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Determination of Trace Level Nitrofuran Metabolites
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
acidic solution and derivatized to nitrobenzyl- (NB-)
Nitrofurans (furazolidione, furaltadone, nitrofurazone
derivatives with 2-nitrobenzyladehyde (2-NBA). Figure 1illustrates the transformation. LC-MS/MS utilizing selected
and nitrofurantoin) are a group of veterinary antibiotics
reaction monitoring (SRM) of the corresponding four
banned in many countries because of human health
metabolite derivatives has become the method of choice.
concerns. The ban has stimulated significant interest indevelopment of analytical methods for detecting trace
In this note we describe a sensitive and selective
levels of these drug residues in animal products.
LC-MS/MS method for detecting trace level nitrofuran
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
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
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
Analytical column: Hypersil GOLD,™ 5 µm,
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
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
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
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
+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
+33 1 60 92 48 00
+49 6103 4080
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
+1 512 251 1503
meat samples, have mostly been obtained from extrapo-
batch of homogenized crawfish meat samples fortified
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
+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
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.
+41 61 48784 00
samples at 0.05 µg/kg level, far lower than the MRPL
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
marks are the property ofThermo Electron Corporation
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.
Thermo Finnigan LLC,San Jose, CA USA is ISO Certified.
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