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

The performances of three enzyme linked immunosorbent assays (ELISAs) and one radioimmunoassay (RIA) in 17 alpha-19-nortestosterone (17 alpha-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 microgram l-1. The mean sample blank values in urine analysis were close to 1.5 micrograms l-1 for one ELISA, and 0.4-0.8 microgram 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 5.0 micrograms 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 micrograms 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).