High-performance liquid chromatographic assay for the determination of a semisynthetic avermectin analog (eprinomectin) in bovine milk at parts per billion levels—method development and validation

A sensitive and automated method has been developed and validated to determine marker residue eprinomectin B1a in bovine milk. Extraction of eprinomectin B1a from milk is accomplished with acetonitrile after the addition of an internal standard. The extract containing the analytes is evaporated to dryness and reconstituted in a solution containing 30% 1-N-methylimidazole in acetonitrile. Online derivatization is carried out with trifluoroacetic anhydride. Determination of eprinomectin B1a and its internal standard is carried out by HPLC using a reversed-phase C18 column with a mobile phase consisting of methanol, acetonitrile, water, triethylamine and phosphoric acid. The overall extraction recovery of eprinomectin B1a is 94% with milk supplemented between 2 and 50 ng/ml eprinomectin B1a. Precision RSD averaged 3.0% in Laboratory 1 (n=25) compared to 4.3% in Laboratory 2 (n=35). The limit of quantitation is approximately 2 ng/ml eprinomectin B1a, the limit of detection is approximately 0.25 ng/ml using this method.