Immunofiltration as sample cleanup for the immunochemical detection of β-agonists in urine

Abstract

Despite the ban of the European Union on use of drugs to improve animal growth, occasionally β-agonist drugs are still found in samples from cattle. Over time, the specified limits for the detection of these illegal drugs have been lowered. To improve the immunochemical screening of urine samples to detect lower levels of several β-agonists, immunofiltration (IF) was applied for sample cleanup in combination with a β-agonist-ELISA. In the applied IF format, free (non-immobilised) anti-salbutamol polyclonal antibodies were mixed with the urine sample in an ultra-filtration device (cut off 30 kDa) and the sample was removed by centrifugation. The antibody bound β-agonists were freed from the antibodies by the addition of a mixture of methanol and 0.1 M acetic acid (1∶1; v/v) and centrifugation. The filtrate, containing the free β-agonists, was evaporated to dryness and the residue dissolved in buffer, an aliquot of which was analysed with the β-agonist ELISA. Compared with the direct β-agonist ELISA, this IF cleanup procedure resulted in a 30-times lower limit of detection (LOD) of 0.14 ng ml⁻¹ (salbutamol equivalents). The anti-salbutamol antibodies recognised several β-agonists and the combination of IF with the β-agonist ELISA was found suitable for the detection of at least ten β-agonists in urine with comparable LODs.