Development and validation of an HPLC-FLD method for milbemectin quantification in dog plasma.

Milbemectin is a widely used veterinary antiparasitic agent. A high-performance liquid chromatography with fluorescent detection (HPLC–FLD) method is described for the determination of milbemectin in dog plasma. The derivative procedure included mixing 1-methylimizole [MI, MI-ACN (1:1, v/v), 100μL], trifluoroacetic anhydride [TFAA, TFAA-ACN (1:2, v/v), 150μL] with a subsequent incubation for 3s at the room temperature to obtain a fluorescent derivative, which is reproducible in different blood samples and the derivatives proved to be stable for at least 80h at room temperature. HPLC method was developed on C18 column with FLD detection at an excitation wavelength of 365nm and emission wavelength of 475nm, with the mobile phase consisting of methanol and water in the ratio of 98:2 (v/v). The assay lower limit of quantification was 1ng/mL. The calibration curve was linear over concentration range of 1–200ng/mL. The intra- and inter-day accuracy was >94% and precision expressed as % coefficient of variation was <5%. This method is specific, simple, accurate, precise and easily adaptable to measure milbemycin in blood of other animals.