Determination of α-and β-boldenone sulfate, glucuronide and free forms, and androstadienedione in bovine urine using immunoaffinity columns clean-up and liquid chromatography tandem mass spectrometry analysis

The debate about the origins of boldenone in bovine urine is ongoing for two decades in Europe. Despite the fact that its use as a growth promoter has been banned in the European Union (EU) since 1981, its detection in bovine urine, in the form of α‐boldenone conjugate, is considered fully compliant up to 2 ng mL−1. The conjugated form of β‐boldenone must be absent. In recent years, the literature about boldenone has focused on the identification of biomarkers that can indicate an illicit treatment. β‐boldenone sulfate is a candidate molecule, even if the only studies currently available have taken place in small populations. In this study, a method for the determination of sulfate and glucuronate conjugates of β‐boldenone was developed and validated according to the European Commission Decision 2002/657/EC and applied to α‐ boldenone sulfate and glucuronide, α‐ and β‐boldenone free forms and androstadienedione (ADD), too. The clean‐up with immunoaffinity columns enabled the direct determination of the conjugates and free forms and allowed specific and sensitive analyses of urine samples randomly selected to verify this method. The decision limits (CCα) ranged between 0.07 and 0.08 ng mL−1, the detection capabilities (CCβ) between 0.08 and 0.1 ng mL−1. Recovery was higher than 92% for all the analytes. Intra‐day repeatability was between 5.8% and 17.2%, and inter‐day repeatability was between 6.0% and 21.8% for the studied free and conjugated forms. This method has been developed as a powerful tool with the aim to study the origin of boldenone in a trial on a significant number of animals.