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High sensitivity quantitation of metabolites of nitrofuran antibiotics in animal tissue using LC/MS/MS

Furazolidone, nitrofurantoin, furaltadone, and nitrofurazone were analysed by detecting 3-amino-2-oxazolidinon (AOZ), 1-aminohydantoin (AH), 3-amino- 5-morpholinomethyl-1, 3-oxazolidin- 2one (AMOZ), and semicarbazide (SC), respectively.

Materials and methods

The animal tissue was homogenized and centrifuged. The protein-bound residue pellet was cleaned up with a set of organic solvent washes. The pellet was hydrolyzed followed by addition of internal standards. The mixture was derivatized using 2-nitrobenzaldehyde at 37C overnight. After the pH adjusting, the ethylacetate extracted metabolites were dissolved in a mobile phase before injections. The mixed external calibration standards were made by spiking the metabolite stock standards into blank chicken meat followed by the derivatisation described above. The isotopic internal standard D4-AOZ and D5-AMOZ were spiked to correct peak area responses. The D4-AOZ was used for AH, AOZ, and SC and the D5-AMOZ for AMOZ. MS instrument: An API 3000 LC/MS/MS System with a TurboIonSpray source is used in a positive mode at 450C. Two pair of MRM transitions are used for each metabolite for quantitation and confirmation. The parameters were auto-optimised and the quantitation was done using Analyst Software. HPLC: The LC system was composed of an Agilent 1100 binary pump with an autosampler, a C18 Symmetry column (4.6 x 150 mm). The mobile phase was methanol and 20 mM ammonium acetate at a flow rate of 700 μl/min with 50/50 split and a step-linear gradient from 5 - 95% organic in 4 minutes for a total run time of 15 minutes. The injection volume was 25 μl.

Conclusions

The new LC/MS/MS method allows the high sensitive detection of four nitrofuran antibiotics in chicken tissue via their metabolites. The one step derivatization improves non-specific fragmentation
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