Int J App Pharm, Vol 9, Issue 1, 2017, 30-36Original Article


A UPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF SOFOSBUVIR FROM HUMAN PLASMA

DARSHAN BHATTa,* B. RAJKAMALb

aMewar University, Gangrar, Chittorgarh 312901, Rajasthan, India, bKVK College of Pharmacy, Surmajiguda, Hyderabad 501512, Telangana, India
Email: darshanbhatt1984@gmail.com

Received: 13 Oct 2016, Revised and Accepted: 05 Dec 2016


ABSTRACT

Objective: The present work aimed to develop a simple, rapid, specific and precise ultra-performance liquid chromatography-tandem mass spectrophotometric (LC–MS/MS) validated method for quantification of sofosbuvir and internal standard (ISTD) Sofosbuvir-d3 in human plasma.

Methods: Samples prepared by employing liquid-liquid extraction (LLE) using 2.5 ml of ethyl acetate. Chromatographic separation was achieved on Gemini 5µ C18, 50 x 4.6 mm column using a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 30:70 v/v as the mobile phase. The flow rate was 0.50 ml/min. The LC eluent was split, and approximately 0.1 ml/min was introduced into Tandem mass spectrometer using turbo Ion Spray interface at 325 °C. Quantitation was performed by transitions of 428.35/279.26 (m/z) for sofosbuvir and 431.38/282.37 (m/z) for sofosbuvir-d3.

Results: The concentrations of ten working standards showed linearity between 4.063 to 8000.010ng/ml (r2 ≥ 0.9985). Chromatographic separation was achieved within 2 min. The average extraction recoveries of three quality control concentrations were 75.36% for sofosbuvir and were within the acceptance limits. The coefficient of variation was ≤15% for intra-and inter-batch assays. The %CV of ruggedness ranges 0.35% and 3.09%. The % stability of short term and long term stock solution stability studies was found to be 97.25% and 98.81% respectively.

Conclusion: The results obtained for specificity, linearity, accuracy, precision, ruggedness and stability studies were within the acceptance limits. Thus the validated economical method was applied for pharmacokinetic studies of sofosbuvir.

Keywords: Sofosbuvir, LC-MS/MS, Human plasma, Stability studies


INTRODUCTION

Sofosbuvir, a phosphoramidate prodrug, is chemically described as (S)-Isopropyl 2-((S) ((2R, 3R, 4R, 5R)-5-(2, 4-dioxo-3,4-dihydro-pyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4 methyl tetrahydrofuran-2-yl) methoxy)-(phenoxy) phosphorylamino) propanoate [1-2]. Literature survey reveals two HPLC methods for determination of sofosbuvir from its bulk and pharmaceutical dosage forms [3-4]. Three UPLC-MS/MS method were reported for quantification of sofosbuvir from its metabolites and along with other drugs from human plasma [5-7]. Described here is a simple, sensitive, and selective UPLC-MS/MS method for sofosbuvir in the human plasma concentration range of 4.063 to 8000.010ng/ml. As there is no literature on stability and validation details of sofosbuvir estimation from human plasma, this study performed assay validations, according to the FDA guidelines [8]. While this method with validation details were economical and applied for pharmacokinetic studies of sofosbuvir.

MATERIALS AND METHODS [5]

Apparatus and software

The UPLC (Waters, Model Acquity) was coupled with Mass spectrometer (Waters Quattro Premier XE) having Turbo Ion Spray (Waters Quattro Premier XE). The chromatographic integration was performed by MassL ynx V4.1 software.

Chemicals and reagents

Sofosbuvir and Sofosbuvir-d3 (IS) were procured from Mylan Laboratories Ltd, Hyderabad, Formic acid, Methanol and ethyl acetate was procured from Merck Specialities Pvt. Ltd, Mumbai, India. Water used was collected from water purification systems (Milli Q, MilliPore, USA) installed in the laboratory. Pooled drug-free expired frozen human plasma (K2-EDTA as anticoagulant) was obtained froma Blood Bank, Hyderabad, was used during validation and study sample analysis. The plasma was stored into-70±5 °C.

Standards and working solutions

Calibration standard solutions

Stock solutions of sofosbuvir and Sofosbuvir-d3 internal standard (IS) were prepared in methanol. Further dilutions were carried out in 50% methanol. Calibration standards often concentration levels were prepared freshly by spiking drug-free plasma with a sofosbuvir stock solution to give the concentrations of 4.063, 8.125, 62.5, 125.0, 250, 500, 1000, 2000, 4000 and 8000ng/ml.

Quality control standards

Lowest quality control standards, Median quality control standards and highest quality control standards were prepared by spiking drug-free plasma with sofosbuvir to give a solution containing 11.488, 522.180 and 7252.503 ng/ml respectively. They were stored at-20 °C till the time analysed.

Chromatographic conditions

Chromatographic separation was performed on Gemini 5µ C18, 50 x 4.6 mm, analytical column and the mobile phase was a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 30:70 v/v. Injection volume was 10μL. The flow rate was 0.50 ml/min. Total analysis time of single injection was 2.0 min. Column oven temperature and autosampler temperature was set to 30 °C and 10 °C, respectively.

Mass spectrometric conditions

The LC eluent was split, and approximately 0.100 ml/min was introduced via electrospray ionisation using a Turbo Ion Spray interface set at 325 °C to generate positive ions [M+H]+. The Mass spectrometric parameters were optimised as shown in table no 1.

Table 1: Mass spectrometric conditions

Capillary voltage 3500V
Nozzle voltage 1500V
Delta EMV(+) 500 Positive
Gas flow 5 L/min
Gas temperature 350 °C
Nebulizer pressure 25 psi
Sheath gas temperature 300 °C
Sheath gas flow 11L/min
Acquisition
Parameters Sofosbuvir
Transition 428.35/279.26 (m/z)
Polarity Positive
MS1 resolution Unit
MS2 resolution Unit
Dwell time (millisec) 200
Fragmentor (V) 100
Collision energy (V) 8

Sample preparation method

To 250 µl of plasma, 50 µl of ISTD (1µg/ml) and 50 µl of 0.1% formic acid was added and vortexed. The drug was extracted with 2.5 ml of ethyl acetate, followed by centrifugation at 2000 rpm/min on a cooling centrifuge for 15 min at 4 °C. The supernatant of 2 ml was withdrawn and evaporated at 50 °C 15 psi of nitrogen until dryness at LV evaporator. The residue was reconstituted with 500 µl of mobile phase, and respective samples were injected into the column.

Validation [9-13]

Specificity

A solution containing 4.063ng/ml was injected onto the column under optimised chromatographic conditions to show the separation of sofosbuvir from impurities and plasma. The specificity of the method was checked for the interference from plasma.

Linearity

Spiked concentrations were plotted against peak area ratios of sofosbuvir to the internal standard and the best fit line was calculated. Wide range calibration was determined by solutions containing4.063 to 8000.010ng/ml.

Recovery studies

The % mean recoveries were determined by measuring the responses of the extracted plasma Quality control samples at HQC, MQC and LQC against unextracted Quality control samples at HQC, MQC and LQC.

Precision and accuracy

The between-run (Inter-day) accuracy and precision evaluation were assessed by the repeated analysis of human K3 EDTA plasma samples containing different concentrations of sofosbuvir on separate occasions. A single run consisted of a calibration curve plus six replicates of the lower limit of quantitation, low, medium and high-quality control samples.

Within-run (Intraday) accuracy and precision evaluations were performed by analysing replicate concentrations of sofosbuvir in human K3 EDTA plasma. The run consisted of a calibration curve plus a total of 24 spiked samples, six replicates of each of the LLOQ, lower, medium and higher quality control samples.

Matrix effect

The matrix effect for the intended method was assessed by using chromatographically screened human plasma. Concentrations equivalent to LLOQ of Sofosbuvir were prepared with seven different plasma batches/lots. Samples were analysed along with one set of freshly spiked CC Standards prepared in the screened biological matrix.

Ruggedness

The ruggedness of the method was assessed by analysing a precision and accuracy batch using a different column, by the different analyst in another instrument.

Stability studies

Short-term stock solution stability of sofosbuvir

Solutions of sofosbuvir were prepared in methanol (Stability Samples) and were kept at room temperature for 6 h 30 min. A freshly prepared solution of sofosbuvir (Comparison Samples) and stability samples were diluted at approximately the same analyte concentration and analysed in a single run; analyte responses were used to determine % stability over time.

Short-term stock solution stability of internal standard

Solutions of internal standard (Sofosbuvir-d3) were prepared in methanol (Stability Samples) and were kept at room temperature for 6 h 30 min. A freshly prepared solution of internal standard (Comparison Samples) and stability samples were diluted at approximately the same analyte concentration and analysed in a single run; Analyte responses were used to determine % stability over time.

Long-term stock solution stability of sofosbuvir

Solutions of Sofosbuvir were prepared in methanol (Stability Samples) and were kept at refrigerator (2-8 °C) for 10 D 02 H. A freshly prepared solution of sofosbuvir (Comparison Samples) and stability samples were diluted at approximately the same analyte concentration and analysed in a single run.

Long-term stock solution stability of internal standard

Solutions of Internal standard were prepared in methanol (Stability Samples) and were kept at refrigerator (2-8 °C) for 10 D 02 H. A freshly prepared solution of internal standard (Comparison Samples) and stability samples were diluted at approximately the same analyte concentration and analysed in a single run.

Freeze-thaw stability

Samples were prepared at low and high-quality control levels, aliquoted and frozen at-70 °C. Some of the aliquots of quality control samples were subjected to five freeze-thaw cycles (stability samples). A calibration curve and quality control samples were freshly prepared (Comparison Samples) and processed with 6 replicates of stability samples and analysed in a single run.

RESULTS AND DISCUSSION

The chromatography observed during the course of validation was acceptable and representative chromatograms of standard blank, HQC, MQC, LQC and LLOQ are shown in fig. 1-3.

Fig. 1: Chromatograms of standard blank and HQC matrix


Fig. 2: Chromatograms of MQC and LQC


Fig. 3: Chromatograms of LLOQ

The method developed was validated for specificity, accuracy and precision, linearity, ruggedness and stability as per FDA guidance [9-11]. The results of validating parameters are given below.

Specificity

Nine different lots of plasma were analysed to ensure that no endogenous interferences were present at the retention time of sofosbuvir and Sofosbuvir-d3. Nine LLOQ (4.063 ng/ml) level samples along with plasma blank from the respective plasma lots were prepared and analysed. (table 2). In all plasma blanks, the response at the retention time of sofosbuvir was less than 20% of LLOQ response and at the retention time of IS, the response was less than 5% of mean IS response in LLOQ. The typical chromatogram of plasma blank and the chromatogram of LLOQ was shown in (fig. 1).

Table 2: Results of specificity for sofosbuvir and sofosbuvir-d3 (ISTD)

S. No.

Drug response

ISTD response

STD BL

LLOQ

% Interference

STD BL

LLOQ

% Interference

Area

RT

Area

RT

01

0

298

0.800

NIL

0

61776

0.800

NIL

02

0

290

0.800

NIL

0

66613

0.800

NIL

03

0

334

0.800

NIL

0

70621

0.800

NIL

04

0

267

0.807

NIL

0

64807

0.800

NIL

05

0

271

0.800

NIL

0

67694

0.800

NIL

06

0

303

0.800

NIL

0

65249

0.800

NIL

07

0

281

0.800

NIL

0

68774

0.800

NIL

08

0

255

0.800

NIL

0

62927

0.800

NIL

09

0

147

0.800

NIL

0

37012

0.800

NIL

10

0

283

0.800

NIL

0

66641

0.800

NIL


Linearity

The calibration curve (peak area ratio Vs Concentration) was linear over working range of 4.063 to 8000.010ng/ml with ten point calibration used for quantification by linear regression, shown in (fig. 2). The regression equation for the analysis was Y=0.0011227x-0.000164437 with coefficient of correction (r2) = 0.9985.

Recovery

The % mean recovery for sofosbuvir in LQC, MQC and HQC was 75.47%, 74.37% and 76.26% respectively (table 3).

Fig. 3: Spiked concentrations (4.063 to 8000.010ng/ml) were plotted against calculated concentration Vs concentration with ten point calibration used for quantification by linear regression


Table 3: The % mean recovery of sofosbuvir for LQC, MQC and HQC

S. No.

HQC

MQC

LQC

Aqueous
area ratio

Extracted area ratio

Aqueous area ratio

Extracted area ratio

Aqueous area ratio

Extracted area ratio

01

13.466

8.226

0.981

0.598

0.021

0.013

02

13.541

8.082

1.010

0.590

0.022

0.013

03

13.318

7.995

0.995

0.571

0.021

0.012

04

13.133

8.248

1.001

0.599

0.021

0.013

05

12.997

7.994

0.985

0.600

0.021

0.013

Mean

13.2910

8.1090

0.9944

0.5916

0.0212

0.0128

SD

0.22652

0.12243

0.01178

0.01218

0.00045

0.00045

% CV

1.70

1.51

1.18

2.06

2.11

3.49

% Mean Recovery

76.26

74.37

75.47

%Global Recovery

 

75.36

 


Intraday (within run) and Inter-day (between run) precision and accuracy

The within-run coefficients of variation ranged between 1.06% and 5.06% for sofosbuvir. The within-run percentages of nominal concentrations ranged between 97.21% and 105.93% for sofosbuvir. Results are presented in table 4.

The between-run coefficients of variation ranged between 2.04% and 5.48% for sofosbuvir. The between-run percentages of nominal concentrations ranged between 98.34% and 100.58% for sofosbuvir. Results are presented in table 4.

Matrix effect

The % accuracy of LLOQ samples prepared with the different biological matrix lots were found within the range of 89.49 to 97.49% which were found within the range of 80.00-120.00% for the seven different plasma lots. % CV for LLOQ samples was observed as 2.87% which are within 20.00% of the acceptance criteria. Results are presented in table 6.

Table 4: Intraday and interday precision and accuracy

QC ID HQC MQC LQC LLOQ QC
Concentration (ng/ml) 7252.503 522.180 11.488 4.136
Within Batch Precision and Accuracy
PandA I Calculated Concentration (ng/ml)
6910.342 511.080 11.630 4.290
7009.484 518.984 10.484 3.998
7189.506 514.176 11.501 4.116
7156.740 511.840 11.892 4.132
6984.985 504.031 11.887 4.477
Mean 7050.211 512.0222 11.4788 4.2026
SD 118.5622 5.42876 0.58102 0.18526
% CV 1.68 1.06 5.06 4.41
% Mean Accuracy 97.21 98.05 99.92 101.61
PandA II 7234.610 533.688 12.086 4.263
7192.185 531.929 12.605 4.266
7272.508 523.890 12.009 4.246
7351.433 522.452 11.705 4.070
7380.960 535.319 12.440 4.172
Mean 7286.339 529.4556 12.1690 4.2034
SD 78.93435 5.88296 0.35753 0.08377
% CV 1.08 1.11 2.94 1.99

%

Mean Accuracy

100.47 101.39 105.93 101.63
PandA III 7161.887 520.892 11.414 4.123
7036.505 514.024 11.006 4.395
6960.208 497.103 10.554 4.354
7181.121 521.290 11.095 4.168
6960.064 522.273 11.006 4.342
Mean 7059.957 515.1164 11.0150 4.2764
SD 106.7109 10.58730 0.30752 0.12214
% CV 1.51 2.06 2.79 2.86
% Mean Accuracy 97.35 98.65 95.88 103.39
Between Batch Precision and Accuracy
Mean 7132.169 518.8647 11.5543 4.2275
SD 147.64818 10.58931 0.63313 0.13174
% CV 2.07 2.04 5.48 3.12
% Mean Accuracy 98.34 99.37 100.58 102.21

Table 5 : Results of matrix effect

LLOQ nominal concen (4.063ng/ml)

S. No.

Calculated LLOQ  concn (ng/ml)

% accuracy

1

3.937

96.9

2

3.808

93.73

3

3.823

94.09

4

3.961

97.49

5

3.636

89.49

6

3.867

95.17

7

3.766

92.69

 

% Mean accuracy

94.223

 

 SD

2.7003

 

% CV

2.87


Ruggedness

The coefficients of variation ranged between 0.35% and 3.09% for sofosbuvir. The percentages of nominal concentrations ranged between 93.2% and 99.29% for sofosbuvir. Results are presented in table 7.

Stability studies

Short-term stock solution stability of sofosbuvir and internal standard

Sofosbuvir and internal standard were found to be stable in methanol for 6 h 30 min at room temperature with a % stability of 97.25% and 97.0% respectively. Results are presented in table 8.

Long-term stock solution stability of sofosbuvir and internal standard

Sofosbuvir and internal standard were found to be stable in methanol 10 D 02 H at refrigerator (2-8 °C) with a % stability of 98.81% and 107.96% respectively. Results are presented in table 9.

Freeze-thaw stability

Sofosbuvir is found to be stable in human K3 EDTA plasma after five freeze-thaw cycles at-70 °C with coefficients of variation of 3.27% (LQC) and 3.86% (HQC) for sofosbuvir, and the percentages of nominal concentrations for sofosbuvir were found to be 103.17% (LQC) and 101.23% (HQC). Results are presented in table 10.

Table 6 : Results of ruggedness with different column

QC ID HQC MQC LQC LLOQ QC
Conc.(ng/ml) 7252.503 522.180 11.488 4.136
PandA ID Calculated concentration (ng/ml)

Different

Column

Acquisition batch ID: 031008PandADC01
6980.672 523.650 11.419 4.044
7005.431 518.262 11.463 3.725
7243.518 521.038 11.403 3.853
7100.206 527.007 11.307 3.861
7312.115 516.714 11.443 3.790
Mean 7128.3884 521.3342 11.4070 3.8546
SD 145.55342 4.13570 0.06040 0.11925
% CV 2.04 0.79 0.53 3.09
% Mean Accuracy 98.29 99.84 99.29 93.20

Table 7 : Short-term stock solution stability of drug and ISTD

S. NO.

Drug

ISTD

Nominal Conc (ng/ml)

Nominal Conc (µg/ml)

396675.19

400000.4

4.034

4.075

Area ratio

Area ratio

comparison samples

Stability samples

Comparison samples

Stability samples

01

9.134

9.076

0.116

0.115

02

9.181

8.829

0.117

0.114

03

9.147

9.090

0.115

0.117

04

9.082

8.973

0.117

0.113

05

9.231

8.946

0.114

0.111

06

9.197

8.996

0.117

0.112

Mean

9.1620

8.9850

0.1160

0.1137

SD

0.05245

0.09532

0.00126

0.00216

% CV

0.57

1.06

1.09

1.90

% Mean Stability

97.25

97.00


Table 8 : Long-term stock solution stability of drug and internal standard

S. No.

DRUG

ISTD

Nominal Conc (ng/ml)

Nominal Conc (µg/ml)

400000.480

398186.240

4.214

4.075

Area ratio

Area ratio

Comparison samples

Stability samples

Comparison samples

Stability samples

01

9.219

9.049

0.108

0.111

02

9.116

9.111

0.107

0.110

03

9.228

9.026

0.108

0.115

04

8.918

9.141

0.112

0.119

05

9.208

9.073

0.111

0.119

06

9.138

9.022

0.113

0.114

Mean

9.1378

9.0703

0.1098

0.1147

SD

0.11700

0.04777

0.00248

0.00383

% CV

1.28

0.53

2.26

3.34

% Mean Stability

98.81

107.96


Table 9 : Freeze-thaw stability at-70 °C

S. No.

HQC

 

LQC

 

Nominal Conc (ng/ml)

 

Nominal Conc (ng/ml)

 

7252.503

 

11.488

 

Calculated Conc (ng/ml)

% accuracy

Calculated Conc (ng/ml)

% accuracy

1

7255.363

100.04

11.571

100.72

2

6985.35

96.32

11.547

100.51

3

7017.724

96.76

12.168

105.92


CONCLUSION

Chromatographic separation was performed on Gemini 5µ C18, 50 x 4.6 mm, analytical column and the mobile phase was a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 30:70 v/v. The drug was extracted from the sample with 2.5 ml of ethyl acetate. The specificity of the method was checked for the interference from plasma. Wide range calibration was determined by solutions containing 4.063 to 8000.010ng/ml. The % mean recovery for sofosbuvir in LQC, MQC and HQC was 75.47%, 74.37% and 76.26% respectively. The within-run coefficients of variation ranged between 1.06% and 5.06% for sofosbuvir. The between-run coefficients of variation ranged between 2.04% and 5.48% for sofosbuvir the % accuracy of LLOQ samples prepared with the different biological matrix lots were found within the range of 89.49 to 97.49%. Stability test were performed to assess the long term and short term stability of sofosbuvir sample solutions, internal standard solutions. The developed method was validated for the quantitative determination of sofosbuvir from plasma was simple, rapid, specific, sensitive, accurate and precise. Hence, the method is quite suitable to detect the drug from plasma samples of human volunteers.

ACKNOWLEDGEMENT

I am also grateful to my scholars and my friends for their kind help from time to time at each and every step of my project work.

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. https://pubchem.ncbi.nlm.nih.gov/compound/45375808. Last accessed on 12 Jun 2016]
  2. Hoofnagle J, Di Bisceglie A. The treatment of chronic viral hepatitis. N Eng J Med 1997;336:347-56.
  3. Ravikumar V, Subramanyam CVS, Veerabhadram G. Estimation and validation of sofosbuvir in bulk and tablet dosage form by RP-HPLC. Int J Pharm 2016;6:121-7.
  4. Mohan Vikas P, Satyanarayana T, Vinod Kumar E, Mounika E, Sri Latha M, Anusha R, et al. Development and validation of new RP-HPLC method for the determination of sofosbuvir in pure form. World J Pharm Pharm Sci 2016;5:775-81.
  5. Rezk MR, Basalious EB, Karim IA. Development of a sensitive UPLC-ESI-MS/MS method for quantification of sofosbuvir and its metabolite, GS-331007, in human plasma: application to a bioequivalence study. J Pharm Biomed Anal 2015;114:97-104.
  6. Rezk MR, Basalious EB, Amin ME. Novel and sensitive UPLC-MS/MS method for quantification of sofobuvir in human plasma: application to a bioequivalence study. Biomed Chromatogr 2016;30:1354-62.
  7. Shi X, Zhu D, Lou J, Gan D. Evaluation of a rapid method for the simultaneous quantification of ribavirin, sofobuvir and its metabolites in rat plasma by UPLC-MS/MS method. J Chromatogr B: Anal Technol Biomed Life Sci 2015;1002:353-7.
  8. Analytical procedures and methods validation for drugs and biologics guidance for industry. U. S. Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER); 2015.
  9. Sindhusri M, Swetha T, Ramadevi A, Ashok Kumar A. A novel rapid rp-hplc method development and validation for the quantitative estimation of balofloxacin in tablets. Int J Pharm Pharm Sci 2014;7:319-22.
  10. Raveendra Babu G, Lakshmana Rao A, Venkateswara Rao J. A rapid RP-HPLC method development and validation for the quantitative estimation ribavirin in tablets. Int J Pharm Pharm Sci 2014;7:60-3.
  11. Srinidhi M, Mushabbar Basha MD, V Raj Kumar, Rajendra Kumar J. Stability indicating RP-HPLC method development and validation for the estimation of sumatriptan in bulk and pharmaceutical dosage form. J Appl Pharm Sci 2016;6:20-5.

How to cite this article

  • Darshan Bhatt, B Rajkamal. A UPLC-MS/MS method development and validation for the estimation of sofosbuvir from human plasma. Int J Appl Pharm 2017;9(1):30-36.


About this article

Title

A UPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF SOFOSBUVIR FROM HUMAN PLASMA

Keywords

Sofosbuvir, LC-MS/MS, Human plasma, Stability studies

DOI

10.22159/ijap.2017v9i1.15652

Date

31-12-2016

Additional Links

Manuscript Submission

Journal

International Journal of Applied Pharmaceutics
Vol 9, Issue 1, 2017 Page: 30-36

Online ISSN

0975-7058

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Authors & Affiliations

Darshan Bhatt
Mewar University, Gangrar, Chittorgarh 312901, Rajasthan, India
India

B. Rajkamal
KVK college of pharmacy, Surmajiguda, Hyderabad 501512, Telangana, India
India


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