IAC) was prepared and its application to the selective extraction of MA from actual samples was evaluated. The employed antibody exhibited good sensitivity and specificity towards MA with an IC₅₀ value of 7.89 ng/mL and less than 1% cross-reactivity towards structurally related compounds. By coupling CNBr-activated Sepharose with the antibody, an IAC was prepared. Two percent methanol and 70% methanol were selected, respectively, as loading and eluting solution by optimisation. The maximum binding capacity of the column for MA reached 4760 ng/mL gel. The average recovery of 50, 250 and 500 ng MA standards from IACs was 88.6% with a relative standard deviation (RSD) among columns of 8.5%. After three times usage, the binding capacity and recovery still remained 93.4% and 81.1%, respectively. To further verify the effect of matrices on the extraction efficiency, the IACs were challenged with MA-fortified animal tissue and feed samples and the recovery were found to be in the range of 78.7%–95.1%.