Levels of Sulfonamides in Local Food of Animal Origin (Muscle Tissues, Kidney and Liver) Using HPLC-PDA

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European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

Levels of Sulfonamides in Local Food of Animal Origin (Muscle Tissues, Kidney and Liver) Using HPLC-PDA

Mahmoud Alawia, Mohammad Abu Othmana, and Rafat Ahmadb a The University of Jordan, Chemistry Department, P.O.Box13003, Amman-11942, Jordan

b Royal Scientific Society, Amman Al-Jubaiha, 11941, Jordan

ABSTRACT

The study was performed to determine the residues of sulfonamides in food of animal origin (Muscle tissue, Kidney and Liver). A high performance liquid chromatographic method with photo diode array detection at 265 nm was developed and validated in terms of accuracy, precision and selectivity for quantitative determination of the residue of four of the most used sulfonamides in Jordan, namely: Sulfathiazole, Sulfamerazine, Sulfadimidine and Sulfadimethoxine. Extraction was performed using acetonitrile and no halogenated solvents were used followed by clean up on solid phase extraction cartridges (oasis HLB). Separation was performed using RP-C18, (250×4.6 mm, 5µm particle size) under isocratic elution conditions using 0.01M Potassium dihydrogen Phosphate (KH2PO4) buffer and acetonitrile (70:30, v/v) as mobile phase at a flow rate of 1.0 ml/min. The limits of detection were 0.01 mg/kg for STZ, SMR and SDD and 0.02 mg/kg for SDM. Mean recoveries of sulfonamides using kidney samples spiked at three levels were 90.8-107.5 % for sulfathiazole, 81.4-92.1 % for sulfamerazine, 91.0-103.9 % for Sulfadimidine, and 74.6-95.9 % for Sulfadimethoxine and RSD values were lower than 11 %. It was found that 3 out of 36 samples collected randomly from Amman slaughterhouse do not fit human’s consumption requirments because their concentrations exceeded the recommended maximum residual limit.

Keywords: Veterinary antibiotics, residues, sulfonamides, HPLC-PDA, Jordan.

1. INTRODUCTION

The growth of the veterinary antibiotic pharmaceuticals business sector, which provides farms with low cost veterinary antibiotic, reflects these challenges. However, recent research reveals many concerns about the side effects accompanying the use of these antibiotics for the human health which include allergic and toxic reactions as reported by (Kunihiro, 2007) that were found to be carcinogenic (Nue, 1992). The use of antimicrobial medicines in animals may unnecessarily result in increased human morbidity, increased human mortality, reduced efficacy of related antibiotics used for human medicine, increased healthcare costs, increased potential for carriage and dissemination of pathogens within human population and facilitated the emergence of resistant human pathogens (World Health Organization Study Group, 2002). Such side effects on humans are a result of the lack of bio-security tests and governmental control over the use of antibiotics, which continue to affect food safety. 84

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

The research of Saschenbrecker and Fish, (1980) shows that each antibiotic has a withdrawal time which is the time required to assure that the drug residues in the marketable animal products are below the determined maximum residue limit (MRL) after the administration of the drug to a dairy animal The Maximum Residue Limit (MRL) represents the maximum concentration of an antibiotic allowed to be present in an animal tissue at the time of slaughter. The withdrawal time for antibiotics varies from 15 to 80 days to ensure that the residues are below MRL level (Saschenbrecker and Fish, 1980). Observations show that some slaughterhouses and farm keepers do not monitor the time between the administrations of a drug and the planned slaughter time. The withdrawal period for some antibiotics highlights that farmers should stop using drugs at specific time ~ 80 days in some antibiotics before the livestock is slaughtered; otherwise they face the risk of high residue values.

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

2. EXPERIMENTAL

2.1 Instrumentation High Performance Liquid Chromatographic (HPLC) System consists of column oven (CTO-20A/20AC), PDA-Detector (SPD-M20A), Auto sampler (SIL-20A /20AC), VACUUM Degasser (DGU-20AS) and Software (Shimadzu LC Solution, Japan) was used.

2.1.1 HPLC conditions RP-C18, LiChrospher (MERCK, Germany) (250×4.6 mm, 5 µm particle size) column was used for the separation of the 4 sulfonamides. A mixture of 0.01 M potassium dihydrogen phosphate buffer and acetonitrile at the ratio of 70:30% (v/v) was used as the mobile phase at a flow rate of 1.0 ml /min and at 27 °C. The injection volume was 20 µL and the PDA- detector at wavelength of 265 nm was applied.

2.2 Materials Bond elutes SPE-cartridge Oasis HLB; Waters, 3 ml were used

2.3 Reagents

The following reagents were used, Potassium dihydrogen phosphate (KH2PO4) was purchased from CHEM-LAB NV, Belgium. Methanol and acetonitrile were of HPLC -grade quality, and purchased from Scharlau- Spain. The water was purified and deionized by Pacific-UP/UPW pure water system. The mobile phase was filtered through 0.45 µm pore size, Chrom Tech (PALL). Formic acid, extra pure quality purchased from (Scharlau -Spain).

2.4 Preparation of standard solutions Individual stock sulfonamide solutions (100 µg/ml) were prepared by dissolving 5.0 mg of each compound in 50 ml of solvent (acetonitrile: water) (1:1)). Appropriate dilution of these stock solutions was used to prepare various concentrations, 10, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.08, 0.06, 0.04, 0.02 and 0.01 µg/ml.

2.5 Sampling and sample handling Samples of muscle tissue, liver and kidney of 50-100 g were collected from the same animal from Amman slaughterhouse as shown in table 1. Each sample was cut into small pieces and minced in blender. Ten grams of the blended sample and 10 ml of deionized water were added into 50 ml polypropylene centrifuge tube. The mixture was blended using Ultra-Turrax for 3 min at 4000 rpm. , and then preserved in a deep freezer at a temperature of (-20 °C).

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

Table1

2.6 Preparation and extraction of meat samples Five hundred milligram (500 mg) of homogenate meat was accurately weighed in a 50 ml disposable polypropylene centrifuge tube and 1 ml of acetonitrile was added to it. The tube was held for 10 minutes at room temperature. After 10 minutes, the tube was vortexed using vortex mixer (RVM-101 Rexmed, Taiwan) at a high speed for 2 minutes and centrifuged (Eppendorf 580AR, Germany) at10000 rpm for 5 minutes. The supernatant was collected into a separate test tube and the residues in the first tube were extracted once again with 1 ml of a deionized water/acetonitrile mixture (1:1) followed by centrifugation once again as mentioned. The supernatant was added to the previous tube. The pooled extracts were evaporated under a mild stream of nitrogen at 45 °C.

The residue was dissolved in 2 ml of 0.01 M KH2PO4 buffer (PH 3.9), and then loaded onto Oasis HLB Cartridge (3 ml, Waters). The conditions for SPE are given in Table 2 after that, it was evaporated again under a mild stream of nitrogen at 45 °C. The residue was dissolved in 500 µl of acetonitrile /water mixture (1:1) and filtered through a disposable syringe filter Nylon (PA) membrane into HPLC auto sampler vials for HPLC analysis.

Table 2

3. METHOD OPTIMIZATION

Each sample of animal origin has it is own characteristics and the interfering compounds vary from sample to sample which makes it hard to develop a robust method.

3.1 The optimal wavelength of detection Photo Diode Array (PDA) detector, with deuterium and tungsten lamps as light sources, was used. Sulfonamides standard solution showed maximum absorption at 260-271 nm. Therefore, UV at 265 nm was used for the detection of sulfonamide residues in the samples of this study. The calibration curves were linear in the range of 0.02 to 0.8 µg /ml for sulfathiazole, sulfamerazine, sulfadimidine, and from 0.04 - 0.8 µg/ml for sulfadimethoxine.

3.2 Limit of detection (LOD) The present method provided the limit of detection of sulfathiazole, sulfamerazine, sulfadimidine at 0.02 and of sulfadimethoxine at 0.03 µg/ml respectively which were below the MRL (0.1 µg/g).

3.3 Selection of extraction procedure

Acetonitrile was found to be favorable for the extraction of sulfonamide compounds (Batzias, et al., 2002). This solvent was used to have the advantages over many of other organic solvents in term of deproteination

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

(removal of > 99% of protein and fat), chromatographic interferences, recovery, emulsion formation (McMaster, 1994) and easy to evaporate (Bui, 1993). The disadvantage of using acetonitrile was binding with highly polar compounds from the sample matrices, which come first in the reverse phase chromatography (Kao, 2001). To avoid the undesirable impact, acetonitrile/water (1:1) was used

3.4 Optimization of SPE and clean up with C18-LiChrospher, Oasis HLB and Plexa The pKa values of all sulfonamide drugs are within 5.5-7.5, they will be completely extracted by increasing the solvent pH. At pH of 9.0 (only acetonitrile using), more polar compounds are dissolved in the solvent and shown as interferences in the chromatogram, while at pH 7.0 (water only), extraction was not sufficient to extract all compounds. Therefore when acetonitrile /water (1:1) was used at (pH 8.0), higher extraction efficiency of sulfonamides was gained (Biswas, et al., 2007). Supernatant is taken, evaporated and then reconstituted with 0.01 M buffer at pH of 3.9 and finally loaded into SPE (Oasis HLB) cartridge at the conditions shown in Table 2. The eluate was then evaporated and reconstituted with the eluent and injected into the HPLC.

4. METHOD VALIDATION

The described method was validated in terms of linearity, selectivity, precision, and accuracy according to the European Union regulation for live animals and animal products Decision 2002/657/EC guidelines.

4.1 Linearity Calibration curves of peak area (y) versus concentration (x) of standards were obtained. Linearity between the peak areas and sulfonamides’ concentrations are described by the regression equations given in Table 3 and Figures 1-4.

Table 3

The calibration curves were linear in the range of 0.02 to 0.8 µg /ml for sulfathiazole, sulfamerazine, sulfadimidine, and from 0.04 to 0.8 µg/ml for sulfadimethoxine.

4.2 Selectivity Blank samples of muscle tissue, liver and kidney were analyzed in order to verify selectivity of the planned method and no interferences were detected. A small unknown endogenous peak appeared in the chromatogram of all samples at 7.5 minutes, but it elutes at quite different retention times from the examined SAs. However, the analyte of interests eluted at 4.6, 5.5, 6.2 and 15.0 minutes and was sufficiently separated for quantitation.

4.3 Method precision In order to evaluate the precision of the method as shown in Table 4, four kidney samples (fortified with 100 µg /L with sulfadimethoxine) were analyzed and RSD% is calculated.

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

Table 4

4.5 Accuracy During sample preparation and clean up procedure, the drug may be lost, so it was important to calculate the percent recovery of the drug in spiked samples. This prove that sample preparation procedure (which is similar in principles to procedure of Biswas, et al., (2007)) suitable for analysis of sulfonamide residues in samples. To check the efficiency of procedure of Biswas, et al., (2007), blank kidney samples were spiked to yield concentrations of 0.06, 0.1, 0.2 mg/kg of sulfonamides. Tables 5, 6, 7, and 8 show the recoveries for the four compounds (The QC solutions were prepared in blank matrix to avoid the bias due to matrix mismatch between test materials and standard solution).

Table 5

Table 6

Table 7

Table 8

5. RESULTS AND DISCUSSION

This study was performed to separate combination of four sulfonamides mostly used in Jordan in one run as shown in figure 5.

Figure 5

Thirty six samples were analyzed for sulfonamide residues, 3 samples which represents (8%) of them were positive and exceeded recommended MRL, as shown in Table 9. Peak identification is more reliable using the diode array detector in addition to the position of the peak (retention time), checking the maximum wavelength of the analyte in each of standard and sample.

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

Table 9

Sulfadimidine was found in Local sample - 6 in muscle tissue, kidney and liver as shown in Table 9, it was eluted at retention time 5.8 minutes as shown in Figures 6, 7 and 8 and in spike sample at retention time 5.9 as shown in Figure 9.

Comparing our results with the results of other countries, like for instance the USA where contamination rates due to sulfonamides amounted in some years to over 4 % (Weiss, et al., 2006) and in Islamabad (Pakistan) a total 30 poultry meat samples were analyzed for sulfonamide residues, 7 (23%) exceeded the recommended MRL of 0.12- 0.8 mg/kg (Mehtabuddin, et al., 2012). In Italy the overall rate of residue violation detected in the study was 0.33 % (1/299), where only Sulfaquinoxaline was present in one violative sample (0.12 mg/kg). (Weiss, et al. 2006). It was observed that sulfonamide drugs stay mostly in the liver. This observation is in agreement with the findings of Saschenbrecker and Fish, (1980). They demonstrated that liver residual level of sulfadimidine requires nine days to go back to the 0.1 mg/kg tolerance limit, while kidney and muscle tissue require seven and one-half and five days, respectively. Therefore it is advisable, in case of animal poisoning with these drugs, to analyze the liver.

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

6. REFERENCES

Batzias, G.C., Botsoglou, N .A., Lotsaki-kovatst, V. P., Kounesnis, G. (2002). New simple liquid chromatographic method for determination of trimethoprim ,sulfadiazine and N4 –acetyl sulfadiazine in plasma of broilers . Journal of Chromatography B, 769,253-259.

Biswas, A. k., Rao, G .S., Kondalan, N., Anjaneyulu, A.S.R. and Malik. J .K. (2007). Simple Multiresidue Method for Monitoring of Trimethoprim and Sulfonamide Residue in Buffalo Meat by High-Performance Liq uid Chromatography.Agricultural and Food Chemistry,55,8845-8850.

Bui, L.V. (1993). Liquid chromatographic determination of six sulfonamide residues in animal tissues using post column derivatization. J.AOAC Int, 76, 966-977.

Companyό, R. (2010). Optimization of an SPE Clean-up Approach for the analysis of sulfonamides in animal feed) unpublished Master thesis, university of Barchalona,Spain.

European Council Regulation (EEC) (1990). Community procedure for the establishment of maximum residue limit of veterinary medicinal products in food stuff of animal origin (L, 224, 1-8).

Kao, T. M., Chang, M. H., Cheng, C. C., Chou, S. S.(2001). Multiresidue determination of veterinary drugs in chicken and swine muscles by high performance liquid chromatography. Journal of Food and Drug Analysis, 9, 84-95.

Kunihiro, K. (2007). Quantitation and confirmation of six sulphonamides in meat by liquid chromatography–mass spectrometry with photodiode array detection. Food Control, 18, 301-305.

McMaster, M. C. (1994) Sample preparation and methods developments, In HPLC-A Practical User Guid, 135-143.

Mehtabuddin, A. A., Mian, T., Ahmad, S., Nadeem, Z. I., Tanveer and Arshad, J. (2012). Sulfonamide residues determination in commercial poultry meat. The Journal of Animal and plant science, 22, 473- 478.

Nue, H. C. (1992). The crisis in antibiotic resistance. Science, 257, 1064-1072.

Saschenbrecker, P.W., and Fish, N.A. (1980). Sulfamethazine Residues in Uncooked Edible Tissues of Pork Following Recommended Oral Administration and Withdrawal, Can. J. Comp. Med, 44, 338-345.

Weiss, C., Conte, A., Milandri, C., Scortchini, G., Semprini, P., Usberti, R. and Migliorati,G. (2006). Veterinary drugs residue monitoring in Italian poultry: Current strategies and possible developments. Food Control, 18, 1068-1076.

World Health Organization Study Group (2002). Future trends in veterinary public 707, 1-85.

European International Journal of Apppplied Science and Technology Vol.l. 1 No. 3; May 2014

Figure 1: Standard calibration curve fofor sulfathiazole

Figure 2: Standard calibration curve fofor sulfamerazine

European International Journal of Apppplied Science and Technology Vol.l. 1 No. 3; May 2014

Figure 3: Standard calibration curve fofor sulfadimidine

Figure 4: Standard calibration curvrve for sulfadimethoxine

European International Journal of Apppplied Science and Technology Vol.l. 1 No. 3; May 2014

Intensity mAU

ReRetention time (min)

Figure 5: Separation of 600µ0µg/l sulfonamides using acetonitrile / 0.01 M pototassium Dihydrogen phosphate bufferfer (30:70 %) as mobile phase and (RP-C18, LiChhrospher (Merck, Germany) (250× 4.6.6 mm, 5 µm particle size) column with injectionn volume 20 µl.

European International Journal of Apppplied Science and Technology Vol.l. 1 No. 3; May 2014

Intensity mAU IntensitymAU

Retention time (min)

Figure 6: Chromatogram off MMuscle tissue of Local sample -6

Intensity mAU mAU Intensity

Figure 7: Chromatogramam of Kidney of Local sample -6

European International Journal of Apppplied Science and Technology Vol.l. 1 No. 3; May 2014

Intensity mAU IntensitymAU

Retention time (min)

Figure 8: Chromatogram of Liver of LocalL sample-6

Intensity mAU Intensity mAU

Retention time (min)

Figure 9: Chromatogram of spiked kikidneys sample using Oasis HLP

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

Table1: Details about the collected samples from Amman slaughterhouse

Sample No. Sample ID No. of Type of sample Source Date of collection Animals 1-18 Local Sheep 6 6 Muscle tissue Jordan 08.08.2012 6 Kidney 6 Liver 19-24 Imported Bovine 2 2 Muscle tissue Ethiopia 08.08.2012 2 Kidney 2 Liver 25-30 Imported Sheep 2 2 Muscle tissue Australia 08.08.2012 2 Kidney 2 Liver 31-36 Imported Sheep 2 2 Muscle tissue Romania 08.08.2012 2 Kidney 2 Liver

Table 2: Condition of loading the extract to SPE cartridge in method 2

SPE sorbents (Cartridges) used – Isolute C18XL Column, Plexa PCX and Oasis HLB.

Conditioning 2 ml of methanol

Conditioning 2 ml of water

Loading solvent 2 ml of sample solution prepared with 0.01 M KH2PO4 at pH 3.9

Washing solvent 2 ml of 10% methanol in water

Elution 2 ml of methanol

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

Table 3: Regression equations and coefficient of determination of the calibration curves

Analytes Regression equations Coefficient of determination (R2)

Sulfathiazole y = 69246x - 339.64 0.9992

Sulfamerazine y = 86979x - 388.01 0.9986

Sulfadimidine y = 83821x - 417.16 0.9991

Sulfadimethoxine y = 77187x - 552.13 0.9991

Y refers to the peak area, and x refers to the concentration

Table 4: Method precision (%RSD) using spiked kidney at 100mg/kg

Preparation No. %Recovery 1 95.7 2 100.8 3 92.4 4 94.2 (mean ± SD) 95.8 ± 3.6 RSD 3.8%

Table 5: Recoveries for sulfathiazole in kidney examined at QC-levels: low 0.06 mg/kg, mid 0.1 mg/kg, and high 0.2 mg/kg (n = 6)

Spiked concentration of QC level 0.06 0.1 0.2 (mg/kg)

Recovery (%, mean ± SD) 96.7 ± 9.4 90.8±6.9 107.5 ± 5.6

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

Table 6: Recoveries for sulfamerazine in kidney examined at QC-levels: low 0.06 mg/kg, mid 0.1 mg/kg, and high 0.2 mg/kg (n = 6)

Spiked concentration of QC level 0.06 0.1 0.2 (mg/kg)

Recovery (%, mean ± SD) 82.5 ± 7.7 92.1 ± 4.8 81.4 ± 3.0

Table 7: Recoveries for Sulfadimidine in kidney examined at QC-levels: low 0.06 mg/kg, mid 0.1 mg/kg, and high 0.2 mg/kg (n = 6)

Spiked concentration of QC level 0.06 0.1 0.2 (mg/kg)

Recovery (%, mean ± SD) 91.0 ± 7.9 102.4 ± 9.1 103.9 ± 6.6

Table 8: Recoveries for Sulfadimethoxine in kidney examined at QC-levels: low 0.06 mg/kg, mid 0.1 mg/kg, and High 0.2 mg/kg (n = 6)

Spiked concentration of QC level 0.06 0.1 0.2 (mg/kg)

Recovery (%, mean ± SD) 74.6 ± 5.8 95.9 ± 3.3 95.2 ± 6.0

European International Journal of Applied Science and Technology Vol. 1 No. 3; May 2014

Table 9: Concentration of sulfonamide residues in food of animal origin in Jordan

Concentration (mg/kg) Sample Sample identity Mixed Muscles (Loin, No. Liver Kidney Shoulder and leg) 1-3 Local Sheep-1 < d. L. a < d. L. a < d. L. a 4-6 Local Sheep-2 < d. L. a < d. L. a < d. L. a 7-9 Local Sheep-3 < d. L. a < d. L. a < d. L. a 10-12 Local Sheep-4 < d. L. a < d. L. a < d. L. a 13-15 Local Sheep-5 < d. L. a < d. L. a < d. L. a 16-18 Local Sheep-6 1.043 b 1.002b 0.840b 19-21 Ethiopian Bovine-1 < d. L. a < d. L. a < d. L. a 22-24 Ethiopian Bovine-2 < d. L. a < d. L. a < d. L. a 25-27 Australian Sheep-1 < d. L. a < d. L. a < d. L. a 28-30 Australian Sheep-2 < d. L. a < d. L. a < d. L. a 31-33 Romanian Sheep -1 < d. L. a < d. L. a < d. L. a 34-36 Romanian Sheep -2 < d. L. a < d. L. a < d. L. a

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