Test strip enzyme immunoassays and the fast screening of nortestosterone and clenbuterol residues in urine samples at the parts per billion level.

The preliminary results of an investigation into the development of "on-site" test strip enzyme immunoassays for the screening of urine samples for the presence of growth promoters, such as 17 beta, 19-nortestosterone and clenbuterol at the parts per billion level are described. Urine samples, enzyme-labelled analyte and a nitrocellulose test strip, containing immobilized antibodies, are incubated together, after which the strip is placed in a chromogen-containing substrate solution for colour reaction. Using prefabricated strips, the tests can be performed in 45-60 min. A similar assay was worked out using a dot-blotting device, allowing the test to be performed in 20-50 min. The tests are simple and easy to perform outside the laboratory. Urine samples identified positive by gas chromatography mass spectrometry were also found to be positive with these test strips and, so far, no false-positive results have been encountered. With standard additions to blank urine samples, positive samples could be distinguished above the 5 ng ml level. However, samples from treated calves contain one or more metabolites of the parent compound, which increase the sensitivity of the assays. Although the tests described can be improved and still have to be evaluated further by analysing more urine samples, the preliminary results are very promising and give a lead to further research into the applicability of such "on-site" tests in residue analysis.

Rapid determination of clenbuterol residues in urine by high-performance liquid chromatography with on-line automated sample processing using immunoaffinity chromatography.

A liquid chromatographic column-switching system for automated sample pretreatment and determination of clenbuterol in calf urine, using an immunoaffinity precolumn with Sepharose-immobilized polyclonal antibodies against clenbuterol, is described. A second precolumn packed with C18-bonded silica was used for the reconcentration of desorbed clenbuterol prior to the analytical separation. Urine, after 2-fold dilution with buffer (pH 7.4), was loaded directly onto the immuno precolumn, where clenbuterol was trapped by the immobilized antibodies. This immuno precolumn has been used for more than 200 runs with standard solutions and samples. Bound analyte was desorbed with 0.01 M acetic acid and transferred, via the second precolumn, to the analytical column. The total runtime per sample was 35 min. Using a sample load of 27 ml of dilute urine and UV detection at 244 nm, the detection limit was 0.5 ng/ml. The mean recovery of clenbuterol added to a blank urine sample at the 5 ng/ml level was 82 +/- 2% (n = 5) as determined with standard solutions loaded onto the same system. Urine samples from treated animals were analysed and the clenbuterol concentrations were comparable to those obtained by high-performance liquid chromatography using solid-phase extraction for sample clean-up.