Development of a confirmatory method for detecting recombinant bovine somatotropin in plasma by immunomagnetic precipitation followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry.

Recombinant bovine somatotropin (rbST), a synthetic growth hormone, is used to stimulate growth and enhance milk production in dairy cows. Both its use and the sale of dairy products from treated animals are prohibited in the European Union, as well as in Australia, Canada, Japan, and New Zealand, but authorised in several countries (e.g. Brazil, USA). Screening methods involve detecting anti-rbST antibodies (biomarkers) in treated cows. Confirmatory methods are required to prove rbST abuse. The major challenges in determining rbST are its potentially low levels, its high similarity to native bST, and matrix interferences. To overcome these obstacles, we have developed a method involving immunomagnetic precipitation followed by UHPLC-MS/MS for rbST detection. Briefly, protein G magnetic beads pre-coated with an in-house produced monoclonal antibody were added to plasma. Incubation at room temperature allowed rbST present in the sample to bind to the magnetic beads. After that, magnetic beads were isolated by centrifugation and thoroughly washed (PBS, PBS + 0.2% Tween 20). Finally, rbST was released by alkalinisation and the samples were trypsin digested prior to UHPLC-MS/MS analysis in the MRM mode. Validation was done in accordance with European Commission Decision 2002/657/CE. Matrix-matched calibration with internal standards was used. The decision limit (CCα) reached with this approach was 0.11 µg l-1.