Determination of fenoterol and ractopamine in urine by enzyme immunoassay

Abstract

Fenoterol and ractopamine are phenethanolamines with β-adrenergic agonist activity. An enzyme immunoassay (EIA) for these compounds was developed using antibodies raised in a New Zealand white rabbit against fenoterol–bovine serum albumin and fenoterol coupled to horseradish peroxidase (HRP). The calibration graphs of fenoterol and ractopamine showed linearity over the concentration ranges 0.1–5 and 0.2–25 ng ml^–1, respectively. Isoxsuprine showed a cross-reactivity of 0.7% while the cross-reactivity of other β-agonists was <0.1%. The screening assay was used to detect fenoterol in urine samples obtained from an animal experiment in which male calves were treated with fenoterol (100 µg of fenoterol per kg of bodymass per meal for a period of four weeks). Using a direct method, without sample preparation, fenoterol concentrations ranged from 22 to 210 ng ml^–1. The mean concentration of fenoterol after extraction in isobutanol was 3.5 times lower compared with the direct method. On applying enzymic hydrolysis in combination with isobutanol extraction, the mean concentration was eight times higher than that obtained when using extraction only. Gas chromatography–mass spectrometry (GC–MS) analysis confirmed the presence of fenoterol in most of these samples. However, probably owing to the absence of a proper GC–MS internal standard, the correlation between GC–MS and EIA concentrations was low (r= 0.7976). In general, the concentrations found by the EIA are much higher than those found by GC–MS, which might be caused by the presence of metabolites detected with the EIA. Fenoterol is excreted in urine mostly (about 85%) as glucuronidated–sulfated conjugate. The antibodies partly recognize the conjugated fenoterol, which makes it possible to use a direct screening assay. In blank calf urine the detection limits, mean background + 3 times the standard deviation, are 1.3 (fenoterol) and 2.6 ng ml^–1(ractopamine). In bovine urine, however, owing to matrix effects, the detection limits are 20 times higher.