β₂-Agonist abuse in food producing animals: use of in vitro liver preparations to assess biotransformation and potential target residues for surveillance

Abstract

1. The biotransformation of [³H]clenbuterol, [³H]salbutamol, [¹⁴C]salmeterol and 7-ethoxycoumarin by bovine liver was investigated by incubation with freshly prepared microsomes, suspension and monolayer cultures of isolated hepatocytes, precision-cut (250 μm) and chopped (600 μm) tissue slices. 2. Radio-HPLC analysis indicated that the saligenin β₂-agonists salmeterol and salbutamol were extensively metabolized by all intact cell preparations. A single major product (SmM1) was evident for salmeterol and two unresolved products for salbutamol (SbM1 and SbM2). Differential enzyme hydrolysis studies with Helix pomatia β-glucuronidase/aryl sulphatase indicated that the main metabolites were glucuronide conjugates. Consistent with this, analysis of metabolites by liquid chromatography-mass spectrometry showed molecular ions ([M+H]⁺) at m/z 592 for Sm1 and 416 for both Sb1 and Sb2. 3. Comparable studies with clenbuterol revealed three minor metabolites. Prolonged incubations generated products representing, at maximum, 27% biotransformation. Two of the products have been identified as a glucuronide ([M+H]⁺, m/z 453) and hydroxyclenbuterol ([M+H]⁺, m/z 293). 4. These findings indicate that in vitro studies provide simple and cost-effective means of evaluating xenobiotic metabolism, and thus of identifying potential target residues to enable surveillance of use of unlicensed veterinary drugs, or prohibited substances in farm animals.