Investigation of the presence of prednisolone in bovine urine

Over the past 2 years low levels of prednisolone have been reported in bovine urine by a number of laboratories in EU member states. Concentrations vary, but are reported to be below approximately 3 g/l. In 40% of bovine urine samples from the Dutch national control plan had concentrations of prednisolone between 0.112.04 g/l. In this study the mechanism of formation of prednisolone was investigated. In-vitro conversion of cortisol by bacteria from faeces and soil, bovine liver enzymes and stability at elevated temperatures were studied. In-vitro bovine liver S9 incubation experiments showed a significant 20% decrease of cortisol within 6 hours, and formation of prednisolone was observed from 0.2 g/l at t = 0 to 0.5 g/l at t = 6. Under the influence of faeces, the stability of cortisol in urine is reduced and cortisol breaks down within 50 h. Prednisolone is formed up to 4 g/l at 70C after 15 h. However, this decreases again to 0 after 50 h. With soil bacteria, a slower decrease of cortisol was observed, but slightly higher overall formation of prednisolone, up to 7 g/l at 20C. As opposed to incurred urine, in fortified urine incubated with faeces or soil bacteria no prednisolone was detected. This difference may be explained by the presence of natural corticosteroids in the incurred sample. With UPLC-QToF-MS experiments, in urine and water samples incubated with faeces, metabolites known from the literature could be (tentatively) identified as 20ß-hydroxy-prednisolone, cortisol-21-sulfate, oxydianiline, tetrahydrocortisone-3-glucuronide and cortexolone, but for all compounds except 20ß-hydroxy-prednisolone no standards were available for confirmation. Based on the results of this study and literature data, for regulatory purposes a threshold of 5 g/l for prednisolone in bovine urine is proposed. Findings of prednisolone in concentrations up to 5 g/l in bovine urine can, most likely, originate from other sources than illegal treatment with growth promoters.