A high-throughput screening (HTS) immunochemical method for the analysis of stanozolol metabolites in cattle urine samples ☆

Abstract A high-throughput immunosorbent solid-phase extraction (HTS-IS-SPE) procedure coupled to enzyme-linked immunosorbent assay (ELISA) has been established for the analysis of stanozolol (St) and its main metabolite in cattle, 16β-hydroxy-stanozolol (16βOH-St), in cow urine samples. The chemical structure of the immunizing hapten 2′H-androst-2-eno[3,2-c]-pyrazol-17-hemiglutarate 5 (hapten A) has been designed to accomplish simultaneous detection of St and 16βOH-St. The antibodies obtained have been used to establish a microplate ELISA method able to detect these metabolites with IC 50 values of 0.57 μg L −1 and 1.46 μg L −1 , respectively in PBST. Immunosorbents prepared by covalently attaching the antibodies to Sepharose, efficiently removed the matrix interferences caused by the cattle urine samples. Moreover, St and 16βOH-St were efficiently extracted from urine samples as demonstrated by LC–MS/MS analysis. The immunosorbents are filled on small mini-columns arranges on a 96-SPE-setup compatible with the microplate based ELISA methods. Samples and standards can be run in parallel which increment considerably the speed of the screening method. The recovery values of the whole HTS-IS-SPE-ELISA procedure has found to be 112 ± 10% and St can be detected in hydrolyzed urine samples with LOD of 1.26 ± 0.46 μg L −1 using just 1 mL of sample. As proof-of-concept the urinary excretion profile of St treated animals has been investigated by analyzing individual sampling points. Results from pooled urine samples have also been compared with the results obtained by GC–MS analysis demonstrating the StIR equiv. measured with the HTS-IS-SPE-ELISA protocol are in accordance with the St and 16βOH-St levels found with the chromatographic method. The analytical procedure is rapid, effective and the detectability achieved is below the MPRL (minimum performance required levels) recommended by CRL (Community Reference Laboratory) to the European Community.