beta(2)-agonist abuse in food producing animals: use of in vitro liver preparations to assess biotransformation and potential target residues for surveillance

1. The biotransformation of [H-3]clenbuterol, [H-3]salbutamol, [C-14]salmet erol and 7-ethoxycoumarin by bovine liver was investigated by incubation wi th freshly prepared microsomes, suspension and monolayer cultures of isolat ed hepatocytes, precision-cut (250 mu m) and chopped (600 mu m) tissue slic es. 2. Radio-HPLC analysis indicated that the saligenin beta(2)-agonists salmet erol and salbutamol were extensively metabolized by all intact cell prepara tions. A single major product (SmM1) was evident for salmeterol and two unr esolved products for salbutamol (SbM1 and SbM2). Differential enzyme hydrol ysis studies with Helix pomatia beta-glucuronidase/aryl sulphatase indicate d that the main metabolites were glucuronide conjugates. Consistent with th is, analysis of metabolites by liquid chromatography-mass spectrometry show ed 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. Pr olonged incubations generated products representing, at maximum, 27 % biotr ansformation. 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-ef fective means of evaluating xenobiotic metabolism, and thus of identifying potential target residues to enable surveillance of use of unlicensed veter inary drugs, or prohibited substances in farm animals.