Detection of boldenone, its conjugates and androstadienedione, as well as five corticosteroids in bovine bile through a unique immunoaffinity column clean-up and two validated liquid chromatography–tandem mass spectrometry analyses

The presence of β-boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or natural origin or illicit treatment, is under debate within the European Union. The detection of β-boldenone conjugates, α-boldenone conjugates at concentrations higher than 2 ng mL−1 and prednisolone above the cut-off level of 5 ng mL−1 in urine have been, until now, critical in deciding if illegal drug use has occurred. The use of urine sometimes is not entirely satisfactory, especially when the drug is administrated at low doses or when its metabolic conversion is very fast. This subsequently would hamper its detection in urine. The introduction of a new, advantageous matrix where the illicit treatment can be investigated would be highly appreciated. In this study, we have developed and validated a simple and unique immunoaffinity clean-up procedure, which was applied to bovine bile samples, followed by two different analytical liquid chromatography-electrospray-tandem mass spectrometry methods. The first method tests androstadienedione, α- and β-boldenone sulphate, glucuronate and related free forms, while the other method assays prednisolone, prednisone, dexamethasone, cortisone, and cortisol. The methods were validated according to European Commission Decision 2002/657/EC. The evaluated parameters were linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit and detection capability. The decision limits (CCα) were between 0.38 and 0.45 ng mL−1 for anabolic steroids, and 0.13 and 0.15 ng mL−1 as far as corticosteroids were concerned. Intra- and inter-day repeatability was below 15.8 and 19.9% for all analytes, respectively. The methods were applied to the analysis of some bile samples collected from untreated young bulls in order to investigate the presence of the studied steroids in this matrix.