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Metabolism and depletion of nifursol in broilers
Analytica Chimica Acta 529 (2005) 339–346
Metabolism and depletion of nifursol in broilers T. Zuidemaa,∗ , P.P.J. Muldera , J.A. van Rhijna , N.G.M. Keestraa , L.A.P. Hoogenbooma , B. Schata , D.G. Kennedyb b
a RIKILT – Institute of Food Safety, Bornsesteeg 45, P.O. Box 230, 6700 AE Wageningen, The Netherlands Chemical Surveillance Department, Veterinary Sciences Division, Department of Agriculture and Rural Development, Stoney Road, Stormont, Belfast BT4 3SD, Northern Ireland, UK
Received 14 June 2004; received in revised form 17 August 2004; accepted 17 August 2004 Available online 18 December 2004
Abstract Nifursol has recently been prohibited for use as a feed additive. Considering the similarity in structure between nifursol and the other nitrofurans, an analogous metabolism could be expected. To study the formation of tissue-bound residues in poultry, broilers were treated orally with nifursol during a period of 7 consecutive days, via medicated feed at a dosage of 50 mg/kg feed. Muscle, kidney, liver, bile and plasma samples were collected at the day of cessation of medication (day 0) and at days 3, 7, 14 and 21 after the end of medication. Samples were analysed for nifursol and the acid-hydrolysable side-chain of nifursol (DNSH; 3,5-dinitro salicylhydrazide). Samples were also analysed for the ratio between free (solvent-extractable) metabolites and tissue-bound (non-extractable) metabolites. The results obtained clearly indicate the formation of tissue-bound residues in poultry. Concentrations of non-extractable residue at zero withdrawal time averaged to 900 g/kg in liver tissue, 2000 g/kg in kidney tissue, 225 g/kg in muscle tissue, 1000 g/kg in bile and 1000 g/kg in plasma. Taking into account an LoD of 1 g/kg, non-extractable residues of DNSH can be detected for at least 3 weeks after administration in liver, kidney, bile and plasma and for up to 2 weeks in muscle tissue. The amounts of extractable residues were relatively low, in many instances less than 10% of the total amount of residue. In general terms the depletion data obtained show a similar behaviour of nifursol in broilers as previously found for furazolidone and furaltadone in broilers. © 2004 Elsevier B.V. All rights reserved. Keywords: Nifursol; Nitrofurans; Metabolism; Depletion; DNSH; Tissue-bound residue; Broilers
1. Introduction Nifursol (3,5-dinitro-N -(5-nitrofurfurylidene)salicylhydrazide) has been used extensively as a feed additive for the prevention of histomoniasis (black head disease) in turkeys. Nifursol belongs to the family of nitrofurans, substances harbouring a 5-nitrofuran group, a structure that is suspected of imparting genotoxic characteristics to these substances. Extensive research has been performed on the mutagenicity and genotoxicity of the nitrofurans and especially on furazolidone and its marker metabolite, the acid-hydrolysable side-chain 3-amino-2-oxazolidinone (AOZ). Vroomen et al. ∗
Corresponding author. Tel.: +31 317 475580; fax: +31 317 417717. E-mail address: [email protected] (T. Zuidema).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.08.044
[1], Hoogenboom et al. [2], McCracken et al. [3] and Polman et al. [4] showed a rapid depletion in pig tissues of the parent drug and the formation of persistent metabolites. Because of this rapid metabolism, residues of the parent drugs cannot be detected even within a few hours after cessation of treatment. Treatment of two pigs with 75 mg 14 C-labelled furazolidone per kg body weight per day for a period of 10 days showed that furazolidone as such cannot be detected in the tissues, while even after 14 days after cessation of treatment in tissues and plasma radioactivity comparable to ppb levels of furazolidone could be detected [1]. Due to the fact that part of the 14 C-label could not be extracted from the tissues even when organic solvents were used, the conclusion was made that tissue-bound residues were formed. In vitro research clarified that these tissue-bound residues originated
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from furazolidone [2]. In general, metabolism will result in compounds that can be depleted more effectively compared to the parent compound. In some cases, as for furazolidone, reactive intermediates are formed that can be bound covalently to DNA or proteins. Such adducts will have a longer lifetime in the animal and as such will be the ideal marker residue for control purposes. Residues of tissue-bound metabolites of nitrofurans can be detected in tissues of pigs even weeks after cessation of treatment [1,5]. One of the characteristics of furazolidone is that in the presence of a weak acid (stomach), hydrolysis of the imine bond takes place yielding the side-chain, 3-amino2-oxazolidinone (AOZ). Furazolidone and AOZ have been studied intensively in relation to their toxicity [6,7] and both have been found to posses mutagenic and carcinogenic properties. Combined with the lack of data to determine a No Observed Effect Level (NOEL) in 1993 the nitrofurans as a group (with the exclusion of furazolidone) were included in Annex IV of Council Regulation (EEC) no 2377/90 [8]. Furazolidone was included in Annex IV in 1997 [8]. In addition, AOZ has been identified as the official marker metabolite of furazolidone [9]. In contrast, the other nitrofurans are much less intensively studied, and especially for nifursol, to date no reports are published concerning its metabolism and the possible formation of reactive intermediates and tissue-bound residues. Recently, nifursol has been prohibited for use as a feed additive in Council Regulation 2002/1756/EC [10]. Until then,
nifursol had been the last antibiotic that was available and permitted for the prevention of histomoniasis (black head disease) in turkeys. Considering the similarity in structure between nifursol and the other nitrofurans (Fig. 1), the acid-hydrolysable sidechain of nifursol, 3,5-dinitro-salicylhydrazide (DNSH), can be used as a marker metabolite to determine whether or not nifursol has been used. To date no studies have been performed to verify this assumption. At the EuroResidue V Conference (10–12 May 2004, Noordwijkerhout, The Netherlands), results of two smallscale animal studies were presented. Both studies independently showed the formation of DNSH in nifursol-treated turkeys [11,12]. The study described by Kaufmann and Butcher [11] was part of the method validation of the analysis of nifursol and because of that only two animals were part of this study. The main goal of the study described by Bock et al. [12] was to determine the possible metabolites in turkey after treatment of the animals with nifursol and their residue behaviour in muscle tissue. Both studies did not address the depletion of nifursol and tissue-bound residues of DNSH explicitly. In this paper, we present a study on the formation of tissue-bound residues and depletion of nifursol and the acid-hydrolysable side-chain of nifursol (DNSH) in broilers. Research has been focussed on DNSH and not on other possible metabolites, especially because we wanted to make the comparison to the other nitrofurans, for which the acid-
Fig. 1. Molecular structures of the nitrofurans, nifursol, furazolidone and furaltadone, their acid-hydrolysable side-chains DNSH, AOZ and AMOZ and their corresponding nitrophenyl derivatives NPDNSH, NPAOZ and NPAMOZ.
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hydrolysable side-chain is being used as a marker residue. To be able to make a good comparison between the depletion of nifursol and the more studied furazolidone and furaltadone, broilers were used as target animal. This study is performed in support of implementing methods for official control using DNSH as a marker residue, which is currently underway.
2. Experimental 2.1. Animal experiments The clinical phase of the study was carried out at Zodiac Animal Facilities (Wageningen, The Netherlands). The stable unit was equipped with continuous ventilation and heating system. The temperature was adjusted according to the age of the animals. The light scheme was 23 h of light and 1 h of darkness. The feed consumption during the medication period was recorded. During the study, the animals were fed with a standard commercial feed (Research Diet Services BV, Wijk bij Duurstede, The Netherlands). No antibiotics (feed additives) were present in the feed. One-day broilers (n = 40) were housed on straw and randomly divided into two different groups. A wing tag identified all broilers. Group 1 consisted of 30 animals (15 males and 15 females). Group 2, the control group, consisted of 10 animals (five males and five females). The clinically healthy broilers were kept for 14 days on a non-medicated starter ration. During the acclimation period, one animal (female) of group 1 died and was replaced by an animal from group 2 (female). From the start of the acclimation period until the moment of slaughter, the animals were monitored daily for general health by qualified personnel supervised by a veterinarian. The animals were in good health and free from apparent abnormalities and malformations. The broilers were vaccinated for Infectious Bronchitis and Newcastle’s Disease (NCD) prior to shipping to the test facility. The second NCD vaccination was given during the acclimation period at the test facility. After the acclimation period animals of group 1 were fed for 7 consecutive days on a feed ration containing 50 mg/kg of nifursol (Research Diet Services BV). Animals of group 2 were given non-medicated feed. Drinking water and feed was given ad libitum. Seven days after start of the treatment, the medicated feed was replaced by non-medicated feed. Taking the mean daily feed consumption (120 g/kg body weight) and the amount of nifursol in the feed (50 mg/kg) into account, the average dosage was 6.0 mg nifursol per kg body weight. Groups of six animals were sacrificed at days 0, 3, 7, 14 and 21 after cessation of the treatment. The broilers were slaughtered at the Zodiac Animal Facilities and qualified personnel collected tissue samples as soon as possible after slaughter. During collection, precautionary measures were taken to prevent mutual contamination of the samples. Samples of muscle, liver, kidney, bile and plasma from each animal were collected separately and stored in plastic
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bags or tubes marked with the animal ID code. Samples were stored on dry ice in an insulating transport box immediately after collection and were transported to the laboratory as soon as possible (within 0.5–3 h, depending on the order of slaughter). In the laboratory, samples were snap-frozen using liquid nitrogen and subsequently stored below −70 ◦ C until analysis. Samples were analysed for both nifursol and the acidhydrolysable side-chain of nifursol, DNSH. The analytical method will be described elsewhere [13]. A brief description will be given in the following paragraphs. 2.2. Materials All reagents and solvents were of analytical grade or better unless stated otherwise. Ethyl acetate and methanol were from Biosolve (Valkenswaard, The Netherlands). Acetone, hydrochloric acid, trisodium phosphate dodecahydrate, sodium hydroxide and acetic acid were from Merck (Darmstadt, Germany). Ortho-nitrobenzaldehyde (o-NBA), 3,5-dinitrosalicylic acid (DNSA), hydrazine, N-ethoxycarbonyl-2-ethoxy-1,2dihydroquinoline (EEDQ), 4-hydroxy-3,5-dinitro-benzoic acid (HBA) and 5-nitrofurfural were from Sigma (St. Louis, MO, USA). Water was demineralised using a Millipore purification system (Millipore, Billerica, MA, USA). Nifursol standard was obtained from Solvay Pharmaceuticals (Weesp, The Netherlands). 3,5-Dinitro salicylhydrazide (DNSH) was synthesised in-house from DNSA and hydrazine using EEDQ as a coupling agent [14]. 4-Hydroxy3,5-dinitro-benzohydrazide (HBH) was synthesised in-house by conversion of HBA with hydrazine and EEDQ. The nitrophenyl derivatives of DNSH and HBH were prepared by reaction with o-NBA in 1 M HCl. The 5-nitrofuran derivative of HBH (NFHBH) was prepared by reaction with 5-nitrofurfural in 1 M HCl. Individual stock solutions were prepared by dissolving the standard substances in the appropriate volume of methanol to obtain solutions of 100 g/ml. Individual standard solutions were prepared by diluting the stock solutions using methanol to obtain solutions of 1 g/ml and 0.1 g/ml. Standard solutions were stored in the dark at +4 ◦ C for up to 3 months. LC–MS/MS analysis was carried out using an Agilent 1100 series gradient HPLC system (Palo Alto, CA, USA) equipped with a 50 × 3 mm Waters XTerra® C18 column. Fifty microlitres of each sample extract was injected. Chromatographic separation was achieved by gradient elution using a flow rate of 0.4 ml/min and a linear gradient from 5% methanol containing 0.05% ammonium hydroxide to 90% methanol containing 0.05% ammonium hydroxide in 7 min allowing a good separation between the internal standard and the analyte. For the analysis, chromatographical separation is necessary due to the similarity in molecular weight and fragmentation pathway of the analyte and the internal standard. The column was connected to a Micromass Quattro Micro (Waters-Micromass, Manchester, UK) triple quadrupole
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Table 1 Diagnostic ions of nifursol, NFHBH, NPDNSH and NPHBH m/z
Nifursol NFHBH NPDNSH NPHBH
Precursor ion
Product ion 1
Product ion 2
364 364 374 374
226 183 226 183
182 182
Dwell time (s)
Collision energy (eV)
0.2 0.2 0.2 0.2
20 20 20 20
mass spectrometer equipped with an electrospray interface operated in negative ion mode. The interface was operated at settings optimised for optimum sensitivity (capillary voltage 2.7 kV, cone voltage 35 V, source temperature 100 ◦ C, desolvation temperature 300 ◦ C, desolvation gas flow 700 l/h, cone gas flow 100 l/h) that were modified slightly on a regular basis to maintain optimum sensitivity. Deprotonated molecules of the analytes of interest were selected as precursor ions and fragmented to compound-specific product ions using collision-induced dissociation with argon as the collision gas at a pressure of 3.2 Pa. Data acquisition is performed in MRM mode using the settings listed in Table 1.
Samples (washed pellets as well as the acetone extracts) were extracted and purified with the method described for the acid-hydrolysable metabolites of the other nitrofurans [15]. In brief, after addition of 25 g of a structure-related internal standard (4-hydroxy-3,5-dinitro-benzohydrazide, HBH (Fig. 2)), samples were hydrolysed and derivatised overnight in 5 ml diluted hydrochloric acid (0.2 M) in the presence of 100 mM ortho-nitrobenzaldehyde. The aqueous sample was subsequently adjusted to pH 7.0 ± 0.5 and extracted twice with 4 ml ethyl acetate. The combined ethyl acetate fractions were evaporated to dryness and redissolved in 500 l methanol/water (10/90), filtered and analysed by LC–MS/MS. Quantification of the ortho-nitrophenyl derivative of DNSH was performed by the internal standard method. Matrix-matched standards were prepared by adding known amounts of the stock solutions to blank sample matrix to obtain concentration levels of 1, 2.5, 10, 25 and 100 g/kg in the matrix. Identification of the analytes was carried out according to EU requirements for banned substances [16]. The observed limit of detection (LoD; signal to noise ratio of 3 or more) ranged from 0.2 g/kg for extractable residues to 1 g/kg in case of non-extractable residues.
2.3. Analysis of nifursol 3. Results and discussion The parent substance nifursol was extracted from the tissue and plasma samples (1 g) using acetone (3 times 8 ml). After evaporation and reconstitution in 500 l HPLC mobile phase, nifursol was analysed without further sample processing by LC–MS/MS. Quantification was performed using matrix-matched standards prepared by adding known amounts of the stock solutions to blank sample matrix to obtain concentration levels of 1, 2.5, 10, 25 and 100 g/kg in the matrix. NFHBH is a structural analogue of nifursol and is used as the internal standard (Fig. 2). NFHBH was added at a concentration of 25 g/kg in the matrix. The observed LoD for nifursol in tissues and plasma (signal to noise ratio of 3 or more) was 0.25 g/kg for plasma, 1 g/kg for kidney and 2 g/kg for liver, muscle and bile. 2.4. Analysis of DNSH Samples were analysed for the ratio between extractable metabolites and non-extractable metabolites. To this end samples (1 g) were extracted three times with 8 ml acetone to remove any free metabolites before analysing the samples for non-extractable residues.
3.1. Nifursol The parent drug, nifursol, could only be detected in bile and plasma samples of broilers at days 0 and 3 after cessation of treatment (Table 2). In the liver, kidney and muscle samples of broilers, no nifursol could be detected above the LoD of the method applied (1–2 g/kg for tissues), despite careful storage below −70 ◦ C. For liver, kidney and muscle samples this observation is in agreement with observations for furazolidone and furaltadone in pig [3,4] and broilers [17], with the exception of muscle tissue of furaltadone-treated broilers. In the latter study, low amounts of furaltadone were detected at zero withdrawal time. For bile and plasma substantial concentrations of nifursol were detected at zero withdrawal time. Quantification of these elevated amounts is disputable considering the concentration range of the matrix-matched standards used (1–100 g/kg), but the presence of high concentrations of residues of nifursol cannot be questioned. For bile and plasma samples, these results are in agreement with observations for both furazolidone and furaltadone in broilers [17]. Bile samples were not analysed in the pig studies [3,4].
Fig. 2. Molecular structures of HBH, NPHBH and NFHBH.
T. Zuidema et al. / Analytica Chimica Acta 529 (2005) 339–346 Table 2 Average residue of nifursol in g/kg in liver, kidney, muscle, bile and plasma of broilers treated with feed containing 50 mg/kg nifursol during a period of 7 consecutive days Days after cessation of medication
Liver Kidney Muscle Bile Plasma
0
3
7
14
21
<2 <1 <2 16285 281
<2 <1 <2 20 0.3
<2 <1 <2 <2 <0.25
<2 <1 <2 <2 <0.25
<2 <1 <2 <2 <0.25
Polman et al. [4] did analyse plasma samples of furaltadonetreated pigs but this concerned samples that were taken during the medication period and samples taken at days 1 and 14 after cessation of treatment. Residues of furaltadone with levels up to approximately 2500 g/l could be detected in the plasma samples taken during medication, but at day 1 after cessation of treatment no residues of furaltadone could be detected. 3.2. DNSH The depletion curves for tissue-bound (non-extractable) and free (extractable) residues of DNSH in liver, kidney, muscle, bile and plasma of broilers treated with nifursol are given in Fig. 3A–E, respectively. For matter of comparison, in Fig. 4 depletion curves are included for the non-extractable residues of AOZ and AMOZ in liver tissue of broilers treated with furazolidone and furaltadone, respectively [17]. At zero withdrawal time, non-extractable residue concentrations determined as DNSH average to approximately 900 g/kg in liver (n = 6; RSD = 26%), 2000 g/kg in kidney (n = 6; RSD = 31%) and 225 g/kg in muscle tissue (n = 6; RSD = 16%). In bile and plasma (n = 3 because the limited sample amount), the DNSH non-extractable residue concentrations average to approximately 1000 g/kg (RSD = 58%) and 1000 g/kg (RSD = 43%), respectively. In a similar experiment broilers have been treated with 200 mg/kg furaltadone and furazolidone in feed [17]. The observed residue concentrations of DNSH in liver, kidney and muscle tissue correlate quite well to those observed for AMOZ, the metabolite of furaltadone, especially taking into account the four-fold higher concentration of furaltadone in the feed. An exception is the non-extractable residue detected in plasma. The non-extractable residue of DNSH in plasma (1000 g/kg) is considerably higher compared to the nonextractable residue of AMOZ and AOZ in plasma (10 and 2 g/kg, respectively). The residue levels of DNSH in the various tissues, bile and plasma decrease steadily after cessation of medication, with levels declining in muscle, kidney, bile and plasma somewhat faster than those in liver tissue. A half-life of approximately 3 days could be estimated for non-extractable DNSH in liver tissue. In muscle tissue, kidney tissue, bile and plasma a halflife of approximately 2 days can be established, even though
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the non-extractable residue concentrations in kidney tissues at zero withdrawal time are 2 times higher compared to nonextractable residue levels of DNSH in liver tissues. Residue levels of DNSH in muscle reach the LoD (1 g/kg) after approximately 14 days. In liver tissue, however, concentrations exceeding 10 g/kg (average 11.2 g/kg; n = 5; RSD = 60%) can still be found 21 days after the end of treatment. Taking the half-life and the LoD (1 g/kg) of non-extractable DNSH in liver tissues into account, it can be estimated that residues can at least be detected up to 30 days after cessation of treatment. However, due to the fact that the presented study ended at 21 days after cessation of treatment, this figure is based on extrapolation of the available data. For comparison, 21 days after cessation of medication, the nonextractable concentrations of acid-hydrolysable residues of furaltadone and furazolidone in liver tissues amounted to 50 and 20 g/kg, respectively. The half-life of non-extractable residues of AMOZ and AOZ in liver tissues is approximately 3.5 days. In contrast to nifursol, at 21 days substantial amounts of non-extractable residues of furaltadone and furazolidone metabolites could still be detected in muscle tissue (30 and 10 g/kg, respectively; the calculated half-life for both AMOZ and AOZ in muscle tissue is 4 days). In kidney tissue as well as in bile and plasma, nonextractable residues of DNSH can be found above the LoD 21 days after cessation of treatment (kidney: 4.6 g/kg (n = 5; RSD = 56%); bile: 2.5 g/kg (n = 3; RSD = 33%); plasma: 2.0 g/kg (n = 3; RSD = 63%)). It is evident from these results that from the perspective of regulatory control, the detection of nifursol abuse should preferably focus at the occurrence of residues in liver tissue, rather than in muscle tissue, which in practice is often chosen as the target tissue. Considering the regular withdrawal time of 25 days for turkeys [11], residues can be expected in treated animals. The preference of residues to bind to liver tissue compared to muscle tissue was also observed in the small-scale animal study performed by Kaufmann and Butcher [11]. In this study two turkeys were each treated daily with 14.1 mg nifursol per animal. One animal was sacrificed during treatment and one animal was sacrificed 25 days (withdrawal time) after cessation of treatment. Muscle and liver tissues were sampled and analysed for nifursol and DNSH. In the muscle and liver tissue samples of the animal sacrificed during treatment residues of DNSH could be detected at total residue concentrations of 6 and 68 g/kg, respectively. In the muscle and liver tissue of the animal sacrificed at day 25 no residues of nifursol and DNSH could be detected (LoD 0.2 g/kg). In contrast, the presented study in broilers resulted in considerable higher concentrations of non-extractable, DNSH in muscle and liver tissues, 225 and 900 g/kg, respectively. Because no details of the animal study described by Kaufmann and Butcher [11] are given no sufficient explanation can be given for this discrepancy in concentrations of DNSH in muscle and liver tissue. During the study of Kaufmann and Butcher [11], commercial samples of turkey were analysed
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Fig. 3. Depletion curves of non-extractable DNSH and extractable DNSH in liver (A), muscle (B), kidney (C), bile (D) and plasma (E) of broilers treated with feed containing 50 mg/kg nifursol during a period of 7 consecutive days. Error bars of the average residue concentrations are indicated.
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Fig. 4. Depletion curves of non-extractable AOZ and AMOZ in liver of broilers treated with feed containing 200 mg/kg furazolidone and 200 mg/kg furaltadone, respectively, during a period of 7 consecutive days. Error bars of the average residue concentrations are indicated.
and one sample was found positive for residues of DNSH, even though the withdrawal time was respected. The formation of tissue-bound residues after treatment with nifursol was also shown in turkeys [11,12], but both studies did not address the depletion of nifursol and tissuebound residues of DNSH explicitly. 3.3. Non-extractable compared to extractable DNSH Intensive research has proven that acetone is the most effective extraction solvent for removal of residues of nifursol and its free metabolites [13]. From Fig. 3A–E, it can be derived that, with the exception of bile, even with this solvent the amount of residue that can be extracted from tissues and plasma is rather small. For bile clearly a different situation is observed, as the majority of residues is solvent extractable.
Fig. 5. Fraction of non-extractable residue of DNSH in liver, kidney, muscle, bile and plasma of broilers treated with feed containing 50 mg/kg nifursol during a period of 7 consecutive days. Error bars of the average residue concentrations are indicated.
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Fig. 6. Fraction of non-extractable residue of AOZ in liver, kidney, muscle and plasma of broilers treated with feed containing 200 mg/kg furazolidone, during a period of 7 consecutive days. Error bars of the average residue concentrations are indicated.
Very likely the majority of these extractable residues is nifursol itself. In Fig. 5 the fraction of non-extractable residue of DNSH in liver, kidney and muscle tissue and bile and plasma as function of the withdrawal time is shown. Approximately 85% of the DNSH residues in liver tissue can be considered non-extractable at zero withdrawal time. This increases to 95% a few days after the end of medication. The relative amount of non-extractable residue in muscle and kidney tissues is already close to 95% at zero withdrawal time and remains that high at longer withdrawal times. The concentration of extractable residues in muscle drops below the LoD (0.2 g/kg) after 1 week after the end of treatment. The relative amount of non-extractable residue in plasma increases from approximately 75% at zero withdrawal time to 95% a few days after the end of medication. As mentioned before, in bile the relative amount of non-extractable residue is completely different. Starting at 15% (day 0), increasing to 65% (day 3) and decreasing again to 35% (day 21). It should be noted that bile at day 0 was found to contain high concentrations of parent nifursol that, under the conditions used is transformed to DNSH. For comparison relative amounts of non-extractable residue of AOZ in tissues and plasma of broilers treated with furazolidone, are presented in Fig. 6. For this compound, as for furaltadone, the percentage non-extractable residue varies between approx. 50 and 75% depending on the withdrawal time and matrix [18]. It is clear that the relative fraction of non-extractable residues is considerably higher for nifursol than for other nitrofurans like furazolidone and furaltadone. Perhaps the low amounts of extractable metabolites present is related to the fact that nifursol is significantly more lypophillic than furazolidone and furaltadone. This is also substantiated by the observation that extraction with water and water/methanol mixtures is less efficient in removal of free residues than extraction with polar organic solvents like acetone.
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4. Conclusions Broilers treated with medicated feed containing nifursol at a therapeutic dose yielded considerable amounts of residues of the acid-hydrolysable side-chain of nifursol (DNSH) in liver, kidney and muscle tissue and in bile and plasma. Taking into account an LoD of 1 g/kg for non-extractable residues of DNSH, detection is possible for at least 3 weeks in liver, kidney, bile and plasma and for up to 2 weeks in muscle tissue. The vast majority of the DNSH residues were found to be non-extractable. From a regulatory control perspective, residue monitoring should be directed at the analysis of nonextractable residues in liver tissue, rather than in muscle tissue. Despite some individual differences, in general terms the previously postulated similarity in disposition and depletion of nifursol with furazolidone and furaltadone is supported by this study. Consequently it is clear that control of the ban on veterinary use of nifursol can only be effectively enforced when regulatory control laboratories implement methods for the detection of non-extractable DNSH, preferably in liver tissue.
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[3] R.J. McCracken, W.J. Blanchflower, C. Rowan, M.A. McCoy, D.G. Kennedy, Analyst 120 (1995) 2347. [4] Th.H.G. Polman, P.L.M. Berende, L.A.P. Hoogenboom, RIKILT Report 94.04, Wageningen, The Netherlands, 1994. [5] D.W. Gottschall, R. Wang, J. Agric. Food Chem. 43 (1995) 2520. [6] Angelis, I. De, L. Rossi, J.Z. Pedersen, A.L. Vignoli, O. Vincentini, L.A.P. Hoogenboom, T.H.G. Polman, A. Stammati, F. Zucco, Xenobiotica 11 (1999) 1157. [7] Food and Drug Administration, Part V, New Animal Drugs, Furazolidon (NF-180), Fed. Register 41, 19906, 1976. [8] Annex IV of Council Regulation (EEC) No. 2377/90, 26 June 1990 Off. J. L224, p. 1. [9] Furazolidone Summary Report. www.emea.eu.int. [10] Council Regulation (EC) No. 1756/2002, 23 September 2002 Off. J. L265, p. 1. [11] A. Kaufmann, P. Butcher, in: R.W. Stephany, A.A. Bergwerff (Eds.), Proceedings of EuroResidue V, Part 2, May 2004, p. 607. [12] C. Bock, J. Polzer, F. Hamann, C. Stachel, P. Gowik, in: L.A. van Ginkel, A.A. Bergwerff (Eds.), Proceedings of EuroResidue V, Part 1, May 2004, p. 135. [13] P.P.J. Mulder, T. Zuidema, N.G.M. Keestra, P.J.F. Kooij, I.J.W. Elbers, J.A. van Rhijn, Analyst, submitted for publication. [14] J. Schl¨osser, A. Mehlich, F. Ballwanz, M. Petz, Fres. J. Anal. Chem. 360 (1998) 498. [15] J.A. van Rhijn, D.G. Kennedy, P.P.J. Mulder, I.J.W. Elbers, T. Zuidema, J.J.P. Lasaroms, Analyst, submitted for publication. [16] Commission Decision (CD) 2002/657/EC, 12 August 2002 Off. J. L221, p. 8. [17] T. Zuidema, J.A. van Rhijn, B. Schat, P.P.J. Mulder, Y.J.C. Bolck, L.A.P. Hoogenboom, D.G. Kennedy, in: R.W. Stephany, A.A. Bergwerff (Eds.), Proceedings of EuroResidue V, Part 2, May 2004, p. 996. [18] T. Zuidema, J.A. van Rhijn, B. Schat, P.P.J. Mulder, Y.J.C. Bolck, L.A.P. Hoogenboom, D.G. Kennedy, in preparation.
Metabolism and depletion of nifursol in broilers T. Zuidemaa,∗ , P.P.J. Muldera , J.A. van Rhijna , N.G.M. Keestraa , L.A.P. Hoogenbooma , B. Schata , D.G. Kennedyb b
a RIKILT – Institute of Food Safety, Bornsesteeg 45, P.O. Box 230, 6700 AE Wageningen, The Netherlands Chemical Surveillance Department, Veterinary Sciences Division, Department of Agriculture and Rural Development, Stoney Road, Stormont, Belfast BT4 3SD, Northern Ireland, UK
Received 14 June 2004; received in revised form 17 August 2004; accepted 17 August 2004 Available online 18 December 2004
Abstract Nifursol has recently been prohibited for use as a feed additive. Considering the similarity in structure between nifursol and the other nitrofurans, an analogous metabolism could be expected. To study the formation of tissue-bound residues in poultry, broilers were treated orally with nifursol during a period of 7 consecutive days, via medicated feed at a dosage of 50 mg/kg feed. Muscle, kidney, liver, bile and plasma samples were collected at the day of cessation of medication (day 0) and at days 3, 7, 14 and 21 after the end of medication. Samples were analysed for nifursol and the acid-hydrolysable side-chain of nifursol (DNSH; 3,5-dinitro salicylhydrazide). Samples were also analysed for the ratio between free (solvent-extractable) metabolites and tissue-bound (non-extractable) metabolites. The results obtained clearly indicate the formation of tissue-bound residues in poultry. Concentrations of non-extractable residue at zero withdrawal time averaged to 900 g/kg in liver tissue, 2000 g/kg in kidney tissue, 225 g/kg in muscle tissue, 1000 g/kg in bile and 1000 g/kg in plasma. Taking into account an LoD of 1 g/kg, non-extractable residues of DNSH can be detected for at least 3 weeks after administration in liver, kidney, bile and plasma and for up to 2 weeks in muscle tissue. The amounts of extractable residues were relatively low, in many instances less than 10% of the total amount of residue. In general terms the depletion data obtained show a similar behaviour of nifursol in broilers as previously found for furazolidone and furaltadone in broilers. © 2004 Elsevier B.V. All rights reserved. Keywords: Nifursol; Nitrofurans; Metabolism; Depletion; DNSH; Tissue-bound residue; Broilers
1. Introduction Nifursol (3,5-dinitro-N -(5-nitrofurfurylidene)salicylhydrazide) has been used extensively as a feed additive for the prevention of histomoniasis (black head disease) in turkeys. Nifursol belongs to the family of nitrofurans, substances harbouring a 5-nitrofuran group, a structure that is suspected of imparting genotoxic characteristics to these substances. Extensive research has been performed on the mutagenicity and genotoxicity of the nitrofurans and especially on furazolidone and its marker metabolite, the acid-hydrolysable side-chain 3-amino-2-oxazolidinone (AOZ). Vroomen et al. ∗
Corresponding author. Tel.: +31 317 475580; fax: +31 317 417717. E-mail address: [email protected] (T. Zuidema).
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[1], Hoogenboom et al. [2], McCracken et al. [3] and Polman et al. [4] showed a rapid depletion in pig tissues of the parent drug and the formation of persistent metabolites. Because of this rapid metabolism, residues of the parent drugs cannot be detected even within a few hours after cessation of treatment. Treatment of two pigs with 75 mg 14 C-labelled furazolidone per kg body weight per day for a period of 10 days showed that furazolidone as such cannot be detected in the tissues, while even after 14 days after cessation of treatment in tissues and plasma radioactivity comparable to ppb levels of furazolidone could be detected [1]. Due to the fact that part of the 14 C-label could not be extracted from the tissues even when organic solvents were used, the conclusion was made that tissue-bound residues were formed. In vitro research clarified that these tissue-bound residues originated
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from furazolidone [2]. In general, metabolism will result in compounds that can be depleted more effectively compared to the parent compound. In some cases, as for furazolidone, reactive intermediates are formed that can be bound covalently to DNA or proteins. Such adducts will have a longer lifetime in the animal and as such will be the ideal marker residue for control purposes. Residues of tissue-bound metabolites of nitrofurans can be detected in tissues of pigs even weeks after cessation of treatment [1,5]. One of the characteristics of furazolidone is that in the presence of a weak acid (stomach), hydrolysis of the imine bond takes place yielding the side-chain, 3-amino2-oxazolidinone (AOZ). Furazolidone and AOZ have been studied intensively in relation to their toxicity [6,7] and both have been found to posses mutagenic and carcinogenic properties. Combined with the lack of data to determine a No Observed Effect Level (NOEL) in 1993 the nitrofurans as a group (with the exclusion of furazolidone) were included in Annex IV of Council Regulation (EEC) no 2377/90 [8]. Furazolidone was included in Annex IV in 1997 [8]. In addition, AOZ has been identified as the official marker metabolite of furazolidone [9]. In contrast, the other nitrofurans are much less intensively studied, and especially for nifursol, to date no reports are published concerning its metabolism and the possible formation of reactive intermediates and tissue-bound residues. Recently, nifursol has been prohibited for use as a feed additive in Council Regulation 2002/1756/EC [10]. Until then,
nifursol had been the last antibiotic that was available and permitted for the prevention of histomoniasis (black head disease) in turkeys. Considering the similarity in structure between nifursol and the other nitrofurans (Fig. 1), the acid-hydrolysable sidechain of nifursol, 3,5-dinitro-salicylhydrazide (DNSH), can be used as a marker metabolite to determine whether or not nifursol has been used. To date no studies have been performed to verify this assumption. At the EuroResidue V Conference (10–12 May 2004, Noordwijkerhout, The Netherlands), results of two smallscale animal studies were presented. Both studies independently showed the formation of DNSH in nifursol-treated turkeys [11,12]. The study described by Kaufmann and Butcher [11] was part of the method validation of the analysis of nifursol and because of that only two animals were part of this study. The main goal of the study described by Bock et al. [12] was to determine the possible metabolites in turkey after treatment of the animals with nifursol and their residue behaviour in muscle tissue. Both studies did not address the depletion of nifursol and tissue-bound residues of DNSH explicitly. In this paper, we present a study on the formation of tissue-bound residues and depletion of nifursol and the acid-hydrolysable side-chain of nifursol (DNSH) in broilers. Research has been focussed on DNSH and not on other possible metabolites, especially because we wanted to make the comparison to the other nitrofurans, for which the acid-
Fig. 1. Molecular structures of the nitrofurans, nifursol, furazolidone and furaltadone, their acid-hydrolysable side-chains DNSH, AOZ and AMOZ and their corresponding nitrophenyl derivatives NPDNSH, NPAOZ and NPAMOZ.
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hydrolysable side-chain is being used as a marker residue. To be able to make a good comparison between the depletion of nifursol and the more studied furazolidone and furaltadone, broilers were used as target animal. This study is performed in support of implementing methods for official control using DNSH as a marker residue, which is currently underway.
2. Experimental 2.1. Animal experiments The clinical phase of the study was carried out at Zodiac Animal Facilities (Wageningen, The Netherlands). The stable unit was equipped with continuous ventilation and heating system. The temperature was adjusted according to the age of the animals. The light scheme was 23 h of light and 1 h of darkness. The feed consumption during the medication period was recorded. During the study, the animals were fed with a standard commercial feed (Research Diet Services BV, Wijk bij Duurstede, The Netherlands). No antibiotics (feed additives) were present in the feed. One-day broilers (n = 40) were housed on straw and randomly divided into two different groups. A wing tag identified all broilers. Group 1 consisted of 30 animals (15 males and 15 females). Group 2, the control group, consisted of 10 animals (five males and five females). The clinically healthy broilers were kept for 14 days on a non-medicated starter ration. During the acclimation period, one animal (female) of group 1 died and was replaced by an animal from group 2 (female). From the start of the acclimation period until the moment of slaughter, the animals were monitored daily for general health by qualified personnel supervised by a veterinarian. The animals were in good health and free from apparent abnormalities and malformations. The broilers were vaccinated for Infectious Bronchitis and Newcastle’s Disease (NCD) prior to shipping to the test facility. The second NCD vaccination was given during the acclimation period at the test facility. After the acclimation period animals of group 1 were fed for 7 consecutive days on a feed ration containing 50 mg/kg of nifursol (Research Diet Services BV). Animals of group 2 were given non-medicated feed. Drinking water and feed was given ad libitum. Seven days after start of the treatment, the medicated feed was replaced by non-medicated feed. Taking the mean daily feed consumption (120 g/kg body weight) and the amount of nifursol in the feed (50 mg/kg) into account, the average dosage was 6.0 mg nifursol per kg body weight. Groups of six animals were sacrificed at days 0, 3, 7, 14 and 21 after cessation of the treatment. The broilers were slaughtered at the Zodiac Animal Facilities and qualified personnel collected tissue samples as soon as possible after slaughter. During collection, precautionary measures were taken to prevent mutual contamination of the samples. Samples of muscle, liver, kidney, bile and plasma from each animal were collected separately and stored in plastic
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bags or tubes marked with the animal ID code. Samples were stored on dry ice in an insulating transport box immediately after collection and were transported to the laboratory as soon as possible (within 0.5–3 h, depending on the order of slaughter). In the laboratory, samples were snap-frozen using liquid nitrogen and subsequently stored below −70 ◦ C until analysis. Samples were analysed for both nifursol and the acidhydrolysable side-chain of nifursol, DNSH. The analytical method will be described elsewhere [13]. A brief description will be given in the following paragraphs. 2.2. Materials All reagents and solvents were of analytical grade or better unless stated otherwise. Ethyl acetate and methanol were from Biosolve (Valkenswaard, The Netherlands). Acetone, hydrochloric acid, trisodium phosphate dodecahydrate, sodium hydroxide and acetic acid were from Merck (Darmstadt, Germany). Ortho-nitrobenzaldehyde (o-NBA), 3,5-dinitrosalicylic acid (DNSA), hydrazine, N-ethoxycarbonyl-2-ethoxy-1,2dihydroquinoline (EEDQ), 4-hydroxy-3,5-dinitro-benzoic acid (HBA) and 5-nitrofurfural were from Sigma (St. Louis, MO, USA). Water was demineralised using a Millipore purification system (Millipore, Billerica, MA, USA). Nifursol standard was obtained from Solvay Pharmaceuticals (Weesp, The Netherlands). 3,5-Dinitro salicylhydrazide (DNSH) was synthesised in-house from DNSA and hydrazine using EEDQ as a coupling agent [14]. 4-Hydroxy3,5-dinitro-benzohydrazide (HBH) was synthesised in-house by conversion of HBA with hydrazine and EEDQ. The nitrophenyl derivatives of DNSH and HBH were prepared by reaction with o-NBA in 1 M HCl. The 5-nitrofuran derivative of HBH (NFHBH) was prepared by reaction with 5-nitrofurfural in 1 M HCl. Individual stock solutions were prepared by dissolving the standard substances in the appropriate volume of methanol to obtain solutions of 100 g/ml. Individual standard solutions were prepared by diluting the stock solutions using methanol to obtain solutions of 1 g/ml and 0.1 g/ml. Standard solutions were stored in the dark at +4 ◦ C for up to 3 months. LC–MS/MS analysis was carried out using an Agilent 1100 series gradient HPLC system (Palo Alto, CA, USA) equipped with a 50 × 3 mm Waters XTerra® C18 column. Fifty microlitres of each sample extract was injected. Chromatographic separation was achieved by gradient elution using a flow rate of 0.4 ml/min and a linear gradient from 5% methanol containing 0.05% ammonium hydroxide to 90% methanol containing 0.05% ammonium hydroxide in 7 min allowing a good separation between the internal standard and the analyte. For the analysis, chromatographical separation is necessary due to the similarity in molecular weight and fragmentation pathway of the analyte and the internal standard. The column was connected to a Micromass Quattro Micro (Waters-Micromass, Manchester, UK) triple quadrupole
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Table 1 Diagnostic ions of nifursol, NFHBH, NPDNSH and NPHBH m/z
Nifursol NFHBH NPDNSH NPHBH
Precursor ion
Product ion 1
Product ion 2
364 364 374 374
226 183 226 183
182 182
Dwell time (s)
Collision energy (eV)
0.2 0.2 0.2 0.2
20 20 20 20
mass spectrometer equipped with an electrospray interface operated in negative ion mode. The interface was operated at settings optimised for optimum sensitivity (capillary voltage 2.7 kV, cone voltage 35 V, source temperature 100 ◦ C, desolvation temperature 300 ◦ C, desolvation gas flow 700 l/h, cone gas flow 100 l/h) that were modified slightly on a regular basis to maintain optimum sensitivity. Deprotonated molecules of the analytes of interest were selected as precursor ions and fragmented to compound-specific product ions using collision-induced dissociation with argon as the collision gas at a pressure of 3.2 Pa. Data acquisition is performed in MRM mode using the settings listed in Table 1.
Samples (washed pellets as well as the acetone extracts) were extracted and purified with the method described for the acid-hydrolysable metabolites of the other nitrofurans [15]. In brief, after addition of 25 g of a structure-related internal standard (4-hydroxy-3,5-dinitro-benzohydrazide, HBH (Fig. 2)), samples were hydrolysed and derivatised overnight in 5 ml diluted hydrochloric acid (0.2 M) in the presence of 100 mM ortho-nitrobenzaldehyde. The aqueous sample was subsequently adjusted to pH 7.0 ± 0.5 and extracted twice with 4 ml ethyl acetate. The combined ethyl acetate fractions were evaporated to dryness and redissolved in 500 l methanol/water (10/90), filtered and analysed by LC–MS/MS. Quantification of the ortho-nitrophenyl derivative of DNSH was performed by the internal standard method. Matrix-matched standards were prepared by adding known amounts of the stock solutions to blank sample matrix to obtain concentration levels of 1, 2.5, 10, 25 and 100 g/kg in the matrix. Identification of the analytes was carried out according to EU requirements for banned substances [16]. The observed limit of detection (LoD; signal to noise ratio of 3 or more) ranged from 0.2 g/kg for extractable residues to 1 g/kg in case of non-extractable residues.
2.3. Analysis of nifursol 3. Results and discussion The parent substance nifursol was extracted from the tissue and plasma samples (1 g) using acetone (3 times 8 ml). After evaporation and reconstitution in 500 l HPLC mobile phase, nifursol was analysed without further sample processing by LC–MS/MS. Quantification was performed using matrix-matched standards prepared by adding known amounts of the stock solutions to blank sample matrix to obtain concentration levels of 1, 2.5, 10, 25 and 100 g/kg in the matrix. NFHBH is a structural analogue of nifursol and is used as the internal standard (Fig. 2). NFHBH was added at a concentration of 25 g/kg in the matrix. The observed LoD for nifursol in tissues and plasma (signal to noise ratio of 3 or more) was 0.25 g/kg for plasma, 1 g/kg for kidney and 2 g/kg for liver, muscle and bile. 2.4. Analysis of DNSH Samples were analysed for the ratio between extractable metabolites and non-extractable metabolites. To this end samples (1 g) were extracted three times with 8 ml acetone to remove any free metabolites before analysing the samples for non-extractable residues.
3.1. Nifursol The parent drug, nifursol, could only be detected in bile and plasma samples of broilers at days 0 and 3 after cessation of treatment (Table 2). In the liver, kidney and muscle samples of broilers, no nifursol could be detected above the LoD of the method applied (1–2 g/kg for tissues), despite careful storage below −70 ◦ C. For liver, kidney and muscle samples this observation is in agreement with observations for furazolidone and furaltadone in pig [3,4] and broilers [17], with the exception of muscle tissue of furaltadone-treated broilers. In the latter study, low amounts of furaltadone were detected at zero withdrawal time. For bile and plasma substantial concentrations of nifursol were detected at zero withdrawal time. Quantification of these elevated amounts is disputable considering the concentration range of the matrix-matched standards used (1–100 g/kg), but the presence of high concentrations of residues of nifursol cannot be questioned. For bile and plasma samples, these results are in agreement with observations for both furazolidone and furaltadone in broilers [17]. Bile samples were not analysed in the pig studies [3,4].
Fig. 2. Molecular structures of HBH, NPHBH and NFHBH.
T. Zuidema et al. / Analytica Chimica Acta 529 (2005) 339–346 Table 2 Average residue of nifursol in g/kg in liver, kidney, muscle, bile and plasma of broilers treated with feed containing 50 mg/kg nifursol during a period of 7 consecutive days Days after cessation of medication
Liver Kidney Muscle Bile Plasma
0
3
7
14
21
<2 <1 <2 16285 281
<2 <1 <2 20 0.3
<2 <1 <2 <2 <0.25
<2 <1 <2 <2 <0.25
<2 <1 <2 <2 <0.25
Polman et al. [4] did analyse plasma samples of furaltadonetreated pigs but this concerned samples that were taken during the medication period and samples taken at days 1 and 14 after cessation of treatment. Residues of furaltadone with levels up to approximately 2500 g/l could be detected in the plasma samples taken during medication, but at day 1 after cessation of treatment no residues of furaltadone could be detected. 3.2. DNSH The depletion curves for tissue-bound (non-extractable) and free (extractable) residues of DNSH in liver, kidney, muscle, bile and plasma of broilers treated with nifursol are given in Fig. 3A–E, respectively. For matter of comparison, in Fig. 4 depletion curves are included for the non-extractable residues of AOZ and AMOZ in liver tissue of broilers treated with furazolidone and furaltadone, respectively [17]. At zero withdrawal time, non-extractable residue concentrations determined as DNSH average to approximately 900 g/kg in liver (n = 6; RSD = 26%), 2000 g/kg in kidney (n = 6; RSD = 31%) and 225 g/kg in muscle tissue (n = 6; RSD = 16%). In bile and plasma (n = 3 because the limited sample amount), the DNSH non-extractable residue concentrations average to approximately 1000 g/kg (RSD = 58%) and 1000 g/kg (RSD = 43%), respectively. In a similar experiment broilers have been treated with 200 mg/kg furaltadone and furazolidone in feed [17]. The observed residue concentrations of DNSH in liver, kidney and muscle tissue correlate quite well to those observed for AMOZ, the metabolite of furaltadone, especially taking into account the four-fold higher concentration of furaltadone in the feed. An exception is the non-extractable residue detected in plasma. The non-extractable residue of DNSH in plasma (1000 g/kg) is considerably higher compared to the nonextractable residue of AMOZ and AOZ in plasma (10 and 2 g/kg, respectively). The residue levels of DNSH in the various tissues, bile and plasma decrease steadily after cessation of medication, with levels declining in muscle, kidney, bile and plasma somewhat faster than those in liver tissue. A half-life of approximately 3 days could be estimated for non-extractable DNSH in liver tissue. In muscle tissue, kidney tissue, bile and plasma a halflife of approximately 2 days can be established, even though
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the non-extractable residue concentrations in kidney tissues at zero withdrawal time are 2 times higher compared to nonextractable residue levels of DNSH in liver tissues. Residue levels of DNSH in muscle reach the LoD (1 g/kg) after approximately 14 days. In liver tissue, however, concentrations exceeding 10 g/kg (average 11.2 g/kg; n = 5; RSD = 60%) can still be found 21 days after the end of treatment. Taking the half-life and the LoD (1 g/kg) of non-extractable DNSH in liver tissues into account, it can be estimated that residues can at least be detected up to 30 days after cessation of treatment. However, due to the fact that the presented study ended at 21 days after cessation of treatment, this figure is based on extrapolation of the available data. For comparison, 21 days after cessation of medication, the nonextractable concentrations of acid-hydrolysable residues of furaltadone and furazolidone in liver tissues amounted to 50 and 20 g/kg, respectively. The half-life of non-extractable residues of AMOZ and AOZ in liver tissues is approximately 3.5 days. In contrast to nifursol, at 21 days substantial amounts of non-extractable residues of furaltadone and furazolidone metabolites could still be detected in muscle tissue (30 and 10 g/kg, respectively; the calculated half-life for both AMOZ and AOZ in muscle tissue is 4 days). In kidney tissue as well as in bile and plasma, nonextractable residues of DNSH can be found above the LoD 21 days after cessation of treatment (kidney: 4.6 g/kg (n = 5; RSD = 56%); bile: 2.5 g/kg (n = 3; RSD = 33%); plasma: 2.0 g/kg (n = 3; RSD = 63%)). It is evident from these results that from the perspective of regulatory control, the detection of nifursol abuse should preferably focus at the occurrence of residues in liver tissue, rather than in muscle tissue, which in practice is often chosen as the target tissue. Considering the regular withdrawal time of 25 days for turkeys [11], residues can be expected in treated animals. The preference of residues to bind to liver tissue compared to muscle tissue was also observed in the small-scale animal study performed by Kaufmann and Butcher [11]. In this study two turkeys were each treated daily with 14.1 mg nifursol per animal. One animal was sacrificed during treatment and one animal was sacrificed 25 days (withdrawal time) after cessation of treatment. Muscle and liver tissues were sampled and analysed for nifursol and DNSH. In the muscle and liver tissue samples of the animal sacrificed during treatment residues of DNSH could be detected at total residue concentrations of 6 and 68 g/kg, respectively. In the muscle and liver tissue of the animal sacrificed at day 25 no residues of nifursol and DNSH could be detected (LoD 0.2 g/kg). In contrast, the presented study in broilers resulted in considerable higher concentrations of non-extractable, DNSH in muscle and liver tissues, 225 and 900 g/kg, respectively. Because no details of the animal study described by Kaufmann and Butcher [11] are given no sufficient explanation can be given for this discrepancy in concentrations of DNSH in muscle and liver tissue. During the study of Kaufmann and Butcher [11], commercial samples of turkey were analysed
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Fig. 3. Depletion curves of non-extractable DNSH and extractable DNSH in liver (A), muscle (B), kidney (C), bile (D) and plasma (E) of broilers treated with feed containing 50 mg/kg nifursol during a period of 7 consecutive days. Error bars of the average residue concentrations are indicated.
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Fig. 4. Depletion curves of non-extractable AOZ and AMOZ in liver of broilers treated with feed containing 200 mg/kg furazolidone and 200 mg/kg furaltadone, respectively, during a period of 7 consecutive days. Error bars of the average residue concentrations are indicated.
and one sample was found positive for residues of DNSH, even though the withdrawal time was respected. The formation of tissue-bound residues after treatment with nifursol was also shown in turkeys [11,12], but both studies did not address the depletion of nifursol and tissuebound residues of DNSH explicitly. 3.3. Non-extractable compared to extractable DNSH Intensive research has proven that acetone is the most effective extraction solvent for removal of residues of nifursol and its free metabolites [13]. From Fig. 3A–E, it can be derived that, with the exception of bile, even with this solvent the amount of residue that can be extracted from tissues and plasma is rather small. For bile clearly a different situation is observed, as the majority of residues is solvent extractable.
Fig. 5. Fraction of non-extractable residue of DNSH in liver, kidney, muscle, bile and plasma of broilers treated with feed containing 50 mg/kg nifursol during a period of 7 consecutive days. Error bars of the average residue concentrations are indicated.
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Fig. 6. Fraction of non-extractable residue of AOZ in liver, kidney, muscle and plasma of broilers treated with feed containing 200 mg/kg furazolidone, during a period of 7 consecutive days. Error bars of the average residue concentrations are indicated.
Very likely the majority of these extractable residues is nifursol itself. In Fig. 5 the fraction of non-extractable residue of DNSH in liver, kidney and muscle tissue and bile and plasma as function of the withdrawal time is shown. Approximately 85% of the DNSH residues in liver tissue can be considered non-extractable at zero withdrawal time. This increases to 95% a few days after the end of medication. The relative amount of non-extractable residue in muscle and kidney tissues is already close to 95% at zero withdrawal time and remains that high at longer withdrawal times. The concentration of extractable residues in muscle drops below the LoD (0.2 g/kg) after 1 week after the end of treatment. The relative amount of non-extractable residue in plasma increases from approximately 75% at zero withdrawal time to 95% a few days after the end of medication. As mentioned before, in bile the relative amount of non-extractable residue is completely different. Starting at 15% (day 0), increasing to 65% (day 3) and decreasing again to 35% (day 21). It should be noted that bile at day 0 was found to contain high concentrations of parent nifursol that, under the conditions used is transformed to DNSH. For comparison relative amounts of non-extractable residue of AOZ in tissues and plasma of broilers treated with furazolidone, are presented in Fig. 6. For this compound, as for furaltadone, the percentage non-extractable residue varies between approx. 50 and 75% depending on the withdrawal time and matrix [18]. It is clear that the relative fraction of non-extractable residues is considerably higher for nifursol than for other nitrofurans like furazolidone and furaltadone. Perhaps the low amounts of extractable metabolites present is related to the fact that nifursol is significantly more lypophillic than furazolidone and furaltadone. This is also substantiated by the observation that extraction with water and water/methanol mixtures is less efficient in removal of free residues than extraction with polar organic solvents like acetone.
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4. Conclusions Broilers treated with medicated feed containing nifursol at a therapeutic dose yielded considerable amounts of residues of the acid-hydrolysable side-chain of nifursol (DNSH) in liver, kidney and muscle tissue and in bile and plasma. Taking into account an LoD of 1 g/kg for non-extractable residues of DNSH, detection is possible for at least 3 weeks in liver, kidney, bile and plasma and for up to 2 weeks in muscle tissue. The vast majority of the DNSH residues were found to be non-extractable. From a regulatory control perspective, residue monitoring should be directed at the analysis of nonextractable residues in liver tissue, rather than in muscle tissue. Despite some individual differences, in general terms the previously postulated similarity in disposition and depletion of nifursol with furazolidone and furaltadone is supported by this study. Consequently it is clear that control of the ban on veterinary use of nifursol can only be effectively enforced when regulatory control laboratories implement methods for the detection of non-extractable DNSH, preferably in liver tissue.
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