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ISSN: 2157-7064
Journal of Chromatography & Separation Techniques
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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

*Corresponding Author:
Hanwen Sun
College of Chemistry and Environmental Science, Hebei University
Key Laboratory of Analytical Science and Technology of Hebei Province
Baoding, 071002, China
Tel: +86 312 5079719
Fax: +86 312 5079739
E-mail: [email protected]

Received date: November 19, 2012; Accepted date: November 27, 2012; Published date: November 30, 2012

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

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 use, distribution, and reproduction in any medium, provided the original author and source are credited.

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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; High performance liquid chromatography; Sulfonamide; Sarafloxacin; Pork; Chicken

Introduction

The residues of sulphonamides (SAs) and fluoroquinolones (FQs) in foods of animal origin are a major concern because they are harmful to the consumer’s health, and could induce pathogens to develop resistance. It has been approved for the treatment of bacterial infections in poultry. To ensure food safety for consumers, the European Community, and China Agriculture Department have laid down the maximum residue level (MRL) of 100 μg.kg–1 for SAs, and 10-1900 μg.kg–1 for FQs in animal origin food, in particular MRL of sarafloxacin is lowest as 10 μg.kg–1 for pork and chicken muscle [1,2]. Therefore, it is urgently in need of developing rapid and effective method for simultaneous determination of SAs and sarafloxacin residues at the low concentrations normally present in food in meats.

A series of analytical methods were reported for the determination of residual antibacterials in food [3]. Recently, several LC-UV/DAD methods were reported for the determination of residual SAs in food [4-8]. LC–tandem mass spectrometry (MS/MS) detection was developed for multiresidue analysis of SAs in milk samples [9-12]. A capillary electrophoresis-MS/MS method was presented for the determination 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 of 5 μg.kg–1 for sarafloxacin [14], and in milk food or infant foods with MS/MS detection [15-17]. A LC method with programmable fluorescence-ultraviolet detection was described for the determination of sulfadiazine, sulfapyridine, sulfathiazole, sulfadimidine, and 7 FQs 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 of 6 FQs without sarafloxacin and 3 SAs residues in chicken was described with the LOQ of 15.29-22.85 μg.kg–1 for the SAs [19]. In general, HPLC-MS/MS method has lower limit of detection (LOD) for detection of SAs and FQs than HPLC-UV/DAD method [20], but it is not widely available in general laboratories because of its high price. A multiresidue determination of enoxacin, lomefloxacin, sulfanilamide, sulfamethoxazole, and tetracycline in porcine tissue was reported by the matrix solid-phase dispersion extraction (MSPD)-HPLC-DAD with the LOQ of 7-34 μg.kg-1 [21].

Sample pretreatment is always a crucial step in deciding the levels of detection limits of the overall method, especially when large number of aquatic product with complex matrices is involved, and rapid extraction becomes even more essential. To extract SAs in matrices, various traditional methods have been studied but most of them required large volumes of solvents and were time-consuming [7,18]. A SPE column was used for clean-up extracts of SAs after traditionally extraction with solvent [5,18], but the recovery and precision need to be improved. An ionic liquid aqueous two-phase system was developed for the separation of sulfadiazine and sulfamethoxazole in water samples and aquaculture products [8]. A simplified method with single-step extraction was developed for the extraction and determination of seven FQs residues and three quinolones in porcine muscle, table eggs, and commercial whole milk [14]. Recently, we reviewed the application of accelerated solvent extraction (ASE) in multiresidue analysis for food and feed [22]. It is becoming increasingly important as a sample preparation technique in food analysis, combining the benefits of high-throughput, automation and low solvent consumption. Water was used as a solvent of ASE for extraction of SAs from cattle and trout muscle tissues and of veterinary drugs from bovine muscle tissues [23,24]. Methanol and CAN were used for ASE of SAs in animal feed [25], and enrofloxacin and ciprofloxacin in table eggs [26], respectively. The analysis of two classes of antibiotics in meat is quite complex as they have different performances and may bind to the lipoproteins. To achieve trace residue analysis with speediness, high-throughput and high sensitivity, it is important to develop a new and effective extraction-cleanup system for simultaneous determination of SAs and FQs.

Sarafloxacin could be combined with SAs in animal treatments. The MRL of sarafloxacin in pork and chicken muscle is lowest for FQs in animal foods. However, to our knowlege there were few reports for simultaneous determination of SAs and sarafloxacin in pork and chicken muscle by HPLC. This work selected the three SAs and sarafloxacin in pork and chicken muscle as target, and used ASE–SPE for sample preparation and cleanup. The developed method was used for the determination of 4 residual drugs in pork and chicken muscle at lower than their MRL levels.

Experimental

Chemicals and solutions

Sulfadiazine, sulfamerazine, sulfametoxydiazine, and sarafloxacin (purity: ≥ 99%) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All chemicals and reagents were of analytical grade except specific statements. Methanol (HPLC grade), acetonitrile (HPLC grade), ethyl acetate, triethylamine, and phosphoric acid were obtained from Beijing Chemical Factory (Beijing, China). Doubly deionized water was used throughout. The methanol was filtered through a 0.22 μm microporous membrane of polyvinylidene fluoride before use. Phosphate buffer solution with 0.1% triiethylamine was filtered through a 0.45 μm microporous membrane of mixed cellulose ester for HPLC analysis.

Single sulfonamide stock standards of 400 mg/L were prepared in methanol. The stock solutions were stored at -4°C. Mixed standard working solutions were prepared by diluting the standard stock solution just before use.

Instrumentation

The chromatographic system consisted of a Shimadzu HPLC system equipped with an LC-10A Multisolvent Delivery System, a DGU-12A online degasser, an SCL-10Avp gradient controller, a CTO-10Avp column thermostat, and a multi-wavelength SPD-M10Avp photodiode-array detector (DAD) covering the range 190-800 nm, which was interfaced to a computer for data acquisition using a CLASS-VP workstation (Shimadzu, Kyoto, Japan). The extraction equipment was an APLE 2000 automatic accelerated solvent extraction apparatus (Beijing Titan Instruments Co., Ltd, China) equipped with 33 mL stainless-steel extraction cells. A centrifuge TGL-16M (Xiangyi Centrifuge Co., Hunan, China), an ultrasonic cleaner (Ultrasonic Instrument Co., Kunshan, China), RE-2000A rotary evaporator (Asia Rong Biochemical Instrument Factory, Shanghai, China) and a PHS-3C pH meter (Shanghai Precision & Scientific Instrument Co., Shanghai, China) were used in sample treatment.

ASE and SPE

Pork and chicken samples were purchased from a local market in Baoding, and after being homogenized in a high-speed food blender, they were stored below -20°C in a freezer until the time of analysis.

Samples were extracted with the APLE 2000 automatic accelerated solvent extraction apparatus. Approximately 10 g of the blank/spiked sample material mixed with 5 g of diatomite was packed in a 33 mL stainless-steel extraction cell. Each cell was locked with stainless-steel screw caps equipped with Teflon O-ring sealing and circular glass microfiber filters of 1.98 cm diameter were placed above and below the packing. Acetic acid-acetonitrile (2:98, v/v) was used as extraction solvent. Conditions used in the extraction were: an oven temperature 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 40% of the extraction cell volume. The extracted analytes were purged from the sample cell using pressurized nitrogen for 2 min. Finally, each resulting extract was centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant evaporated to dryness under vacuum distillation at 37°C, the residue was rinsed with water, evaporated to dryness under a nitrogen flow at 45°C and re-dissolved in 10 mL water for clean up.

The obtained solution was applied to an Oasis HLB SPE cartridge (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, the SPE cartridge was washed with 5 mL of water. The analyte was eluted with ammonia-methanol (5:95, v/v). The collected eluate was evaporated to dryness under vacuum distillation under a nitrogen flow at 45°C, and the residue was re-dissolved in 1.0 mL of phosphate buffer with 0.01% triethylamine-methanol (75:25,v/v). The solution was filtered through a 0.22 μm microporous membrane of polyvinylidene fluoride for HPL Canalysis.

HPLC analysis

The separation of sulfadiazine, sulfamerazine, sulfametoxydiazine, and sarafloxacin was carried out using a Kromasil 100-5 C18 analytical column (250×4.6 mm ID, 5 μm, AKZO NOBEL, Bohus, Sweden), and a six-port valve with a 20 μL sample loop injector was used. A mobile phase is composed of 50 mM potassium dihydrogen phosphate (adjusted the pH to 3.05 with phosphoric acid) with 0.01% triethylamine and different proportion of methanol. The flow rate of the mobile phase was set at 1.0 mL min-1. A 20 μL volume of sample solution was 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 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 solution was monitored at 267 nm for sulfadiazine, sulfamerazine and sulfametoxydiazine, and at 280 nm for sarafloxacin. The linear equations for the relationship between the peak areas of analytes and their concentration were determined by least-squares method. Quantification was carried out by using standard curve calibration based on peak area toward concentration in 7 concentration points.

Results and Discussion

Optimization of LC conditions

Effects of gradient program: The initial test showed that the separation of the four analytes could achieved using a mobile phase composed of 50 mM potassium dihydrogen phosphate (pH 3.05) with 0.01% diethylamide (reagent for eliminating peak tails) and different proportion of methanol. The sensitivity increased and retention time decreased with increase in the ratio of methanol from 25% to 40% (v/v) (Figure 1). The subsequent test for the spiked sample analysis showed that low sensitivity of sarafloxacin was observed when 25% (v/v) methanol was used, and the peaks of sulfamerazine and matrices could not be separated when 30-40% methanol was used. So that it is necessary to use gradient program (Figure 1).

chromatography-separation-techniques-Chromatograms-analyte-mobile

Figure 1: Chromatograms of the analyte standard solutions with the mobile phase composed of 50 mM potassium dihydrogen phosphate (pH 3.05) with 0.01% diethylamide and different proportion of methanol.

Three gradient programs were compared by changing proportion of methanol in mobile phase. Figure 2 shows that a better gradient program was as follows: methanol set at 25% for 0→5 min, increased from 25% to 40% for 5→7 min, retained 40% between 7 and 11 min, and returned from 40% to 25% for 11→13 min. Using this program the peaks of sulfamerazine and matrices could be separated and high sensitivity for sarafloxacin could be achieved (Figure 2).

chromatography-separation-techniques-Chromatograms-analyte-gradient

Figure 2: Chromatograms of the analyte standard solutions using gradient programs.

Optimization of buffer pH

The pH value of buffer solution influenced mainly the resolution and the apparent mobility of the target analytes. Generally, for compounds with lower pKa values, the dissociation ratio is higher and acidity is stronger. The pH of buffer influences ionization of the analytes. The mobile phase constituted of 50 mM potassium dihydrogen phosphate 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 in figure 3.

chromatography-separation-techniques-Effect-different-separation

Figure 3: Effect of different pH of buffer solution on separation.

It is shown that when pH from 2.55 to 4.05 the four compounds can be separated. The migration order is related to the order (from high to low) of pKa. Using buffer of pH 3.05, high sensitivity for sarafloxacin was achieved, and the separation of the four analytes could be achieved within 14 min, so it was selected in this work.

Selection of detection wavelength

UV-DAD detection in the range of 190-800 nm was investigated. The result showed that higher sensitivity was achieved by detection of the SAs at 267 nm and sarafloxacin at 280 nm (Figure 4). For the 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).

chromatography-separation-techniques-Chromatograms-analyte-solutions

Figure 4: Chromatograms of the analyte standard solutions.

Optimization of ASE conditions

Effect of extraction solvent: The selection of a suitable extraction solvent is the first challenge in ASE method development. Several solvents have been used to ASE for the preparation of food samples. The polarity of the extraction solvent should closely match that of the target compounds. In this work, the extraction efficiency for using acetonitrile and 2% (v/v) acetic acid in acetonitrile as extraction solvent was investigated and compared for simultaneous extraction of the SAs and sarafloxacin from a meat sample (Figure 5 left).

chromatography-separation-techniques-solvent-temperature-recover

Figure 5: Effect of solvent, temperature, and flush volume on recovery.

The polarity of the extraction solvent should closely match that of the target compounds. Using methanol the recovery of three SAs was 80-90%, but the recovery of sarafloxacin was only 70%. Use of 2% (v/v) acetic acid in acetonitrile has higher extraction efficiency than methanol, the recovery for the four compounds achieved to 90-100%.

Effect of extraction temperature: Temperature is one of the most important parameter for ASE. The extraction efficiency with 2% (v/v) acetic acid in acetonitrile as extraction solvent for simultaneous extraction of the four analytes from pork muscle sample was compared under temperature of 60-90°C at 10.3 MPa (Figure 5 middle). The result showed that highest extraction efficiency (90-105%) was achieved for each compound under temperature of 70°C.

Effect of extraction time: The extraction process can be conducted in a static or dynamic mode. In the static extraction mode, the critical factors are the temperature and time of the extraction. During this process, the analytes are isolated from the sample under stable static conditions. The static process can be repeated several times if low recoveries are obtained in a single stage. In this work, the efficiency of the extraction was investigated using static time of 5, 10, 15 min and two cycles. The result showed that significant amounts of the analytes 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 recovery during the extraction step, three consecutive extractions were made further to optimize the number of cycles. To save time, two extraction cycles were used with extraction efficiency of ≥ 90%. So 5 min static times and 2 static cycles were selected in further work.

Effect of flush volume: Flush volume is the percentage of fresh volume introduced into the cell after the static time to drag the analytes towards the collection vial. This volume ensures that all analytes are 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, a flush volume of 40% (cell volume, 33 mL) was enough to push the analytes extracted out of the cell with higher extraction efficiency (92-108%).

Optimization of cleanup: The solution obtained by ASE was applied for cleanup. Cleanup procedure was investigated by using AccuBONDII ODS-C18 SPE columns (100 mg/3 mL, Agilent, USA) and Oasis HLB (500 mg /6 mL, Waters, USA). The SPE columns were preconditioned with 5 mL of methanol and 5 mL of water. After sample loading, the column was washed with 5 mL water, and the analytes were eluted with ammonia-methanol (5:95, v/v). The collected eluate was evaporated to dryness under vacuum distillation under a nitrogen flow at 45°C, and the residue was re-dissolved in 1.0 mL of phosphate buffer with 0.01% triethylamine-methanol (75:25,v/v). Oasis HLB column and AccuBONDII ODS-C18 column allowed to achieving the recoveries of >92% and >80%, respectively. The HLB column was selected in this work.

Performance of the method

Selectivity: Under the optimized conditions, selectivity was determined for each analyte in the assay. Blank meat sample and spiked meat sample were treated with the ASE-SPE procedure. The chromatograms were obtained, as shown in figure 6.

chromatography-separation-techniques-Chromatograms-blank-spiked

Figure 6: Chromatograms of blank meat sample and spiked meat sample.

It was shown that the four drugs could be baseline separated. The unknown peak does not interfere with the separation and determination of the four analytes. There was no interference peak in real samples.

Linearity and detection limit: The linearity for analysis of the studied analytes was evaluated with concentrations of calibration standards (7 concentration point) against measured peak areas under the optimized conditions. The equations of calibration curves obtained 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 coefficient (r) greater than 0.999.

Analyte Linear range ?μg.mL–1? Regression equation Correlation coefficient (r) LOD (μg.mL–1) LOQ (μg.mL–1)
Sulfadiazine 0.12–100 A=2.120×104C –1.038×104 0.9999 0.038 0.12
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
Sarafloxacin 0.04–40 A=9.710×104C –3.739×104 0.9998 0.013 0.04

Table 1: Linearity regression equation, correlation coefficient, linear range, limit of detection (LOD) and limit of quantification (LOQ).

The limit of detection (LOD) was determined as the sample concentration that produces a peak with a height three times the level of the baseline noise, and the limit of quantification (LOQ) was calculated that produced a peak with 10 times the signal-to-noise ratio. Table 1 gives the instrument LODs and LOQs. For 10 g of sample and 1 mL of final test solution, the method LOD values were 3.8, 1.5 and 3.0 μg.kg–1 for sulfadiazine, sulfamerazine, and sulfametoxydiazine, and 1.3 μg.kg–1 for sarafloxacin in meat sample, and their method LOQ values were 12, 5, 10 μg.kg–1 and 4 μg.kg–1. The obtained LOQs values were lower than those of recently reported LC [5,18,20] and CE-MS/MS [13] for the SAs, and LC-MS/MS methods for sarafloxacin [17]. The proposed method permits simultaneous detection of the SAs and sarafloxacin in meat samples at lower than the required MRLs.

Repeatability: The precisions of the method were investigated by analyzing the four analytes in spiked blank pork muscle. Under the optimized conditions the intraday variability (RSD) of peak area for 6 determinations and inter-day RSD for 2 determinations per a day within 3 days were investigated. The results are listed in table 2.

Analyte Spiked level (μg.kg–1) Intra-day?RSD, n=6? (%) Inter-day (RSD,n=6) (%)
Sulfadiazine 10 0.5 0.7
20 0.8 0.9
50 0.8 1.0
Sulfamerazine 10 0.9 1.3
20 1.1 1.1
50 0.6 1.3
Sulfametoxydiazine 10 0.3 0.9
20 0.5 0.7
50 0.3 1.0
Sarafloxacin 5 1.2 1.7
10 1.5 1.8
25 1.1 1.5

Table 2: Intra-day and inter-day precision as relative standard deviayion (RSD).

The intra- and inter-day RSDs of the four analytes were in the range of 0.3-1.5% and 0.7-1.8%, respectively. It was shown that the repeatability of the method is satisfactory for the residue determination of the studied SAs and sarafloxacin in meat samples.

Sample analysis: Under the optimized conditions of ASE and HPLC, pork and chicken muscles were analyzed. The studied four drugs were not detected in meat samples. The recovery test of the assays for the four drugs was carried out by adding known amounts of these drugs to the meat samples. The recoveries are listed in table 3.

Analyte Spiked level (μg.kg–1) Pork Chicken
Recoveries (%) RSD(n=6) (%) Recoveries (%) RSD(n=6) (%)
Sulfadiazine 10 104 0.6 108 0.5
20 106 0.5 104 0.8
50 97 0.9 103 0.8
100 99 0.8 96 0.8
Sulfamerazine 10 92 1.0 92 0.9
20 96 0.7 95 1.1
50 98 0.9 96 0.6
100 98 0.8 98 0.7
Sulfametoxydiazine 10 93 0.3 92 0.3
20 95 0.4 90 0.5
50 96 0.6 95 0.3
100 100 0.5 97 0.4
Sarafloxacin 5 90 0.9 89 1.2
10 92 1.2 85 1.5
50 88 1.0 91 1.1
100 98 0.5 97 0.7

Table 3: Determination of the SAs and sarafloxacin in pork and chicken muscle spiked at three concentation levels for each analyte.

The data showed that the average recovery was 88-106% with the RSD of 0.3-1.2% for pork muscle, and 85-108% with the RSD of 0.3-1.5% for chicken muscle. It is indicated that the proposed method has high recovery, and can be used for the determination of the studied drugs in real samples.

Conclusions

A simple, selective and sensitive strategy for the determination of three SAs and sarafloxacin in two complex matrixes has been developed, showing the usefulness of ASE as a powerful tool for extraction without cleanup. The proposed ASE method provides faster extraction and reduces the volume of solvent required than conventional exaction techniques. The HPLC analysis combined with ASE provides a rapid and simple extraction procedure, good repeatability, high sensitivity, and effective separation. The proposed method permits the detection of the studied SAs and sarafloxacin in meat samples at lower than MRLs.

Acknowledgements

This work was supported by the Natural Science Found of Hebei Province (B2008000583) and the sustentation Plan of Science and Technology of Hebei Province (No. 10967126D).

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