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LC-MS/MS methods for the confirmation of nitrofuran metabolites

Liquid chromatography, coupled to tandem mass spectrometry, (LC-MS/MS) is now firmly established as the method of choice for the detection, confirmation and quantification of a wide range of veterinary drug residues in food. A method to detect the stable, tissue-bound side chain metabolites of the nitrofuran drugs was one of the main objectives of the FoodBRAND project. The developed method was used, initially in two FoodBRAND partner laboratories to uncover global misuse of the nitrofuran drugs in poultry and aquaculture. The FoodBRAND method (in the form of two Standard Operating Procedures) was disseminated to the EU NRL/CRL network and 3rd countries during 2001 - 2003.

In brief, the method consists of a simple derivatisation step (that may be preceded by a series of pre-washing steps if only tissue-bound residues are to be measured, followed by liquid:liquid extraction and detection using LC-MS/MS. Minced tissues (1.00 +/- 0.02 g) were homogenized in ice-cold methanol (8ml) and water (1ml). Following centrifugation (2000rpm at 4°C for 10min) the supernatant was discarded and the sample repeatedly washed by vortex mixing for 10 seconds in ice cold methanol (3 x 4ml), ethanol (2 x 4ml) and diethyl ether (2 x 4ml). Mixed internal standard (50ml of a solution) was added equally to all samples, controls and standards. Control tissues from nitrofuran-free pigs were fortified with mixed standard (100ml of a solution) to act as recovery control samples. Blank tissues were also included in every analytical run. To all tubes were added de-ionized water (4ml), 1M hydrochloric acid (0.5ml) and 2-nitrobenzaldehyde (150ml, 50mM in DMSO). After vortex mixing, tubes were incubated overnight (approximately 16 h) in a water bath held at 37°C. All tubes were then adjusted to pH 7.4 +/- 0.2 with 0.1M di-potassium hydrogen orthophosphate (5ml) and 1M sodium hydroxide (approximately 0.4ml). Liquid-liquid extraction was carried out using ethyl acetate (2x5ml, mixing for 1min and centrifugation at 2000rpm for 15min at 4°C) and the organic phase evaporated to dryness at 50°C under nitrogen. Residues were re-dissolved in methanol: water (50:50 v/v; 200 ml) and transferred to HPLC microvials (200ml). LC-MS/MS analysis was carried out using electrospray ionisation.

The analytical methods were validated according to Commission Decision 2002/657/EC. In all cases, the determined values of CCa and CCb were well below the Minimum Required Performance Limit of 1.0 µg/kg established for nitrofuran metabolites in poultrymeat and aquaculture products by Commission Decision 2003/181/EC and amended by Commission Decision 2005/34/EC to cover all matrices.

This method, with minor variations, is now in use throughout the world for the control of the nitrofuran antibiotics in food animal production.

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Queen's University Belfast
Stoney Road, Stormont
BT4 3SD Belfast
United Kingdom
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