Simultaneous analysis of tiaramide metabolites in horse urine and plasma by solid-phase extraction and reversed-phase...

A simple method for the simultaneous analysis of tiaramide (TRA) metabolites in the horse is described. The sample preparation method using a Bond-Elut PH cartridge and stepwise elution with ice-cold, 30% aqueous methanol followed by additional methanol is effective for recovering the metabolites with different properties. The extraction method gives good recoveries (greater than 80%) and reproducibility. Each metabolite is well separated by high-performance liquid chromatography using an octadecyl-type column of polymer-based packing with a solvent system of 20 mM phosphate buffer (pH 6.5)-acetonitrile-tetrahydrofuran (6:1:0.2) containing a quaternary ammonium salt as an ion-pairing reagent. The detection limit of each metabolite is in the range of 20-80 ng/ml. The major metabolites of TRA in equine urine and plasma are N-acetic acid (TRAA) and N-oxide. The N-oxide of TRAA is detected as a metabolite in the urine. A dealkylation product PER) is detected as a minor metabolite. Unchanged TRA is detected only at low concentrations in the urine. Several minor metabolites of DER or TRAA or both are detected in the urine.