Feng Liang, Peibin Hu, and Ping Li Agilent Technologies, Inc. Beijing, China
analytical detection of the metabolites and not the parent compounds are required in samples of animal origin. The criteria for detection and confirmation of veterinary drugs in animal and animalproducts established by the European Union (EU)  has been accepted in much of the world. This criteria mandates a separation technique combined with a spectrometric technique. For banned substances such as the nitrofurans, no maximum residue limit (MRL) could be set. Therefore a minimum required performance level (MRPL) was set at 1 µg/kg for each metabolite . Only LC/MS could meet thesecriteria, and very good methods have been reported [4–6]. However, the most widely accepted methodology employs triple quadrupole tandem mass spectrometers. This is the first report showing analysis of these metabolites using the new Agilent triple quadrupole LC/MS system.
The metabolites of nitrofuran antibiotics banned in meat and meat products are analyzed by LC/MS/MS with the newAgilent 6410 triple quadrupole. The method is shown to be highly sensitive, to 0.01 ppb (10 ppt), for each of the four analytes. Calibration from 0.1 ppb to 10 ppb is presented with all criteria for confirmation as set by the European Union decisions for analytical method performance. Extracts of tilapia are used to show the performance of the LC/MS/MS method for aquaculture samples.Introduction
Nitrofurans are inexpensive antibiotics used for Gram positive and Gram negative bacteria. They have been used to treat gastrointestinal and dermatological infections in farm animals and fish. In addition, they have been used to treat bacteria in bees. Because both parent compounds and their metabolites are suspect carcinogens, they have been banned around the world. The Rapid Alert Systemfor Food and Feed Annual report for 2005  shows that these compounds continue to be detected in food samples and remains a major concern for food safety. The four compounds–furazolidone, furaltadone, nitrofurantoin, and nitrofurazone–have been found to metabolize rapidly, and the metabolites bind to muscle tissue. Thus the
Chemicals Derivatized standards of nitrofuranmetabolites and all chemicals for sample preparation were received from a food manufacturing company. Acetonitrile was HPLC grade from Merck (Darmstadt, Germany). Formic acid was reagent grade from Merck (Darmstadt, Germany). Sample Preparation The accepted procedure for sample preparation was followed. To 2 g of tilapia was added 15 mL 0.125 M HCl and the mixture homogenized.
To this solution, a 50-µLsolution of 2-nitrobenzaldehyde (50 mM in DMSO) was added and shaken. The solution was then incubated at 37 °C for 16 hours. This was followed by neutralization to pH ~7 with NaOH and K2HPO4. The neutral derivatized sample was then extracted with ethyl acetate, concentrated to dryness, and reconstituted in 100 L of initial LC mobile phase. Standards of the four metabolites were spiked into 0.125M HCl, derivatized, and extracted for calibration using the same procedure as was used for the samples of tilapia. LC/MS/MS Method
LC Conditions Instrument: Column: Column temp.: Mobile phase: Gradient: Agilent 1100 LC C18, 2.1 mm × 150 mm, 3 µm 40 °C A = 0.1% formic acid in water B = acetonitrile 22% B at 0 min 99% B at 6 min 99% B at 9 min 0.3 mL/min 50 nL
Quantitation Quantitativeanalysis was done with the first transition listed in the MRM parameter table. The second transition was used as a qualifier ion for confirmation as per the confirmation criteria. Quantitative results were performed with the new MassHunter quantitative analysis software.
Results and Discussion
The instrument sensitivity is an important performance parameter for this analysis when considering the...