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Sulphamethazine residue depletion study in piglets after oral administration
Analytica Chimica Acta 529 (2005) 325–329
Sulphamethazine residue depletion study in piglets after oral administration E.P. Papapanagiotou∗ , D.J. Fletouris, I.E. Psomas Department of Food Hygiene and Technology, School of Veterinary Medicine, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece Received 31 May 2004; received in revised form 2 September 2004; accepted 2 September 2004 Available online 7 December 2004
Abstract The depletion profile of sulphamethazine (SMZ) was studied in healthy piglets after oral administration of a premix, containing 100 g SMZ/kg, at a rate of 1 kg/t of feed for 30 consecutive days. A total of 50 piglets were used in the experiment, 42 of which received the medication and the remaining 8 acted as control animals. The piglets were of 42 days of age and of a mean bodyweight of 28 kg at the beginning of the experiment. Six medicated and one control piglet were sacrificed at days 5, 10, 15, 20, 25, 30 and 35 after the cessation of the treatment and muscle, liver, kidney and fat tissues were sampled and stored at −75 ◦ C pending analysis. The samples were analysed using a liquid chromatographic method, which was fully validated for SMZ residue analysis prior to use. Quite high mean concentrations of SMZ residues of 3.84 mg kg−1 for muscle, 8.14 mg kg−1 for liver, 6.42 mg kg−1 for kidney and 2.88 mg kg−1 for fat tissues were attained 5 days post-medication. SMZ residues were still detected even 20 days post-medication at levels higher than 1.12 mg kg−1 in all tissues examined. In descending order, the SMZ residues concentrations found in all examined tissues were liver > kidney > muscle > fat. The time needed for the concentration of SMZ to drop below the EU established MRL of 100 g/kg, was 30 days. The statistically estimated withdrawal period was calculated to be 41 days. © 2004 Elsevier B.V. All rights reserved. Keywords: Sulphamethazine; Depletion; Residues; Piglet tissues; Oral administration
1. Introduction Sulphonamides are a group of synthetic organic compounds that have played an important role as effective chemotherapeutics in bacterial and protozoal infections in veterinary medicine. Indications for sulphonamides are wide against both Gram negative (Pasteurella spp., Escherichia coli, Bordetella bronchiseptica, Haemophilus spp.) and Gram positive (Streptococcus spp., Staphylococcus spp.) bacteria, owing to their wide spectrum of activity. They cover infectious diseases of the digestive and respiratory tracts, secondary infections, mastitis, metritis and foot rot [1]. All drugs of the sulphonamide group are currently included in Annex
∗
Corresponding author. Tel.: +30 2310 999827; fax: +30 2310 999803. E-mail address: [email protected] (E.P. Papapanagiotou).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.09.042
I of the Council Regulation 2377/90. The existing EU MRL for all drugs of the sulphonamide group is 100 g/kg in all food-producing species [2]. Sulphamethazine (SMZ) is perhaps one of the most widely used sulphonamides. It is employed largely in mass medication for therapeutic or prophylactic use against atrophic rhinitis and other infections of piglets at concentrations of 400 or 100 g/t feed, respectively. However, information on the depletion of SMZ residues from edible tissues of piglets after medication is quite limited. Kuiper et al. [3] reported that 18 days were required for the elimination of SMZ residues from the tissues of piglets affected with atrophic rhinitis, after oral administration of medicated feed containing 1075 g SMZ/t for 3 weeks, a dose which is much higher than normally used. Nevertheless, this study did not aim to establish the withdrawal period of SMZ, since it used a limited number of diseased animals and an exceptionally high dose of SMZ in feed.
326
E.P. Papapanagiotou et al. / Analytica Chimica Acta 529 (2005) 325–329
In this paper, the depletion profile of SMZ residues from edible piglet tissues is reported together with a large-scale experiment, which was carried out in order to establish the withdrawal period of SMZ after preventive oral administration to healthy piglets.
pure and dichloromethane were of analytical grade and were all obtained from Merck (Darmstadt, Germany). Sulphamethazine (SMZ) was obtained from Sigma Chem. Co. (St. Louis, MO, USA). 2.5. Standard solutions
2. Experimental 2.1. Animals Fifty healthy commonly used fattening piglets of both sexes (at a ratio of 1:1), of 42 days of age were used in this study, eight of which served as control animals. The animals were kept under common farm conditions, with continuous ventilation and heating systems. The piglets received normal fattening pig feed consisting of mixed-grain pellets composed of cereals, oleaginous fats, vitamins and minerals. Feed and water was administered to the piglets ad libitum. All animals were monitored during the experimental period to observe apparent clinical abnormalities and/or changes of behaviour.
Stock standard solution was prepared in a 25 ml volumetric flask by dissolving ca. 10 mg of SMZ in acetonitrile. This solution was stable for at least 1 month if stored in a refrigerator (4 ◦ C). Intermediate standard solution was also prepared by appropriately diluting an aliquot from the stock standard solution with acetonitrile. Working standard solutions in the range of 25–250 ng ml−1 of SMZ were prepared daily by appropriately diluting aliquots from the intermediate standard solution with 3 M hydrochloric acid/3.8 M sodium acetate (1:1). Working standard solutions were stable for at least 24 h at ambient temperature and 4 days at 4 ◦ C.
2.2. Drug administration and sampling
2.6. Stationary phase—mobile phase
The experiment included the administration of a sulphonamide premix containing 100 g SMZ/kg of product for 30 days to piglets at a rate of 1 kg/t of feed. Six medicated and one control animal were sacrificed at days 5, 10, 15, 20, 25, 30 and 35 after the cessation of the medication period. The tissue samples (muscle, liver, kidney and fat) obtained, were deep frozen at −75 ◦ C pending analysis.
The stationary phase was a Macherey–Nagel (Germany) Nucleosil 100-5 C18, 5 m column (250 mm × 4.6 mm i.d.). During runs, the stationary phase was kept equilibrated at ambient temperature. The mobile phase consisted of acetonitrile and 10 mM ortho-phosphoric acid (16:84, v/v). Following its preparation, the mobile phase was passed through a 0.2 m Nylon-66 filter (Schleicher & Schuell, Germany) and degassed using helium. The mobile phase was delivered to the system at a rate of 1 ml min−1 . Under the established conditions, SMZ eluted at 7.0 min.
2.3. Equipment A modular liquid chromatography (LC) system (Gilson Medical Electronics, Villiers-le-Bel, France) consisting of a Model 305 piston pump, a Model 805 manometer, and a Model TC 831 column oven was used in this study. Injections were made through a Model 7125 Rheodyne valve (Cotati, CA) equipped with 20 l loop. A Model 119, variable wavelength UV–vis detector (Gilson, Middleton, WI) with wavelength set at 271 nm, was used to detect the signal response. The sensitivity of the detector was set at 0.005 a.u.f.s. The detector was linked to a Model BD 111 Kipp & Zonen (Delft, Holland) pen recorder. The chart speed of the recorder was set at 2 mm min−1 . A Model D7402 EasyPure UV compact system (Barnstead/Thermolyne Corp., Dubuque, I) was used for ultrapurification of tap water, whereas a Model G-560E vortex mixer (Scientific Industries Inc., Bohemia, NY), an UltraTurrax T25 (Janle & Kunkel, IKA Labortechnik, Germany) and a Model Centra-MP4 centrifuge (IEC, Needman Heights, MA) were used for sample treatment. 2.4. Chemicals Acetonitrile was of HPLC grade, whereas orthophosphoric acid, hydrochloric acid, sodium acetate supra-
2.7. Sample extraction and cleanup The sample preparation procedure used in this study was based on that previously reported by Papapanagiotou et al. [4] for the determination of sulphadiazine and trimethoprim in gilthead sea bream tissues. The method was fully validated for all piglet tissues examined. Three grams of ground piglet tissue (muscle, fat, liver and kidney) were accurately weighed in a 50 ml centrifuge tube. Thirty milliliters of dichloromethane were added and the sample was homogenized with an Ultra Turrax for 1 min and centrifuged at 3000 × g for 10 min. The supernatant phase was filtered and 10 ml of the filtrate were transferred to a 15 ml glass centrifuge tube. Following addition of 1 ml of 3 M hydrochloric acid, the tube was vortexed for 15 s, and centrifuged for 5 min at 3000 × g. From the upper aqueous layer, 250 l were transferred to a 2 ml glass centrifuge tube and another 250 l of 3.8 M sodium acetate solution were added and vortexed for 15 s. Finally, a 20 l aliquot was injected into the LC system and analysed at ambient temperature.
E.P. Papapanagiotou et al. / Analytica Chimica Acta 529 (2005) 325–329
Fig. 1. Chromatograms of a blank muscle sample (a), a blank muscle sample fortified with SMZ (1) at 150 ng/g (b) and an incurred (1427 ng/g) muscle sample at 20 days post-treatment (c).
2.8. Estimation of the withdrawal period The withdrawal period was estimated by linear regression analysis of the log-transformed tissue concentrations, and determined at the time when the upper one-sided tolerance limit, with a confidence of 95%, was below the MRL of 100 g/kg established by the European Union for sulphonamides in all edible animal products [5].
3. Results and discussion 3.1. Liquid chromatographic method validation The method was validated for use with respect to linearity, specificity, sensitivity, accuracy, precision and stability aspects [6]. The linearity of the detector response for the test compound was evaluated by injecting, seven standard solutions of various concentrations covering the working range of the assay (0.5–5 ng/20 l of SMZ). The correlation coefficient (r) for the analyte was better than 0.999 (requirement: >0.99). The method was also evaluated for its specificity to ensure that there was no interference with the test compound from matrix co-extractives. Chromatograms obtained from blank tissue extracts and blank tissue fortified with SMZ showed that the peak attributable to the test compound was resolved sufficiently from any other peaks to enable reliable quantification in all tissues examined. Typical chromatograms of blank, fortified and incurred piglet muscle tissues are presented in Fig. 1.
327
The limit of quantification (LOQ), defined as the lowest concentration of SMZ in tissues that has a precision (expressed as relative standard deviation, RSD%) below or equal to 20% and a mean recovery within the range of 80–110%, was 50 g/kg for all four tissues examined. The accuracy of the method, in terms of recovery efficiency, was studied by fortifying blank tissue samples with SMZ at four fortification levels and analyzing six replicates per fortification level each day, for three consecutive days. Blank tissue samples were fortified at half the MRL value (50 g/kg), at the MRL value (100 g/kg), at one-and-a-half time the MRL value (150 g/kg) and at two times the MRL value (200 g/kg) for the test compound. The mean overall recoveries determined for SMZ were 78.78%, 78.20%, 82.70% and 80.64%, for muscle, liver, kidney and fat tissues, respectively. The precision of the method for SMZ was evaluated utilizing the data generated during assessment of the method accuracy at three consecutive days. The results showed that the overall value of the relative standard deviation for betweenor inter-day variability of SMZ was 8.91%, 10.29%, 13.14% and 10.45%, for muscle, liver, kidney and fat tissues, respectively. The stability experiments carried out, referred to the stability of SMZ in incurred tissue samples stored at −20 and −75 ◦ C. The results indicated that the test compound was stable during the storage of incurred tissue samples at −20 and −75 ◦ C for at least 3 and 5 months, respectively. 3.2. Sulphamethazine residue depletion The SMZ residues concentrations in the animals slaughtered 5 days after the cessation of the medication ranged between 1.85 and 4.51 mg kg−1 in muscle tissue, 4.59 and 9.92 mg kg−1 in liver tissue, 4.06 and 8.72 mg kg−1 in kidney tissue and 1.53 and 3.97 mg kg−1 in fat tissue. It is worth noting that even 20 days after the end of treatment, the mean SMZ residues concentrations remained at levels higher than 1.12 mg kg−1 . More specifically, the corresponding mean (n = 6) SMZ residues levels were, in descending order, 3.71 mg kg−1 in liver, 2.17 mg kg−1 in kidney, 1.99 mg kg−1 in muscle and 1.12 mg kg−1 in fat (Table 1). SMZ residues concentrations in all tissues examined dropped below the EU set MRL of 0.1 mg kg−1 , 30 days after the end of the medication. This finding is clearly not in agreement with the 18 days period required for the elimination of SMZ from pig tissues reported by Kuiper et al. [3]. This period, however, is not validated for use as a withdrawal period since diseased piglets were used, the number of animals per slaughter day was small and the levels of SMZ on the final sampling day were still above the MRL, which rendered an extrapolation inevitable. Thirty-five days post-treatment, the SMZ concentrations in all muscle and fat samples, four out of six liver samples and two out of six kidney samples, dropped below the LOQ of the method (Table 1).
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Table 1 SMZ elimination over time from piglet tissues after cessation of feed medication with 100 g SMZ/t feed, for 30 consecutive days Days post-medication
5 10 15 20 25 30 35 a b
Meana SMZ concentration (mg kg−1 ) ± S.D. Muscle
Liver
Kidney
Fat
3.84 ± 0.99 3.69 ± 0.71 2.92 ± 0.82 1.99 ± 0.64 0.18 ± 0.02 0.07 (1/6)b
8.14 ± 1.97 7.05 ± 1.48 5.54 ± 1.71 3.71 ± 1.08 0.44 ± 0.11 0.07 ± 0.005 0.05 (2/6)b
6.42 ± 1.67 4.92 ± 1.03 4.43 ± 1.38 2.17 ± 1.11 0.30 ± 0.05 0.06 (2/6)b 0.05 (4/6)b
2.88 ± 0.90 2.32 ± 1.14 1.48 ± 0.16 1.12 ± 0.25 0.15 ± 0.03 0.06 (2/6)b
Six replicates. Number of individual animals with SMZ concentrations above the LOQ.
Fig. 2. Plot of withdrawal period calculation for muscle.
The LC method used in this study incorporated an acidic extraction procedure, which converts the N-4-acetyl metabolites of SMZ to parent SMZ. The same observation was also reported by Rose et al. [7]. However, according to Kuiper et al. [3] for the determination of a safe withdrawal period for the SMZ treatment, the total amount of SMZ and N4-acetyl-SMZ residues in edible organs and tissues should be taken into account, since N-4-acetyl-SMZ may be con-
Fig. 3. Plot of withdrawal period calculation for fat.
Fig. 4. Plot of withdrawal period calculation for liver.
verted to the parent compound after consumption by the consumers. 3.3. Estimation of the withdrawal period Residual levels of SMZ in muscle, fat, liver and kidney samples were estimated (95% tolerance limit and 95% confidence) to fall below the MRL after a withdrawal period of 40.92, 38.18, 40.76 and 40.12 days, respectively (Figs. 2–5).
Fig. 5. Plot of withdrawal period calculation for kidney.
E.P. Papapanagiotou et al. / Analytica Chimica Acta 529 (2005) 325–329
Since these time points do not make up full days, the withdrawal periods have to be rounded up to the next days. However, the longest withdrawal period of 41 days has to be selected as the conclusive withdrawal period to guarantee consumer safety [5].
4. Conclusions The results of the present study clearly indicate that a 30-day treatment of the piglets with SMZ at a dose of 100 g/t feed, results in residue levels that are cleared quite slowly. The concentrations of SMZ residues were below the existing EU MRLs in all examined piglet tissues 30 days after the cessation of the administration of SMZ. However, a 41 days withdrawal period was estimated, by using the statistical method suggested by EMEA [5]. It becomes quite evident that the 11 additional days calculated with the statistical method represent a safety factor aiming at offering the consumer a significantly higher level of protection.
329
Therefore, a 41-day withdrawal period is proposed to safeguard the health of the consumer from violative SMZ residues in edible tissues of piglets. Furthermore, this outcome would prove to be very useful in risk analysis methodology implementation in view of the protection of Public Health. References [1] N.A. Botsoglou, D.J. Fletouris (Eds.), Drug Residues in Foods: Pharmacology, Food Safety and Analysis, Marcel Dekker, New York, 2001. [2] EEC Regulation 90/2377/EEC incorporating amending regulation 92/675/EEC, Official Journal of the European Communities, No. L224, Brussels, 1990, p. 1. [3] H.A. Kuiper, R.M.L. Aerts, N. Haagsma, H. van Gogh, J. Agric. Food Chem. 36 (1988) 822–825. [4] E.P. Papapanagiotou, E. Iossifidou, I. Psomas, G. Photis, J. Liq. Chromatogr. Rel. Technol. 23 (2000) 2839. [5] EMEA/CVMP/036/95, London, 1995. [6] EC Decision 2002/657/EC, Official Journal of the European Communities, No. L221, Brussels, 2002, p. 8. [7] M.D. Rose, W.H.H. Farrington, G. Shearer, Food Addit. Contam. 12 (1995) 739.
Sulphamethazine residue depletion study in piglets after oral administration E.P. Papapanagiotou∗ , D.J. Fletouris, I.E. Psomas Department of Food Hygiene and Technology, School of Veterinary Medicine, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece Received 31 May 2004; received in revised form 2 September 2004; accepted 2 September 2004 Available online 7 December 2004
Abstract The depletion profile of sulphamethazine (SMZ) was studied in healthy piglets after oral administration of a premix, containing 100 g SMZ/kg, at a rate of 1 kg/t of feed for 30 consecutive days. A total of 50 piglets were used in the experiment, 42 of which received the medication and the remaining 8 acted as control animals. The piglets were of 42 days of age and of a mean bodyweight of 28 kg at the beginning of the experiment. Six medicated and one control piglet were sacrificed at days 5, 10, 15, 20, 25, 30 and 35 after the cessation of the treatment and muscle, liver, kidney and fat tissues were sampled and stored at −75 ◦ C pending analysis. The samples were analysed using a liquid chromatographic method, which was fully validated for SMZ residue analysis prior to use. Quite high mean concentrations of SMZ residues of 3.84 mg kg−1 for muscle, 8.14 mg kg−1 for liver, 6.42 mg kg−1 for kidney and 2.88 mg kg−1 for fat tissues were attained 5 days post-medication. SMZ residues were still detected even 20 days post-medication at levels higher than 1.12 mg kg−1 in all tissues examined. In descending order, the SMZ residues concentrations found in all examined tissues were liver > kidney > muscle > fat. The time needed for the concentration of SMZ to drop below the EU established MRL of 100 g/kg, was 30 days. The statistically estimated withdrawal period was calculated to be 41 days. © 2004 Elsevier B.V. All rights reserved. Keywords: Sulphamethazine; Depletion; Residues; Piglet tissues; Oral administration
1. Introduction Sulphonamides are a group of synthetic organic compounds that have played an important role as effective chemotherapeutics in bacterial and protozoal infections in veterinary medicine. Indications for sulphonamides are wide against both Gram negative (Pasteurella spp., Escherichia coli, Bordetella bronchiseptica, Haemophilus spp.) and Gram positive (Streptococcus spp., Staphylococcus spp.) bacteria, owing to their wide spectrum of activity. They cover infectious diseases of the digestive and respiratory tracts, secondary infections, mastitis, metritis and foot rot [1]. All drugs of the sulphonamide group are currently included in Annex
∗
Corresponding author. Tel.: +30 2310 999827; fax: +30 2310 999803. E-mail address: [email protected] (E.P. Papapanagiotou).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.09.042
I of the Council Regulation 2377/90. The existing EU MRL for all drugs of the sulphonamide group is 100 g/kg in all food-producing species [2]. Sulphamethazine (SMZ) is perhaps one of the most widely used sulphonamides. It is employed largely in mass medication for therapeutic or prophylactic use against atrophic rhinitis and other infections of piglets at concentrations of 400 or 100 g/t feed, respectively. However, information on the depletion of SMZ residues from edible tissues of piglets after medication is quite limited. Kuiper et al. [3] reported that 18 days were required for the elimination of SMZ residues from the tissues of piglets affected with atrophic rhinitis, after oral administration of medicated feed containing 1075 g SMZ/t for 3 weeks, a dose which is much higher than normally used. Nevertheless, this study did not aim to establish the withdrawal period of SMZ, since it used a limited number of diseased animals and an exceptionally high dose of SMZ in feed.
326
E.P. Papapanagiotou et al. / Analytica Chimica Acta 529 (2005) 325–329
In this paper, the depletion profile of SMZ residues from edible piglet tissues is reported together with a large-scale experiment, which was carried out in order to establish the withdrawal period of SMZ after preventive oral administration to healthy piglets.
pure and dichloromethane were of analytical grade and were all obtained from Merck (Darmstadt, Germany). Sulphamethazine (SMZ) was obtained from Sigma Chem. Co. (St. Louis, MO, USA). 2.5. Standard solutions
2. Experimental 2.1. Animals Fifty healthy commonly used fattening piglets of both sexes (at a ratio of 1:1), of 42 days of age were used in this study, eight of which served as control animals. The animals were kept under common farm conditions, with continuous ventilation and heating systems. The piglets received normal fattening pig feed consisting of mixed-grain pellets composed of cereals, oleaginous fats, vitamins and minerals. Feed and water was administered to the piglets ad libitum. All animals were monitored during the experimental period to observe apparent clinical abnormalities and/or changes of behaviour.
Stock standard solution was prepared in a 25 ml volumetric flask by dissolving ca. 10 mg of SMZ in acetonitrile. This solution was stable for at least 1 month if stored in a refrigerator (4 ◦ C). Intermediate standard solution was also prepared by appropriately diluting an aliquot from the stock standard solution with acetonitrile. Working standard solutions in the range of 25–250 ng ml−1 of SMZ were prepared daily by appropriately diluting aliquots from the intermediate standard solution with 3 M hydrochloric acid/3.8 M sodium acetate (1:1). Working standard solutions were stable for at least 24 h at ambient temperature and 4 days at 4 ◦ C.
2.2. Drug administration and sampling
2.6. Stationary phase—mobile phase
The experiment included the administration of a sulphonamide premix containing 100 g SMZ/kg of product for 30 days to piglets at a rate of 1 kg/t of feed. Six medicated and one control animal were sacrificed at days 5, 10, 15, 20, 25, 30 and 35 after the cessation of the medication period. The tissue samples (muscle, liver, kidney and fat) obtained, were deep frozen at −75 ◦ C pending analysis.
The stationary phase was a Macherey–Nagel (Germany) Nucleosil 100-5 C18, 5 m column (250 mm × 4.6 mm i.d.). During runs, the stationary phase was kept equilibrated at ambient temperature. The mobile phase consisted of acetonitrile and 10 mM ortho-phosphoric acid (16:84, v/v). Following its preparation, the mobile phase was passed through a 0.2 m Nylon-66 filter (Schleicher & Schuell, Germany) and degassed using helium. The mobile phase was delivered to the system at a rate of 1 ml min−1 . Under the established conditions, SMZ eluted at 7.0 min.
2.3. Equipment A modular liquid chromatography (LC) system (Gilson Medical Electronics, Villiers-le-Bel, France) consisting of a Model 305 piston pump, a Model 805 manometer, and a Model TC 831 column oven was used in this study. Injections were made through a Model 7125 Rheodyne valve (Cotati, CA) equipped with 20 l loop. A Model 119, variable wavelength UV–vis detector (Gilson, Middleton, WI) with wavelength set at 271 nm, was used to detect the signal response. The sensitivity of the detector was set at 0.005 a.u.f.s. The detector was linked to a Model BD 111 Kipp & Zonen (Delft, Holland) pen recorder. The chart speed of the recorder was set at 2 mm min−1 . A Model D7402 EasyPure UV compact system (Barnstead/Thermolyne Corp., Dubuque, I) was used for ultrapurification of tap water, whereas a Model G-560E vortex mixer (Scientific Industries Inc., Bohemia, NY), an UltraTurrax T25 (Janle & Kunkel, IKA Labortechnik, Germany) and a Model Centra-MP4 centrifuge (IEC, Needman Heights, MA) were used for sample treatment. 2.4. Chemicals Acetonitrile was of HPLC grade, whereas orthophosphoric acid, hydrochloric acid, sodium acetate supra-
2.7. Sample extraction and cleanup The sample preparation procedure used in this study was based on that previously reported by Papapanagiotou et al. [4] for the determination of sulphadiazine and trimethoprim in gilthead sea bream tissues. The method was fully validated for all piglet tissues examined. Three grams of ground piglet tissue (muscle, fat, liver and kidney) were accurately weighed in a 50 ml centrifuge tube. Thirty milliliters of dichloromethane were added and the sample was homogenized with an Ultra Turrax for 1 min and centrifuged at 3000 × g for 10 min. The supernatant phase was filtered and 10 ml of the filtrate were transferred to a 15 ml glass centrifuge tube. Following addition of 1 ml of 3 M hydrochloric acid, the tube was vortexed for 15 s, and centrifuged for 5 min at 3000 × g. From the upper aqueous layer, 250 l were transferred to a 2 ml glass centrifuge tube and another 250 l of 3.8 M sodium acetate solution were added and vortexed for 15 s. Finally, a 20 l aliquot was injected into the LC system and analysed at ambient temperature.
E.P. Papapanagiotou et al. / Analytica Chimica Acta 529 (2005) 325–329
Fig. 1. Chromatograms of a blank muscle sample (a), a blank muscle sample fortified with SMZ (1) at 150 ng/g (b) and an incurred (1427 ng/g) muscle sample at 20 days post-treatment (c).
2.8. Estimation of the withdrawal period The withdrawal period was estimated by linear regression analysis of the log-transformed tissue concentrations, and determined at the time when the upper one-sided tolerance limit, with a confidence of 95%, was below the MRL of 100 g/kg established by the European Union for sulphonamides in all edible animal products [5].
3. Results and discussion 3.1. Liquid chromatographic method validation The method was validated for use with respect to linearity, specificity, sensitivity, accuracy, precision and stability aspects [6]. The linearity of the detector response for the test compound was evaluated by injecting, seven standard solutions of various concentrations covering the working range of the assay (0.5–5 ng/20 l of SMZ). The correlation coefficient (r) for the analyte was better than 0.999 (requirement: >0.99). The method was also evaluated for its specificity to ensure that there was no interference with the test compound from matrix co-extractives. Chromatograms obtained from blank tissue extracts and blank tissue fortified with SMZ showed that the peak attributable to the test compound was resolved sufficiently from any other peaks to enable reliable quantification in all tissues examined. Typical chromatograms of blank, fortified and incurred piglet muscle tissues are presented in Fig. 1.
327
The limit of quantification (LOQ), defined as the lowest concentration of SMZ in tissues that has a precision (expressed as relative standard deviation, RSD%) below or equal to 20% and a mean recovery within the range of 80–110%, was 50 g/kg for all four tissues examined. The accuracy of the method, in terms of recovery efficiency, was studied by fortifying blank tissue samples with SMZ at four fortification levels and analyzing six replicates per fortification level each day, for three consecutive days. Blank tissue samples were fortified at half the MRL value (50 g/kg), at the MRL value (100 g/kg), at one-and-a-half time the MRL value (150 g/kg) and at two times the MRL value (200 g/kg) for the test compound. The mean overall recoveries determined for SMZ were 78.78%, 78.20%, 82.70% and 80.64%, for muscle, liver, kidney and fat tissues, respectively. The precision of the method for SMZ was evaluated utilizing the data generated during assessment of the method accuracy at three consecutive days. The results showed that the overall value of the relative standard deviation for betweenor inter-day variability of SMZ was 8.91%, 10.29%, 13.14% and 10.45%, for muscle, liver, kidney and fat tissues, respectively. The stability experiments carried out, referred to the stability of SMZ in incurred tissue samples stored at −20 and −75 ◦ C. The results indicated that the test compound was stable during the storage of incurred tissue samples at −20 and −75 ◦ C for at least 3 and 5 months, respectively. 3.2. Sulphamethazine residue depletion The SMZ residues concentrations in the animals slaughtered 5 days after the cessation of the medication ranged between 1.85 and 4.51 mg kg−1 in muscle tissue, 4.59 and 9.92 mg kg−1 in liver tissue, 4.06 and 8.72 mg kg−1 in kidney tissue and 1.53 and 3.97 mg kg−1 in fat tissue. It is worth noting that even 20 days after the end of treatment, the mean SMZ residues concentrations remained at levels higher than 1.12 mg kg−1 . More specifically, the corresponding mean (n = 6) SMZ residues levels were, in descending order, 3.71 mg kg−1 in liver, 2.17 mg kg−1 in kidney, 1.99 mg kg−1 in muscle and 1.12 mg kg−1 in fat (Table 1). SMZ residues concentrations in all tissues examined dropped below the EU set MRL of 0.1 mg kg−1 , 30 days after the end of the medication. This finding is clearly not in agreement with the 18 days period required for the elimination of SMZ from pig tissues reported by Kuiper et al. [3]. This period, however, is not validated for use as a withdrawal period since diseased piglets were used, the number of animals per slaughter day was small and the levels of SMZ on the final sampling day were still above the MRL, which rendered an extrapolation inevitable. Thirty-five days post-treatment, the SMZ concentrations in all muscle and fat samples, four out of six liver samples and two out of six kidney samples, dropped below the LOQ of the method (Table 1).
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Table 1 SMZ elimination over time from piglet tissues after cessation of feed medication with 100 g SMZ/t feed, for 30 consecutive days Days post-medication
5 10 15 20 25 30 35 a b
Meana SMZ concentration (mg kg−1 ) ± S.D. Muscle
Liver
Kidney
Fat
3.84 ± 0.99 3.69 ± 0.71 2.92 ± 0.82 1.99 ± 0.64 0.18 ± 0.02 0.07 (1/6)b
8.14 ± 1.97 7.05 ± 1.48 5.54 ± 1.71 3.71 ± 1.08 0.44 ± 0.11 0.07 ± 0.005 0.05 (2/6)b
6.42 ± 1.67 4.92 ± 1.03 4.43 ± 1.38 2.17 ± 1.11 0.30 ± 0.05 0.06 (2/6)b 0.05 (4/6)b
2.88 ± 0.90 2.32 ± 1.14 1.48 ± 0.16 1.12 ± 0.25 0.15 ± 0.03 0.06 (2/6)b
Six replicates. Number of individual animals with SMZ concentrations above the LOQ.
Fig. 2. Plot of withdrawal period calculation for muscle.
The LC method used in this study incorporated an acidic extraction procedure, which converts the N-4-acetyl metabolites of SMZ to parent SMZ. The same observation was also reported by Rose et al. [7]. However, according to Kuiper et al. [3] for the determination of a safe withdrawal period for the SMZ treatment, the total amount of SMZ and N4-acetyl-SMZ residues in edible organs and tissues should be taken into account, since N-4-acetyl-SMZ may be con-
Fig. 3. Plot of withdrawal period calculation for fat.
Fig. 4. Plot of withdrawal period calculation for liver.
verted to the parent compound after consumption by the consumers. 3.3. Estimation of the withdrawal period Residual levels of SMZ in muscle, fat, liver and kidney samples were estimated (95% tolerance limit and 95% confidence) to fall below the MRL after a withdrawal period of 40.92, 38.18, 40.76 and 40.12 days, respectively (Figs. 2–5).
Fig. 5. Plot of withdrawal period calculation for kidney.
E.P. Papapanagiotou et al. / Analytica Chimica Acta 529 (2005) 325–329
Since these time points do not make up full days, the withdrawal periods have to be rounded up to the next days. However, the longest withdrawal period of 41 days has to be selected as the conclusive withdrawal period to guarantee consumer safety [5].
4. Conclusions The results of the present study clearly indicate that a 30-day treatment of the piglets with SMZ at a dose of 100 g/t feed, results in residue levels that are cleared quite slowly. The concentrations of SMZ residues were below the existing EU MRLs in all examined piglet tissues 30 days after the cessation of the administration of SMZ. However, a 41 days withdrawal period was estimated, by using the statistical method suggested by EMEA [5]. It becomes quite evident that the 11 additional days calculated with the statistical method represent a safety factor aiming at offering the consumer a significantly higher level of protection.
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Therefore, a 41-day withdrawal period is proposed to safeguard the health of the consumer from violative SMZ residues in edible tissues of piglets. Furthermore, this outcome would prove to be very useful in risk analysis methodology implementation in view of the protection of Public Health. References [1] N.A. Botsoglou, D.J. Fletouris (Eds.), Drug Residues in Foods: Pharmacology, Food Safety and Analysis, Marcel Dekker, New York, 2001. [2] EEC Regulation 90/2377/EEC incorporating amending regulation 92/675/EEC, Official Journal of the European Communities, No. L224, Brussels, 1990, p. 1. [3] H.A. Kuiper, R.M.L. Aerts, N. Haagsma, H. van Gogh, J. Agric. Food Chem. 36 (1988) 822–825. [4] E.P. Papapanagiotou, E. Iossifidou, I. Psomas, G. Photis, J. Liq. Chromatogr. Rel. Technol. 23 (2000) 2839. [5] EMEA/CVMP/036/95, London, 1995. [6] EC Decision 2002/657/EC, Official Journal of the European Communities, No. L221, Brussels, 2002, p. 8. [7] M.D. Rose, W.H.H. Farrington, G. Shearer, Food Addit. Contam. 12 (1995) 739.