Multiresidue analysis of β 2-agonists in human and calf urine using multimodal solid-phase extraction and high-performance liquid chromatography with electrochemical detection

Abstract

Various β 2-agonists are used as illegal growth promoters in man and in animals. We developed a multiresidue procedure for the analysis of four β-agonists in human and calf urine. The sample was pre-extracted with an Extrelut column at alkaline pH. The β-agonists were eluted with a mixture of tert.-butylmethyl ether and hexane. Then the extract was further cleaned with a mixed mode SPE column, or with a combination of immunoaffinity chromatography (IAC) and the mixed mode SPE column. The IAC column contained antibodies against salbutamol, which were suitable for multiresidue extractions. The extract was then brought onto a mixed mode SPE column at an acidic pH. The column was washed with 70% methanol in water. Thereafter, the β-agonists were eluted with ammoniated ethanol–hexane. The extract was analysed with an HPLC method with electrochemical detection. The β-agonists were separated on a reversed-phase column using a mobile phase buffered at pH 5.5 and containing an ion-pair reagent. Recoveries were higher when the IAC procedure was not performed (90–105% vs. 65–75%), but the extracts were cleaner when the latter step was included. Detection limits in human and calf urine were in the low ng/ml range. The study indicated that β 2-agonists can be analysed in human and calf urine without the selectivity of a mass spectrometer, but that comprehensive clean-up is required to avoid the interference of urine matrix components.