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Qualitative and Quantitative Analysis of Dan-Deng-Tong-Nao Capsules by HPLC-DAD-MS/MS

Yunliang Zheng, Xingjiang Hu, Meihua Lin, You Zhai, Lihua Wu and Jianzhong Shen-Tu*

Research Center for Clinical Pharmacy, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, PR China

*Corresponding Author:
Jianzhong Shen-Tu
Research Center for Clinical Pharmacy
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases
First Affiliated Hospital, Zhejiang University
79# Qingchun Road, Hangzhou 310003, PR China
Tel: 86-571-87236537
E-mail: [email protected]

Received date: February 19, 2016; Accepted date: February 29, 2016; Published date: March 05, 2016

Citation: Zheng Y, Hu X, Lin M, Zhai Y, Wu L, et al. (2016) Qualitative and Quantitative Analysis of Dan-Deng-Tong-Nao Capsules by HPLC-DAD-MS/MS. Mass Spectrom Purif Tech 2:110. doi:10.4172/2469-9861.1000110

Copyright: © 2016 Zheng Y. 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

This review article discussed application of ionic liquids as matrices (ILMs) for mass spectrometry (MS). ILMs were applied for matrix assisted laser desorption/ionization, (ILMALDI-MS), electrospray ionization (ILMs-ESI-MS) and desorption corona beam ionization (DCBI-MS). Ionic liquids matrices provided several advantages such as low vapor pressure, have high stability for storage and under vacuum, extremely high sensitivity and showed low background or interferences. They are non-explosive, non-flammable and are thermally stable. The materials are promising for real measurements and require further investigations to improve the current performance. The combination between the conventional matrices and organic bases leaded to high ionization performance. The proton transfer efficiency of ILMs is higher than conventional matrices as the proton take place from the salt form. They were applied for many analytes such as biomarker, protein, peptides, polymers (synthetic and nature), small organic compounds and pharmaceuticals drugs.

Keywords

Dan-Deng-Tong-Nao capsules; Quantitative analysis; Chemical fingerprint; Quality control; HPLC-DAD-MS/MS

Introduction

“Dan-Deng-Tong-Nao capsules” (DDTNCs) is a commonly used traditional Chinese formula medicine, which is prepared (extracted, purified and combined) from four commonly used Chinese herb drugs, including Radix Salvia miltiorrhiza, Radix Pueraria lobata, Erigeron breviscapus and Rhizoma chuanxiong. DDTNCs have the ability of activating blood circulation to dissipate blood stasis, removing obstruction in the meridians and heart-nourishing effects [1,2]. It was usually used for the treatment of apoplexy and the syndrome of apoplexy involving both collaterals and meridians caused by obstruction of collaterals by blood stasis [3,4].

Chemical constituents of the four component herbs of DDTNCs have been intensively studied [5-8]. However, little is known about the chemical composition of DDTNCs, and the current analysis methods could not reflect the quality of it [9,10]. Therefore, practicable and reliable quality evaluation method to monitor the systematic chemical profile changes of DDTNCs in the procedure from preparation and storage to clinic usage is of significant importance.

In this paper, an HPLC-DAD-MS/MS method was established for rapidly identifying and reliably determining multiple components in DDTNCs, and was then successfully applied to quantitative and fingerprint analysis of fifteen batches of DDTNCs samples. This study represents the first detailed investigation of the components of DDTNCs and provides an applicable method for its comprehensive quality evaluation.

Experiment

Apparatus, materials, chemicals and reagents

HPLC-DAD analysis was carried out on an Agilent 1200 Series HPLC with diode-array detector; MS/MS was performed on an Agilent 6410 Triple Quad MS with an electrospray ionization (ESI) interface (Agilent Technologies, Inc., USA). Fifteen batches of DDTNCs were purchased from different pharmacies in China. Standards of danshensu, 5-hydroxymethylfurfural, 3,4-dihydroxybenzaldehyde, epicatechin, puerarin and salvianolic acid A (Structures shown in Figure 1) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products(Beijing, China), their purities (>98%) were determined by normalization of the peak area detected by HPLC–UV. HPLC grade reagents (methanol, acetonitrile and formic acid) were obtained from Merck (Darmstadt, Germany). Water was purified using a Milli-Q system (Milford, MA, USA).

mass-spectrometry-purification-techniques-Structures-identified-compounds

Figure 1: Structures of the identified compounds from DDTNCs.

Chromatographic and MS conditions

The separation was performed on an Agilent Zorbax SB-Aq column (250 mm × 4.6 mm i.d., 5 μm) with a C18 guard column (10 mm × 4.6 mm i.d., 5 μm) at 35°C with a flow rate of 0.8 ml/min. Elution was achieved with a linear gradient solvent program of methanol (A) in water–0.2% acetic acid (B) (v/v): 0–40 min, 20–55% A; 40–41 min, 55–95% A; 41–45 min, 95% A; 45–46 min, 95-20% A. The UV spectra (DAD) were recorded between 190 and 400 nm, and the wavelength of 280 nm was chosen for determination.

The ESI-MS/MS detection was acquired directly after DAD measurement in positive ionization mode, and recorded on a mass range of m/z 100-1000. Drying gas (Nitrogen) temperature was set at 350°C with a flow rate of 10.00 L/min and nebulizer pressure of 45.00 psi. Capillary voltage was 4000 V.

Preparation of standard and sample solutions

Accurately weighed solid portion of each standard was dissolved in methanol to prepare stock solutions: 1.60 mg/mL for danshensu, 2.50 mg/mL for 5-hydroxymethylfurfural, 2.88 mg/ mL for 3,4-dihydroxybenzaldehyde, 2.90 mg/mL for puerarin, 2.50 mg/mL for epicatechin and 3.00 mg/mL for salvianolic acid A. A mixture stock solution containing the six standards (300 μg/mL for danshensu, 125 μg/mL for 5-hydroxymethylfurfural, 140 μg/mL for 3,4-dihydroxybenzaldehyde, 580 μg/mL for puerarin, 125 μg/mL for epicatechin and 300 μg/mL for salvianolic acid A) was prepared in 50% methanol and stored away from light at 4°C until used. Working solutions of the lower concentration were prepared by appropriate dilution of the stock solution with 50% methanol. The chromatogram of the six standards is shown in Figure 2.

mass-spectrometry-purification-techniques-representative-chromatogram-samples

Figure 2: The representative HPLC-DAD chromatogram of DDTNCs samples at 280 nm (A: Standards; B: Sample).

Sample solution preparation

The powder of DDTNCs (about 50 mg) was extracted with 20.0 mL solvent composed of 80% methanol in water (v/v) for 20 min in an ultrasonic bath. 1.0 mL of the extract solution was centrifugation at 15,000 rpm for 5 min and 5 μL of the supernatant was injected into the HPLC-DAD-MS/MS system. Content for each component was calculated from its corresponding calibration curve.

Results

Identification of components from DDTNCs

The traditional methods to isolate different standard compounds from herb drugs are restricted and laborious. However, HPLC coupled The traditional methods to isolate different standard compounds from herb drugs are restricted and laborious. However, HPLC coupled

ESI in both negative and positive mode was tried to detect components in DDTNCs. The results indicated that more peaks could be detected in positive mode than in negative mode. For identification of components contained in the crude extracts of DDTNCs, mass fragmentation with MS2 spectrometry data provided valuable information. An efficient transmission of molecular ions and fragment ions was obtained by ramping the collision energy from 10 V to 50 V for the rapid identification of trace level components in the crude extracts with large sample throughput.

In the positive ESI-MS spectra of DDTNCs, the quasi-molecular ion peaks of components always appeared as [M+H]+ ion, except for peak 17. Most peaks of components had the fragments of [M+H-18]+, [M+H-28]+ and [M+H-15]+ corresponding to the loss of “H2O”, “CO” and “CH3”, respectively. Furthermore, there were some fragments of [M+H-176]+ which corresponded to the loss of “Glu” and indicated the structure belonged to glycosides.

Through on-line DAD and ESI–MS spectra, six major peaks: 1, 2, 4, 7, 9 and 26 in the chromatogram of DDTNCs (Figure 2) were identified by comparing their retention time, ultraviolet absorption and MS/MS spectra with standard compounds. Finally, 26 compounds were characterized by comparing their retention times, UV spectra and MS data with those of reference compounds or literature data [11-16], and most of them belonged to flavonoids, the representative cleavage pathway of flavonoid is shown in Figure 3. The results of the MS/MS and UV data are listed in Table 1, and the chemical structures of the identified compounds are shown in Figure 1.

mass-spectrometry-purification-techniques-representative-cleavage-pathway

Figure 3: The representative MS/MS cleavage pathway of flavonoids in DDTNCs.

Peak No. Rt (min) UV MS [M+H]+ MS/MS Identify
1 6.24 282, 288 199 181, 137, 125, 75 Danshensu
2 6.99 285 127 109, 99, 81,69,53 5 - Hydroxymethylfurfural
3 8.52 242, 295, 325 399 355, 337, 295, 283, 237, 181, 163 9-COAa
4 10.97 220, 280, 309 139 121, 93, 67 Protocatechualdehyde
5 11.80 250, 315 463 287, 17,159, 115 Scutellarin
6 12.56 198, 249, 310 433 397, 367, 313, 283 3’-Hydroxypuerarin
7 13.26  / 291 247, 205, 181, 127, 111 Epicatechin
8 15.36 240, 300, 340 603 559, 517, 441, 397, 379, 235 1-Malonyl-3,5-diCQAb
9 16.03 198, 250, 304 417 399, 381, 363, 351, 321, 297, 267 Puerarin
10 17.37 225, 260 447 432, 411, 327, 285, 270, 253 3’-Methoxydaidzin
11 17.90 215 475 299 Sophoraside A
12 18.91 235, 368 419 257, 137 Isoliquiritin
13 20.22 260, 325 433 415, 397, 313, 273, 257 Genistin
14 21.19 250, 300 417 255 Daidzein-4'-glucoside
15 22.74 280 447 285 Sissotorin
16 24.04 238, 290, 320 195 151, 119, 85, 57 Ferulic acid
17 25.10 250 471 [2M+Na]+ 247 [M+Na]+ Senkyunolide I
18 25.93 250, 290, 325 419 / Salvianolic acid D
19 26.55 195, 238 431 416, 365, 311, 255 Ononin
20 27.02 250 447 429, 411, 393, 351, 327, 297 3-Methoxypuerarin
21 28.14 248, 270, 306 417 399, 381, 351, 297, 255 Daidzin
22 29.58 215, 240 447 414, 383, 337, 297 '-Hydroxyl-4'methyldaidzin
23 30.16 275 247 188, 170, 146, 118 Unknown
24 32.02 210,280 447 429,414, 393, 337, 297, 4’-Methoxypuerarin
25 33.32 /  431 269 Unknown
26 35.20 230, 285, 340 495 385, 297, 187, 137 Salvianolic acid A
27 36.51 249, 310 255 227, 199, 173, 153, 137 Daidzein
28 38.17 250 285 270, 252, 225, 270, 213, 185 Biochanin A

Table 1: The on-line detected UV and MS spectrometric data of the identified and deduced compounds.

Validation of the method

Calibration curve, limits of detection and quantification: Solutions containing the six standards at six different concentrations were analyzed. The calibration curve of each analyte was constructed by plotting the peak area versus the corresponding concentration. The mixed standards solution was further diluted to a certain concentration to explore the limits of detection (LOD) and quantification (LOQ). The LOD and LOQ were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively. The satisfactory results regarding regression equations, correlation coefficients, linear ranges, LODs and LOQs were listed in Table 2. The high correlation coefficient values (R2>0.9992) indicated good linearity between their peak areas (y) and investigated compound concentration (x, μg/ml) in relatively wide concentration ranges.

Analytes Regressive equation R2 Test range (μg/ml) LOD (μg/ml) LOQ (μg/ml)
Danshensu y=3.333x-5.215 0.9992 1.60~160 0.45 1.00
5-Hydroxymethylfurfural y=43.555x-17.189 1.0000 1.25~125 0.10 0.41
3,4-Dihydroxybenzaldehyde y=18.995x-28.514 0.9996 0.35~70 0.11 0.32
Epicatechin y=3.798x-2.690 0.9994 1.25~125 0.12 0.40
Puerarin y=9.008x-19.794 0.9999 5.80~580 0.60 1.50
Salvianolic acid A y=7.392x-29.247 0.9995 3.00~300 0.45 1.30

Table 2: Linear regression data, LOD and LOQ of the six investigated components from DDTNC.

Precision, stability and accuracy of the method: The intra- and inter-day precisions were determined by continuously injecting the sample solution for six replicates on the same day and by measuring it once a day for three consecutive days, respectively. Stability was assessed by analyzing the same sample solution at different time within 24 h (0, 8, 12, 24 h) at room temperature.

Recoveries test that reflecting method’s accuracy was performed employing the method of standard addition. Three different quantities (low, medium and high) of the authentic standards were added into the known real sample. The recovery was figured out according to the following formula: Recovery (%)=(amount detected−original amount)/ amount spiked × 100%, and RSD (%)=(SD/mean) × 100%. All the results were estimated on the ground of relative standard deviation (RSD%).

Tables 3 and 4 list the results of precision, stability and recovery test, which suggested that the RSDs for the six compounds are less than 5.0%, the average recoveries of the six compounds is 94.6–104.8%. These data indicated that the established HPLC-DAD method was precise, accurate and sensitive enough for simultaneously quantitative determination of these six compounds in DDTNCs.

Analytes Intra-day precision (n=6) Inter-day precision (n=3 days) Stability (24 h)
Mean ± SD (μg/ml) RSDa (%) Mean ± SD (μg/ml) RSD (%) RSD (%)
Danshensu 7.16 ± 0.12 1.68 7.12 ± 0.29 4.03 1.00
5-Hydroxymethylfurfural 7.56 ± 0.29 3.84 7.62 ± 0.33 4.33 1.06
3,4-Dihydroxybenzaldehyde 0.48 ± 0.008 1.68 0.49 ± 0.02 4.49 1.91
Epicatechin 3.81 ± 0.11 2.89 3.91 ± 0.15 3.84 0.57
Puerarin 157.6 ± 4.77 3.03 159.3 ± 5.1 3.20 1.11
Salvianolic acid A 3.25 ± 0.07 2.15 3.27 ± 0.11 3.36 0.83

Table 3: Intra-day and inter-day precisions of the method and the stability of the extract samples.

Analytes Initial (μg/25 mg) Added (μg) Found (μg) Recoverya (%) RSD (%)
Danshensu 71.6 55.0 128.4 103.3 3.24
70.0 141.1 99.3 1.44
80.0 155.4 104.8 2.32
5-Hydroxymethylfurfural 75.6 60.0 137.1 102.5 3.34
75.0 150.1 99.3 4.31
90.0 165.3 99.7 3.45
3,4-Dihydroxybenzaldehyde 4.8 4.0 8.8 101.0 4.32
4.8 9.5 97.9 2.57
5.6 10.2 97.1 3.12
Epicatechin 38.1 30.0 68.4 101.0 1.87
37.5 75.2 98.9 2.33
45.0 83 99.8 2.36
Puerarin 1575.5 1250.0 2806.8 98.5 3.32
1550.0 3119.2 99.6 1.36
1900.0 3461.8 99.3 3.91
Salvianolic acid A 32.5 26.0 58.1 98.5 3.35
35.0 65.6 94.6 1.34
40.0 72.5 100.0 2.76

Table 4: Recovery of analytes from DDTNC through the current HPLC–DAD method.

Fingerprint analysis of DDTNCs: The chromatograms of different samples have to be standardized, so as to perform fingerprint analysis. The process of standardization included the selection of ‘‘common peaks’’ in chromatograms and the normalization of retention times of all the common peaks [17]. In this study, the extracts of fifteen samples of DDTNCs were analyzed. More than thirty peaks appeared in the HPLC-DAD chromatographic fingerprints as shown in Figure 2. The peak of puerarin (peak 9) was selected as a reference peak because it is an intense peak situated nearly in the middle of chromatogram. Among all the peaks observed, fifteen of them (1-5, 7, 9, 10, 14, 16, 17, 21, 22, 26 and 27) were defined as common peaks.

Similarity analysis was performed by using the software “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicines’’ (Version 2004A), which used to generate the mean chromatogram, and calculate the similarities of the sample chromatograms with mean chromatogram with cosine method. The average chromatogram of the fifteen batches of commercial samples was taken as the standard characteristic fingerprint of DDTNCs. It was subsequently compared with the chromatograms from fifteen batches of DDTNCs to generate the similarity values which were in the range of 0.671~0.991 (Table 5). It was found that correlation coefficients of DDTNCs samples varied considerably, indicating the quality of DDTNCs was inconsistent.

Batch no. Content of investigated components (μg/mg) Similaritya
Danshensu 5 –Hydroxyl- methylfurfural 3,4-Dihydroxy- benzaldehyde Epicatechin Puerarin Salvianolic acid A
C1710002 3.15 2.39 1.20 0.87 59.30 2.42 0.986
C1710011 2.73 1.33 0.21 0.80 65.60 1.76 0.945
C1710013 5.04 6.35 0.20 1.69 64.35 1.27 0.752
C1710017 2.48 1.24 1.19 0.89 58.35 3.06 0.983
C1710025 2.80 7.96 0.89 1.03 72.70 1.29 0.947
C1711006 3.00 3.33 0.20 2.07 63.80 1.27 0.976
C1711017 3.14 1.39 0.20 3.93 59.45 1.12 0.957
C1711011 3.00 3.16 0.19 1.66 65.55 4.33 0.991
C1711021 3.23 2.34 0.23 0.79 58.32 1.27 0.909
C1712001 4.15 3.26 0.29 1.54 54.23 2.10 0.866
C1712005 3.45 3.19 0.43 2.43 65.24 2.41 0.721
C1712012 5.28 2.35 0.32 1.34 61.26 3.09 0.671
C1713005 4.21 4.27 0.87 0.98 71.21 1.35 0.947
C1713014 3.68 1.96 0.24 2.31 49.38 1.49 0.952
C1713021 3.46 3.8 1.09 1.24 60.12 1.27 0.982

Table 5: Contents of the six investigated components and similarity of DDTNCs from fifteen different batches.

Quantitative analysis of DDTNCs: The proposed HPLC method was applied to analyze six components in fifteen batches of DDTNC. The quantitative analyses were performed by means of the external standard method. Samples were prepared as described in section “2.3”. The typical chromatogram is given in Figure 2. The results, shown in Table 5, indicate that puerarin showed the highest amount (43.60- 72.70 μg/mg), followed by 5-hydroxymethylfurfural (1.24-7.96 μg/mg), danshensu (1.48-5.04 μg/mg) and salvianolic acid A (0.85-4.33 μg/mg). Our results showed that the contents of the six analytes in different batches are noticeably different, which may occurred due to various factors such as cultivation, harvest, storage and so on.

Discussion

Optimization of HPLC conditions

The selection of the HPLC conditions was guided by the requirement for obtaining chromatograms with better resolution of adjacent peaks. The optimization of HPLC conditions was performed using standard mixture and sample extract. Different HPLC parameters including mobile phase (acetonitrile–water, methanol– water, acetonitrile–acid aqueous solution, methanol–acid aqueous solution), category of column (Agilent Zorbax SB-C18 250 mm × 4.6 mm i.d., 5 μm, Agilent Zorbax Extended-C18 250 mm × 4.6 mm i.d., 5 μm, Agilent Zorbax SB-Aq column 250 mm × 4.6 mm i.d., 5 μm) and column temperature (25, 35, 45°C) were examined and compared. The results showed that the best resolution and retention behavior were achieved when the methanol–water/ acetic acid (100/0.2, v/v) system and the column (Agilent Zorbax SB-Aq column 250 mm × 4.6 mm i.d., 5 μm) were used. Finally, an optimized HPLC condition was developed by comprehensively evaluating the resolution, baseline, elution time. Meanwhile, linear gradient was applied in HPLC procedure to obtain a perfect resolution, which could be examined by DAD and ESI-MS spectrometer. DAD detection was employed at wavelength range of 190–400 nm to investigate the UV spectra of the compounds in DDTNCs. It was found that UV at 280 nm was the best wavelength for the detection, because the most peaks can be detected under 280 nm. The optimal HPLC condition was shown in section “2.2”, and the representative HPLC-DAD chromatograms of DDTNCs samples are shown in Figure 2.

Optimization of extraction conditions

In order to obtain as many peaks as possible, related extraction conditions were examined and evaluated. Firstly, different extraction solvents (water and aqueous methanol of 20%, 50%, 70%, 80%, 90% and methanol (v/v)) were compared, the total number of peaks and the content of the six quantitative indicators were used as response to evaluate the extraction solvents. The results showed that the peak intensities of middle eluted constituents increased with the increase of methanol concentration in the extraction solution, while the peak intensities of early eluted constituents decreased with the increase of methanol concentration. The sample extracted with 80% methanol exhibited higher contents of the indicators than those with other tested solvents, which was further confirmed by the HPLC-DAD quantitative analysis method (Figure 4). Therefore, 80% methanol was selected as the extraction solvent for both identification and quantitative analysis of DDTNCs. Then extraction repetitions (1, 2, 3 times) and extraction time (10 min, 20 min, and 30 min) were also evaluated. Finally, by comparing the areas of characteristic peaks in chromatograms obtained under different conditions, the optimal condition for extraction of DDTNCs was selected and presented in detail in section “2.3”.

mass-spectrometry-purification-techniques-Comparison-extraction-efficiencies

Figure 4: Comparison of the extraction efficiencies of different solvents.

Conclusion

In the last decades, only one or two markers or pharmacologically active components in TCMs were usually employed for evaluating the quality of them, which could not give a complete picture of TCMs, because multiple constituents are usually responsible for its therapeutic effects. The proposed HPLC-DAD-MS/MS method makes it possible to comprehensively evaluate the quality of the commonly used TCM (DDTNCs) through a simultaneous qualitative and quantitative analysis of multi-components.

This method has been applied successfully to simultaneously identify and quantify the main components in different batches of DDTNCs. Results indicated: (1) the established method is rapid, simple and accuracy; (2) most ingredients of DDTNCs belong to flavonoids and phenolic acids; (3) the quality of the collected samples was inconsistent, which could be easily affected by the exterior factors, not only the processing methods, but also the harvested time and habitats of the four raw herbs.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (No. 81402807) and the Doctoral Fund of the Ministry of Education of China (No. 20130101120136).

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