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Research Article OpenOpen Access Access A Rapid and Effective Method for Simultaneous Determination of Residual Sulfonamides and Sarafloxacin in Pork and Chicken Muscle by High Performance Liquid Chromatography with Accelerated Solvent Extraction–Solid Phase Extraction Cleanup Hanwen Sun*, Yun Hao and Xingqiang Wu College of Chemistry and Environmental Science, Hebei University, Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding, 071002, China

Abstract A rapid and effective high performance liquid chromatographic (HPLC) method with accelerated solvent extraction (ASE)-solid phase extraction cleanup was developed for the determination of sulfonamide and sarafloxacin residues in pork and chicken muscle. Four residues were extracted with acetic acid-acetonitrile at 70°C under 10.3 MPa pressure and 5 min static times with 2 static cycles. The standard curves for the determination of four residues have good linearity (r>0.999). The method limits of quantification (LOQs) were 4-12 μg.kg-1. Intra- and inter-day precisions (RSDs) were 0.3-1.5% and 0.7-1.8%, respectively. Their average recoveries with four spiked levels ranged from 83% to 108% with the RSD of 0.3-1.5%. This method provides an effective extraction procedure and high sensitivity, and can be applied for the determination of 4 residual drugs in pork and chicken muscle at lower than their MRL levels.

Keywords: Accelerated solvent extraction; Solid phase extraction; sulfamethoxazole, and tetracycline in porcine tissue was reported by High performance liquid chromatography; Sulfonamide; Sarafloxacin; the matrix solid-phase dispersion extraction (MSPD)-HPLC-DAD Pork; Chicken with the LOQ of 7-34 μg.kg-1 [21]. Introduction Sample pretreatment is always a crucial step in deciding the levels of detection limits of the overall method, especially when large number of The residues of sulphonamides (SAs) and fluoroquinolones (FQs) aquatic product with complex matrices is involved, and rapid extraction in foods of animal origin are a major concern because they are harmful becomes even more essential. To extract SAs in matrices, various to the consumer’s health, and could induce pathogens to develop traditional methods have been studied but most of them required large resistance. It has been approved for the treatment of bacterial infections volumes of solvents and were time-consuming [7,18]. A SPE column in poultry. To ensure food safety for consumers, the European was used for clean-up extracts of SAs after traditionally extraction with Community, and China Agriculture Department have laid down the solvent [5,18], but the recovery and precision need to be improved. An –1 maximum residue level (MRL) of 100 μg.kg for SAs, and 10-1900 ionic liquid aqueous two-phase system was developed for the separation –1 μg.kg for FQs in animal origin food, in particular MRL of sarafloxacin of sulfadiazine and sulfamethoxazole in water samples and aquaculture –1 is lowest as 10 μg.kg for pork and chicken muscle [1,2]. Therefore, products [8]. A simplified method with single-step extraction was it is urgently in need of developing rapid and effective method for developed for the extraction and determination of seven FQs residues simultaneous determination of SAs and sarafloxacin residues at the low and three quinolones in porcine muscle, table eggs, and commercial concentrations normally present in food in meats. whole milk [14]. Recently, we reviewed the application of accelerated A series of analytical methods were reported for the determination solvent extraction (ASE) in multiresidue analysis for food and feed of residual antibacterials in food [3]. Recently, several LC-UV/DAD [22]. It is becoming increasingly important as a sample preparation methods were reported for the determination of residual SAs in food [4- technique in food analysis, combining the benefits of high-throughput, 8]. LC–tandem mass spectrometry (MS/MS) detection was developed automation and low solvent consumption. Water was used as a solvent for multiresidue analysis of SAs in milk samples [9-12]. A capillary of ASE for extraction of SAs from cattle and trout muscle tissues and electrophoresis-MS/MS method was presented for the determination of veterinary drugs from bovine muscle tissues [23,24]. Methanol and of 12 SAs in pork meat with the LOQ of 46.5 μg.kg–1 [13]. Several LC methods were described for the determination of residual FQs in muscle, eggs, and milk with fluorescence detection with the LOQ *Corresponding author: Hanwen Sun, College of Chemistry and Environmental of 5 μg.kg–1 for sarafloxacin [14], and in milk food or infant foods Science, Hebei University, Key Laboratory of Analytical Science and Technology with MS/MS detection [15-17]. A LC method with programmable of Hebei Province, Baoding, 071002, China, Tel: +86 312 5079719; Fax: +86 312 5079739; E-mail: [email protected] fluorescence-ultraviolet detection was described for the determination of sulfadiazine, sulfapyridine, sulfathiazole, sulfadimidine, and 7 FQs Received November 19, 2012; Accepted November 27, 2012; Published November 30, 2012 without sarafloxacin in chicken muscle with the limit of quantification of 15 μg.kg–1 for the SAs [18]. A LC-MS/MS method for the determination Citation: Sun H, Hao Y, Wu X (2012) A Rapid and Effective Method for Simultaneous Determination of Residual Sulfonamides and Sarafloxacin in Pork and Chicken of 6 FQs without sarafloxacin and 3 SAs residues in chicken was Muscle by High Performance Liquid Chromatography with Accelerated Solvent described with the LOQ of 15.29-22.85 μg.kg–1 for the SAs [19]. In Extraction–Solid Phase Extraction Cleanup. J Chromat Separation Techniq 3:154. general, HPLC-MS/MS method has lower limit of detection (LOD) for doi:10.4172/2157-7064.1000154 detection of SAs and FQs than HPLC-UV/DAD method [20], but it is Copyright: © 2012 Sun H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted not widely available in general laboratories because of its high price. A use, distribution, and reproduction in any medium, provided the original author and multiresidue determination of enoxacin, lomefloxacin, sulfanilamide, source are credited.

J Chromat Separation Techniq ISSN:2157-7064 JCGST, an open access journal Volume 3 • Issue 7 • 1000154 Citation: Sun H, Hao Y, Wu X (2012) A Rapid and Effective Method for Simultaneous Determination of Residual Sulfonamides and Sarafloxacin in Pork and Chicken Muscle by High Performance Liquid Chromatography with Accelerated Solvent Extraction–Solid Phase Extraction Cleanup. J Chromat Separation Techniq 3:154. doi:10.4172/2157-7064.1000154

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CAN were used for ASE of SAs in animal feed [25], and enrofloxacin sample material mixed with 5 g of diatomite was packed in a 33 mL and ciprofloxacin in table eggs [26], respectively. The analysis of two stainless-steel extraction cell. Each cell was locked with stainless-steel classes of antibiotics in meat is quite complex as they have different screw caps equipped with Teflon O-ring sealing and circular glass performances and may bind to the lipoproteins. To achieve trace microfiber filters of 1.98 cm diameter were placed above and below residue analysis with speediness, high-throughput and high sensitivity, the packing. Acetic acid-acetonitrile (2:98, v/v) was used as extraction it is important to develop a new and effective extraction-cleanup system solvent. Conditions used in the extraction were: an oven temperature for simultaneous determination of SAs and FQs. of 70°C with 3 min heat-up time at a pressure of 10.3 MPa and two static cycles with a static time of 5 min. The flush volume amounted to Sarafloxacin could be combined with SAs in animal treatments. 40% of the extraction cell volume. The extracted analytes were purged The MRL of sarafloxacin in pork and chicken muscle is lowest for FQs from the sample cell using pressurized nitrogen for 2 min. Finally, each in animal foods. However, to our knowlege there were few reports resulting extract was centrifuged at 8000 rpm and 4°C for 10 min, and for simultaneous determination of SAs and sarafloxacin in pork the supernatant evaporated to dryness under vacuum distillation at and chicken muscle by HPLC. This work selected the three SAs and 37°C, the residue was rinsed with water, evaporated to dryness under sarafloxacin in pork and chicken muscle as target, and used ASE–SPE a nitrogen flow at 45°C and re-dissolved in 10 mL water for clean up. for sample preparation and cleanup. The developed method was used for the determination of 4 residual drugs in pork and chicken muscle at The obtained solution was applied to an Oasis HLB SPE cartridge lower than their MRL levels. (6 mL/500 mg, Waters, USA) which had been previously conditioned with 5 mL of methanol and 5 mL of water. After the extract was drained, Experimental the SPE cartridge was washed with 5 mL of water. The analyte was Chemicals and solutions eluted with ammonia-methanol (5:95, v/v). The collected eluate was evaporated to dryness under vacuum distillation under a nitrogen flow Sulfadiazine, sulfamerazine, sulfametoxydiazine, and sarafloxacin at 45°C, and the residue was re-dissolved in 1.0 mL of phosphate buffer (purity: ≥ 99%) were purchased from National Institute for the with 0.01% triethylamine-methanol (75:25,v/v). The solution was Control of Pharmaceutical and Biological Products (Beijing, China). filtered through a 0.22 µm microporous membrane of polyvinylidene All chemicals and reagents were of analytical grade except specific fluoride for HPL Canalysis. statements. Methanol (HPLC grade), acetonitrile (HPLC grade), ethyl acetate, triethylamine, and phosphoric acid were obtained from Beijing HPLC analysis Chemical Factory (Beijing, China). Doubly deionized water was used The separation of sulfadiazine, sulfamerazine, sulfametoxydiazine, throughout. The methanol was filtered through a 0.22 µm microporous and sarafloxacin was carried out using a Kromasil 100-5 C18 analytical membrane of polyvinylidene fluoride before use. Phosphate buffer column (250×4.6 mm ID, 5 µm, AKZO NOBEL, Bohus, Sweden), solution with 0.1% triiethylamine was filtered through a 0.45 μm and a six-port valve with a 20 µL sample loop injector was used. A microporous membrane of mixed cellulose ester for HPLC analysis. mobile phase is composed of 50 mM potassium dihydrogen phosphate Single sulfonamide stock standards of 400 mg/L were prepared (adjusted the pH to 3.05 with phosphoric acid) with 0.01% triethylamine in methanol. The stock solutions were stored at -4°C. Mixed standard and different proportion of methanol. The flow rate of the mobile -1 working solutions were prepared by diluting the standard stock solution phase was set at 1.0 mL min . A 20 µL volume of sample solution was just before use. injected in the column at 35°C, and then eluted in gradient with 25% of methanol in the mobile phase from 0 to 5 min, 25%→40% from 5 Instrumentation to 7 min, 40% from 7 to 11 min, and 40%→25% from 11 to 13 min. There was a re-equilibration time (about 20 min) after each procedure. The chromatographic system consisted of a Shimadzu HPLC The solution was monitored at 267 nm for sulfadiazine, sulfamerazine system equipped with an LC-10A Multisolvent Delivery System, a and sulfametoxydiazine, and at 280 nm for sarafloxacin. The linear DGU-12A online degasser, an SCL-10Avp gradient controller, a CTO- equations for the relationship between the peak areas of analytes 10Avp column thermostat, and a multi-wavelength SPD-M10Avp and their concentration were determined by least-squares method. photodiode-array detector (DAD) covering the range 190-800 nm, Quantification was carried out by using standard curve calibration which was interfaced to a computer for data acquisition using a based on peak area toward concentration in 7 concentration points. CLASS-VP workstation (Shimadzu, Kyoto, Japan). The extraction equipment was an APLE 2000 automatic accelerated solvent extraction Results and Discussion apparatus (Beijing Titan Instruments Co., Ltd, China) equipped with 33 mL stainless-steel extraction cells. A centrifuge TGL-16M (Xiangyi Optimization of LC conditions Centrifuge Co., Hunan, China), an ultrasonic cleaner (Ultrasonic Effects of gradient program: The initial test showed that the Instrument Co., Kunshan, China), RE-2000A rotary evaporator (Asia separation of the four analytes could achieved using a mobile phase Rong Biochemical Instrument Factory, Shanghai, China) and a PHS-3C composed of 50 mM potassium dihydrogen phosphate (pH 3.05) with pH meter (Shanghai Precision & Scientific Instrument Co., Shanghai, 0.01% diethylamide (reagent for eliminating peak tails) and different China) were used in sample treatment. proportion of methanol. The sensitivity increased and retention time decreased with increase in the ratio of methanol from 25% to 40% ASE and SPE (v/v) (Figure 1). The subsequent test for the spiked sample analysis Pork and chicken samples were purchased from a local market in showed that low sensitivity of sarafloxacin was observed when 25% Baoding, and after being homogenized in a high-speed food blender, (v/v) methanol was used, and the peaks of sulfamerazine and matrices they were stored below -20°C in a freezer until the time of analysis. could not be separated when 30-40% methanol was used. So that it is necessary to use gradient program (Figure 1). Samples were extracted with the APLE 2000 automatic accelerated solvent extraction apparatus. Approximately 10 g of the blank/spiked Three gradient programs were compared by changing proportion

J Chromat Separation Techniq ISSN:2157-7064 JCGST, an open access journal Volume 3 • Issue 8 • 1000154 Citation: Sun H, Hao Y, Wu X (2012) A Rapid and Effective Method for Simultaneous Determination of Residual Sulfonamides and Sarafloxacin in Pork and Chicken Muscle by High Performance Liquid Chromatography with Accelerated Solvent Extraction–Solid Phase Extraction Cleanup. J Chromat Separation Techniq 3:154. doi:10.4172/2157-7064.1000154

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Optimization of buffer pH 8 1 6 1 25% methanol 30% methanol The pH value of buffer solution influenced mainly the resolution and 6 4 4 3 2 3 the apparent mobility of the target analytes. Generally, for compounds 2 4 with lower pKa values, the dissociation ratio is higher and acidity is 2 2 stronger. The pH of buffer influences ionization of the analytes. The

0 0 mobile phase constituted of 50 mM potassium dihydrogen phosphate 4 6 8 10 12 14 16 18 20 4 5 6 7 8 9 10 11 12 with 0.01% triethylamine-methanol was adjusted with phosphoric acid to different pH. The effect of pH on the peak of the analytes is shown 1 10 1 40% methanol 4 8 35% methanol 4 in figure 3. 8 3 Absorbance / mAU / Absorbance 6 3 2 6 2 It is shown that when pH from 2.55 to 4.05 the four compounds can 4 4 be separated. The migration order is related to the order (from high to 2 2 low) of pKa. Using buffer of pH 3.05, high sensitivity for sarafloxacin 0 0 was achieved, and the separation of the four analytes could be achieved 4.0 5.0 6.0 7.0 3.0 3.5 4.0 4.5 5.0 within 14 min, so it was selected in this work. Time / min Selection of detection wavelength –1 –1 1. Sulfadiazine, 2 μg mL ; 2. sulfamerazine, 0.48 μg mL ; 3. sulfametoxydiazine, UV-DAD detection in the range of 190-800 nm was investigated. 1 μg mL–1; 4. sarafloxacin, 2 μg mL–1 The result showed that higher sensitivity was achieved by detection Figure 1: Chromatograms of the analyte standard solutions with the mobile of the SAs at 267 nm and sarafloxacin at 280 nm (Figure 4). For the phase composed of 50 mM potassium dihydrogen phosphate (pH 3.05) with 0.01% diethylamide and different proportion of methanol. sake of simplicity a wavelength of 267 nm was used only for above condition selection. In real sample analysis, the wavelengths of 267 nm and 280 nm were used, respectively, for the detection of the SAs and sarafloxacin, with higher sensitivity (Figure 4). a 10 4 10 b c 4 15 4 8 1 8 1 Optimization of ASE conditions 6 6 10 3 2 3 1 4 2 4 Effect of extraction solvent: The selection of a suitable extraction 5 3 2 2 2 solvent is the first challenge in ASE method development. Several Absorbance / mAU / Absorbance 0 0 0 solvents have been used to ASE for the preparation of food samples. 4 6 8 10 12 14 4 6 8 10 12 14 4 6 8 10 12 The polarity of the extraction solvent should closely match that of the Time / min target compounds. In this work, the extraction efficiency for using 1. Sulfadiazine, 2 μg mL-1; 2. sulfamerazine, 0.48 μg mL-1; 3. sulfametoxydiazine, acetonitrile and 2% (v/v) acetic acid in acetonitrile as extraction solvent 1 μg mL-1; 4. sarafloxacin, 2 μg mL-1; (a) 0~5 min: 25% B; 5~7 min: 25% B→30% was investigated and compared for simultaneous extraction of the SAs B; 7~11 min: 30% B; 11~13 min: 30% B→25% B. (b) 0~5 min: 25% B; 5~7 min: and sarafloxacin from a meat sample (Figure 5 left). 25% B→35% B; 7~11 min: 35% B; 11~13 min: 35% B→25

Figure 2: Chromatograms of the analyte standard solutions using gradient 1.50 1.50 programs. 4 4 1.25 267 nm 1.25 280 nm 1.00 1.00 0.75 0.75 15 pH 2.55 4 pH 3.05 4 15 0.50 0.50 1 2 3 10 mAU / Absorbance 0.25 1 2 3 1 10 0.25 1 0.00 5 2 3 0.00 5 2 3 6.0 7.5 8.0 12.0 16.0 6.0 7.5 8.0 12.0 16.0 0 0 Time / min 4 6 8 10 12 14 4 6 8 10 12 14 -1 -1 15 15 1. Sulfadiazine, 0.1 μg mL ; 2. sulfamerazine, 0.05 μg mL ; 3. sulfametoxydiazine, pH 3.55 pH 4.05 0.1 μg mL-1; 4. sarafloxacin, 0.07 μg mL-1 4 4 Absorbance / mAU / Absorbance 10 10 Figure 4: Chromatograms of the analyte standard solutions. 1 1 5 2 3 5 2 3

1 4 1 4 1 4 1 4 1 4 0 0 100 1 4 1 4 1 4 1 4 4 6 8 10 12 14 4 6 8 10 12 14 80 Time / min 60 40 1.Sulfadiazine, pKa=6.52, 2 μg mL-1; 2. sulfamerazine, pKa=6.77, 0.48 μg mL-1; 3. sulfamethoxydiazine, pKa=7.0, 1 μg mL-1; 4. sarafloxacin, pKa =7.14, 2 μg mL-1 / % Recovery 20 0 Figure 3: Effect of different pH of buffer solution on separation. Acetonitrile Acetonitrile- 40 70 80 90 20 40 60 acetic acid Solvent Temperatute /°C Flush volume / % of methanol in mobile phase. Figure 2 shows that a better gradient 1. Sulfadiazine, 2. sulfamerazine, 3. sulfametoxydiazine, 4. sarafloxacin; blank program was as follows: methanol set at 25% for 0→5 min, increased pork muscle spiked with 50 μg.kg-1 for each analyte; 2 times to each pork sample, from 25% to 40% for 5→7 min, retained 40% between 7 and 11 min, and the chromatographic measurement carried out 3 times; recovery data are the average value of three measurements and contains less than 2% of relative and returned from 40% to 25% for 11→13 min. Using this program standard deviation. the peaks of sulfamerazine and matrices could be separated and high Figure 5: Effect of solvent, temperature, and flush volume on recovery. sensitivity for sarafloxacin could be achieved (Figure 2).

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The polarity of the extraction solvent should closely match that A 3 of the target compounds. Using methanol the recovery of three SAs 4 C 2 was 80-90%, but the recovery of sarafloxacin was only 70%. Use of 2% 2 1 (v/v) acetic acid in acetonitrile has higher extraction efficiency than 0 methanol, the recovery for the four compounds achieved to 90-100%. 0 -2 -1 Effect of extraction temperature: Temperature is one of the most important parameter for ASE. The extraction efficiency with 2% 4 6 8 10 12 14 16 4 6 8 10 12 14 16 (v/v) acetic acid in acetonitrile as extraction solvent for simultaneous 3 D extraction of the four analytes from pork muscle sample was compared 4 B 2 4 4 under temperature of 60-90°C at 10.3 MPa (Figure 5 middle). The result mAU / Absorbance 2 2 2 3 showed that highest extraction efficiency (90-105%) was achieved for 1 1 3 0 each compound under temperature of 70°C. 0 -2 Effect of extraction time:The extraction process can be conducted -1 in a static or dynamic mode. In the static extraction mode, the critical 4 6 8 10 12 14 16 4 6 8 10 12 14 16 factors are the temperature and time of the extraction. During this Time / min process, the analytes are isolated from the sample under stable static conditions. The static process can be repeated several times if low A. Blank chicken sample; B. spiked blank chicken sample; C. blank pork sample; recoveries are obtained in a single stage. In this work, the efficiency of D. spiked blank chicken sample; spiked levels: 1. sulfadiazine,10 μg.kg–1; 2. sulfamerazine, 10 μg.kg–1; 3. sulfametoxydiazine, 10 μg.kg–1; 4. sarafloxacin, 5 the extraction was investigated using static time of 5, 10, 15 min and μg.kg–1 two cycles. The result showed that significant amounts of the analytes Figure 6: Chromatograms of blank meat sample and spiked meat sample. were found with 5 min of static time in the first extract. In order to evaluate the number of extraction cycles needed for an appropriate Analyte Linear Regression equation Correlation LOD LOQ recovery during the extraction step, three consecutive extractions range coefficient (r) (μg.mL-1) (μg.mL-1) were made further to optimize the number of cycles. To save time, two (μg.mL-1) extraction cycles were used with extraction efficiency of ≥ 90%. So 5 Sulfadiazine 0.12-100 A=2.120×104C-1.038×104 0.9999 0.038 0.12 min static times and 2 static cycles were selected in further work. Sulfamerazine 0.05-50 A=6.351×104C-1.993×104 0.9998 0.015 0.05 Sulfametoxydiazine 0.10-100 A=3.356×104C-1.130×104 0.9999 0.030 0.10 Effect of flush volume: Flush volume is the percentage of fresh Sarafloxacin 0.04-40 A=9.710×104C-3.739×104 0.9998 0.013 0.04 volume introduced into the cell after the static time to drag the analytes towards the collection vial. This volume ensures that all analytes are Table 1: Linearity regression equation, correlation coefficient, linear range, limit of detection (LOD) and limit of quantification (LOQ). eluted and is closely related to the final volume. Different flush volumes were used to extract analytes (Figure 5 right). To save solvent and time, Analyte Spiked level Intra-day(RSD, n=6) Inter-day (RSD,n=6) a flush volume of 40% (cell volume, 33 mL) was enough to push the (μg.kg-1) (%) (%) analytes extracted out of the cell with higher extraction efficiency (92- Sulfadiazine 10 0.5 0.7 108%). 20 0.8 0.9 Optimization of cleanup: The solution obtained by ASE was 50 0.8 1.0 applied for cleanup. Cleanup procedure was investigated by using Sulfamerazine 10 0.9 1.3 20 1.1 1.1 AccuBONDII ODS-C18 SPE columns (100 mg/3 mL, Agilent, USA) and Oasis HLB (500 mg /6 mL, Waters, USA). The SPE columns were 50 0.6 1.3 preconditioned with 5 mL of methanol and 5 mL of water. After sample Sulfametoxydiazine 10 0.3 0.9 loading, the column was washed with 5 mL water, and the analytes were 20 0.5 0.7 eluted with ammonia-methanol (5:95, v/v). The collected eluate was 50 0.3 1.0 evaporated to dryness under vacuum distillation under a nitrogen flow Sarafloxacin 5 1.2 1.7 at 45°C, and the residue was re-dissolved in 1.0 mL of phosphate buffer 10 1.5 1.8 with 0.01% triethylamine-methanol (75:25,v/v). Oasis HLB column 25 1.1 1.5 Intra-day and inter-day precision as relative standard deviayion (RSD). and AccuBONDII ODS-C18 column allowed to achieving the recoveries Tabie 2: of >92% and >80%, respectively. The HLB column was selected in this standards (7 concentration point) against measured peak areas under work. the optimized conditions. The equations of calibration curves obtained Performance of the method based on three parallel measurements for standard solution are listed in table 1. It can be seen that the linearity is satisfactory with a correlation Selectivity: Under the optimized conditions, selectivity was coefficient (r) greater than 0.999. determined for each analyte in the assay. Blank meat sample and spiked meat sample were treated with the ASE-SPE procedure. The The limit of detection (LOD) was determined as the sample chromatograms were obtained, as shown in figure 6. concentration that produces a peak with a height three times the level of the baseline noise, and the limit of quantification (LOQ) was calculated It was shown that the four drugs could be baseline separated. The that produced a peak with 10 times the signal-to-noise ratio. Table 1 unknown peak does not interfere with the separation and determination gives the instrument LODs and LOQs. For 10 g of sample and 1 mL of of the four analytes. There was no interference peak in real samples. final test solution, the method LOD values were 3.8, 1.5 and 3.0 μg.kg–1 Linearity and detection limit: The linearity for analysis of the for sulfadiazine, sulfamerazine, and sulfametoxydiazine, and 1.3 μg.kg–1 studied analytes was evaluated with concentrations of calibration for sarafloxacin in meat sample, and their method LOQ values were

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Analyte Spiked level Pork Chicken Acknowledgements (μg.kg–1) Recoveries RSD(n=6) RSD(n=6 ) This work was supported by the Natural Science Found of Hebei Province (%) (%) Recoveries (%) (B2008000583) and the sustentation Plan of Science and Technology of Hebei (%) Province (No. 10967126D). Sulfadiazine 10 104 0.6 108 0.5 References 20 106 0.5 104 0.8 50 97 0.9 103 0.8 1. Council regulation 2377/90/EC (1990) laying down a Community procedure for 100 99 0.8 96 0.8 the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off J Eur Commun 224: 1-124. Sulfamerazine 10 92 1.0 92 0.9 20 96 0.7 95 1.1 2. China Agriculture Ministry, Establishment of Maximum Residue Levels of Veterinary Medical Products in Foodstuffs of Animal Origin, PR China, 50 98 0.9 96 0.6 Regulation No. 235, 2002. 100 98 0.8 98 0.7 3. Blasco C, Picó Y (2007) Progress in analysis of residual antibacterials in food. Sulfametoxydiazine 10 93 0.3 92 0.3 Trac Trends Anal Chem 26: 895-913. 20 95 0.4 90 0.5 50 96 0.6 95 0.3 4. H You, H Yu, Y Wu, Y Wang, C Liu, S Chen (2010) Simultaneous determination of 5 sulfonamides residues in beef by pressurized solvent extraction–high 100 100 0.5 97 0.4 performance liquid chromatography. Chemistry 73: 854-857. Sarafloxacin 5 90 0.9 89 1.2 5. Shi X, Meng Y, Liu J, Sun A, Li D, et al. (2011) Group-selective molecularly 10 92 1.2 85 1.5 imprinted polymer solid-phase extraction for the simultaneous determination 50 88 1.0 91 1.1 of six sulfonamides in aquaculture products. J Chromatogr B Analyt Technol 100 98 0.5 97 0.7 Biomed Life Sci 879: 1071-1076.

Table 3: Determination of the SAs and sarafloxacin in pork and chicken muscle spiked 6. Chitescu CL, Nicolau AI, Csuma A, Moisoiu C (2011) Simultaneous analysis of at three concentation levels for each analyte. four sulfonamides in chicken muscle tissue by HPLC. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 28: 1013-1020. –1 –1 12, 5, 10 μg.kg and 4 μg.kg . The obtained LOQs values were lower 7. Huang J, Liu J, Zhang C, Wei J, Mei L, et al. (2012) Determination of than those of recently reported LC [5,18,20] and CE-MS/MS [13] for sulfonamides in food samples by membrane-protected micro-solid phase the SAs, and LC-MS/MS methods for sarafloxacin [17]. The proposed extraction coupled with high performance liquid chromatography. J Chromatogr A 1219: 66-74. method permits simultaneous detection of the SAs and sarafloxacin in 8. Li YF, Han J, Yan YS, Chen B, Zhang GC, et al. (2012) Simultaneous extraction meat samples at lower than the required MRLs. and determination of sulfadiazine and sulfamethoxazole in water samples and aquaculture products using [Bmim]BF4/(NH4)3C6H5O7 aqueous two-phase Repeatability: The precisions of the method were investigated by system coupled with HPLC. J Iran Chm Soc. analyzing the four analytes in spiked blank pork muscle. Under the 9. Yu H, Tao Y, Chen D, Wang Y, Huang L, et al. (2011) Development of a high optimized conditions the intraday variability (RSD) of peak area for performance liquid chromatography method and a liquid chromatography– 6 determinations and inter-day RSD for 2 determinations per a day tandem mass spectrometry method with the pressurized liquid extraction for within 3 days were investigated. The results are listed in table 2. the quantification and confirmation of sulfonamides in the foods of animal origin. J Chromatogr B Analyt Technol Biomed Life Sci 879: 2653-2662.

The intra- and inter-day RSDs of the four analytes were inthe 10. Won SY, Lee CH, Chang HS, Kim SO, Lee SH, et al. (2011) Monitoring of 14 range of 0.3-1.5% and 0.7-1.8%, respectively. It was shown that the sulfonamide antibiotic residues in marine products using HPLC-PDA and LC- repeatability of the method is satisfactory for the residue determination MS/MS. Food Control 22: 1101-1107. of the studied SAs and sarafloxacin in meat samples. 11. Lu Y, Shen Q, Dai Z, Zhang H, Wang H (2011) Development of an on-line matrix solid-phase dispersion/fast liquid chromatography/tandem mass spectrometry Sample analysis: Under the optimized conditions of ASE and system for the rapid and simultaneous determination of 13 sulfonamides in HPLC, pork and chicken muscles were analyzed. The studied four grass carp tissues. J Chromatogr A 1218: 929-937. drugs were not detected in meat samples. The recovery test of the assays 12. Yu C, Hu B (2012) C18-coated stir bar sorptive extraction combined with high for the four drugs was carried out by adding known amounts of these performance liquid chromatography– electrospray tandem mass spectrometry drugs to the meat samples. The recoveries are listed in table 3. for the analysis of sulfonamides in milk and milk powder. Talanta 90: 77-84. 13. Font G, Juan-García A, Picó Y (2007) Pressurized liquid extraction combined The data showed that the average recovery was 88-106% with the with capillary electrophoresis–mass spectrometry as an improved methodology RSD of 0.3-1.2% for pork muscle, and 85-108% with the RSD of 0.3- for the determination of sulfonamide residues in meat. J Chromatogr A 1159: 1.5% for chicken muscle. It is indicated that the proposed method has 233-241. high recovery, and can be used for the determination of the studied 14. Cho HJ, Yi H, Cho SM, Lee DG, Cho K, et al. (2010) Single-step extraction drugs in real samples. followed by LC for determination of (fluoro)quinolone drug residues in muscle, eggs, and milk. J Sep Sci 33: 1034-1043.

Conclusions 15. Kantiani L, Farré M, Barceló D (2011) Rapid residue analysis of fluoroquinolones A simple, selective and sensitive strategy for the determination of in raw bovine milk by online solid phase extraction followed by liquid chromatography coupled to tandem mass spectrometry. J Chromatogr A 1218: three SAs and sarafloxacin in two complex matrixes has been developed, 9019- 9027. showing the usefulness of ASE as a powerful tool for extraction without cleanup. The proposed ASE method provides faster extraction and 16. Herrera-Herrera AV, Hernández-Borges J, Rodríguez-Delgado MA, Herrero M, Cifuentes A (2011) Determination of quinolone residues in infant and reduces the volume of solvent required than conventional exaction young children powdered milk combining solid-phase extraction and ultra- techniques. The HPLC analysis combined with ASE provides a rapid performance liquid chromatography–tandem mass spectrometry. J Chromatogr and simple extraction procedure, good repeatability, high sensitivity, A 1218: 7608-7614. and effective separation. The proposed method permits the detection of 17. Rodriguez E, Villoslada FN, Moreno-Bondi MC, Marazuela MD (2010) the studied SAs and sarafloxacin in meat samples at lower than MRLs. Optimization of a pressurized liquid extraction method by experimental design

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methodologies for the determination of fluoroquinolone residues in infant foods 22. Sun H, Ge X, Lv Y, Wang A (2012) Application of accelerated solvent extraction by liquid chromatography. J Chromatogr A 1217: 605-613. in the analysis of organic contaminants, bioactive and nutritional compounds in food and feed. J Chromatogr A 1237: 1-23. 18. Li C, Jiang HY, Zhao SJ, Zhang SX, Ding SY, et al. (2008) Simultaneous Determination of Fluoroquinolones and Sulfonamides in Chicken Muscle by LC 23. Bogialli S, Curini R, Di Corcia A, Nazzari M, Samperi R (2003) A liquid with Fluorescence and UV Detection. Chromatographia 68: 117-121. chromatography−mass spectrometry assay for analyzing sulfonamide antibacterials in cattle and fish muscle tissues. Anal Chem 75: 1798-1804. 19. Liu P, Jiang N, Wang Y, Yan L (2008) Simultaneous determination of sulfonamides and fluoroquinolones residues in chicken by high performance 24. Blasco C, Masia A, Morillas FG, Picó Y (2011) Comparison of the effectiveness liquid chromatography-electrospray tandem mass spectrometry. Chin J of recent extraction Procedures for antibiotic residues in bovine muscle tissues. Chromatogr 26: 348-352. J AOAC Int 94: 991-1003.

20. Li WH, Shi YL, Gao LH, Liu JM, Cai YQ (2010) Simultaneous Determination 25. Kantiani L, Farré M, Freixiedas JM, Barceló D (2010) Development and validation of quinolones, sulfonamides and macrolides in fish samples using accelerated of a pressurised liquid extraction liquid chromatography–electrospray–tandem solvent extraction followed by high performance liquid chromatography- mass spectrometry method for β-lactams and sulfonamides in animal feed. J electrospray ionization tandem mass spectrometry. J Inst Anal 29: 987-992. Chromatogr A.

21. Yu H, Mun H, Hu Y (2012) Determination of fluoroquinolones, sulfonamides, 26. Herranz S, Moreno-Bondi MC, Marazuela MD (2007) Development of a new and tetracyclines multiresidues simultaneously in porcine tissue by MSPD and sample pretreatment procedure based on pressurized liquid extraction for the HPLC–DAD. J Pharm Anal 2: 76-8. determination of fluoroquinolone residues in table eggs. J Chromatogr A 1140: 63-70.

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