GR CALUX assay detects synthetic glucocorticoids in calf urine: a validation study

ABSTRACT

Member States of the EU are required to monitor the use of pharmacologically active substances in food-producing animals. There is evidence, however, that the target-based approach currently applied in official monitoring plans might under-estimate the real incidence of growth promoter abuse in livestock. As demonstrated for sex hormones, the association of effect-oriented biological screening with chemical confirmatory techniques could be the best strategy in revealing the abuse of veterinary drugs. Here we demonstrate the reliability of a cell-based assay to screen calf urine samples for synthetic glucocorticoids. The validation included the most widely used synthetic drugs (flumethasone, dexamethasone, betamethasone, methylprednisolone and prednisolone) and was developed according to the Commission Decision 2002/657/EC, thus including the verification of cut-off level, the β error, the specificity, ruggedness and stability. The study was carried out using prednisolone as representative substance at 5 ng mL⁻¹ concentration. All blank and spiked urine fulfilled the EU criteria, moreover the method resulted in being specific and sound, and the analytes in urine were stable for at least 30 days. The assay results indicated its suitability for a qualitative analysis of calf urine samples. This method enabled the detection of low doses of synthetic glucocorticoids (GCs) in matrix (<2 ng mL⁻¹ for flumethasone, dexamethasone, betamethasone; < 4 ng mL⁻¹ for methylprednisolone; 5 ng mL⁻¹ for prednisolone), with the possibility of detecting new or unknown molecules and cumulative effects of low-level mixtures with glucocorticoid bioactivity.

KEYWORDS:

• Validation
• reporter gene assay
• illicit treatment
• glucocorticoids
• calves
• urine samples

Acknowledgments

Experiment supported by the Italian Ministry of Health under Grant IZSPLVA 03/10RC and Grant IZSPLVA 14M01.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Italian Ministry of Health [Grant IZSPLVA 03/10RC and Grant IZSPLVA 14M01].