Development and optimisation of an improved derivatisation procedure for the determination of avermectins and milbemycins in bovine liver

Abstract

A robust procedure has been developed to overcome the instability problems experienced with the fluorescent derivative of eprinomectin. The procedure involves addition of acetic acid, together with the typical reagents methylimidazole and trifluoroacetic anhydride, to produce a fluorescent molecule that can be determined by high performance liquid chromatography (HPLC) with fluorescence detection. Derivatisation is completed in 30 min at 65 °C. This derivatisation procedure was shown to be suitable, also, for the related compounds, moxidectin, abamectin, doramectin and ivermectin. A multi-residue method for these compounds in bovine liver has been developed using the derivatisation procedure. Samples are extracted with acetonitrile; followed by clean-up on deactivated alumina and C18 solid phase extraction (SPE) cartridges. The method was validated using bovine liver fortified at levels of 4 and 20 μg kg⁻¹ with the drugs. The mean recovery ranged between 73 and 97%. The intra- and inter-assay variations showed relative standard deviations typically of <6% and <14%, respectively. The limit of quantitation of the method is 2 μg kg⁻¹ (ppb).