Multiresidue confirmatory method for determination of quinolones in milk by HPLC: method development and validation according to the criteria of Commission Decision 2002/657/EC

Abstract

Veterinary drugs have become an integral part of the livestock production and play an important role in maintaining animal welfare. The use of veterinary medicines may be cause of the presence of drug residues in animal food products if appropriate withdrawal periods are not respected or if contaminated feeds are used. This work presents the development of an high performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD) method for the quantitative detection of eight quinolones – norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, difloxacin, oxolinic acid, nalidixic acid, and flumequine – in bovine milk. After deproteination and extraction with a metaphosphoric acid 1% w/v/methanol/acetonitrile (60/20/20 v/v/v) solution, the sample is partially evaporated and cleaned up on a reversed phase solid phase extraction (SPE) cartridge. The extract is analyzed using an HPLC-FLD. Mean recovery ranged between 65-88%. The method is validated as a confirmatory method according to Decision 2002/657/EC. All the verified parameters (linearity, selectivity/specificity, trueness, precision, CC, ruggedness and stability) were satisfactory and the method is able to quantify all the analytes in milk in the concentration range 15-60 μg/Kg for danofloxacin and 25-150 μg/Kg for the other quinolones.