Quantitation of Nitrofuran Metabolites in

Shrimp and Poultry by LC/MS/MS Using

the Agilent LC/MSD Trap XCT
Application

Food

Authors shrimp, and poultry. In 1995 the four drugs Fura-

zolidone, Furaltadone, Nitrofurantoin, and Nitrofu-
Bernhard Wüst, Christian Sauber razone were banned by the European Union (EU)
Agilent Technologies GmbH, for their usage in food-producing animals. Due to
Hewlett-Packardstr. 8, 76337 their rapid metabolism nitrofuran parent sub-
Waldbronn stances are not suitable for monitoring and typi-
Germany cally their metabolites are analyzed. A liquid
chromatography /mass spectrometry/mass spec-
Hans (J.) A. van Rhijn
trometry (LC/MS/MS) method was developed for
RIKILT -Institute of Food Safety
the sensitive, qualitative, and quantitative analysis
Bornsesteeg 45,6700 AE Wageningen of four derivatized nitrofuran metabolites found in
the Netherlands shrimp and poultry. See Figure 1.

Abstract Experimental
An LC/MS/MS method was developed for the qualitative All liquid chromatography/mass spectrometry
and quantitative measurement of nitrofuran metabolites in (LC/MS) experiments were performed using an
chicken and shrimp using the Agilent LC/MSD XCT Ion Agilent 1100 Series LC system coupled to a mass
Trap. The limit of quantitation for all four nitrofurans selective detector (MSD) Ion Trap XCT mass
investigated easily met the specified European Union spectrometer. The Ion Trap was operated with an
Minimum Required Performance Level of 1 µg/kg and orthogonal electrospray source in positive ion
ranged from 0.125 µg/kg to 0.5 µg/kg. mode using multiple reaction monitoring (MRM).
A gradient method was used for chromatography.
Deuterated NBA-AMOZ was used as the internal
Introduction standard (ISTD) for NBA-AMOZ and deuterated
NBA-AOZ was used as the ISTD for the other
Nitrofuran antibiotics are widely used for the
metabolites.
treatment of infectious diseases in cattle, pigs,
 _ O O
O N
O O O
N+ O N
N
O N N
N O H2N O O

Furazolidone AOZ NBA-AOZ

_
O
O
N+ H2N
O O N N
O N
N N
N O
O O
O
O N
O N
N
O
O
Furaltadone O AMOZ NBA-AMOZ

_ O O O H
O O O N
N
O O
N+ N NH
N NH H2N
O N N
O O

Nitrofurantoin AHD NBA-AHD

_ O O
O H N
O H N O H
N+ N O H2N N O
O N N
NH2
NH2 NH2

Nitrofuranzone SEM NBA-SEM

Drugs Metabolites Derivatized metabolites

Figure 1. Chemical structures of nitrofurans, nitrofuran metabolites, and corresponding derivatives.

Sample Preparation The sample was then neutralized with 500 µL of a

0.3 M Na3PO4 solution in water and adjusted to
One (1) g of homogenized tissue from shrimp or pH7 with 2 M NaOH solution. After the addition of
poultry was mixed with 5 mL of a 0.2 M hydrochlo- 4-mL ethyl acetate, the sample was centrifuged
ric acid and 50 µL of 2-nitrobenzaldehyde (2-NBA, and the organic layer was transferred to a clean
100 mM in methanol) and incubated overnight at tube. The sample was further extracted with a
37 °C. This is a protocol from the State Institute 4-mL aliquot of ethyl acetate, centrifuged, and the
for Quality Control of Agricultural Products organic layer added to the first extract. After evap-
(RIKILT, The Netherlands). Using this protocol, oration to near dryness, the sample was reconsti-
tissue-bound residues of the nitrofurans with an tuted in 500-µL solvent (50-mL acetonitrile, 80-mL
intact side chain are released through acidic water and 0.5-mL acetic acid) for subsequent
hydrolysis of the imine bond. The free amino LC/MS analysis.
groups of the corresponding metabolites are
derivatized with 2-NBA to form an aromatic imine Calibration standards were made by spiking
bond. known concentrations of all four underivatized
analytes into shrimp and poultry prior to sample
preparation.

2
Chemicals
Methanol (Biosolve, 13683502)
Hydrochloric acid, HCL 32% (Merck, 100319)
2-Nitrobenzaldehyde (2-NBA), C7H5NO3 (Sigma, N6001)
Tri-sodiumphosphate-dodecahydrate, Na3PO4 12(H2O) (Merck, 106578)
Sodium hydroxide, NaOH (Merck,106498)
Ethyl acetate, CH3COOC2H5 (Biosolve 05402602)
Acetic acid, CH3COOH (Merck 10063)
Acetonitrile, CH3CN (Biosolve, 01203502)
Methanol-d, 99.5% D (Aldrich, 15.193-9)

LC/MS/MS Method Details

HPLC: Agilent 1100
Flow rate: 0.4 mL/min
Column: Zorbax XDB-C18, 2.1 mm × 150 mm, 3.5 µm
Mobile phases: A: Water + 0.1% acetic acid
B: Acetonitrile + 0.1% acetic acid
Gradient: 0–14 min: 10% A - 45% A; 14–16 min: 45% A - 90% A
Injection: 50 µL out of 500 µL

MS 1100 LCMSD XCT Ion Trap

Ionization mode: Positive ESI
Nebulizer pressure: 45 psi
Drying gas flow: 12 L/min
Drying gas temperature: 350 °C
Skimmer: 20 V
Capillary exit: 55 V
Trap drive: 55
ICC: On
MRM mode
4 Segments: 0–2.2 min, 2.2–7.4 min, 7.4–10.6 min, 10.6–13.9 min
Segment 1: Divert valve: to waste, no spectra
Segment 2: MS/MS of m/z 335, Isolation width: 2.0, Cut-off: 140; Amplitude: 1.16
MS/MS of m/z 340, Isolation width: 2.0, Cut-off: 140; Amplitude: 1.16
Segment 3: MS/MS of m/z 209, Isolation width: 2.0, Cut-off: 120; Amplitude: 1.28
MS/MS of m/z 249, Isolation width: 2.0, Cut-off: 100; Amplitude: 1.25
Segment 4: MS/MS of m/z 236, Isolation width: 2.0, Cut-off: 100; Amplitude: 1.25
MS/MS of m/z 240, Isolation width: 2.0, Cut-off: 100; Amplitude: 1.25

Quantitation
NBA-AMOZ: EIC of 261 + 291 (MS/MS of 335), Ret. Time: 4.5 min
NBA-dAMOZ: EIC of 266 + 296 (MS/MS of 340), Ret. Time: 4.5 min
NBA-SEM: EIC of 166 + 192 (MS/MS of 209), Ret. Time: 9.9 min
NBA-AHD: EIC of 134 (MS/MS of 249), Ret. Time: 10.0 min
NBA-AOZ: EIC of 134 (MS/MS of 236), Ret. Time: 10.8 min
NBA-dAOZ: EIC of 134 (MS/MS of 240), Ret. Time: 10.8 min

Maximum accumulation time: 150 ms

Smart target: 100.000
Scan: 100–350

3
Results and Discussion
Very low limits of detection (LOD) are required for
nitrofuran metabolites and the derivatization
method increased the ionization efficiency, as well
as improving the chromatographic separation. A
liquid-liquid extraction procedure was used which
resulted in a relatively high concentration factor to
further improve LOD.

The ion trap mass spectrometer was operated in

MRM mode. In this mode, only precursor ions are
chosen and full-scan MS/MS-spectra of the corre-
sponding analytes are acquired. These full scan-
MS/MS spectra are then used for identification by
comparing them with MS/MS-spectra stored in a
library. No further qualifier ion has to be
monitored. See Figure 2.

Intensity Intensity
x104
x104 3
3
2A NBA-AMOZ 2B NBA-AMOZ 290.9
2
2
1 1
192.9 261.9 317.8 333.9
0 0

x105
x104
0.8 NBA-d5AMOZ 6 NBA-d5AMOZ 295.9
4
0.4
2
264.9
0.0 0

x104 x104
2.0 3.0
164.7 217.7 235.8
2.0
1.0 NBA-AOZ 133.8 NBA-AOZ
1.0 206.7
119.9 148.8 190.7
171.8
0.0 0.0

x104 x104
2.0 2.0
133.8
NBA-d4AOZ NBA-d4AOZ
1.0 1.0
158.9 176.8 239.9
122.9 148.8 166.7 204.8 222.8
0.0 0.0
8000
6000 162.7
6000 133.8
4000 230.7
4000 248.8
NBA-AHD 120.8 188.7 NBA-AHD
2000 2000 144.8 200.7212.8
138.7 170.7
0 0

x104
1.2 8000
6000 165.7
0.8
NBA-SEM 4000 NBA-SEM
0.4 190.8 207.8
2000 135.8 148.7
217.8 228.7
0.0 0
2 4 6 8 10 12 Time [min] 100 125 150 175 200 225 250 275 300 325 m/z

Figure 2. Representative chromatograms (2A) and MS/MS spectra (2B) for all analytes plus ISTDs (1 µg/kg).

4
Quantitation is performed by selecting one or more
product ions to create extracted ion chromatograms
for each analyte and ISTD. The product ions used
for quantitation were selected for best signal-to-
noise (S/N) ratio post-acquisition. See
Figures 3 and 4.

4000

3000
Intensity

2000

1000

0
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
Time [min]

Figure 3. Limit of quantitation (LOQ) for NBA-AMOZ, 0.125 µg/kg in shrimp matrix.

1.0 1.0

0.8 0.8
ISTD Response x106
Relative response

0.6 0.6

0.4 0.4

0.3 0.3

0.0 0.0
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50
Relative concentratiion

Figure 4. Calibration curve for NBA-AMOZ, 0.125 µg/kg – 2 µg/kg poultry matrix, three replicates.

5
NBA-AMOZ and NBA-SEM were quantified using
the sum of two product ions, while NBA-AHD and
NBA-AOZ were quantified using one product ion.

The European Union has set a Minimum Required

Performance Level (MRPL) of 1 µg/kg for nitrofu-
ran metabolites. These detection limits are easily
reached using this method with LOQs ranging from
0.125 µg/kg for NBA-AMOZ to 0.25 µg/kg for
NBA-AOZ and NBA-SEM, and 0.5 µg/kg for
NBA-ADH. See Figure 5.

Intensity
x104
4
NBA-AMOZ
3
2
1
0

x104
6
4 NBA-d5 AMOZ
2
0

x104
1.50

1.00
NBA-AOZ
0.50

0.00

x104
1.2

0.8 NBA-d4 AOZ

0.4

0.0

6000
5000
4000
NBA-AHD
3000
2000
1000
0

x104
4 NBA-SEM
3
2
1
0
2 4 6 8 10 12 Time [min]

Figure 5. Representative chromatogram of a positive shrimp sample at a level of 0.25 µg/kg.

6
Linearity of the method was evaluated up to twice
the MRPL (2 µg/kg) and showed a linear weighted
regression (1/×) with coefficients of correlation of
0.99 or better. Intraday relative standard devia-
tions (RSDs) were below 10% for all analytes at all
concentrations. See Table 1.

Table 1. Method Reproducibility and Accuracy for the Four Target Derivatized Metabolites

NBA-SEM NBA-AOZ NBA-AMOZ NBA-AHD

Standard Accuracy % Accuracy % Accuracy % Accuracy %
(µg/kg) SD % (average) SD % (average) SD % (average) SD % (average)
0.125 3.77 98.81 2.29 98.94 4.34 101.10
0.25 2.26 102.72 2.52 101.76 5.87 95.55 6.05 98.31
0.5 3.40 100.72 3.35 100.58 6.21 105.19 4.59 103.87
1 3.56 96.62 3.01 101.90 5.46 99.11 6.53 100.22
2 2.94 101.12 2.13 96.82 5.77 99.05 6.97 97.60
All calibration curves linear weighted 1/x n=6

Conclusions
An LC/MS/MS method was developed for the quali-
tative and quantitative measurement of nitrofuran
metabolites in chicken and shrimp using the
Agilent XCT Ion Trap. The LOQ for all four nitrofu-
rans investigated easily met the specified EU MRPL
of 1 µg/kg and ranged from 0.125 µg/kg to 0.5 µg/kg.

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© Agilent Technologies, Inc. 2004

Printed in the USA

March 25, 2004
5989-0738EN