Determination of prednisolone metabolites in beef cattle

Prednisolone is a synthetic corticosteroid acting on both hydrosaline balance and metabolism that is liable to fraudulent administration to meat-producing animals for growth-promoting purposes. Its use outside strict therapeutic control and prescription is banned by the European legislation, but official controls are hampered by its negligible direct excretion into the urinary matrix. Recent studies reported on a potential endogenous origin of prednisolone in animals subjected to stressful conditions, accounting for its occasional detection in control urines. The objective of the present study was the identification and quantification of prednisolone urinary metabolites, to be used as illicit treatment biomarkers, in place of the parent drug. An LC-MS/MS screening was conducted on urine samples collected from a bullock i.m. administered with prednisolone acetate using a therapeutic protocol (2 x 0.52 mg kg-1 at 48h interval). Four prednisolone metabolites were identified, including 20β-dihydroprednisolone, 20α-dihydroprednisolone, 6β-hydroxyprednisolone and 20β-dihydroprednisone; the first was detected at relatively high concentrations. An existing quantitative LC-MS/MS method was expanded and re-validated, to include these metabolites. The new analytical method proved sensitive (LODs: 0.35-0.42 ng ml-1 ) and specific, and was applied to urine samples collected from eight beef cattle subjected to low-dosage oral administration of prednisolone acetate for a 35-days period, as in standard growth-promoting treatments. 20β-dihydroprednisolone was detected in all urine samples collected during the treatment, at relatively high concentration (1.2-27 ng ml-1 ), whereas the prednisolone concentration was virtually negligible (<0.7 ng ml-1 ). 20β-dihydroprednisolone was no longer present in almost all samples collected six days after the end of the treatment, but trace amounts of this metabolite were found in two urine samples from control animals. 20β-dihydroprednisolone is proposed as an effective biomarker 24 to test illegal growth-promoting treatments with prednisolone in meat cattle, alternatively to the parent drug.