Optimization of the HPLC Method of Trimethoprim Determination in Additive Premixes and Medicated Fodders

Abstract

An HPLC method was developed for rapid determination of trimethoprim in additive premixes, in final fodders, and in contaminated final fodders. Trimethoprim is extracted from the sample with acidified aqueous methanol or dichloromethane and, after dilution of the methanolic extract or purification of the dichloromethane extract by re-extraction into 0.15 mol.l^-1 H₂SO₄ and on the solid phase C18, it is determined by reverse-phase chromatography on C₁₈ with UV detection. The composition of the mobile phase was optimized. The limit of determination (2 mg.kg^-1) and the yield of the method at trimethoprim concentrations 15 - 45 mg.kg^-1 (99.4 ± 5.7 %) were determined. The repeatability was not determined due to a low number of real samples of final fodders. To lower the limit of determination, fluorescent detection was used after precolumn derivatization of trimethoprim with phthalaldehyde. The calculated limit of detection was then 15 µg.kg^-1