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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Determination of tetracycline residues in chicken meat by liquid chromatography-tandem mass spectrometry a
a
a
b
c
F. Cetinkaya , A. Yibar , G.E. Soyutemiz , B. Okutan , A. Ozcan & M.Y. Karaca
c
a
Faculty of Veterinary Medicine, Department of Food Hygiene and Technology , University of Uludag , 16059 Gorukle Campus, Bursa , Turkey b
Pendik Veterinary Control and Research Institute , 34890 Pendik , Istanbul , Turkey
c
Central Food Control and Research Institute , 16036 Hurriyet , Bursa , Turkey Accepted author version posted online: 10 Jan 2012.Published online: 01 Mar 2012.
To cite this article: F. Cetinkaya , A. Yibar , G.E. Soyutemiz , B. Okutan , A. Ozcan & M.Y. Karaca (2012) Determination of tetracycline residues in chicken meat by liquid chromatography-tandem mass spectrometry, Food Additives & Contaminants: Part B: Surveillance, 5:1, 45-49, DOI: 10.1080/19393210.2012.655782 To link to this article: http://dx.doi.org/10.1080/19393210.2012.655782
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives and Contaminants: Part B Vol. 5, No. 1, March 2012, 45–49
Determination of tetracycline residues in chicken meat by liquid chromatography-tandem mass spectrometry F. Cetinkayaa*, A. Yibara, G.E. Soyutemiza, B. Okutanb, A. Ozcanc and M.Y. Karacac a Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, University of Uludag, 16059 Gorukle Campus, Bursa, Turkey; bPendik Veterinary Control and Research Institute, 34890 Pendik, Istanbul, Turkey; cCentral Food Control and Research Institute, 16036 Hurriyet, Bursa, Turkey
Downloaded by [McMaster University] at 12:42 27 October 2014
(Received 10 May 2011; final version received 4 January 2012) Analysis of residual levels of tetracyclines (TCs) in chicken meat was performed using a validated liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS) technique. Overall, the recoveries for TCs ranged from 56.9% to 101.2%, with standard deviations of 4.5–13.2%. Detection limits ranged from 7.9 to 14.6 mg kg1. In four of 60 samples, doxycycline (DXC) was determined in a range from 19.9 to 35.6 mg kg1; and in one sample tetracycline was detected at 17.2 mg kg1. Chlortetracycline (CTC) and oxytetracycline (OTC) were not detected in any of the tested samples. This study indicates that chicken meat sold in Bursa, Turkey, contained some residues of TCs. Therefore, stricter regulations for the use of antibiotics in the poultry industry and the monitoring of drug residues in chicken meat prior to marketing are needed. Finally, this method has been applied successfully for the confirmation of TCs in chicken meat. Keywords: meat; veterinary drug residues; veterinary drug residues – antibiotics; veterinary drug residues – tetracycline; veterinary drug residues – chloramphenicol
Introduction Tetracycline antibiotics (TCs), represented by oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DXC), are commonly used worldwide in veterinary therapy, especially in poultry farming and medicine (Nakazawa et al. 1999; Berendsen and van Rhijin 2006; Miranda et al. 2009). However, the abundant and improper use of TCs may result in the presence of their residues in edible animal tissues, which can be toxic and dangerous for human health and potentially cause allergic reactions. In addition, low-level doses of antibiotic in foodstuffs consumed for long periods can lead to the spread of drug-resistant microorganisms (Shalaby et al. 2001; Masawat and Slater 2007; Yu et al. 2011). To ensure human food safety, the European Union laid down maximum residue limits (MRLs) for oxytetracyclin, TC, CTC and DXC, including 100 mg kg1 in muscle (European Commission 1990). Monitoring of antibiotic residues is very important in controlling the safety of foods for human consumption (Koesukwiwat et al. 2007). During recent years, many analytical methods to detect TCs in foods have been described (Shalaby et al. 2001; Miranda et al. 2009). There are two types of analytical methods including screening methods, immunoassay-based
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/19393210.2012.655782 http://www.tandfonline.com
methods and confirmatory methods performed based on gas-chromatography or liquid chromatography (Tajik et al. 2010). Mass spectrometry (MS) is also used as a highly sensitive detection method of TCs (de Wasch et al. 1998; Gentili et al. 2005; MartinsJunior et al. 2007; Granelli et al. 2009; McDonald et al. 2009). MS techniques can confirm the residual TCs with high sensitivity and selectivity; therefore, a method combining a simple and precise chromatographic separation with an appropriate MS determination technique would offer a significant advantage for the absolute confirmation of the residual TCs (Oka et al. 2000; Alfredsson et al. 2005). This study aimed to survey the occurrence and quantification of TC residues in chicken meat samples marketed in Bursa, Turkey. To achieve these goals, we used the LC-MS/MS method validated according to European Commission Decision 2002/657/EC for confirmatory assay (European Commission 2002).
Materials and methods Samples From August 2009 to January 2010, a total of 60 chicken meat samples were collected in Bursa
46
F. Cetinkaya et al.
province, Turkey. Samples were purchased from large supermarkets and smaller units including butcher shops and poulterer shops, and transported to the laboratory immediately after sampling. Chemicals and reagents
Downloaded by [McMaster University] at 12:42 27 October 2014
All chemicals used were of analytical grade from Merck (Darmstadt, Germany). Deionised water used in all procedure steps was prepared by the Milli-Q Ultra Pure System (Millipore, Bedford, MA, USA). TC standards were supplied by Dr. Ehrenstorfer (Augsburg, Germany). Standard solutions Stock standard solutions (1 mg ml1) were prepared in methanol and stored at 4 C. Working standard solutions were made by appropriate dilution of the stock standard solutions with methanol. Sample extraction and clean-up A total of 4 g of chicken thigh and breast was weighed into 50 ml plastic centrifuge tubes. A total of 15 ml methanol were added to the centrifuge tube and capped tightly. The tube was shaken vigorously for 5 min, using a Multi Reax Vortex (Heidolph, Schwabach, Germany). Afterwards 4 ml of 1% formic acid and 400 ml of 0.01 M EDTA were added to the centrifuge tube and the tube was shaken for 3 and 2 min, respectively, by vortex. The tube was then centrifuged at 2000g for 10 min at 15–20 C. After centrifugation, the supernatant was obtained with a injector, filtered through a 0.45 mm membrane filter and 50 ml were injected into the LC-MS/MS system. Preparation of calibration curves Quantification of TCs in chicken meat was carried out using matrix-matched calibration curves. For the preparation of standard matrix samples, 4 g of the three blank tissue samples were weighted into 15 ml centrifuge tubes. To each of these tubes were added 50, 100 and 150 ml of the standard solution; then prepared blank samples were subjected to the extraction procedure as described above. Calibration curves were drawn by plotting concentration against peak area, obtained from chromatograms of the matrix standard samples. Instrumentation LC-MS analysis was carried out on a Tandem Gold Triple Quadrapole mass spectrometer (Zivak, Kocaeli, Turkey). The mass spectrometer was operated in the positive electrospray ionisation (ESI) mode. The
Table 1. MS/MS conditions. Ionisation mode API nebulising gas pressure Drying gas temperature Drying gas pressure Scan time SIM width Needle Shield Capillary Detector CID gas pressure Spray chamber temperature
ESIþ 50 psi 300 C 35 psi 0.3 s 1.5 amu 5000 V 600 V 60 V 1600 V 2.25 mTorr 60 C
MS/MS conditions are presented in Table 1 and the specific monitoring parameters for the TCs in Table 2. Chromatography was performed on a 75 4.6 mm LC column packed with 5 mm diameter C18. Mobile phase A consisted of water with 1% formic acid and mobile phase B was methanol containing 0.1% formic acid. A gradient elution procedure was used at a flow rate of 0.5 ml min1. A linear gradient from 100% A at 0 min to 15% A and 85% B at 4 min was run. At 10 min, A was increased to 100%. The column temperature was maintained at 32 C and the injection volume was 50 ml.
Results and discussion Recently there has been an increasing international and local awareness of the danger of consuming food products with drug residues. Many of them are now classified as carcinogenic, toxic and allergenic (Mahgoub et al. 2006). Growing concern among consumers and public health authorities regarding the presence of antibiotic residues in animal origin foods demands better control in the use of antimicrobials in animal production (Popelka et al. 2005). The presence of antibiotic residues, which are used on a large scale in poultry farming, in chicken muscle and in liver samples has been demonstrated by some previously published data (de Wasch et al. 1998; Tajick and Shohreh 2006; Miranda et al. 2009; Tajik et al. 2010). The goal of this work was to identify and quantify TC residues in raw chicken meat using the LC-MS/MS method. In the present study, 60 chicken samples were subjected to LC-MS/MS for confirmatory analysis of the TC compounds. LC-MS/MS chromatograms of chicken meat samples positive for TC (17.2 mg kg1) and DXC (19.9 mg kg1) are shown in Figures 1a and 1b, respectively. The limits of detection (LOD ¼ 3.3*SD/m) and quantification (LOQ ¼ 10*SD/m) were determined by analysing the chicken meat samples spiked with standard solutions of the TCs. Table 3 shows the results of the linearity assay for TC, DXC, CTC and OTC in the samples. The R2 values for the system results were
Food Additives and Contaminants: Part B
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Table 2. Parameters for MS/MS monitoring of TCs. Compound
MW
MS MHþ (m/z)
MS/MS (m/z)
Collision energy (eV)
Tetracycline (TC) Doxycycline (DXC) Oxytetracycline (OTC) Chlortetracycline (CTC)
444.4 444.4 460.4 478.9
445.0 445.0 461.0 479.0
410.0 428.0 426.0 444.0
15 15 15 20
Figure 1. Typical chromatograms of chicken meat samples positive for tetracycline (a) and doxycycline (b).
Table 3. Data summary showing the LC-MS/MS method results.
Antibiotics TC DXC OTC CTC
LOD (mg kg1)
LOQ (mg kg1)
Measurement uncertainty
R2
8.3 13.2 7.9 14.6
25.3 39.8 24.2 44.3
3.3 5.7 2.2 2.0
0.9990 0.9971 0.9996 0.9996
all 40.99 for the linear regression equations in the concentration ranges (50–200 mg kg1) tested. The repeatability of the method and recovery were calculated using chicken meat samples spiked with three different concentrations of 50, 100 and 150 mg kg1 of each of the TCs. The results are presented in Table 4. After LC-MS/MS analysis (Table 5), DXC was found in four of the chicken samples, while TC was found in one sample. The levels of DXC (LOD ¼ 13.2 mg kg1) ranged from 19.9 to 35.6 mg kg1 and TC level (LOD ¼ 8.3 mg kg1) was determined as
17.2 mg kg1 in the samples. The levels of residues were less than the international levels set by the European Union and limits allowed in Turkey. According to Turkish legislation on veterinary drug residues, MRLs for the sum of parent drug and its 4-epimers should be 5100 mg kg1 in muscle for all food-producing species (Turkish Food Codex 2002). The remaining 55 chicken samples were negative with LC-MS/MS detection. The abundant and unsuitable use of TCs and improper observance of withdrawal times in poultry production may result in the presence of their residues in the samples. Although the residual TC levels were below the limit, their hazards for human health still exist. In the preliminary study realised by ELISA using the same samples (unpublished data), it has been found that from 60 chicken meat samples, 11 (18.3%) contained TC residues from 6.1 to 26.6 mg kg1, with a mean of 10.5 mg kg1. Thereafter, to evaluate and confirm these results further, analysis by LC-MS/MS was conducted. In comparison with the preliminary results, DXC was found in one of the 11 ELISApositive and in three of the 49 ELISA-negative chicken
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F. Cetinkaya et al. Table 4. Recovery, repeatability and standard deviation for the confirmation of tetracyclines in chicken meat using LC-MS/MS. Added
Antibiotics
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1
Mean (mg kg1)
SD
Repeatability (%)
Recovery (%)
50 mg kg
TC DXC OTC CTC
39.0 28.5 38.8 30.2
5.8 4.9 5.4 4.5
14.7 17.1 13.8 14.9
78.1 56.9 77.6 60.3
100 mg kg1
TC DXC OTC CTC
88.8 77.9 87.2 70.2
8.1 7.7 7.1 6.2
9.1 9.9 8.1 8.9
88.8 77.9 87.2 70.2
150 mg kg1
TC DXC OTC CTC
151.8 134.5 145.9 120.2
13.2 9.2 11.1 9.9
8.7 6.8 7.6 8.2
101.2 89.7 97.3 80.1
Notes: Negative samples were spiked with 50, 100 and 150 mg kg1 of each tetracycline. Six replicates were measured at each level on 3 different days.
Table 5. Levels of antibiotic residues detected by the LC-MS/MS technique. LC-MS/MS results Number of samples analysed 11a 49b
TC
DXC
OTC
CTC
58.3 1 (17.2)
1 (35.6)c 3 (19.9–24.7)
57.9 57.9
514.6 514.6
Notes: aNumber of samples previously found positive by ELISA. b Number of samples previously found negative by ELISA. c Number of positive samples (antibiotic levels, mg kg1). TC, tetracycline; DXC, doxycycline; OTC, oxytetracycline; CTC, chlortetracycline.
samples; while TC was found in one of the ELISAnegative samples. False-positive and -negative results associated with the presence of TCs in chicken meat were obtained by the ELISA test. Similar false-positive and -negative results in ELISA were also reported previously (de Wasch et al. 1998; Karbancioglu and Heperkan 2009). False-positive results might be related to the lower detection limit of ELISA compared with LC-MS/MS. Moreover, by using the ELISA technique one may detect the total TC level in chicken meat, whereas by the LC-MS/MS technique each of TCs may be determined. Another cause raised by these arguments can be related to the possible cross-reactivity between several compounds from the TC family. The percentage crossreactivities (CR) given by the manufacturer for the ELISA kit (Ridascreen, R-Biopharm, Germany) between TC and other relevant compounds such as rolitetracycline (RTC), CTC, demeclocycline, OTC and DXC residues are 100%, 42%, 27%, 13% and 8%, respectively. A previous report (Chafer-Pericas
et al. 2010) suggested high cross-reactivity (10%) of OTCs towards TC (1028%), CTC (28%) and RTC (446%) in fish samples as revealed by ELISA. Furthermore, Ferguson et al. (2005) reported similarly high cross-reactivity for chloramphenicol (CAP) and chloramphenicol–glucuronide in milk, honey and poultry meat when they used Qflex Kit Chloramphenicol.
Conclusions The results for the chicken meat samples obtained from supermarkets and smaller markets suggest that chicken meat marketed in Bursa contain some residues of TCs. Even though these levels are lower than those established by the European Union and Turkey, their presence may still be regarded as a health hazard for consumers since they may cause allergic reactions or contribute to the generation of antibiotic-resistant strains of bacteria which became a worldwide problem in the treatment of human infectious diseases. For this reason, national authorities have to set up strict monitoring plans for controlling veterinary drug residues in animals and their products. Finally, the present method was successfully applied to confirm and quantify TCs in chicken meat samples. The results described also indicate that ELISA can be used as a screening assay for detecting the presence of TCs in chicken meat; however, samples need to be confirmed by chromatographic methods such as LC-MS/MS to eliminate false-positive or -negative results.
Acknowledgements This work was financially supported by Uludag University (Grant Number V(U)-2009/20).
Food Additives and Contaminants: Part B
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References Alfredsson G, Branzell C, Granelli K, Lundstro¨m A. 2005. Simple and rapid screening and confirmation of tetracyclines in honey and egg by a dipstick test and LC-MS/MS. Analyt Chim Acta 529:47–51. Berendsen BJA, van Rhijin JA. 2006. Residue analysis of tetracyclines in poultry muscle: shortcomings revealed by a proficiency test. Food Addit Contam. 23:1141–1148. Chafer-Pericas C, Maquieira A, Puchades R, Miralles J, Moreno A, Pastor-Navarro N, Espinos F. 2010. Immunochemical determination of oxytetracycline in fish: comparison between enzymatic and time-resolved fluorometric assays. Anal Chim Acta 662:177–185. De Wasch K, Okerman L, Croubels S, de Brabander H, Hoof JV, de Backer P. 1998. Detection of residues of tetracycline antibiotics in pork and chicken meat: correlation between results of screening and confirmatory tests. Analyst 123:2737–2741. European Commission. 1990. Council Regulation (EEC) No. 2377/90. Laying down a community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off J Eur Commun. L224:1–8. European Commission. 2002. Commission Decision 2002/ 657/EC of 14 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J L 221:8–36. Ferguson J, Baxter A, Young P, Kennedy G, Elliott C, Weigel S, Gatermann R, Ashwin H, Steadd S, Sharmand M. 2005. Detection of chloramphenicol and chloramphenicol glucuronide residues in poultry muscle, honey, prawn and milk using a surface plasmon resonance biosensor and Oflex kit chloramphenicol. Anal Chim Acta 529:109–113. Gentili A, Perret D, Marchese S. 2005. Liquid chromatography-tandem mass spectrometry for performing confirmatory analysis of veterinary drugs in animal-food products. Trends Anal Chem. 24:704–733. Granelli K, Elgerud C, Lundstro¨m A, Ohlsson A, Sjo¨berg P. 2009. Rapid multi-residue analysis of antibiotics in muscle by liquid chromatography-tandem mass spectrometry. Analyt Chim Acta 637:87–91. Karbancioglu F, Heperkan GD. 2009. Comparison of enzyme linked immunoassay and high performance liquid chromatography for determination of fumonisin in dried figs. Proc Natl Sci Matica Srpska Novi Sad. 117:37–43. Koesukwiwat U, Jayanta S, Leepipatpiboon N. 2007. Validation of a liquid chromatography-mass spectrometry multi-residue method for the simultaneous determination of sulfonamides, tetracyclines, and pyrimethamine in milk. J Chromatogr A 1140:147–156. Mahgoub O, Kadim IT, Mothershaw A, Al Zasjali SA, Annamalai K. 2006. Use of enzyme linked immunosorbent
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assay (ELISA) for detection of antibiotic and anabolic residues in goat and sheep meat. World J Agric Sci. 2:298–302. Martins-Junior HA, Kussumi TA, Wang AY, Lebre DT. 2007. A rapid method to determine antibiotic residues in milk using liquid chromatography coupled to electrospray tandem mass spectrometry. J Braz Chem Soc. 18:397–405. Masawat P, Slater JM. 2007. The determination of tetracycline residues in food using a disposable screen-printed gold electrode (SPGE). Sensor Actuat B 124:127–132. McDonald M, Mannion C, Rafter P. 2009. A confirmatory method for the simultaneous extraction, separation, identification and quantification of tetracycline, sulphonamide, trimethoprim and dapsone residues in muscle by ultrahigh-performance liquid chromatography-tandem mass spectrometry according to Commission Decision 2002/ 657/EC. J Chromatogr A 1216:8110–8116. Miranda JM, Rodrı´ guez JA, Gala´n-Vidal CA. 2009. Simultaneous determination of tetracyclines in poultry muscle by capillary zone electrophoresis. J Chromatogr A 1216:3366–3371. Nakazawa H, Ino S, Kato K, Watanabe T, Ho Y, Oka H. 1999. Simultaneous determination of residual tetracyclines in foods by high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry. J Chromatogr B 732:55–64. Oka H, Ito Y, Matsumoto H. 2000. Chromatographic analysis in tetracycline antibiotics in foods. J Chromatogr A 882:109–133. Popelka P, Nagy J, Germuska R, Marcincak S, Jevinova P, de Rijk A. 2005. Comparison of various assays used for detection of beta-lactam antibiotics in poultry meat. Food Addit Contam. 22:557–562. Shalaby AR, Salama NA, Abou-Raya SH, Emam WH, Mehaya FM. 2001. Validation of HPLC method for determination of tetracycline residues in chicken meat and liver. Food Chem. 124:1660–1666. Tajick MA, Shohreh B. 2006. Detection of antibiotics residue in chicken meat using TLC. Int J Poultry Sci. 5:611–612. Tajik H, Malekinejad H, Razavi-Rouhani SM, Pajouhi MR, Mahmoudi R, Haghnazari A. 2010. Chloramphenicol residues in chicken liver, kidney and muscle: a comparison among the antibacterial residues monitoring methods of four plate test, ELISA and HPLC. Food Chem Toxicol. 48:2454–2468. Turkish Food Codex. 2002. Communique No. 2002/30 on maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Official Gazette no. 24739 (28 April). Yu H, Tao Y, Chen D, Wang Y, Yuan Z. 2011. Development of an HPLC-UV method for the simultaneous determination of tetracyclines in muscle and liver of porcine, chicken and bovine with accelerated solvent extraction. Food Chem. 124:1131–1138.
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Determination of tetracycline residues in chicken meat by liquid chromatography-tandem mass spectrometry a
a
a
b
c
F. Cetinkaya , A. Yibar , G.E. Soyutemiz , B. Okutan , A. Ozcan & M.Y. Karaca
c
a
Faculty of Veterinary Medicine, Department of Food Hygiene and Technology , University of Uludag , 16059 Gorukle Campus, Bursa , Turkey b
Pendik Veterinary Control and Research Institute , 34890 Pendik , Istanbul , Turkey
c
Central Food Control and Research Institute , 16036 Hurriyet , Bursa , Turkey Accepted author version posted online: 10 Jan 2012.Published online: 01 Mar 2012.
To cite this article: F. Cetinkaya , A. Yibar , G.E. Soyutemiz , B. Okutan , A. Ozcan & M.Y. Karaca (2012) Determination of tetracycline residues in chicken meat by liquid chromatography-tandem mass spectrometry, Food Additives & Contaminants: Part B: Surveillance, 5:1, 45-49, DOI: 10.1080/19393210.2012.655782 To link to this article: http://dx.doi.org/10.1080/19393210.2012.655782
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives and Contaminants: Part B Vol. 5, No. 1, March 2012, 45–49
Determination of tetracycline residues in chicken meat by liquid chromatography-tandem mass spectrometry F. Cetinkayaa*, A. Yibara, G.E. Soyutemiza, B. Okutanb, A. Ozcanc and M.Y. Karacac a Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, University of Uludag, 16059 Gorukle Campus, Bursa, Turkey; bPendik Veterinary Control and Research Institute, 34890 Pendik, Istanbul, Turkey; cCentral Food Control and Research Institute, 16036 Hurriyet, Bursa, Turkey
Downloaded by [McMaster University] at 12:42 27 October 2014
(Received 10 May 2011; final version received 4 January 2012) Analysis of residual levels of tetracyclines (TCs) in chicken meat was performed using a validated liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS) technique. Overall, the recoveries for TCs ranged from 56.9% to 101.2%, with standard deviations of 4.5–13.2%. Detection limits ranged from 7.9 to 14.6 mg kg1. In four of 60 samples, doxycycline (DXC) was determined in a range from 19.9 to 35.6 mg kg1; and in one sample tetracycline was detected at 17.2 mg kg1. Chlortetracycline (CTC) and oxytetracycline (OTC) were not detected in any of the tested samples. This study indicates that chicken meat sold in Bursa, Turkey, contained some residues of TCs. Therefore, stricter regulations for the use of antibiotics in the poultry industry and the monitoring of drug residues in chicken meat prior to marketing are needed. Finally, this method has been applied successfully for the confirmation of TCs in chicken meat. Keywords: meat; veterinary drug residues; veterinary drug residues – antibiotics; veterinary drug residues – tetracycline; veterinary drug residues – chloramphenicol
Introduction Tetracycline antibiotics (TCs), represented by oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DXC), are commonly used worldwide in veterinary therapy, especially in poultry farming and medicine (Nakazawa et al. 1999; Berendsen and van Rhijin 2006; Miranda et al. 2009). However, the abundant and improper use of TCs may result in the presence of their residues in edible animal tissues, which can be toxic and dangerous for human health and potentially cause allergic reactions. In addition, low-level doses of antibiotic in foodstuffs consumed for long periods can lead to the spread of drug-resistant microorganisms (Shalaby et al. 2001; Masawat and Slater 2007; Yu et al. 2011). To ensure human food safety, the European Union laid down maximum residue limits (MRLs) for oxytetracyclin, TC, CTC and DXC, including 100 mg kg1 in muscle (European Commission 1990). Monitoring of antibiotic residues is very important in controlling the safety of foods for human consumption (Koesukwiwat et al. 2007). During recent years, many analytical methods to detect TCs in foods have been described (Shalaby et al. 2001; Miranda et al. 2009). There are two types of analytical methods including screening methods, immunoassay-based
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/19393210.2012.655782 http://www.tandfonline.com
methods and confirmatory methods performed based on gas-chromatography or liquid chromatography (Tajik et al. 2010). Mass spectrometry (MS) is also used as a highly sensitive detection method of TCs (de Wasch et al. 1998; Gentili et al. 2005; MartinsJunior et al. 2007; Granelli et al. 2009; McDonald et al. 2009). MS techniques can confirm the residual TCs with high sensitivity and selectivity; therefore, a method combining a simple and precise chromatographic separation with an appropriate MS determination technique would offer a significant advantage for the absolute confirmation of the residual TCs (Oka et al. 2000; Alfredsson et al. 2005). This study aimed to survey the occurrence and quantification of TC residues in chicken meat samples marketed in Bursa, Turkey. To achieve these goals, we used the LC-MS/MS method validated according to European Commission Decision 2002/657/EC for confirmatory assay (European Commission 2002).
Materials and methods Samples From August 2009 to January 2010, a total of 60 chicken meat samples were collected in Bursa
46
F. Cetinkaya et al.
province, Turkey. Samples were purchased from large supermarkets and smaller units including butcher shops and poulterer shops, and transported to the laboratory immediately after sampling. Chemicals and reagents
Downloaded by [McMaster University] at 12:42 27 October 2014
All chemicals used were of analytical grade from Merck (Darmstadt, Germany). Deionised water used in all procedure steps was prepared by the Milli-Q Ultra Pure System (Millipore, Bedford, MA, USA). TC standards were supplied by Dr. Ehrenstorfer (Augsburg, Germany). Standard solutions Stock standard solutions (1 mg ml1) were prepared in methanol and stored at 4 C. Working standard solutions were made by appropriate dilution of the stock standard solutions with methanol. Sample extraction and clean-up A total of 4 g of chicken thigh and breast was weighed into 50 ml plastic centrifuge tubes. A total of 15 ml methanol were added to the centrifuge tube and capped tightly. The tube was shaken vigorously for 5 min, using a Multi Reax Vortex (Heidolph, Schwabach, Germany). Afterwards 4 ml of 1% formic acid and 400 ml of 0.01 M EDTA were added to the centrifuge tube and the tube was shaken for 3 and 2 min, respectively, by vortex. The tube was then centrifuged at 2000g for 10 min at 15–20 C. After centrifugation, the supernatant was obtained with a injector, filtered through a 0.45 mm membrane filter and 50 ml were injected into the LC-MS/MS system. Preparation of calibration curves Quantification of TCs in chicken meat was carried out using matrix-matched calibration curves. For the preparation of standard matrix samples, 4 g of the three blank tissue samples were weighted into 15 ml centrifuge tubes. To each of these tubes were added 50, 100 and 150 ml of the standard solution; then prepared blank samples were subjected to the extraction procedure as described above. Calibration curves were drawn by plotting concentration against peak area, obtained from chromatograms of the matrix standard samples. Instrumentation LC-MS analysis was carried out on a Tandem Gold Triple Quadrapole mass spectrometer (Zivak, Kocaeli, Turkey). The mass spectrometer was operated in the positive electrospray ionisation (ESI) mode. The
Table 1. MS/MS conditions. Ionisation mode API nebulising gas pressure Drying gas temperature Drying gas pressure Scan time SIM width Needle Shield Capillary Detector CID gas pressure Spray chamber temperature
ESIþ 50 psi 300 C 35 psi 0.3 s 1.5 amu 5000 V 600 V 60 V 1600 V 2.25 mTorr 60 C
MS/MS conditions are presented in Table 1 and the specific monitoring parameters for the TCs in Table 2. Chromatography was performed on a 75 4.6 mm LC column packed with 5 mm diameter C18. Mobile phase A consisted of water with 1% formic acid and mobile phase B was methanol containing 0.1% formic acid. A gradient elution procedure was used at a flow rate of 0.5 ml min1. A linear gradient from 100% A at 0 min to 15% A and 85% B at 4 min was run. At 10 min, A was increased to 100%. The column temperature was maintained at 32 C and the injection volume was 50 ml.
Results and discussion Recently there has been an increasing international and local awareness of the danger of consuming food products with drug residues. Many of them are now classified as carcinogenic, toxic and allergenic (Mahgoub et al. 2006). Growing concern among consumers and public health authorities regarding the presence of antibiotic residues in animal origin foods demands better control in the use of antimicrobials in animal production (Popelka et al. 2005). The presence of antibiotic residues, which are used on a large scale in poultry farming, in chicken muscle and in liver samples has been demonstrated by some previously published data (de Wasch et al. 1998; Tajick and Shohreh 2006; Miranda et al. 2009; Tajik et al. 2010). The goal of this work was to identify and quantify TC residues in raw chicken meat using the LC-MS/MS method. In the present study, 60 chicken samples were subjected to LC-MS/MS for confirmatory analysis of the TC compounds. LC-MS/MS chromatograms of chicken meat samples positive for TC (17.2 mg kg1) and DXC (19.9 mg kg1) are shown in Figures 1a and 1b, respectively. The limits of detection (LOD ¼ 3.3*SD/m) and quantification (LOQ ¼ 10*SD/m) were determined by analysing the chicken meat samples spiked with standard solutions of the TCs. Table 3 shows the results of the linearity assay for TC, DXC, CTC and OTC in the samples. The R2 values for the system results were
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Table 2. Parameters for MS/MS monitoring of TCs. Compound
MW
MS MHþ (m/z)
MS/MS (m/z)
Collision energy (eV)
Tetracycline (TC) Doxycycline (DXC) Oxytetracycline (OTC) Chlortetracycline (CTC)
444.4 444.4 460.4 478.9
445.0 445.0 461.0 479.0
410.0 428.0 426.0 444.0
15 15 15 20
Figure 1. Typical chromatograms of chicken meat samples positive for tetracycline (a) and doxycycline (b).
Table 3. Data summary showing the LC-MS/MS method results.
Antibiotics TC DXC OTC CTC
LOD (mg kg1)
LOQ (mg kg1)
Measurement uncertainty
R2
8.3 13.2 7.9 14.6
25.3 39.8 24.2 44.3
3.3 5.7 2.2 2.0
0.9990 0.9971 0.9996 0.9996
all 40.99 for the linear regression equations in the concentration ranges (50–200 mg kg1) tested. The repeatability of the method and recovery were calculated using chicken meat samples spiked with three different concentrations of 50, 100 and 150 mg kg1 of each of the TCs. The results are presented in Table 4. After LC-MS/MS analysis (Table 5), DXC was found in four of the chicken samples, while TC was found in one sample. The levels of DXC (LOD ¼ 13.2 mg kg1) ranged from 19.9 to 35.6 mg kg1 and TC level (LOD ¼ 8.3 mg kg1) was determined as
17.2 mg kg1 in the samples. The levels of residues were less than the international levels set by the European Union and limits allowed in Turkey. According to Turkish legislation on veterinary drug residues, MRLs for the sum of parent drug and its 4-epimers should be 5100 mg kg1 in muscle for all food-producing species (Turkish Food Codex 2002). The remaining 55 chicken samples were negative with LC-MS/MS detection. The abundant and unsuitable use of TCs and improper observance of withdrawal times in poultry production may result in the presence of their residues in the samples. Although the residual TC levels were below the limit, their hazards for human health still exist. In the preliminary study realised by ELISA using the same samples (unpublished data), it has been found that from 60 chicken meat samples, 11 (18.3%) contained TC residues from 6.1 to 26.6 mg kg1, with a mean of 10.5 mg kg1. Thereafter, to evaluate and confirm these results further, analysis by LC-MS/MS was conducted. In comparison with the preliminary results, DXC was found in one of the 11 ELISApositive and in three of the 49 ELISA-negative chicken
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F. Cetinkaya et al. Table 4. Recovery, repeatability and standard deviation for the confirmation of tetracyclines in chicken meat using LC-MS/MS. Added
Antibiotics
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1
Mean (mg kg1)
SD
Repeatability (%)
Recovery (%)
50 mg kg
TC DXC OTC CTC
39.0 28.5 38.8 30.2
5.8 4.9 5.4 4.5
14.7 17.1 13.8 14.9
78.1 56.9 77.6 60.3
100 mg kg1
TC DXC OTC CTC
88.8 77.9 87.2 70.2
8.1 7.7 7.1 6.2
9.1 9.9 8.1 8.9
88.8 77.9 87.2 70.2
150 mg kg1
TC DXC OTC CTC
151.8 134.5 145.9 120.2
13.2 9.2 11.1 9.9
8.7 6.8 7.6 8.2
101.2 89.7 97.3 80.1
Notes: Negative samples were spiked with 50, 100 and 150 mg kg1 of each tetracycline. Six replicates were measured at each level on 3 different days.
Table 5. Levels of antibiotic residues detected by the LC-MS/MS technique. LC-MS/MS results Number of samples analysed 11a 49b
TC
DXC
OTC
CTC
58.3 1 (17.2)
1 (35.6)c 3 (19.9–24.7)
57.9 57.9
514.6 514.6
Notes: aNumber of samples previously found positive by ELISA. b Number of samples previously found negative by ELISA. c Number of positive samples (antibiotic levels, mg kg1). TC, tetracycline; DXC, doxycycline; OTC, oxytetracycline; CTC, chlortetracycline.
samples; while TC was found in one of the ELISAnegative samples. False-positive and -negative results associated with the presence of TCs in chicken meat were obtained by the ELISA test. Similar false-positive and -negative results in ELISA were also reported previously (de Wasch et al. 1998; Karbancioglu and Heperkan 2009). False-positive results might be related to the lower detection limit of ELISA compared with LC-MS/MS. Moreover, by using the ELISA technique one may detect the total TC level in chicken meat, whereas by the LC-MS/MS technique each of TCs may be determined. Another cause raised by these arguments can be related to the possible cross-reactivity between several compounds from the TC family. The percentage crossreactivities (CR) given by the manufacturer for the ELISA kit (Ridascreen, R-Biopharm, Germany) between TC and other relevant compounds such as rolitetracycline (RTC), CTC, demeclocycline, OTC and DXC residues are 100%, 42%, 27%, 13% and 8%, respectively. A previous report (Chafer-Pericas
et al. 2010) suggested high cross-reactivity (10%) of OTCs towards TC (1028%), CTC (28%) and RTC (446%) in fish samples as revealed by ELISA. Furthermore, Ferguson et al. (2005) reported similarly high cross-reactivity for chloramphenicol (CAP) and chloramphenicol–glucuronide in milk, honey and poultry meat when they used Qflex Kit Chloramphenicol.
Conclusions The results for the chicken meat samples obtained from supermarkets and smaller markets suggest that chicken meat marketed in Bursa contain some residues of TCs. Even though these levels are lower than those established by the European Union and Turkey, their presence may still be regarded as a health hazard for consumers since they may cause allergic reactions or contribute to the generation of antibiotic-resistant strains of bacteria which became a worldwide problem in the treatment of human infectious diseases. For this reason, national authorities have to set up strict monitoring plans for controlling veterinary drug residues in animals and their products. Finally, the present method was successfully applied to confirm and quantify TCs in chicken meat samples. The results described also indicate that ELISA can be used as a screening assay for detecting the presence of TCs in chicken meat; however, samples need to be confirmed by chromatographic methods such as LC-MS/MS to eliminate false-positive or -negative results.
Acknowledgements This work was financially supported by Uludag University (Grant Number V(U)-2009/20).
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