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

A sensitive and simple high performance liquid chromatograph tandem mass spectrometry (LC/MS/MS) method is presented for a simultaneous analysis of the metabolites of four nitrofuran antibiotics in chicken tissue.

Abstract

The animal meat samples were prepared by homogenization, organic solvent washes, hydrolysis and derivatization followed by liquid-liquid (L-L) extraction before analysis. The limits of detection were 0.05-0.15 ng/g animal tissue using a triple-quad mass spectrometer with electrospray ionization in a positive mode. The analyte quantitation and identification was performed according to European Union (EU) guidelines, using multiple reaction monitoring (MRM) with one precursor and two product ions as identifiers. This method was validated and has been successfully used for routine analysis in the government labs.

Introduction

Nitrofurans antibiotics, furazolidone, nitrofuratoin, furaltadone, and nitrofurazone, have been widely used as food additives for the treatment of gastrointerstinal infections in cattle, pigs, and poultry. Due to mutagenic and genotoxicity, these antibiotics are now banned in food-producing animals. The contamination may not only occur from deliberate and direct misuse of the drugs, but also from contaminated feed and environmental sources. Nitrofuran antibiotics are rapidly metabolized. The metabolites are highly stable with a longer residence time bonded to the meat tissue. Due to the complexity of the sample matrix and no maximum residue limits set, a highly selective and sensitive bioanalytical method is needed by regulatory agencies of many countries.

The LC/MS/MS methods reported in the literature usually have poor sensitivity and reproducibility and time-consuming for sample preparation. We improved the LC/MS/MS method with higher sensitivity and reproducibility and simplified the sample preparation. 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 and poor MS detection sensitivity of the small and polar molecules of the drug residues. The sample preparation is simple and fast with good recoveries. To meet the regulatory needs, two fragment ions per analyte are used for more confident confirmation. There are two identical product ion pairs for AH and AOZ chosen for MRM quantitation because they give the best sensitivity. With the TM LC/MS/MS System LINAC collision cell feature, a fast analysis can be achieved without chromatographic separation of the two compounds with the same product ions. There was no cross-talk when they are co-eluted from the LC. The validated method has high sensitivity with the LODs between 0.15-0.05 ng/g chicken tissue and excellent linearity and reproducibility. The method is robust and has been used for routine analysis of nitrofuran antibiotics in animal tissue.

References

1 A. Leitner, P. Zollner, W. Lindner, J. Chromatogr. A, 2001, 939, 49-58.

Acknowledgements

We would like to acknowledge Tum Sutthisak of GeneSystems, Thailand, Prasert Kaewchuchuon, Michael Koenig of Applied Biosystems, SE Asia for their technical and management support.


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