Screening of calf urine for 19-nortestosterone: matrix effect in some immunoassays

Abstract

The performances of three enzyme linked immunosorbent assays (ELISAs) and one radioimmunoassay (RIA) in 17α-19-nortestosterone (17α-19-NT) analysis of bovine urine have been evaluated. Sample preparation was performed by enzymic deconjugation and solid-phase extraction. The main object of the study was the evaluation of the matrix effect, which is generally great in 19-NT immunoassays. Fifty-seven bovine urine samples have been analysed by immunoassay and by gas chromatography–mass spectrometry (GC–MS). The detection limit of GC–MS analysis was 1 µg l^–1. The mean sample blank values in urine analysis were close to 1.5 µg l^–1 for one ELISA, and 0.4–0.8 µg l^–1 for the other assays (n= 55, samples coming from several breeds in different geographic areas). The detection limits, calculated in compliance to the EEC criteria, were 2.67, 1.97, 2.05 and 5.01 µg l^–1 for RIA, ELISAs 1, 2 and 3, respectively. The percentage of false positive results adopting this criteria were: 1.8, 3.6, 1.8 and 3.6%. The probability of the occurrence of false negative results was evaluated by adding 2 µg l^–1 of 19-NT to a negative sample, was high in each assay. Two samples from experimentally treated animals gave positive results both in GC–MS and in immunoassays. The results obtained confirm that by employing immunoassay and solid-phase extraction as sample clean-up, it is not possible to apply the action level (2 ppb) suggested by the European Union (EU).