Food Control xxx (2015) 1e6

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Determination of nitrofuran metabolites in shrimp muscle tissue by

liquid chromatography-photo diode array detection
R. Fernando a, *, D.M.S. Munasinghe b, A.R.C. Gunasena a, P. Abeynayake a
a
Department of Veterinary Public Health and Pharmacology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya,
Peradeniya, Sri Lanka
b
Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka

a r t i c l e i n f o a b s t r a c t

Article history: The use of nitrofurans on any animal in the European Union (EU) and any animal or animal food products
Received 18 April 2015 intended for export to the EU was banned in 1993 (except furazolidone which was banned in 1995) due
Received in revised form to the carcinogenicity of the parent drugs and their metabolites. Thereafter, the screening of food of
16 August 2015
animal origin for nitrofurans and their metabolites became mandatory for all exports to the EU. This
Accepted 31 August 2015
Available online xxx
paper describes a High Performance Liquid Chromatography e Diode Array Detection (HPLC-DAD)
method to detect tissue bound nitrofuran metabolites, namely 3-amino-2-oxazolidinone (AOZ), 3-
amino-5-morpholino-methyl-1,3-oxazolidinone (AMOZ), semicarbazide (SEM) and 1-aminohydantoin
Keywords:
Nitrofuran metabolites
(AHD). The bound residues were hydrolysed and derivatized into the corresponding nitrophenyl de-
AOZ rivatives (NPAOZ, NPAMOZ, NPSEM and NPAHD) with 2-nitrobenzaldehyde and analysed by HPLC-UV at
AMOZ 275 nm. The linearity of the photometric detector response to the four derivatized nitrofuran metabolites
SEM was veried using matrix-matched calibration standards in the range of 1e20 mg/kg and the average
AHD correlation coefcients for NPAOZ, NPAMOZ, NPSEM and NPAHD were 0.9960, 0.9951, 0.9984 and 0.9993,
HPLC respectively. The decision limit of the method was below the Minimum Required Performance Limit
Chemical compounds studied in this article:
(MRPL) of 1 mg/kg, for all four nitrofuran metabolites, which makes it compatible with the EU re-
3-Amino-2-oxazolidinone (PubChem CID: quirements. The average recoveries for (fortied samples between 1 and 5 mg/kg) AOZ, AMOZ, SEM and
65725) AHD were 107%, 107%, 115% and 114%, respectively. This is the rst report of a HPLC-DAD method
3-Amino-5-morpholino-methyl-1,3- detecting nitrofuran metabolites below the MRPL according to our understanding. This method has been
oxazolidinone (PubChem CID: 3016406) successfully used with aquaculture and poultry products; however, currently it is validated according to
Semicarbazide (PubChem CID: 5196) EU guidelines only for shrimp muscle tissue.
1-Aminohydantoin (PubChem CID: 72823) 2015 Published by Elsevier Ltd.

1. Introduction
The nitrofurans are a group of broad-spectrum antimicrobials
active against Salmonella sp., coliforms, Mycoplasma sp., Coccidia
sp. and certain protozoa (Delatour et al., 2003). Furazolidone, fur-
altadone, nitrofurantoin and nitrofurazone are the four main
members of this group (Kennedy, Young, & McCracken, 2003). They
are rapidly metabolized in the body within hours after adminis-
llner, & Lindner, 2001). However, their metabo-
tration (Leitner, Zo
lites reside in animal body for weeks, possibly months, as protein
bound residues (Conneely, Nugent, & O'keeffe, 2002) (Fig. 1). The
use of nitrofurans was banned in the European Union (EU) member
* Corresponding author.
E-mail address: ruchikaf@pdn.ac.lk (R. Fernando).
states in 1993, with the exception of furazolidone due to the car-
cinogenicity of the parent drugs and their metabolites (EC., 1993).
The ban was extended to cover furazolidone in 1995 due to the
carcinogenicity of the parent drug and the unavailability of data
concerning the safety of its metabolites (EC., 1995). Thereafter, it is
prohibited to use nitrofurans on any animal in the EU and any an-
imal or animal food products intended for export to the EU.
In the early 1990's the Laboratory of the Veterinary Science
Division (VSD) in the Department of Agriculture and Rural Devel-
opment, Belfast, developed an LC-MS method to detect furazoli-
done with a detection limit of 2 mg/kg (Kennedy et al., 2003).
However, it was later found that this method has a limited value,
because the tissue concentrations of furazolidone were very low
due to rapid excretion of the drug and instability of the residues. In
1980s RIKILT (State Institute for Quality Control of Agricultural
Products, Wageningen, Netherlands) showed that mild acidic

http://dx.doi.org/10.1016/j.foodcont.2015.08.044
0956-7135/ 2015 Published by Elsevier Ltd.

Please cite this article in press as: Fernando, R., et al., Determination of nitrofuran metabolites in shrimp muscle tissue by liquid
chromatography-photo diode array detection, Food Control (2015), http://dx.doi.org/10.1016/j.foodcont.2015.08.044
2 R. Fernando et al. / Food Control xxx (2015) 1e6

Fig. 1. A-H. Structures of four nitrofuran antibiotics (on the left side) and their respective free metabolites (on the right side).

treatment could release a compound called 3-amino-2-
oxazolidinone (AOZ) from the stable tissue-bound furazolidone
residues (Fig. 2) (Hoogenboom, Berghmans, Polman, Parker, &
Shaw, 1992; Hoogenboom, Van Kammen, Berghmans, Koeman, &
Kuiper, 1991). Later, RIKILT developed an HPLC UV method for
detection of AOZ and showed that it is a more reliable analytical
method for screening tissues of animals treated with furazolidone
than the parent compound. After the European Commission has
launched the FoodBRAND project to overcome the limitations
associated with nitrofuran issue, the project team developed a
sensitive method to detect all four metabolites of the nitrofuran
drugs. The method utilizes a LC-MS/MS and meets the criteria set
out by the EU for unequivocal conrmation of the presence of these
metabolites (EC., 2002; Kennedy et al., 2003). At present a Mini-
mum Required Performance Limit (MRPL) for the nitrofuran me-
tabolites, in poultry and aquaculture products, has been set at
1.0 mg/kg (EC., 2003).
The shrimp farming industry has been one of the most impor-
tant economic activities in the aquaculture sector of many devel-
oping countries including Sri Lanka due to the high demand in the
EU member states, Japan and USA. However, due to fundamental
decits in disease control, management and biosecurity practices
the disease prevalence is high in shrimp farms (Munasinghe,
Stephen, Abeynayake, & Abeygunawardena, 2010). High disease
prevalence, lack of resources and facilities to test food of animal
origin for such banned antimicrobials and inadequate legislative
frame work may increase the risk of abusing nitrofurans by the
shrimp farmers. Therefore, it is essential to screen shrimp (and
other food of animal origin) for banned antimicrobial compounds
including nitrofurans to safeguard the consumers and export in-
dustry. The purpose of this study was to establish a nancially
sustainable method to screen nitrofuran metabolites namely 3-
amino-2-oxazolidinone (AOZ), 3-amino-5-morpholino-methyl-
1,3-oxazolidinone (AMOZ), semicarbazide (SEM) and 1-
aminohydantoin (AHD) in shrimp muscle tissue conforming to
the EU criteria.
2. Material and methods
2.1. Reagents and chemicals
The solvents used were of HPLC grade or above, unless other-
wise stated. Double distilled and deionized water, ltered through a
0.45 mm nylon lter was used. The standard substances (AOZ,

Please cite this article in press as: Fernando, R., et al., Determination of nitrofuran metabolites in shrimp muscle tissue by liquid
chromatography-photo diode array detection, Food Control (2015), http://dx.doi.org/10.1016/j.foodcont.2015.08.044
 R. Fernando et al. / Food Control xxx (2015) 1e6 3

Fig. 2. Hydrolysis and derivatization of the tissue bound residues of nitrofuran drugs by the action of hydrochloric acid. Fig. 2A. Releasing the side chain (AOZ) from the protein
bound furazolidone metabolite in the acidic medium. Fig. 2B. Derivatization of AOZ into NPAOZ with 2-nitrobezaldehyde.

AMOZ, SEM and AHD) and their respective nitrophenyl derivatives
(NPAOZ, NPAMOZ, NPSEM and NPAHD) were supplied by the In-
ternational Atomic Energy Agency (IAEA), Vienna under a Technical
Cooperation Project.
2.2. Apparatus
An Agilent 1100 HPLC system with an autosampler (model No.
G1313A) and a photodiode array detector (model No. G1315B) was
used. The chromatographic separation was carried out using a C18
(Eclipse, XDB, 4.6
150 mm, particle size 5 mm) reverse phase
column. The ChemStation for LC 3D (Rev. A. 10.02) software was
used to control the equipment and analyse the output data.
dissolved in 500 ml reconstitution solvent (water: acetonitrile:
glacial acetic acid, 90:10:0.1, v/v/v). Subsequently, the dissolved
residue was extracted three times with 4 ml of hexane (C6H14) to
remove the excess 2-NBA. Finally, the aqueous layer was trans-
ferred into an amber colour HPLC vial by ltering through a 0.45 mm
PVDF syringe lter (Whatman, Maidstone, UK). The above
described procedures were carried out with minimum exposure to
daylight and under a yellow light since nitrophenyl derivatives of
the nitrofuran metabolites are light sensitive.
Adequate precautions were taken to avoid contact with chem-
icals via skin, inhalation or any other means during the extraction
process. The organic waste material generated were collected in
suitable containers to discard later according to the regulations and
guidelines of the Central Environmental Authority of Sri Lanka.

2.3. Extraction procedure for shrimp muscle tissue samples

Each test sample consisted of homogenized (using an Ultra
Turrax T25 tissue homogenizer, Janke & Kunkel IKA-Labortechnik,
Staufen, Germany) shrimp muscle tissue without the head and
shell/carapace, coming from three individual shrimps that were
randomly picked from a lot/batch. Five grams (5 g 0.05) of the
sample weighed in a 50 ml polypropylene tube was washed with
20 ml of 50% methanol by mixing thoroughly. Then, the sample was
centrifuged at 2500 g for 10 min and the supernatant was dis-
carded. This washing procedure was repeated with 75% and 100%
methanol and nally with water to remove the matrix interferences
that could affect the detection of the nitrophenyl derivatives of the
nitrofuran metabolites.
Next, 10 ml of 0.2 M hydrochloric acid (HCl) and 100 ml of
100 mM 2-nitrobenzaldehyde (2-NBA) solutions were added to the
sample and placed on an orbital shaker (GFL, Burgwedel, Germany)
in an incubator at 37 2  C overnight. On the following day, after
allowing the sample to reach room temperature, 1 ml of 0.3 M
trisodium phosphate dodecahydrate (Na3PO4.12H2O) solution was
added. The pH of the sample was adjusted to 7 0.5 with 2 M
sodium hydroxide (NaOH). Afterwards, the sample was extracted
two times with 8 ml of ethyl acetate (CH3COOC2H5) and the
resulting organic phases (combined) were collected into a clean
glass tube. This extract was evaporated to dryness at 45  C in a heat
block chamber (Dri-Block DB3A, Techne, UK) under a mild ow of
nitrogen gas. The dry residue obtained in the previous step was
2.4. Control samples and calibration standards
2.4.1. Negative controls
Blank shrimp muscle samples without AOZ, AMOZ, SEM and
AHD were used as negative controls.
2.4.2. Positive controls
A known amount of the four nitrofuran metabolites were added
to blank samples after the washing step to remove the matrix in-
terferences as described in Section 2.3. Then, the samples were
allowed to stand for 15 min before continuing with the rest of the
derivatization and extraction process.
2.4.3. Calibration standards
Blank samples were fortied with all four nitrofuran metabo-
lites to obtain nal tissue concentrations of 1, 2, 5, 10 and 20 mg/kg.
These samples were treated exactly in the same manner as the
positive controls described in Section 2.4.2. The linearity of the
photometric detector response coming from calibration standards
was assessed by plotting the respective peak areas against the
concentrations (mg/kg) using a least-square linear regression model
without an intercept (y mx).
2.4.4. Recovery controls
Blank sample extracts subsequently, immediately prior to HPLC-
UV analysis, fortied with NPAOZ, NPAMOZ, NPAHD and NPSEM

Please cite this article in press as: Fernando, R., et al., Determination of nitrofuran metabolites in shrimp muscle tissue by liquid
chromatography-photo diode array detection, Food Control (2015), http://dx.doi.org/10.1016/j.foodcont.2015.08.044
4 R. Fernando et al. / Food Control xxx (2015) 1e6
equivalent to 1.0, 2.0, and 5.0 mg/kg of the respective nitrofuran
metabolite.
2.5. HPLC-diode array detector analysis
A gradient of mobile phase A [water: glacial acetic acid
(100:0.08, v/v)] and mobile phase B [acetonitrile: water: glacial
acetic acid (90:10:0.1, v/v/v)] was used to separate the nitrophenyl
derivatives. The ow rate was set to 1 ml/min, and the total run
time was 27 min. At the beginning of a run (time line 0 min) the
mobile phases A and B were combined at a ratio of 90% and 10%,
respectively. Starting from minute one, over a 19 min period (time
line 20 min) the proportion of mobile phase A was gradually
reduced to 70%. In order to clean the column before the next run,
the mobile phase A was further reduced to 10% during the next
minute (time line 21 min) and continued this mobile phase
combination for another two minutes (time line 23 min). Finally,
over the next one minute (time line 24) the mobile phase combi-
nation was set back to its starting values (A 90% and B 10%) and
maintained until the run comes to an end (time line 27 min).
The injection volume was set to 100 ml. The HPLC-UV analysis
was carried out at 275 nm and the peak spectra were collected in
the range of 190e400 nm.
2.6. Method validation
The method was validated according to the guidelines provided
in the EU Commission Decision 2002/657/EC (EC., 2002). The data
for calculation of decision limit (CCa), detection capability (CCb),
within laboratory coefcient of variation (CV %) and recovery for
the four metabolites were generated on four different days from the
analysis of ve blank samples and blank samples fortied at 1, 2 and
5 mg/kg. The decision limit (CCa), detection capability (CCb), within
laboratory coefcient of variation (CV %) and recoveries were
calculated using the following formulae:
CCa 3
average response (in mg/kg) of the blank
samples 2.33
standard deviation
CCb CCa 1.64
standard deviation of the lowest fortication
level
Recovery% average measured content for each fortication
level
100/fortication level
CV% standard deviation
100/average measured content for
each fortication level
3. Results
The analytes were well separated in 27 min as relatively sharp
and symmetrical peaks. The mean retention times (standard
Fig. 3. Chromatogram of a shrimp sample fortied at 5 mg/kg with the four nitrofuran metabolites.
Please cite this article in press as: Fernando, R., et al., Determination of nitrofuran metabolites in shrimp muscle tissue by liquid
chromatography-photo diode array detection, Food Control (2015), http://dx.doi.org/10.1016/j.foodcont.2015.08.044
deviation), during a single run, were 9.62 0.04, 15.71 0.02,
16.15 0.02 and 18.76 0.03 min for NPAMOZ, NPSEM, NPAHD and
NPAOZ, respectively. Fig. 3 shows an HPLC-UV chromatogram with
the four derivatized nitrofuran metabolites monitored at 275 nm
wavelength. The blank samples (shrimp muscle) did not show any
considerable matrix peaks closer to the retention times of the four
derivatized nitrofuran metabolites (chromatograms not shown).
Also, there was no interference coming from co-eluting compounds
at the retention times of the four derivatized nitrofuran metabolites
in all veried samples.
The linearity of the photometric detector response to the four
derivatized nitrofuran metabolites was veried using matrix-
matched calibration standards in the range of 1e20 mg/kg and the
average correlation coefcients for NPAOZ, NPAMOZ, NPSEM and
NPAHD were 0.9960, 0.9951, 0.9984 and 0.9993, respectively. The
decision limit (CCa), detection capability (CCb), and the mean values
obtained (using 1, 2 and 5 mg/kg fortied muscle tissue samples) for
the within laboratory coefcient of variation (CV%) and percentage
recovery for each metabolite are given in Table 1.
4. Discussion
The derivatization and extraction process of the method
described in this paper was based on the RIKILT HPLC-UV method
developed to detect the tissue-bound furazolidone metabolite AOZ.
However, the present method was extended to cover the other
nitrofuran metabolites of concern in food of animal origin (AMOZ,
SEM and AHD) with a higher sensitivity. The limit of detection
(LOD) of the RIKILT method was 2 mg/kg (Rhijn Van & Mudler,
2002). The decision limits of the present method were below
1 mg/kg for the four nitrofuran metabolites making it compatible
with the MRPL requirements of the EU (EC., 2003). At present, the
EU policy on the tolerance of nitrofuran metabolites in food of
animal origin is kept at zero and therefore presence of any
conrmed concentration of the metabolites in animal tissues is a
non-compliance. Under this circumstance nitrofuran metabolite
screening methods with a higher sensitivity is preferred.
In order to achieve this relatively high sensitivity, below the
MRPL, the sample weight had to be increased up to ve grams. A
Table 1
Average values obtained for CCa, CCb, CV% and recovery for each metabolite for
samples spiked between 1 and 5 mg/kg.
Metabolite CCa (mg/kg) CCb (mg/kg) CV% Recovery%
AMOZ 0.32 0.61 16.9 107
SEM 0.65 0.93 12.7 115
AHD 0.60 0.83 9.3 114
AOZ 0.46 1.69 55.1 107
 R. Fernando et al. / Food Control xxx (2015) 1e6
Fig. 4. UV spectra of the nitrofuran metabolites.
major problem associated with a larger sample weight is the matrix
compounds and derivatizing agent (2-NBA) interfering with the
detection of nitrofuran metabolites (Conneely et al., 2002). The
matrix interferences were mainly removed by serially washing the
sample with 20 ml of 50, 75 and 100% methanol and nally with
water before starting the derivatization process. After derivatiza-
tion, the sample was extracted three times with 4 ml of hexane to
remove the excess 2-NBA. This improved cleaning up procedures
reduced the overall matrix (except a large peak coming around
14 min shown in Fig. 3) and other interferences to a level where a
sensitivity below the MRPL could be obtained for all four nitrofuran
metabolites. According to our experience, the mobile phase com-
bination used in the study (in section 2.5) gives a higher resolution
compared to the earlier used HPLC-UV mobile phase combinations
(Conneely et al., 2002; Conneely et al., 2003).
The specic detection of nitrofuran metabolites were carried out
at 275 nm. However, in complex matrices there may be other co-
eluting compounds which could absorb 275 nm light and inter-
fering with the detection. Therefore, HPLC UV methods may not be
very specic in detecting nitrofuran metabolites. In contrast to
HPLC-UV detectors, HPLC diode array detectors can record the VU
spectra of the peaks in the range of 190e400 nm (Fig. 4). In the event
of a suspected peak appearing in a retention time corresponding to a
nitrophenyl derivative of an unknown sample, the UV spectrum of
the suspected peak can be compared against a positive control to
further verify the suspected peak. This comparison of UV spectra of
peaks using a diode array detector provides a higher condence in
specic detection of nitrofuran metabolites. Currently, Liquid
ChromatographyeMass Spectrometry (LC-MS) methods are
considered as conrmatory due to their higher sensitivity and
specicity (Cooper, McCracken, Buurman, & Kennedy, 2008). In the
recent years, LC-MS methods have signicantly advanced the ca-
pabilities of quantitative determination of nitrofuran metabolites
although the cost of detection is high compared to the HPLC-DAD
methods and still unaffordable to some developing nations.
The average recoveries for the samples fortied (1e5 mg/kg)
with AOZ, AMOZ, SEM and AHD were 107%, 107%, 115% and 114%,
respectively. These recovery values are in agreement with the
performance criteria set for quantitative analytical methods by the
Codex Alimentarius Commission (Codex, 2007). Further, Conneely
et al., 2003 has reported recovery values of 111% and 117% for
prawn samples fortied with AOZ at 5 mg/kg and 25 mg/kg,
respectively.
One observation of this study was that a greater photometric
detector response coming from the samples fortied with nitro-
furan metabolites compared to the same amount of standard
compounds.
Please cite this article in press as: Fernando, R., et al., Determination of nitrofuran metabolites in shrimp muscle tissue by liquid
chromatography-photo diode array detection, Food Control (2015), http://dx.doi.org/10.1016/j.foodcont.2015.08.044
5
Out of all the nitrofuran metabolites, SEM was the highest
notied metabolite by the European Commission Rapid Alert Sys-
tem for Food and Feed (RASFF) during 2004e2006 (Vass, Hruska, &
Franek, 2008). These notications included food of animal origin
and other products such as baby food and our. Later, studies have
conrmed that SEM could present in food due to azodicarbona-
mide, a chemical used in gasket production and an additive in our,
and hypochlorite used in carrageen bleaching and disinfecting eggs
etc. (Becalski, Lau, Lewis, & Seaman, 2006; K Hoenicke, Gatermann,
Hartig, Mandix, & Otte, 2004; Stadler et al., 2004). SEM is also
believed occur naturally in some crustaceans such as prawns, crabs
and shrimp (Katrin Hoenicke & Gatermann, 2006; Pereira, Donato,
& De Nucci, 2004). As a result, the use of SEM as a denitive marker
for the monitoring of nitrofurazone misuse is debatable. However,
according to EU legislation, SEM remains the marker residue to
detect nitrofurazone exposure of food animals (Cooper, Samsonova,
Plumpton, Elliott, & Kennedy, 2007). In our study we did not nd
any blank sample contaminated with SEM.
This HPLC-DAD method will be very useful for developing na-
tions to enforce their regulations on the misuse of nitrofurans by
farmers in a cost effective manner. The conrmatory LC-MS
methods are not affordable to many developing countries due to
extremely high equipment and maintenance costs. A nancially
sustainable method will encourage the regulatory authorities, food
processors and exporters to screen food of animal origin for
nitrofuran metabolites using more representative sampling plans
to assure the chemical quality of the products. Quality and safer
animal products are essential to promote the export industry and
consumer condence.
5. Conclusions
This is the rst report of an HPLC-DAD method detecting all four
nitrofuran metabolites in any food commodity below the MRPL, to
the best of our knowledge. Further, the method can be used to
screen shrimp muscle samples for nitrofuran metabolites in a cost
effective manner. A nancially sustainable and sensitive method is
essential in enforcing export regulations and safeguarding con-
sumers with respect to the misuse of nitrofurans through detecting
non-compliant samples and thereby preventing the malpractices.
The non-compliant samples need to be sent for conrmatory
analysis using LC-MS.
Acknowledgements
The authors thank International Atomic Energy Agency (Grant
numbers - TCP: SRL/5/039, CRP: Contract # 15586), Vienna for
6 R. Fernando et al. / Food Control xxx (2015) 1e6
providing technical and nancial assistance for the project and A.
Cannavan of the IAEA Agriculture and Biotechnology Laboratory, A-
2444 Seibersdorf, Austria for providing technical advice on the
HPLC-DAD method development and validation.
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