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Food Additives and Contaminants

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Depletion of four nitrofuran antibiotics and their

tissue-bound metabolites in porcine tissues and
determination using LC-MS/MS and HPLC-UV
a b b c d
K. M Cooper , P. P. J Mulder , J. A van Rhijn , L Kovacsics , R. J McCracken , P. B
d d
Young & D. G Kennedy Dr
a
Queen's University Belfast, Northern Ireland
b
RIK ILT-Institute of Food Safety, Wageningen, The Netherlands
c
National Food Investigation Institute, Budapest, Hungary
d
Chemical Surveillance Department, Department of Agriculture and Rural Development,
Stoney Road, Stormont, Belfast BT4 3SD, Nothern Ireland
e
Chemical Surveillance Department, Department of Agriculture and Rural Development,
Stoney Road, Stormont, Belfast BT4 3SD, Nothern Ireland E-mail:
Version of record first published: 20 Feb 2007.

To cite this article: K. M Cooper , P. P. J Mulder , J. A van Rhijn , L Kovacsics , R. J McCracken , P. B Young & D. G Kennedy Dr
(2005): Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using
LC-MS/MS and HPLC-UV, Food Additives and Contaminants, 22:5, 406-414

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 Food Additives and Contaminants, May 2005; 22(5): 406–414

Depletion of four nitrofuran antibiotics and their tissue-bound

metabolites in porcine tissues and determination using LC-MS/MS
and HPLC-UV

K. M. COOPER1, P. P. J. MULDER2, J. A. VAN RHIJN2, L. KOVACSICS3, R. J. MCCRACKEN4,

P. B. YOUNG4, & D. G. KENNEDY4
1
Queen’s University Belfast, Northern Ireland, 2RIK ILT-Institute of Food Safety, Wageningen, The Netherlands,
3
National Food Investigation Institute, Budapest, Hungary, and 4Chemical Surveillance Department, Department of
Agriculture and Rural Development, Stoney Road, Stormont, Belfast BT4 3SD, Nothern Ireland

(Received 25 November 2004; revised 15 February 2005; accepted 18 February 2005)

Downloaded by [Florida State University] at 01:54 18 April 2013

Abstract
Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound
metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given
feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400 mg kg
1) for ten days. Animals were
slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These
samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites
(using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period
whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites
accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The
metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes
the benefits of monitoring for the stable metabolites of the nitrofurans.

Keywords: Nitrofurans, pigs, depletion, LC-MS/MS, semicarbazide, FoodBRAND

Introduction 3-amino-2-oxazolidinone (AOZ) is monitored as a

Use of the nitrofuran antibiotics in food-producing
animals was prohibited within the European Union
(Commission Regulation 1442/95) because of their
potentially carcinogenic and mutagenic effects on
human health (Van Koten-Vermeulen et al. 1993).
Previously, nitrofurans had been widely and effec-
tively used for the prevention and treatment of
gastrointestinal infections caused by Escherichia coli,
Salmonella spp., Mycoplasma spp., Coccidia spp.,
coliforms and some other protozoa, and as growth
promoters in livestock.
The four main nitrofuran antibiotics are furazoli-
done, furaltadone, nitrofurantoin and nitrofurazone.
Various studies have demonstrated that the nitro-
furans are rapidly metabolized by animals in vivo
(Vroomen et al. 1986; McCracken et al. 1995) but
that persistent tissue-bound metabolites are formed
which may be released by mild acid hydrolysis and
used as marker residues (Hoogenboom et al. 1991).
For example, the stable tissue-bound metabolite
marker residue for furazolidone. The development of
methods and legislation with regard to the monitor-
ing of nitrofuran residues has recently been reviewed
(Kennedy et al. 2003).
Most research on the accumulation of parent
nitrofurans in food for human consumption and
depletion following withdrawal of medicated feed,
has focussed on furazolidone, largely because it was
the most widely used and the last of the four major
nitrofurans to be banned. McCracken et al. (1995)
measured furazolidone by LC-MS in porcine muscle
at concentrations two orders of magnitude greater
than in liver or kidney 1 h after oral dosing. Winterlin
et al. (1984) failed to detect furazolidone by
HPLC-UV in tissues of pigs receiving medicated
feed, whilst Vroomen et al. (1986, 1987) could not
detect furazolidone in muscle, liver or kidney of
orally dosed piglets or adult swine 2 h after final
treatment. Carignan et al. (1990), however, did
detect furazolidone in muscle of medicated pigs
using LC-ECD but the residues rapidly disappeared

Correspondence: Dr Glenn Kennedy. E-mail: glenn.kennedy@dardni.gov.uk

ISSN 0265–203X print/ISSN 1464–5122 online ß 2005 Taylor & Francis Group Ltd
DOI: 10.1080/02652030512331385218
 Depletion of nitrofuran antibiotics in porcine tissues
with a half-life of 2 h. Zuidema et al. (2004) and
Winterlin et al. (1984) could not detect furazolidone,
by LC-MS/MS or HPLC-UV respectively, in edible
tissues of broiler chickens receiving medicated
feed. However, Parks et al. (1990), using LC-ECD,
detected furazolidone in liver and muscle of treated
chickens, but this disappeared following a 48 h feed
withdrawal period. Furazolidone has also been
detected by HPLC-UV in muscle and liver (but not
kidney or heart) of medicated goats after 24 h
withdrawal (Mustafa et al. 1985).
This instability of furazolidone in animal tissues
prompted the development of analyses for its
metabolite, AOZ. However, the decision to monitor
metabolites of furaltadone (3-amino-5-morpholino-
methyl-2-oxazolidinone, AMOZ), nitrofurantoin
(1-aminohydantoin, AHD) and nitrofurazone
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(semicarbazide, SEM) rather than their parent
compounds was based on limited data suggesting
that the parents were as unstable in vivo as was
furazolidone.
Few data have been published on the accumula-
tion, depletion or stability of these three parent
nitrofurans in animal tissues. Nitrofurazone, furalta-
done and furazolidone were shown by HPLC-UV
analysis to be stable in raw milk in the presence of
chemical preservatives during cold storage for
seven days (Noa et al. 2002). However, furaltadone
concentrations in poultry muscle diminished by 75%
inside four days when stored at
18 C (Kumar et al.
1994). Zuidema et al. (2004) were able to detect
furaltadone residues (3–12 mg kg
1) in treated poul-
try muscle (but not liver) after zero withdrawal when
samples were snap-frozen in liquid nitrogen and
stored at
70 C. Kumar et al. (1994) also detected
furaltadone (362 mg kg
1) in muscle of treated hens
when no withdrawal period was observed. None was
detectable following a 1 day withdrawal period.
Nouws and Laurensen (1990) demonstrated that
the postmortal elimination half-life of furaltadone
ranged from 8 to 63 minutes in the edible tissues of
orally-dosed veal calves.
Almost no information is available on the
accumulation or depletion of nitrofurantoin or
nitrofurazone in edible animal tissues, although
Nouws et al. (1987) demonstrated that nitrofura-
zone exhibited similar pharmacokinetic qualities to
furaltadone. Following oral dosing of pre-ruminant
calves, only 2% of the nitrofuran dose could be
accounted for by renal clearance in the urine
(presumably due to extensive metabolism in vivo),
whilst the elimination half-lives of furaltadone and
nitrofurazone in plasma were 2.5 and 5 h respec-
tively. Furthermore, these same authors demon-
strated that without pH adjustment and addition
of sodium azide, furaltadone and nitrofurazone
dissolved in calves’ urine exhibited half-lives of only
407
2 h at 20 C. Parks and Kubena (1992) detected
parent nitrofurazone in liver and muscle of medi-
cated broiler chickens but none was detected
following two days’ withdrawal.
There is a need for further experimental data
to confirm the rapid breakdown, or otherwise,
of all four nitrofuran antibiotics in the edible tissues
of pigs. The use of furaltadone, and to a lesser
extent furazolidone, has recently been demonstrated
in the pork industries supplying the European
market (O’Keeffe et al. 2004). O’Keeffe described
a survey of retail pork meat carried out within
the EU-funded FoodBRAND research project
(www.afsni.ac.uk/foodbrand) involving and
co-ordinated by one of the authors of the present
paper (DGK). Residues (0.2–3.0 mg kg
1) of AMOZ
and AOZ were identified in 0.8% of pork samples
tested. Furthermore, the EU, via its Rapid Alert
System for Food and Feeds, issued an alert on 13th
May 2004 indicating that AOZ had been detected in
a sample of pork meat originating in Italy (European
Commission 2004).
It is currently unclear for how long tissue-bound
residues of the nitrofuran drugs may be detected in
edible tissues following cessation of medication. The
pharmacokinetics of AOZ residues in pig tissues have
been the subject of various studies. These suggested
that:
(a) Tissue-bound AOZ residues persist for at least
six weeks post-withdrawal;
(b) Tissue-bound AOZ residues have a longer
half-life than solvent extractable AOZ residues
(Horne et al. 1996; McCracken et al. 1997);
and
(c) The highest AOZ concentrations are to be
found in liver tissue but that AOZ in muscle
tissue exhibits a longer depletion half-life
(Hoogenboom et al. 1992; McCracken et al.
1997).
No data have been published regarding the
accumulation or depletion of AMOZ, AHD or
SEM in porcine tissues. However, a recent study
(Zuidema et al. 2004) suggested that depletion
of AOZ and AMOZ residues from broiler chicken
muscle and liver was similar to that seen for AOZ in
porcine tissues. McCracken et al. (2004) have also
recently shown that SEM residues accumulate at
higher concentrations and have a longer half-life than
other nitrofuran metabolites in tissues of broilers
hatched from breeding chickens which received
nitrofuran medicated feed.
This paper provides data to demonstrate the
concentrations and depletion profiles of the parent
compounds and tissue-bound metabolites of the
four major nitrofuran antibiotics in edible tissues
of therapeutically treated pigs.
 408 K. M. Cooper et al.
Materials and methods
Production of incurred pig tissues
Seventy-two weaned piglets, approximately eight
weeks of age, and complete milled pig feed were
purchased from a local supplier. Pigs were divided
into four groups of 18 and housed in four concrete
floored pens within the same building. Four nitro-
furan medicated feeds were prepared using furazoli-
done, furaltadone, nitrofurantoin and nitrofurazone
(all supplied by Sigma-Aldrich, Dorset, UK) at
400 mg per kg feed, the recommended therapeutic
dose for furazolidone preparations (National Office
of Animal Health 1992) prior to it being banned in
livestock in 1995. Each group was fed medicated
feed ad libitum for ten days. On the tenth day, all
medicated feed was removed, pens were thoroughly
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swept out and the feeding area of each pen was hosed
out with water.
All pigs were then fed conventional unmedicated
feed ad libitum for the remainder of the study.
Fresh water was available at all times, pigs were fed
once each morning and faeces were swept from
pens daily.
On 0, 1, 2, 3, 4 and 6 weeks following withdrawal
of medicated feed, three pigs from each feed group
were euthanized by captive bolt and samples of
Longissimus dorsi muscle, liver and kidney removed
and stored at
20 C. Tissues removed on weeks 0
and 1 were sub-sampled, packed in dry ice and
dispatched by overnight courier to the laboratories
performing parent nitrofuran analyses where the
tissues were again stored at
20 C.
LC-MS/MS method for tissue-bound metabolites
Materials and instrumentation
Mixed internal standard solution (100 ng ml
1
methanol) contained D4-3-amino-2-oxazolidinone,
D5-3-amino-5-morpholinomethyl-2-oxazolidinone
and 13C15N2-semicarbazide supplied by Witega
Laboratorien Berlin-Adlershof (Berlin, Germany).
Mixed standard solutions (10 ng ml
1 methanol) con-
tained 3-amino-2-oxazolidinone (AOZ), 3-amino-
5-morpholinomethyl-2-oxazolidinone (AMOZ),
1-aminohydantoin (AHD) and semicarbazide
(SEM) supplied by Sigma-Aldrich (AOZ, SEM)
and Chemical Synthesis Services, Belfast, UK
(AMOZ). AHD was a gift from Proctor and Gamble
Pharmaceuticals USA. Unless stated, all other
chemicals were obtained from Sigma-Aldrich.
An Agilent 1100 Series HPLC system (Agilent
Technologies, USA) coupled to a Quattro UltimaÕ
Platinum tandem mass detector (Micromass/Waters,
Manchester, UK), both operating under MassLynxÕ
software, were used for sample analysis. The mass
spectrometer operated in electrospray positive mode
and data acquisition was in multiple reaction
monitoring mode (MRM). The precursor/product
ions monitored are listed in Table I. The source
settings were as follows: capillary voltage 2.45 kV,
RF lens 25.0 V, source temperature 120 C, desolva-
tion temperature 350 C, cone nitrogen gas flow
80 l h
1, desolvation gas flow 670 l h
1. Argon was
used as the collision gas and the multiplier was
operated at 650 V. The cone voltage (35–60 V) and
collision energy (7–16 eV) changed during analysis
depending on the analyte. The HPLC system was
equipped with a Luna C18(2) 3 mm, 2.0  150 mm
column (Phenomenex). A binary gradient mobile
phase was used at a flow rate of 0.2 ml min
1, solvent
A being 0.5 mM ammonium acetate and methanol
(80:20 v/v mix), solvent B being 100% methanol.
Sample injection volume was 25 ml.
Sample preparation
This method is based on the protocol developed
by RIKILT Wageningen and DARD Belfast as part
of the FoodBRAND project and disseminated to the
EU NRL/CRL network and 3rd countries during
2001–2003.
Tissues were minced in a domestic food blender
(Moulinex Moulinette) and samples (1.0  0.02 g)
weighed into 30 ml glass tubes. A single analysis was
carried out on each tissue from each pig. Ice cold
methanol (8 ml) and water (1 ml) were added and the
samples homogenized for 1 min in a Silverson
laboratory homogenizer. Following centrifugation
(2000 rpm at 4 C for 10 min) the supernatant was
discarded and the sample repeatedly washed by
vortex mixing for 10 seconds in ice cold methanol
(3  4 ml), ethanol (2  4 ml) and diethyl ether
(2  4 ml). Calibration standards were prepared by
addition of mixed standard solutions to clean
30 ml glass tubes. Mixed internal standard (50 ml
of a 100 ng ml
1 solution) was added equally to all
samples, controls and standards. Control tissues
from nitrofuran-free pigs were fortified with mixed
Table I. LC-MS/MS precursor/product ion combinations (base
peaks underlined) monitored in MRM ESI positive mode for the
analysis of the nitrophenyl (2-NP) derivatives of 4 nitrofuran
metabolites.
Precursor Product
Compound ion (m/z) ions (m/z)
2-NP-AOZ 236.3 104.3, 134.3
2-NP-AMOZ 335.3 262.3, 291.4
2-NP-AHD 249.3 104.3, 134.2
2-NP-SEM 209.2 166.2, 192.2
2-NP-D4-AOZ (internal standard) 240.3 134.3
2-NP-D5-AMOZ (internal standard) 340.3 296.4
2-NP-13C15N2-SEM (internal standard) 212.2 195.3
 Depletion of nitrofuran antibiotics in porcine tissues
standard (100 ml of a 10 ng ml
1 solution) to act as
recovery control samples. Blank tissues were also
included in every analytical run. To all tubes were
added de-ionized water (4 ml), 1 M hydrochloric
acid (0.5 ml) and 2-nitrobenzaldehyde (150 ml,
50 mM in DMSO). After vortex mixing, tubes were
incubated overnight (approximately 16 h) in a water
bath held at 37 C.
All tubes were then adjusted to pH 7.4  0.2 with
0.1 M di-potassium hydrogen orthophosphate (5 ml)
and 1 M sodium hydroxide (approximately 0.4 ml).
Liquid-liquid extraction was carried out using ethyl
acetate (2  5 ml, mixing for 1 min and centrifuga-
tion at 2000 rpm for 15 min at 4 C) and the organic
phase evaporated to dryness at 50 C under nitrogen.
Residues were re-dissolved in methanol: water
(50:50 v/v; 200 ml) and transferred to HPLC micro-
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vials (200 ml). LC-MS/MS analysis was carried out
using the conditions described above. Analyte con-
centrations in samples were calculated by comparing
the ratio of an analyte base peak response to its
appropriate internal standard response with the same
ratio in calibration curve standards. Quantification of
AHD utilized the D4-AOZ internal standard. Highly
concentrated samples were diluted as required for
analysis by the above method.
LC-MS/MS method for parent nitrofurans
A LC-MS/MS method developed by RIKILT
Wageningen for the quantification of furazolidone
(FZD), furaltadone (FTD), nitrofurantoin (NFT)
and nitrofurazone (NFZ) in animal tissues is out-
lined below. In order to improve quantification
of parent compounds, isotopically labelled
D4-furazolidone (D4-FZD), D5-furaltadone (D5-
FTD) and 13C15N2-nitrofurazone (13C15N2-NFZ)
internal standards were synthesized by condensation
of 5-nitro-2-furaldehyde with D4-AOZ, D5-AMOZ
and 13C15N2-SEM, respectively. Each of the
isotopically labelled starting materials (10 mg) was
reacted for 1 hour at 60 C, in the presence of 1 M
hydrochloric acid (2 ml), with a 50% molar excess
of 5-nitro-2-furaldehyde. After neutralization
with 1 M sodium hydroxide, the aqueous layer
was extracted several times with ethyl acetate.
The combined organic layers were evaporated
to dryness and the resulting crystal mass was
washed twice with hexane to remove unreacted
5-nitro-2-furaldehyde. Yields of the labelled
products were 85–95%.
Mixed internal standard (5 mg kg
1 D4-FZD and
D5-FTD, 20 mg kg
1 13C15N2-NFZ) was added to
1 g of homogenized tissue sample which was then
extracted with 2  10 ml ethyl acetate. Extracts were
evaporated to dryness at 50 C under nitrogen
and reconstituted in 0.1% acetic acid/acetonitrile
409
(10/90 v:v; 500 ml). Blank liver and muscle samples
and tissues fortified at 0.25–5 mg kg
1 FZD and
FTD, and 1–20 mg kg
1 NFT and NFZ acted as
control samples. Blank tissues were also included
in every analytical run. Analyte concentrations in
samples were calculated by comparing the ratio of
an analyte base peak response to its appropriate
internal standard response with the same ratio in
calibration curve standards. Quantification of NFT
utilized the D4-FZD internal standard.
The HPLC system employed a Waters Symmetry
C18 column (5 mm, 150  3.0 mm) at a flow rate of
0.4 ml min
1 and a solvent split ratio of 1:2. A
binary gradient mobile phase used 0.1% acetic acid
(A) and acetonitrile/1% acetic acid (90:10) (B),
starting at 95% A/5% B and changing linearly to
50% A/50% B in 12 min. Injection volume was
50–100 ml. A Micromass Micro mass spectrometer
operated in electrospray positive mode and data
acquisition was in multiple reaction monitoring
mode (MRM). The source settings were as follows:
Capillary voltage 2.7 kV, cone voltage 30 V, collision
energy 13 to 15 eV, source temperature 110 C,
desolvation temperature 350 C, cone gas flow
100 l h
1 and desolvation gas flow 500 l h
1.
The precursor/product ions monitored were as
follows (base peak underlined): FZD (226 > 139,
95), D4-FZD (230 > 96), FTD (325 > 281, 252),
D5-FTD (330 > 286), NFT (239 > 139, 95), NFZ
(199 > 182, 156) and 13C15N2-NFZ (202 > 185).
HPLC-UV method for parent nitrofurans
A HPLC-UV-PAD method developed by NFII
Budapest for the quantification of FZD, FTD,
NFT and NFZ in animal tissues is outlined below.
To 5 g of homogenous tissue 4 ml phosphate buffer
(0.34 M) and 10 ml acetonitrile were added. The
sample was shaken for 20 min then centrifuged
(2500 rpm, 10 min). The supernatant was transferred
to a clean tube and 5 ml dichloromethane-ethyl
acetate (50:50 v/v) added. After shaking for 20 min,
the tube was centrifuged and the supernatant
removed and evaporated to dryness. This extract
was re-dissolved in 0.5 ml phosphate buffer
(0.34 M). HPLC-UV mobile phase was 20% metha-
nol in 0.1 M sodium acetate (pH 5.0) at a flow rate
of 1 ml min
1 through a Hypersil ODS (C18) 5 mm
(250 mm  4.6 mm) analytical HPLC column.
Injection volume was 20 ml. The Photodiode Array
Detector was set at 365 nm. Quantification was by
reference to peak areas of calibration standards in the
range 2.5–100 mg kg
1 (four parent compounds
obtained from Sigma-Aldrich) and incorporated
correction for recovery efficiency (65–80%) based
on blank samples fortified with each parent
compound at 5 mg kg
1.
 410 K. M. Cooper et al.

Results and discussion Table II. Limits of detection (LoD) of parent nitrofuran drugs
detected by LC-MS/MS and HPLC-UV in porcine tissues.
Parent nitrofuran analysis
LC-MS/MS HPLC
Table II shows the limits of detection (LoD) for the
Muscle Liver/kidney Muscle Liver/kidney
analysis of parent nitrofurans in porcine tissues by mg kg
1 mg kg
1 mg kg
1 mg kg
1
LC-MS/MS and HPLC-UV. LC-MS/MS analysis
Furazolidone 0.2 0.5 2 5
was a factor of ten more sensitive than HPLC-UV Furaltadone 0.2 0.5 2 5
in the detection of furazolidone and furaltadone, a Nitrofurantoin 2 4 2 5
factor of two more sensitive for nitrofurazone, whilst Nitrofurazone 1 2 2 5
the methods were equally sensitive for detection of
nitrofurantoin.
Neither LC-MS/MS nor HPLC-UV detected
5000
any parent drug in muscle, liver or kidney tissues
2000
sampled after one week drug withdrawal period.
1000
However, when no withdrawal period was observed,

AOZ (µg/kg)
furazolidone was detected by LC-MS/MS in one
muscle sample (0.33 mg kg
1) and furaltadone in 100
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one liver sample (0.7 mg kg
1). Neither of these drugs

were detected using HPLC-UV, the concentrations
being below the LoD of the method. These findings
are in agreement with the studies of McCracken et al.
(1995); Winterlin et al. (1984); Vroomen et al.
(1986, 1987) and Carignan et al. (1990) who were
unable to detect furazolidone in medicated pig
tissues or demonstrated furazolidone elimination
within a few hours of drug withdrawal. The detection
of furaltadone in only one tissue sample in the
present study is in agreement with the rapid
clearance and instability of this nitrofuran demon-
strated in poultry and bovine muscle by Kumar et al.
(1994); Nouws and Laurensen (1990) and Zuidema
et al. (2004). The present study is, to our knowledge,
the first report of the elimination of furaltadone from
porcine tissues.
Nitrofurantoin was not detected in any tissue. This
finding supports the anecdotal evidence of this
laboratory, which suggests that nitrofurantoin and
its metabolite AHD are the least stable of the
nitrofuran antibiotics. Preparations containing nitro-
furantoin have been found on shrimp farms in SE
Asia but AHD has rarely been detected in shrimp
tissues during exploratory or routine testing in this
laboratory or elsewhere.
In contrast, nitrofurazone was detected by both
LC-MS/MS (5.4–21.9 mg kg
1) and HPLC-UV
(4–13 mg kg
1) in all three muscle samples at zero
withdrawal (Figure 4), but not in either liver or
kidney. Quantitative correlation between the detec-
tion techniques was acceptable, HPLC-UV nitrofur-
azone results being 59–74% of the corresponding
LC-MS/MS result.
McCracken et al. (1995) demonstrated that
furazolidone was highly unstable in post-mortem
samples. Snap-freezing and storage in liquid nitrogen
was necessary to preserve furazolidone for up to four
weeks. Snap-freezing in liquid nitrogen and storage
at
20 C, however, resulted in loss of 90% of the
Muscle
10
Liver
Kidney
1
0 1 2 3 4 5 6
Time (weeks)
Figure 1. Depletion of AOZ (tissue-bound furazolidone metab-
olite) in tissues of pigs fed furazolidone-medicated feed
(400 mg kg
1) for ten days followed by a six-week drug withdrawal
period. Data are mean of three pigs per time point  standard error
of the mean.
drug within one week. In the present study, the initial
sample freezing and storage conditions (
20 C)
were practical for residues monitoring purposes.
Snap-freezing and storage in liquid nitrogen are not
feasible techniques for use in slaughterhouses. As
would be the case in routine sampling in slaughter-
houses, tissues collected as part of this study were
placed immediately into a þ4 C cold room and
transferred to
20 C within 2 h.
In light of these demonstrations of the instability of
parent nitrofurans, it is unsurprising that parent drug
was detected in only five out of 36 tissue samples
following zero withdrawal period (one liver and four
muscle samples). This probably reflects the higher
metabolic activity of kidney and liver. These findings
give further weight to the decision taken by
EU laboratories to monitor residues of nitrofuran
metabolites rather than their parent nitrofuran
compounds.
Nitrofuran metabolite analysis
Figure 1 illustrates the depletion of the tissue-bound
metabolite AOZ from porcine muscle, liver and
kidney tissues following withdrawal of furazolidone
medicated feed for six weeks.

Table III. Depletion half-lives (days) of tissue-bound nitrofuran
metabolites in tissues of pigs fed nitrofuran-medicated feed
(400 mg kg
1) for ten days followed by a six-week drug
withdrawal period. Half-lives were calculated using all data
points from 0–6 weeks.
Muscle (L. dorsi) Liver
(days) (days)
AOZ 11.5  0.9 7.3  0.3
AMOZ 8.7  1.2 5.4  0.5
AHD 8.3  0.9 5.7  0.5
SEM 15.5  3.1 7.3  0.6
5000
2000
1000
AMOZ (µg/kg)
100
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Muscle
10 Liver
Kidney
1
0 1 2 3 4 5
Time (weeks)
Figure 2. Depletion of AMOZ (tissue-bound furaltadone
metabolite) in tissues of pigs fed furaltadone-medicated feed
(400 mg kg
1) for ten days followed by a six-week drug withdrawal
period. Data are mean of three pigs per time point  standard error
of the mean.
AOZ data were of the same order of magnitude as
those of McCracken et al. (1997) and Hoogenboom
et al. (1992) who also agreed that, with no with-
drawal period, AOZ was most abundant in porcine
liver, followed by kidney, then muscle. Leitner et al.
(2001) also demonstrated AOZ in porcine muscle
at similar concentrations without withdrawal.
Following a six-week withdrawal period AOZ
concentrations were in close agreement with those
of Hoogenboom et al. (1992) and McCracken et al.
(2000) following four and seven weeks’ withdrawal
respectively. However, at six weeks’ withdrawal,
AOZ was equally abundant in muscle and liver.
This can be explained by the longer depletion
half-life exhibited by AOZ in muscle in comparison
with liver and kidney (see Table III).
These differences in half-lives possibly reflect the
lower metabolic rate and slower cellular turn-over
in muscle tissue. Half-life data for AOZ were in
close agreement with McCracken et al. (1997) who
reported half-lives of 9.1 days in muscle, 5.9 days in
kidney and 5.8 days in liver. Hoogenboom et al.
(1992) also reported the longest AOZ half-life was in
porcine muscle and the shortest in kidney.
Figure 2 illustrates the depletion from porcine
tissues of the tissue-bound metabolite AMOZ.
Depletion of nitrofuran antibiotics in porcine tissues 411
5000
2000
1000
AHD (µg/kg)
100
Kidney
(days)
Muscle
6.9  0.8 10 Liver
5.7  0.7 Kidney
5.8  0.7
7.0  0.9 1
0 1 2 3 4 5 6
Time (weeks)
Figure 3. Depletion of AHD (tissue-bound nitrofurantoin
metabolite) in tissues of pigs fed nitrofurantoin-medicated feed
(400 mg kg
1) for ten days followed by a six-week drug withdrawal
period. Data are mean of three pigs per time point  standard error
of the mean.
Leitner et al. (2001) provide the only previously
published data for AMOZ concentrations in
pig tissue without withdrawal. They measured
6 30 mg kg
1 AMOZ in muscle of one pig fed
furaltadone for only three days at a lower dose than
used in the present study. This may account for
the much higher muscle AMOZ concentrations
(1600 mg kg
1) found in the present study. At zero
withdrawal AMOZ was most abundant in liver, but
following six weeks’ withdrawal, liver concentrations
had fallen below those of muscle due to the much
longer depletion half-life in muscle (see Table III).
Figure 3 illustrates the depletion from porcine
tissues of the tissue-bound metabolite AHD. AHD
exhibited similar depletion patterns to AMOZ, being
most abundant in liver at zero withdrawal. However,
the depletion half life in muscle was longer than that
in liver (8.3 versus 5.7 days, Table III) resulting in
the highest AHD concentrations being observed in
muscle following six weeks’ withdrawal. AHD was
the least abundant nitrofuran metabolite, measurable
at 3–8 mg kg
1, giving further evidence of the
instability of this metabolite or the poor absorption
of its parent nitrofurantoin.
Figure 4 illustrates the depletion from porcine
tissues of the tissue-bound metabolite SEM. At zero,
withdrawal SEM was most abundant in kidney but
by six weeks’ withdrawal muscle had the highest
SEM concentrations. SEM half-lives (Table III)
were similar to those of AOZ, however SEM
concentrations were higher than AOZ, particularly
in muscle.
It should be noted that following six weeks’
withdrawal, all four metabolites were still detectable
in all three tissues of their respective animals. Our
data demonstrate that with regard to porcine tissue
type, the pharmacokinetics of AMOZ, AHD and
SEM are similar to AOZ. These are the first
 412 K. M. Cooper et al.
5000
2000
1000
SEM (µg/kg)
100
Muscle
10 Liver
Kidney
Nitrofurazone
1
0 1 2 3 4 5 6 with the ban on nitrofuran use. When furazolidone
Time (weeks)
Figure 4. Depletion of SEM (tissue-bound nitrofurazone metab-
olite) in tissues of pigs fed nitrofurazone-medicated feed
(400 mg kg
1) for ten days followed by a six-week drug withdrawal
period. Data are mean of three pigs per time point  standard error
of the mean. Nitrofurazone was detected in muscle only at zero
Downloaded by [Florida State University] at 01:54 18 April 2013
withdrawel.
published data for the depletion of AMOZ, AHD
and SEM from porcine tissues.
The persistence of these metabolites in porcine
tissues is in agreement with the findings of Zuidema
et al. (2004) who measured depletion of total
(as opposed to tissue-bound residues in the present
study) AOZ and AMOZ in broiler chicken muscle
and liver. The rapid depletion over the first with-
drawal week followed by a lengthening of the
depletion half-life, noted by Zuidema et al. (2004),
is consistent with the findings of McCracken et al.
(1997) and Horne et al. (1996) who demonstrated
that solvent extractable AOZ had a shorter depletion
half-life than tissue-bound AOZ. The same phenom-
enon appears to be present in muscle in the present
study, in which only bound residues were
measured. However, there are too few points on the
depletion curve to enable an accurate calculation
of the depletion parameters associated with a two-
compartment model (that is, a rapid initial phase of
depletion followed by a second, slower depletion
phase). The calculated half lives in the present study
were therefore derived assuming a single compart-
ment model using all data points from zero to six
weeks.
It should be noted that the feeding regime in the
present study was designed to replicate the likely
on-farm situation where medicated animals are
unlikely to be transferred to uncontaminated housing
on cessation of treatment. Medicated feed was
removed, pens were thoroughly swept out and the
feeding area of each pen was hosed out with water.
It is possible that small quantities of nitrofurans
remained on the pigs’ hides and on pen surfaces
during the early weeks of withdrawal. McCracken
et al. (1997) showed that exposure to furazolidone-
contaminated housing led to detectable levels of
AOZ in pig tissues but these were several orders of
magnitude lower than those reported here. House-
to-pig carry over is unlikely to have had a major
influence on half-lives of the tissue-bound metabo-
lites presented here. Nevertheless, any resulting
lengthening of half-lives would represent more
closely the pattern that would be expected to be
seen in farms where these drugs were being abused.
These data emphasize further the need to monitor
residues of metabolites rather than the parent
nitrofuran compounds when monitoring compliance
was used legally in the EU, manufacturers of
veterinary preparations recommended withdrawal
periods of between seven and 28 days following
cessation of medication (National Office of Animal
Health 1992b). This was designed to ensure com-
plete clearance of furazolidone residues from edible
porcine tissues, but, as can be seen from the present
data, such withdrawal is clearly insufficient for
elimination of tissue-bound nitrofuran residues.
Tissue-bound residues of all four nitrofurans are
detectable for at least six weeks post-withdrawal.
Extrapolating the depletion of residues beyond the
six-week experimental period may give erroneous
results as animal growth rates, tissue metabolic rates
and residue stability properties may change over
the ensuing weeks. However, based on the calculated
metabolite half-lives (see Table III) and the limit of
detection (CC) of the LC-MS/MS metabolite
method (0.05 mg kg
1) it can be roughly estimated
that, following treatment with 400 mg kg
1 furazoli-
done in feed, AOZ may be detectable in muscle
tissue for 21 weeks post-withdrawal, AMOZ for
18 weeks, AHD for 14 weeks and SEM for 33 weeks.
Given that in the UK pigs are normally slaughtered
at 5–6 months of age, it is likely that SEM could be
detectable at slaughter in a pig treated therapeutically
with nitrofurazone at any point in its lifetime.
Residues of AOZ, AMOZ and AHD are more
likely to have dropped below detectable concentra-
tions at the time of slaughter if treatment took place
in the very early months of life.
The most striking results of this study are the high
concentrations of SEM (e.g., 1930  80 mg kg
1
in muscle) after treatment with nitrofurazone, and
the remarkably long half-life of SEM in this tissue
(15.5 days). These findings are in keeping with the
report of McCracken et al. (2004) that concentra-
tions of tissue-bound SEM were higher than other
nitrofuran metabolites in broiler chicken tissues.
SEM also persisted in chicken tissue until slaughter
unlike other metabolites, which fell below LC-MS/
MS detection limits.
Semicarbazide, a suspected carcinogen, has
recently been the focus of much coverage in the
scientific literature and news media as a result of
it being found in diverse food products, including
 Depletion of nitrofuran antibiotics in porcine tissues
bread products and baby food, where nitrofurazone
abuse is not suspected. SEM arising from azodicar-
bonamide (ADA) has been implicated (Kennedy
et al. 2004). SEM has been shown to migrate into
food products from plastic gaskets inside bottle lid
seals, which were manufactured using ADA as a
blowing agent. SEM can also arise from the use of
ADA as a flour treatment agent. SEM contained in
bread coatings of value-added food products (for
example, chicken fillets coated in bread crumbs) can
reach several hundred mg kg
1. This led AFSSA,
the Community Reference Laboratory for nitrofuran
drugs, to issue a statement (December 2003) that
‘‘when testing composite food, only analyse the part
of the product which is of animal origin, for example,
only the meat part of breaded products . . . . In the
case of a non-compliant sample for total SEM,
Downloaded by [Florida State University] at 01:54 18 April 2013
a sample must be reanalysed for the bound residues
of SEM only’’ (Sanders 2003). The present study
suggests that if the advice of the CRL is followed
and, for example, bread crumb coatings are removed
from composite foods before analysis, illegal nitro-
furazone treatment of pigs will give rise to SEM
concentrations in edible tissues which are sufficiently
high to dispel any suspicion that SEM derived from
ADA may have contaminated the meat.
In conclusion, we have demonstrated that, whilst
all four major nitrofuran antibiotics are undetectable
in edible pig tissues within one week after cessation
of treatment, their metabolites are detectable at high
concentrations at least six weeks after cessation.
SEM, in particular, was present in tissue at several
hundred parts per million following drug withdrawal
and may be detectable in a pig treated therapeutically
with nitrofurazone at any point in its lifetime.
Acknowledgements
The authors acknowledge the financial support of
the European Commission for the project QLK1-
CT1999-00142 ‘‘FoodBRAND’’ of which this work
is a part. Thanks are expressed to the farm and post-
mortem room staff of DARD Veterinary Sciences
Division.
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