The use of NBoldenone as a growth promoter in meat-producing animals is prohibited in the European Union (Directive 96/22/EC). Boldione (ADD), boldenone, and boldenone esters (primarily the undecylenate form) are all commercially available as anabolic preparations for humans, horses, and cattle. Boldenone metabolites have been discovered naturally in cattle since the late 1990s. According to EU regulations, an unambiguous demonstration of boldenone administration in bovine urine must be provided based on the identification of boldenone in the corresponding conjugate fraction. A method for measuring intact 17-boldenone-sulphate has been developed and validated in accordance with current standards.
The analytical procedure included direct extraction–purification of the target analyte on octadecylsilyl cartridges, as well as direct detection of the phase II metabolite via liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ), or high resolution mass spectrometry (OrbitrapTM). On the triple quadrupole, the decision limit (CC) and detection capability (CC) were 0.2 g L1 and 0.4 g L1 respectively, and 0.1 g L1 and 0.2 g L1 on the hybrid system. The method was successfully used to analyse incurred samples collected in various experiments. 17-Boldenone-sulphate levels were detectable up to 36 hours after boldione administration or 30 days after intramuscular injection of 17-boldenone undecylenate.