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Detection of nitrofurane metabolites in food with HPLC/MS/MS

Furazolidone, Furaltadone, Nitrofurazone and Nitrofurantoine belong to the group of nitrofuranes. As antibiotics they were used for treatment of gastrointestinal infections in cattle and poultry. Due to its proven mutagenic and carcinigenic effect, nitrofuranes (except for furazolidone) was prohibited in 1993 in the European Union (VO (EWG) No. 2901/93). Furazolidone was prohibited in 1995 in the European animal breeding (VO (EWG) No. 1441/95). They are mentioned in appendix IV of VO (EWG) No. 2377/90 as compunds for which no upper limits can be lay down.

Introduction

In 2002 nitrofuranes and their metabolites were frequently detected in imported poultry from Brazilia and shrimp from Asia. In this application note we present a LC/MS/MS method for detecting the nitrofurane metabolites. Since nitrofuranes have in vivo halflife times of a few hours, only their metabolites are discussed here. The metabolites are known to be very stable2: 3-Amino-2-oxazolidinone (AOZ), 3-Amino-5-morpholinomethyl- 2-oxazolidinone (AMOZ), Semicarbazide (SEM) und 1-Amino-hydantoin (AHD). The measurements shown in the following were done both on an API 2000 LC/MS/MS System and an API 4000 LC/MS/MS System. The presented method was developed on an API 2000 system in Laboratory W.E.J. The measurements on the API 4000 system were done in the Application Laboratory of Applied Biosystems (Darmstadt).

Sample Preparation

Due to their small moleculare weight it is neccessary to derivatize the metabolites with 2-nitrobenzaldehyde to achieve selective fragments. The sample treatment is shown here schematically3. AMOZ-D5 was used as an Internal Standard.

HPLC Conditions

Agilent 1100 System Column: Luna C18 (2), 150x3 mm, 3 μm, Phenomenex

Column oven: 25C

Eluent A: 0.5 mMol NH4ac B: MeOH

Flow: 200 μl/min, W.E.J.: during elution isocratic 50/50, AB: gradient from 95/5 to 5/95

Results

Despite their small molecular weight the derivatised metabolites can be quantitated over several orders of magnitude with high accuracy. On the API 4000 system we created for all metabolites a calibration curve from 0.5 pg to 750 pg. We determined linear regression coefficients (weighed by 1/x) of 0.999 and better. Similar coefficient were found on the API 2000 system in a concentration range of 50 pg to 5 ng.

Summary

The observed recovery was found to be 70% for SEM and AHD, 40% for AOZ and 80% for AMOZ. On the API 2000 system the quantification limit was found to be 0.5 μg/kg for AOZ and AMOZ and 1 μg/kg for SEM and AHD. The detection limits were a factor of three lower. At the API 4000 system following quantification limit were found AOZ: 0.02 μg/kg AMOZ: 0.01 μg/kg, SEM: 0.05 μg/kg and AHD: 0.1 μg/kg.

Literature


1 BgVV, Stellungnahmen vom
18.06.02 und 15.07.02
2 A. Conneely et al., Analyst, 2002,
127, 705-709
31 A. Leitner et al., J. Chromatogr.
A, 939, 2001, 49-58


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