Iranian Journal of Pharmaceutical Research (2012), 11 (3): 781-787 Copyright © 2012 by School of Pharmacy Received: May 2011 Shaheed Beheshti University of Medical Sciences and Health Services Accepted: January 2011 Original Article Solid-Phase Extraction and Simultaneous Determination of Tetracycline Residues in Edible Cattle Tissues Using an HPLC-FL Method Mehran Mesgari Abbasia, Mahboob Nematia,b*, Hossein Babaeia,b, Masoud Ansarina and Ashraf-O-Sadat Nourdadgarc aDrug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. bFaculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. cHealth Center, Tabriz University of Medical Sciences, Tabriz, Iran. Abstract In this assay, edible cattle tissues from local markets of Ardabil, a Province of Iran, were examined for residues of tetracycline antibiotics (tetracycline, oxytetracycline and chlortetracycline). In total, 110 samples of triceps, gluteal muscle, diaphragm, kidney and liver were randomly obtained from the local markets of the city of Ardabil. Solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) methods were used to extract and analyze tetracycline antibiotic (TC) residues, respectively. The mean amount of total TC residues in all tested samples was 226.3 ± 112.5 ng/g and the mean amount of the total TC residues in triceps, gluteal muscle, diaphragm, kidney and liver samples were 176.3 ± 46.8, 405.3 ± 219.6, 96.8 ± 26.9, 672.4 ± 192.0 and 651.3 ± 210.1 ng/g, respectively. Additionally, 25.8% of muscle samples, 31.8% of liver samples and 22.7% of kidney samples contained amounts of TC residues beyond the maximum residue limit (MRLs). To reduce the TC residues found in edible cattle tissues, regulatory authorities should ensure that the cattle would undergo the proper withdrawal period from TCs before the slaughtering. Keywords: Tetracycline; Meat; Liver; Kidney; HPLC. Introduction to the enterohepatic circulation, tetracycline antibiotic (TC) residues may persist in the body Tetracyclines are antibiotics with broad long after the administration. The levels of antibacterial spectrums and bacteriostatic activity TC residues in animal products depend on the against both Gram-positive and Gram-negative initial dosage and the duration between the drug bacteria as well as intracellular Mycoplasma, administration and animal product collection. Rickettsia and Chlamydia (1-3). These antibiotics This timeframe is called the withdrawal or are widely used in animal husbandry for both washout period (1). Antibiotic residues, such the prevention and treatment of diseases as well as residues of other drugs, can remain in an as to promote the growth. In food-producing animal’s body even after the slaughtering if animals, tetracyclines may be administrated the antibiotic withdrawal period is insufficient orally in food or drinking water, parenterally, (2). Antibiotic residues in foods can influence or through the intramammary infusion. Due the bacterial composition and the metabolic activity of the intestinal microflora of the * Corresponding author: consumer as well as the consumer’s metabolism E-mail: [email protected] of endogenous compounds (4). TC residues in Mesgari Abbasi M et al. / IJPR (2012), 11 (3): 781-787 meat may stain the teeth of young children (4). above the acceptable limits (16). Additionally, The presence of antibiotic residues in meat, 21.7% of all samples and 5% of kidney and liver milk and other food products may cause allergic samples from slaughterhouses in Tabriz, (Tabriz, reactions in sensitive individuals and drive the Iran) contained TC residues above the MRLs development of resistant strains of bacteria set by the WHO (17). In addition, Ehsani and due to the ingestion of subtherapeutic doses colleagues were reported the results of a study of antibiotics (5-9). To ensure the human food on broiler meats in Ahvaz, Iran. They showed safety, the World Health Organization (WHO) that 60% of the samples had contamination and and the Food and Agriculture Organization tetracycline residue was significantly higher than (FAO) have set standards for acceptable daily European legal concentration (100 ug/Kg) in intake (ADI) and maximum residue limits 10% of samples (18). (MRLs) in foods. Additionally, the European The aim of this study was to investigate the Union (EU), the United States of America presence of TC residues in various marketed (USA), Canada and some other countries have edible cattle tissues in the markets of Ardabil. set their own MRLs (2). The acceptable MRLs for TC residues (individually or in combination), Experimental as recommended by the Joint FAO/WHO Expert Committee on Food Additives, are 200, 600 Chemicals and reagents and 1200 ng/g for muscles, liver and kidney, Analytical standards of TCs, chemicals, and respectively (10). an Oasis HLB cartridge (WAT106202) were Some methods, including microbiological purchased from Sigma-Aldrich (St. Louis, and chromatographic methods, have been USA), Merck (Darmstadt, Germany), and adapted for monitoring the TC residues in edible Waters (Milford, USA), respectively. HPLC- tissues and other food samples. Microbiological grade methanol and acetonitrile were purchased assays are most commonly used to analyze from Merck (Darmstadt, Germany) and double- antibiotic residues are the. However, these distilled and deionized water were prepared using assays are time-consuming, nonspecific and a Millipore water purification unit (Billerica, occasionally produce false positives (11). In USA). contrast, chromatographic techniques, such as thin layer chromatography (TLC), capillary Sampling procedure electrophoresis (CE) and high-performance Sampling was carried out using a stratified liquid chromatography (HPLC), have been random sampling method and 110 edible cattle developed for the quantitative, accurate and tissue samples were obtained from indoor reliable measurements of TC residues in animal markets in different areas of Ardabil. Ultimately, tissues (11). 22 samples of different tissues, including triceps, Oxytetracycline residue was found to be diaphragm, gluteal muscle, kidney and liver present in levels higher than those tolerated by were obtained. At least, 50 g of each sample the EU and FDA in seven of ten cured meat was placed in a sterile polypropylene bag and samples from Turkey analyzed through an HPLC kept in a -70°C deep freezer (Snijders Scientific, method (12). In 2001, 45.6% of meat samples Holland) until analysis. from Nairobi slaughterhouses had detectable TC residues and 20% of them had residue Sample preparation levels above the WHO standard (13). Dipleolu Samples were prepared using the method reported 15.6% positive TC residues of goat reported previously by McDonald and Bouvier meat samples from two states in Nigeria (14). In (19). In brief, the samples were cut into fine a study carried out in 2006 in Hanoi, 5.5% of pieces and 5 g of each was homogenized and all meat samples were positive for TC residues diluted with 20 mL of McIlvaine buffer (mixed and the MRLs were exceeded in 0.69% of the citrate/phosphate with pH of 4.1 and EDTA). The samples (15). Studies in Kuwait showed that mixtures were put in a high-intensity sonicator none of the tested meat samples had TC residues for 12 min followed by shaking for 10 min. 782 Determination of Tetracycline Residues 10 TET U A 5 m OXY CHL 0 0 5 10 15 Time (min) Figure 1. An HPLC chromatogram of a mixed standard solution of TCs (3333 ng/g) The homogenized samples were centrifuged at Mixed standard solutions were prepared for 10,000 g for 10 min at 4°C and then re-diluted simultaneous calibration and calculation of TC with McIlvaine buffer. The mixtures were then residues. The chromatogram of a mixed standard centrifuged again. Next, the supernatants were solution of TCs at 3,333 ng/g concentration is filtered through a 0.45 µ filter (Nalgene, USA). shown in Figure 1. A solid-phase extraction method was used to extract TC residues as follows: HPLC system and procedure An Oasis HLB cartridge (WAT106202) HPLC was carried out on a KNAUER system was conditioned with 3 mL of methanol and (KNAUER, Berlin, Germany). The system was then rinsed with 2 mL of deionized water. The equipped with a quaternary pump (K-1000), filtered supernatant was loaded in the Oasis a BIOTECH model 2003 degasser, a Spark HLB cartridge at a flow rate of 5 mL/min. The Triathlon autosampler and a RF-551 fluorescence cartridge was washed with 2 mL of 5% methanol detector. Data processing was performed using in deionized water. The elution of TC residues the ChromGate V3.1 software. was performed with 3 mL of HPLC grade The mobile phase was a mixture of methanol, methanol at a flow rate of 5 mL/min. Samples acetonitrile and 50 mM oxalic acid (10:20:70, were lyophilized to dry and then reconstituted v/v). The prepared mobile phase was filtered with 1 mL of mobile phase. Concentrations and through a 0.45 µ filter (Nalgene, USA) and then dilution factors were considered to calculate the degassed through sonication for 5 min before the real amount of TC residues in samples after the application. Detection was carried out using 255 analysis (13, 17- 22). nm and 365 nm as the excitation and emission wavelengths, respectively. A Phenomenex Luna Preparation of standard curves 5 µ C18 column (Torrance, CA, USA) was used. Standard solutions were prepared freshly The flow rate was 1 mL/min with an injection through dissolving TCs in a mixture of methanol, volume of 20 µL (17). acetonitrile and 50 mM oxalic acid (10:20:70, The quantitation was based on the linear v/v). Serial dilutions were prepared for each (R2 > 0.999) calibration curves derived from of the TCs (oxytetracycline, tetracycline and the concentrations and measured peak areas of chlortetracycline) to give 10, 50, 100, 500, 1,000, the standard solutions. TC residue levels were 5,000, 10,000 and 50,000 ng/g concentrations, extrapolated from the calibration curves. HPLC which were used to construct the standard curves. chromatograms of two tissue samples containing 783 Mesgari Abbasi M et al. / IJPR (2012), 11 (3): 781-787 Table 1. The recovery, intra-day, and inter-day variations of the method used for the detection of TC residues. Antibiotic Parameter Oxytetracycline Tetracycline Chlortetracycline Total mean n = 9 n = 9 n = 9 n = 27 Mean recovery (%) 76.8 74.8 73.0 74.9 Intra-day variation 3.7 4.0 4.5 4.1 mean C.V. (%) Inter-day variation 4.1 4.5 5.1 4.6 mean C.V. (%) tetracycline and chlortetracycline residues are diaphragm, gluteal muscle, kidney and liver shown in Figure 2. The limit of detection (LOD) samples were 86.0 ± 27.2, 73.8 ± 26.8, 302.9 and the limit of quantification (LOQ) for TC ± 234.9, 187.3 ± 81.9 and 135.3 ± 52.3 ng/g, residues were 2.2 and 6.6 ng/g, respectively. The respectively. The mean value of oxytetracycline recovery, inter-day and intra-day variations of residue in all meat samples was 154.2 ± 79.2 the method were calculated as shown in Table 1. ng/g (Table 2). The mean amount of tetracycline residue Results and Discussion in all analyzed samples was 13.6 ± 6.1 ng/g, with 24.5% of the samples having measurable The mean of oxytetracycline residues in amounts of tetracycline residue, and the all 110 samples was 157.1 ± 51.1 ng/g and maximum amount was 642.7 ng/g. The mean the maximum amount was 4201.8 ng/g. In amounts of tetracycline residue in triceps, total, 45.5% of the samples had detectable diaphragm, gluteal muscle, kidney and liver oxytetracycline residue (Figure 2B). The mean samples were 29.7 ± 29.1, 7.5 ± 4.2, 6.7 ± 4.1, amounts of oxytetracycline residue in triceps, 19.1 ± 6.1 and 5.3 ± 3.5 ng/g, respectively. The OXY 100 U A 50 m A TET 0 OXY 100 U A 50 m B TET 0 100 U mA 50 CHL C 0 0 5 10 15 Time (min) Figure 2. The HPLC chromatograms of a meat sample (A) and a liver sample (B) containing oxytetracycline and tetracycline residues, and a kidney sample (C) containing chlortetracycline residue. 784 Determination of Tetracycline Residues Table 2. The mean amount of TC residues in meat, liver and kidney samples. Data are presented as mean ± standard error of the mean and n indicates the number of samples. Antibiotic Sample Oxytetracycline Tetracycline Chlortetracycline Total TCs (ng/g) (ng/g) (ng/g) (ng/g) Triceps muscle 86.0 ± 27.2 29.7 ± 29.1 60.7 ±19.5 176.3 ± 46.8 n = 22 Diaphragm muscle 73.8 ± 26.8 7.5 ± 4.2 15.4 ± 9.5 96.8 ± 26.9 n = 22 Gluteal muscle 302.9 ± 234.9 6.7 ± 4.1 96.4 ± 77.6 405.3 ± 219.6 n = 22 Mean all muscles 154.2 ± 79.2 14.6 ± 9.8 57.5 ± 26.8 226.3 ± 112.5 n = 66 Liver 187.3 ± 81.9 19.1 ± 6.1 444.8 ± 140 651.3 ± 210.1 n = 22 Kidney 135.3 ± 52.3 5.3 ± 3.5 531.9 ± 162.7 672.4 ± 192.0 n = 22 Total Mean 157.1 ± 51 13.6 ± 6.1 229.8 ± 49.5 396.9 ± 77.5 n = 110 mean value of this antibiotic residue in all meat residue. samples was 14.6 ± 9.8 ng/g (Table 2). In this study, the overall recoveries of TC In the analyzed samples, the mean amount residues from bovine muscle, kidney and liver of chlortetracycline residue was 229.8 ± 49.5 ranged from 73 to 77% with the coefficient of ng/g and 40.9% of samples had detectable variation ranging from 3.7 to 5.1% (n = 27) and chlortetracycline residue. The mean amounts of a calculated limit of detection (LOD) of 2.2 ng/g. chlortetracycline residue in triceps, diaphragm, By comparison, previous studies reported the gluteal muscle, kidney and liver samples were recovery and detection limit of TC residues in 60.7 ±19.5, 15.4 ± 9.5, 96.4 ± 77.6, 444.8 ± 140.1 animal tissues using electrospray tandem mass and 531.9 ± 162.7 ng/g, respectively. The mean spectrometry of 70-115% and 2 ng/g (21) and amount of this drug residue in all meat samples using an HPLC method, were reported to be 78- was 57.5 ± 26.8 ng/g (Table 2). 100%, and 2 - 10 ng/g, respectively (2, 12, 13). The mean amount of total (sum) TC residues Of the meat samples analyzed in this study, in all samples was 396.9 ± 77.5 ng/g and the 9.1% of all samples, 18.2% of triceps, 13.6% maximum amount was 4201.8 ng/g. The mean of diaphragm and 18.2% of gluteal muscle had amount of total TC residues in triceps, diaphragm, oxytetracycline residue levels above the WHO gluteal muscle, kidney and liver samples were standard (200 ng/g). Additionally, 9.1% of the liver 176.3 ± 46.8, 96.8 ± 26.9, 405.3 ± 219.6, 651.3 samples tested in this study had oxytetracycline ± 210.1 and 672.4 ± 192.0 ng/g, respectively residues above the WHO standard. None of the (Table 2). The mean amount of total detected kidney samples had oxytetracycline residues residues in the three muscle samples was 226.3 above the WHO standard. ± 112.5 ng/g and 38.2% of these muscle samples With respect to the tetracycline residue, 1.5% lacked detectable amounts of TC residues. Figure of all meat samples and 4.5% of triceps muscle 2 shows representative chromatograms of a meat samples had levels above the WHO standard sample (A) and a liver sample (B) containing (200 ng/g). None of the diaphragm, gluteal tetracycline and oxytetracycline residues, and a muscle, kidney or liver samples had tetracycline kidney sample (C) containing chlortetracycline residues above the WHO standard level. 785 Mesgari Abbasi M et al. / IJPR (2012), 11 (3): 781-787 Table 3. The number and percent of samples with TC residues above the WHO MRLs. Antibiotic Sample (WHO MRLs) Oxytetracycline Tetracycline Chlortetracycline Total TCs n (%) n (%) n (%) n (%) Triceps muscle 4 (18.2) 1 (4.5) 2 (9.1) 7 (31.8) (> 200 ng/g) Diaphragm muscle 3 (13.6) - - 3 (13.6) (> 200 ng/g) Gluteal muscle 4 (18.2) - 2 (9.1) 6 (27.3) (> 200 ng/g) Mean all muscles 9 (9.1) 1 (1.5) 4 (6.1) 17 (25.8) (> 200 ng/g) Liver 2 (9.1) - 6 (27.3) 7 (31.8) (> 600 ng/g) Kidney - - 5 (22.7) 5 (22.7) (> 1200 ng/g) Regarding the chlortetracycline residue, Conclusions 6.1% of all meat samples, 9.1% of triceps and 9.1% of gluteal muscle samples had levels above This study demonstrates that TC resides are the WHO standard (200 ng/g). Chlortetracycline present in edible cattle tissues marketed in the residues greater than the WHO standard levels city of Ardabil. A considerable percentage of the were observed in 27.3% of liver and 22.7% of tested samples contained TC residues above the kidney samples as well. However, the diaphragm MRLs. This could be a result of the common samples did not have chlortetracycline residues misuse of TCs in Ardabil due to the economical above the WHO standard level. advantages they offer to farmers. The application With respect to TC residue, 25.8% of all meat of antibiotics for treatment and prevention, and samples, 31.8% of triceps, 13.6% of diaphragm as a food additive by farmers without veterinary and 27.3% of gluteal muscle samples had diagnosis, prescription or supervision is frequent amounts (sum) above the WHO standard (200 and must be firmly dealt with. ng/g). In addition, total TC residues were above High levels of TC residues in edible tissues the WHO standard level in 22.7% of kidney and may produce drug resistance, digestive and 31.8% of liver samples, respectively (Table 3). allergic effects, and other harmful effects in consumers. These levels could also change the The mean value of total TC residues in gluteal organoleptic properties of some meat samples. muscle samples was greater than that of triceps Therefore, the edible cattle tissues marketed in samples, and the mean value in triceps samples Ardabil are not optimal given the presence of was greater than that of diaphragm samples. tetracycline residues that are greater than the These levels may correlate with the vicinity of prescribed MRLs. Responsible authorities of the the sampling points to the drug injection points. province should consider these results, be aware The mean level of TC residues was highest in of the health issues they present, collaborate kidney samples. This difference is likely, since with the Ministry of Health and, with clear the major elimination pathway of tetracycline is policies and procedures, undertake a thorough via renal excretion, with approximately 60% of safety review of TC residues. 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