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

Area of Science:

• Veterinary Pharmacology
• Analytical Chemistry
• Biomedical Analysis

Background:

• Milbemycin is a crucial antiparasitic agent in veterinary medicine.
• Accurate quantification of milbemycin in animal plasma is essential for therapeutic drug monitoring.
• Existing analytical methods may lack the sensitivity or specificity required for routine veterinary diagnostics.

Purpose of the Study:

• To develop and validate a sensitive high-performance liquid chromatography with fluorescent detection (HPLC-FLD) method for milbemycin determination in dog plasma.
• To establish a reproducible and stable derivatization procedure for milbemycin analysis.
• To assess the method's accuracy, precision, and linearity for reliable quantification.

Main Methods:

• A novel derivatization procedure using 1-methylimidazole and trifluoroacetic anhydride was employed to create a fluorescent milbemycin derivative.
• HPLC analysis was performed on a C18 column with specific excitation (365 nm) and emission (475 nm) wavelengths.
• The mobile phase consisted of methanol and water (98:2, v/v), and quantification was achieved using fluorescence detection.

Main Results:

• The developed HPLC-FLD method demonstrated a lower limit of quantification of 1 ng/mL.
• Linearity was observed over a concentration range of 1-200 ng/mL, with high accuracy (>94%) and precision (<5% CV).
• The derivatization process was reproducible and the resulting fluorescent derivatives were stable for at least 80 hours at room temperature.

Conclusions:

• The described HPLC-FLD method is a specific, simple, accurate, and precise analytical technique for milbemycin quantification in dog plasma.
• This validated method is readily adaptable for analyzing milbemycin in the blood of other animal species.
• The assay provides a reliable tool for pharmacokinetic studies and therapeutic drug monitoring of milbemycin in veterinary practice.