The derivatisation of avermectins and milbemycins in milk: New insights and improvement of the procedure

Derivatisation of the avermectines ivermectin (IVM), doramectin (DOR), abamectin (ABA) and eprinomectin (EPR), and the milbemycin moxidectin (MOX) to fluorescent derivatives is commonly used for quantitative analysis at relevant levels using high performance liquid chromatography (HPLC) with fluorescence detection. Problems associated with the differences in reactivity towards derivatisation (EPM) and limited stability of the derived products (IVM, DOR, ABA) may seriously hamper the applicability of the method and the reliability of the obtained results. A study was performed to obtain more insight in this derivatisation process from an organic chemistry point of view. This study demonstrated the occurrence of two main fluorescent derivatives: the trifluoroacetyl esters (flu-TFA) and the derivatives with a free hydroxy group at the glycosidic ring (flu-OH). Optimisation of the derivatisation conditions resulted in a fast and reproducible formation of the fluorescent derivatives for all analytes including EPM. The improved procedure involves the addition of 1-methylimidazole (MI), trifluoroacetic anhydride (TFAA), triethylamine (TEA) and trifluoroacetic acid (TFA) with a subsequent incubation for 30 min at 70 °C. With this procedure for IVM, DOR and ABA flu-TFA derivatives are obtained instead of flu-OH derivatives as generally described in literature. The derivatisation is reproducible in different milk samples and the derivatives proved to be stable for at least 80 h at room temperature. Using the optimised procedure a limit of detection (LoD) of 0.1 μg kg−1 in milk was readily obtained.