Determination of Trace Level Nitrofuran Metabolites Note: 361
in Crawfish Meat by Electrospray LC-MS/MSon the Finnigan TSQ Quantum Discovery MAXTao Ding,1 Jingzhong Xu,1 Chongyu Shen1 and Kefei Wang2 1 Food Laboratory, APFIC, Jiangsu Entry-Exit Inspection and Quarantine Bureau of People’s Republic of China, Nanjing, China2 Thermo Electron Corporation, San Jose, CA, USA Introduction
acidic solution and derivatized to nitrobenzyl- (NB-) Key Words
Nitrofurans (furazolidione, furaltadone, nitrofurazone derivatives with 2-nitrobenzyladehyde (2-NBA). Figure 1illustrates the transformation. LC-MS/MS utilizing selected • Finnigan™
and nitrofurantoin) are a group of veterinary antibiotics reaction monitoring (SRM) of the corresponding four TSQ Quantum
banned in many countries because of human health metabolite derivatives has become the method of choice. Discovery MAX
concerns. The ban has stimulated significant interest indevelopment of analytical methods for detecting trace In this note we describe a sensitive and selective • Finnigan
levels of these drug residues in animal products.
LC-MS/MS method for detecting trace level nitrofuran SurveyorHPLC
Due to the rapid in vivo metabolism of the parent metabolites in crawfish using a Finnigan TSQ QuantumDiscovery MAX triple quadrupole mass spectrometer • Food residue
drugs, detection of nitrofurans in meat products relies coupled to a Finnigan Surveyor HPLC module. The limit analysis
on determination of their corresponding tissue-boundmetabolites: 3-amino-2-oxazolidinone (AOZ), 3-amino- of quantitation (LOQ) as low as <0.05 µg/kg has been 5-morpholinomethyl-2-oxazolidinone (AMOZ), semi- clearly demonstrated in fortified crawfish meat for all fournitrofuran metabolites. This LOQ represents twenty-fold • Veterinary drugs
carbazide (SEM) and 1-aminohydantoin (AHD). Thesemetabolites were removed from tissues by hydrolysis in better than the Minimum Required Performance Limit(MRPL) established by European Union (EU) in 2003.
Figure 1: Nitrofurans, their metabolites, and 2-NBA derivatives Experimental Conditions
Note that the sample preparation results in a two-fold concentration that will be factored into the calculation Standards and Reagents
of nitrofuran metabolite concentrations in meat samples.
The following are a list of chemicals used in this work, For fortified samples, the nitrofuran metabolites and their and unless specified all chemicals are of at least reagent internal standards were added into the homogenized meat sample prior to the hydrolysis and derivatization. For calibration, the same procedures were followed except that 2 mL of working standard solutions was used instead Chromatography Conditions
Analytical column: Hypersil GOLD,™ 5 µm, 1-Amino-imidazolidin-2,4-dione-[2,4,5-13C] (WITEGA Laboratorien Berlin-Adlershof GmbH, Berlin, Germany) Eluent: A: 0.5 mM Ammonium Acetate in Water; Methanol (HPLC grade, Fisher, Pittsburgh, PA, USA) Water (in-house distilled water, filtered with a 0.45 µm Analytical Equipment
HPLC: Finnigan Surveyor HPLC module consisting of AS Mass spectrometer: Finnigan TSQ Quantum Discovery MAX Analytical Standard Preparation
Mass Spectrometry Conditions
The primary analytical standard solutions of 1 mg/mL The mass spectrometer was calibrated routinely with were prepared by dissolving the corresponding solid 1,3,6-polytryosine, according to the standard operating standards into methanol. The working standard solutions procedures at the Nanjing laboratory. For method were prepared by serial dilution of the primary standard development, a standard solution containing 1 µg/mL of derivatized nitrofuran metabolites including the internal Sample Preparation
standards was infused at 10 µL/min with 250 µL/min50:50 (A:B) mobile phase into the ESI source. First, the Note: Nitrofuran metabolite derivatives are sensitive to spray voltage, sheath gas, auxiliary gas and tube lens were light; avoid prolonged exposure of sample to direct light optimized with the automated tune of Xcalibur™ software.
Second, the most abundant fragment ions and their opti- The extraction and derivatization of the nitrofurans mized collision energy (CE) values were found in MS/MS from the crawfish were performed at Food Laboratory of optimization. For known SRM transitions, parent and Jiangsu Entry-Exit Inspectional and Quarantine Bureau at product ions can be input directly to obtain the optimized Nanjing, China, following the published procedure with CE value for each SRM transition. Finally, the Source CID (skimmer offset voltage), collision gas pressure, and ion • To 2 g of homogenized crawfish meat inside a 50-mL transfer capillary temperature were adjusted manually for glass tube, add 4 mL water, 0.5 mL of 0.5 M HCl best signal sensitivity. The final operation parameters are solution, and 200 µL of freshly prepared 50 mM 2-NBAin DMSO. Vortex for one minute and store the sample in the dark at 37°C overnight (14-16 hours) • After cooling the sample to room temperature, to 7.0-7.5 with 0.4 M NaOH solution. Centrifuge the Ion transfer capillary temperature: 300°C • Extract the supernatant twice each time with 7 mL ethyl acetate. Combine the ethyl acetate extracts and • Reconstitute the residues in 1.0 mL of water:methanol Collision gas and pressure: Ar at 1.3 mTorr (95:5). Centrifuge the samples and filter the supernatantwith 0.2 µm syringe filter prior to injection to LC-MSsystem For each parent ion, two SRM transitions were used, one for quantitation and one for confirmation, which Ion (m/z)
Ions (m/z)
would give 4 IP (identification points) to meet the EU’s criteria for residue analysis in food. Based on the elution order of the nitrofuran metabolite derivatives, the chromatography run was divided into three segments for data acquisition. Table 1 lists SRM transitions and their Results and Discussion
Figure 2 shows representative chromatograms of a 0.050 µg/kg fortified crawfish sample. As shown, all four nitro- furan metabolite derivatives were detected with excellent Table 1: Segments of chromatography separation and SRM transitions signal quality as measured by signal-to-noise (S/N) ratio.
Note: * SRM transition for quantitation; IS : internal standard; CE: Collision Energy.
For each segment, Scan Time (s) = 0.1 and Scan Width (m/z) = 0.002.
Figure 2: Chromatograms of shrimp meat sample containing 0.050 µg/kg fortified nitrofurans and internal standard in the shrimp meat.
For each panel from the top: TIC (total ion current), SRM for quantitation (bold and red) and confirmation (green), and internal standard (italic and blue).
RT: retention time, AA: peak area counts, SN: signal-to-noise ratio.
Y = 0.08 50 2 6 8+ 1 .0 68 5 7 *X R ^2 = 0 .9 95 6 W: 1/X Y = 0 .0 51 7 60 3+ 0 .692 11 8 *X R ^2 = 0 .99 8 2 W : 1/X Australia
+61 2 8844 9500
Y = 0.1 9 24 35 +1.9 2 75 7*X R ^2 = 0.99 82 W : 1 /X Y = 0.0 56 2 912+ 1.04 3 21 *X R ^2 = 0.9 97 2 W : 1 /X France
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Figure 3: Seven-point calibration curves of nitrofuran metabolite, from 0.025 ng/mL (equivalent to 0.05 ng/mL after sample preparation) to 10 ng/mL The LOQ values reported in literature, ranging from The method accuracy and precision were evaluated 0.02 to 0.1 µg/kg for different nitrofuran metabolites in by performing triplicate preparation and analysis of one Latin America
+1 512 251 1503
meat samples, have mostly been obtained from extrapo- batch of homogenized crawfish meat samples fortified Netherlands
lation based on S/N =10 of the signals of analytes at with nitrofuran metabolites at three different concentra- higher concentrations in standards or fortified samples.
tion levels of 0.05, 0.5 and 2.5 µg/kg. The results are Scandinavia
+46 8 556 468 00
In reality, however, these extrapolated LOQ’s often cannot given in Table 2. As shown, recovery values in the range South Africa
be achieved, because the S/N ratios of the signals deterio- of 79%–110% were obtained with standard deviations rate more than as predicted by the dilution factor. The from 3 to 22%. It should be noted that standard devia- current data in Figure 2 demonstrates that all four nitro- tions include the errors of both the sample preparation furan metabolites can be detected in fortified crawfish (major contributor) and analytical instrument. Switzerland
+41 61 48784 00
samples at 0.05 µg/kg level, far lower than the MRPL Conclusions
Figure 3 shows seven-point calibration curves con- With use of the Finnigan TSQ Quantum Discovery MAX, structed from data from measuring standard solutions a sensitive and reliable LC-MS/MS method using SRM at concentration levels of 0.025, 0.2, 0.5, 1, 2.5, 5.0 and has been developed for detecting trace level nitrofuran
10.0 ng/mL. Excellent linearity was obtained for all four metabolites with a quantitation limit of less than nitrofuran metabolites within the calibration range, with 0.050 µg/kg in crawfish. The sample preparation the correlation coefficient R2 > 0.995 (weight factor = 1/X).
procedure is relatively straightforward and setup of the 2006 Thermo ElectronCorporation. All rights Fortification
Level (µg/kg)
marks are the property ofThermo Electron Corporation References
1. US FDA: Detection of Nitrofuran Metabolites in Shrimp, 2. A. Leitner et al., Journal of Chromatography A, 939 (2001) pp 49-58.
Table 2: Mean recovery values (n=3) of crawfish samples fortified 3. Seu-Ping Khong et al., J. Agric. Food Chem. 2004, 52 pp 5309-5315.
Note: values given after ± are standard deviations.
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