RP-HPLC Method Development and Validation for Velpatasvir and Voxilaprevir  by Simultaneous Determination in Bulk and Their Pharmaceutical Dosage Forms

 

Md. Abdul Sattar*, A. Suneetha

Department of Pharmaceutical Analysis, Hindu College of Pharmacy, Amaravathi Road, Guntur-522002, Andhra Pradesh, India.

*Corresponding Author E-mail: abdulsattar.bph@gmail.com

 

ABSTRACT:

Numerous analysts have endeavored to utilize computer supported structure (Computer assisted design) or CAD and computer helped fabricating (CAM) to know a framework that provides a three-dimensional (3D) condition for recovery of tissues and organs. Therefore, a number of 3D printing advances, as well as stereo lithography, testimony displaying, inkjet-based printing and specific optical maser sintering are created. Since these 3D printing advancements use PCs for structure and manufacture, and that they will produce 3D platforms as structured; as a result, they will be institutionalized. Development of target tissues and organs needs the distance of fitting development factors; therefore manufacture of 3D platform frameworks that discharge these bimolecular has been investigated. The medication was created utilizing Pennsylvania-based Aprecia Pharmaceutical's exclusive ZipDose innovation,that expands once existing 3DP analysis did by MIT into creating fast dissolving materials. Even so empowering the generation of more and more compelling portion organizes; the innovation is at risk of have a key influence within the pharmaceutical business' flip towards low-volume generation and customized prescription. Going higher than and on the far side, on-request medicates printing offices at centers and drug stores, or maybe in patients' homes, may allow specialists to reinforce treatment by creating made-to-order dosing regimens. What are additional, parts might be made-to-order with singular hues, flavors and shapes to talk to person understanding which can support their adherence. As 3D printing capacities grow any, security and body issues ar attended and therefore the expense of the innovation falls besides contract manufacturers and pharmaceutical organizations that explore totally different avenues relating to these 3D printing developments are likely to choose up an aggressive edge. A new method was established for simultaneous estimation of Velpatasvir  and  Voxilaprevir by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of   Velpatasvir  and  Voxilaprevir   by using Thermosil C18 column (4.0×125mm) 5.0µm, flow rate was 1ml/min, mobile phase ratio was (70:30 v/v) methanol: Sodium acetate buffer pH 3 (pH was adjusted with orthophosphoricacid), detection wavelength was 252nm. The retention times were found to be 2.566 mins and 3.417 mins. The linearity study of Velpatasvir  and  Voxilaprevir   was found in concentration range of 5µg-25µg and 50µg-250µg and correlation coefficient (r2) was found to be 0.999 and 0.999, % recovery was found to be 99.56% and 99.48%, %RSD for repeatability was 0.86 and 0.82, % RSD for intermediate precision was 0.44 and 0.19 respectively. The precision study was precise, robust, and repeatable.LOD value was 3.17 and 5.68, and LOQ value was 0.0172 and 0.2125 respectively. Hence the suggested RP-HPLC

 

KEYWORDS: Thermosil C18 column,  Velpatasvir  and  Voxilaprevir  , RP-HPLC

 

 

INTRODUCTION:

Velpatasvir(1) is a Direct-Acting Antiviral (DAA) medication used as part of combination therapy to treat chronic Hepatitis C, an infectious liver disease caused by infection with Hepatitis C Virus (HCV). HCV is a single-stranded RNA virus that is categorized into nine distinct genotypes, with genotype(2)   1 being the most common in the United States, and affecting 72% of all chronic HCV patients. Velpatasvir's mechanism of action is likely similar to other selective NS5A inhibitors which bind domain I of NS5A consisting of amino acids 33-202 . NS5A inhibitors compete with RNA for binding at this site. It is also thought that NS5A inhibitors bind the target during its action in replication when the binding site is exposed. Velpatasvir prevents viral replication by inhibiting non-structural protein 5A (NS5A) .

 

 

 

Structure of velpatasvir:

Voxilaprevir(3)exerts its antiviral action by reversibley binding and inhibiting the NS3/4A serine protease of Hepatitis C Virus (HCV). Viral replication(4) of HCV genetic material and translation into a single polypeptide, Nonstructural Protein 3 (NS3) and its activating cofactor Nonstructural Protein 4A (NS4A) are responsible for cleaving genetic material into the following structural and nonstructural proteins(5) required for assembly into mature virus: NS3, NS4A, NS4B, NS5A, and NS5B . By inhibiting viral protease NS3/4A, Voxilaprevir therefore prevents viral replication and function.

 

 

 

Structure of voxilaprevir:

MATERIALS AND METHODS:

Ortho phosphoric acid, KH2PO4, Acetonitrile, Methanol, Water, K2HPO4

Selection of wavelength:

10 mg of Velpatasvir and Voxilaprevir  was dissolved in mobile phase. The solution was scanned from 200-400 nm the spectrum was obtained.  The overlay spectrum was used for selection of wavelength was 252nm. The isobestic point was taken as detection wavelength.

 

Optimized chromatographic conditions:

Chromatographic conditions

Column                : Thermosil C18 (4.0×125 mm) 5.0µm

Mobile phase ratio: Methanol: Sodium acetate buffer (70: 30 % v/v)

Detection wavelength       :   252 nm

Flow rate                             :   1ml/min

Injection volume                              :    10µl

Column temperature         :    Ambient

Auto sampler temperature              :     Ambient

Run time                             :    8min

Retention time                   :    2.566 & 3.417 mins

 

 

Fig.No.1 Chromatogram showing trial-5 injection

 

Observation:

The separation was good, peak shape was good, so we conclude that there is no required for reduce the retention times of peaks, so it is taken as final method.

 

Specificity: 

The system suitability for specificity was carried out to determine whether there is any interference of any impurities in retention time of analytical peak. The specificity was performed by injecting blank.

 

Linearity:

1 mg of Velpatasvir  and 10 mg of Voxilaprevir working standard were accurately weighed and were transferred into a 10ml clean dry volumetric flask, add about 2ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent.

 

Range:

Based on precision, linearity and accuracy data it can be concluded that the assay method is precise, linear and accurate in the range of 5µg/ml-25µg/ml and 50µg/ml-250µg/ml of Velpatasvir and Voxilaprevir  respectively.

 

Accuracy:

1mg of Velpatasvir and 10mg of Voxilaprevir working standard were accurately weighed and transferred into a 10ml clean dry volumetric flask add about 2ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent.

 

Precision:

Repeatability:

1mg of Velpatasvir  and 10 mg of Voxilaprevir working standard were accurately weighed and transferred into a 10ml clean dry volumetric flask add about 2ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Further pipette out 1ml of the above stock solution into a 10ml volumetric flask and was diluted up to the mark with diluent.

 

IntermediatePrecision/Ruggedness:

To evaluate the intermediate precision (also known as ruggedness) of the method, precision was performed on different days by using different make column of same dimensions. 

 

Limit of detection (LOD):

LOD’s can be calculated based on the standard deviation of the response (SD)and the slope of the calibration curve (S) at levels approximating the LOD according to the formula. The standard deviation of the response can be determined based on the standard deviation of y-intercepts of regression lines.

 

Limit of quantification:

LOQ’s can be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (S) according to the formula. Again, the standard deviation of the response can be determined based on the standard deviation of y-intercepts of regression lines.

 

Robustness:

As part of the robustness, deliberate change in the flow rate, mobile phase composition was made to evaluate the impact on the method.

a)      The flow rate was varied at 0.4ml/min to 0.6 ml/min. Standard solution 15ppm of  Velpatasvir and 150 ppm of Voxilaprevir was prepared and analysed using the varied flow rates along with method flow rate.

b)     The organic composition in the mobile phase was varied from 65% to75 %standard solution 15 µg/ml of  Velpatasvir  and 150 µg/ml of Voxilaprevir  were prepared and analysed using the varied mobile phase composition along with the actual mobile phase composition in the method.

 

 

System suitability:

1mg of  Velpatasvir and 10 mg of Voxilaprevir working standard was accurately weighed and transferred into a 10ml clean dry volumetric flask and add about 2ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution).Further pipette out 1ml of Velpatasvir and Voxilaprevir  from the above stock solution into a 10ml volumetric flask and was diluted up to the mark with diluent.

 

RESULTS:

Table 1: Linearity Results for Velpatasvir :

S.No

Linearity Level

Concentration

Area

1

I

5 ppm

471543

2

II

10 ppm

656277

3

III

15 ppm

794999

4

IV

20 ppm

946124

5

V

25 ppm

1002139

Correlation Coefficient

0.999

 

 

Velpatasvir  r2 = 0.999

Fig.No.2 Showing calibration graph for Velpatasvir

 

Table: 2 Linearity Results for Voxilaprevir

S. No

Linearity Level

Concentration

Area

1

I

50ppm

56472

2

II

100 ppm

73841

3

III

150ppm

92655

4

IV

200ppm

111541

5

V

250ppm

130567

Correlation Coefficient

0.999

 

 

Voxilaprevir r2 = 0.999

Fig.No.3 Showing calibration graph for Voxilaprevir

Accuracy: 

Table 3 Details of Accuracy 50 %

Injection

Peak  Name

RT

Area

Height

1

Velpatasvir

2.553

124366

57028

2

Velpatasvir

2.554

124683

48569

3

Velpatasvir

2.564

128376

24157

4

Voxilaprevir

3.397

132846

24574

5

Voxilaprevir

3.413

136172

24856

6

Voxilaprevir

3.519

134556

86251

Mean

 

 

372548.2

 

Std.Dev

 

 

259486.6

 

% RSD

 

 

0.515

 

Table 4 Details of Accuracy 100 %

Injection

Peak  Name

RT

Area

Height

1

Velpatasvir

2.563

12486

78248

2

Velpatasvir

2.561

12364

78542

3

Velpatasvir

2.559

12458

72345

4

Voxilaprevir

3.431

13744

32815

5

Voxilaprevir

3.467

13784

32894

6

Voxilaprevir

3.431

13887

32577

Mean

 

 

254827.4

 

Std.Dev

 

 

3542781.2

 

% RSD

 

 

0.522

 

 

 

Table 5 Details of Accuracy 150 %

Injection

Peak  Name

RT

Area

Height

1

Velpatasvir

2.574

126647

75842

2

Velpatasvir

2.573

125742

74224

3

Velpatasvir

2.644

128522

72148

4

Voxilaprevir

3.436

126411

73421

5

Voxilaprevir

3.439

127541

72648

6

Voxilaprevir

3.537

128461

78412

Mean

 

 

475826.4

 

Std.Dev

 

 

2758462.2

 

% RSD

 

 

0.543

 

 

Table.No.6. Showing accuracy results for Velpatasvir:

%Concentration

(at specification level)

Average

Area

Amount added

(mg)

Amount found

(mg)

% Recovery

Mean recovery

50%

2630409

5

4.96

99.91%

99.56%

100%

5277055

10

9.98

99.18%

150%

7514836

15

15.02

99.60%

 

Table.No.7 Showing accuracy results for  Voxilaprevir:

%Concentration

(at specification level)

Average

area

Amount added

(mg)

Amount found

(mg)

% Recovery

Mean recovery

50%

1366666

0.5

0.99

99.53%

99.47%

100%

2777487

1.0

1.05

99.38%

150%

4151234

1.5

1.495

99.52%

 

Precision:

Table. No. 8 Showing %RSD results for Velpatasvir

 

Peak Name

Rt

Area

Height

1

velpatasvir

2.755

5223559

541538.3

2

velpatasvir

2.687

5208511

485548.5

3

velpatasvir

2.632

5323569

574440.4

4

velpatasvir

2.612

5259147

557413.5

5

velpatasvir

2.616

5273463

565020.1

6.

velpatasvir

2.613

5276431

587260.1

mean

 

 

5257650

 

Std.dev

 

 

45206.4

 

%RSD

 

 

0.86

 

 

Table. No. 9 Showing% RSD results for Voxilaprevir

 

Peak Name

Rt

Area

Height

1

Voxilaprevir

3.616

2742453

238643.4

2

Voxilaprevir

3.634

2762750

271543.5

3

Voxilaprevir

3.460

2797670

281711.6

4

Voxilaprevir

3.446

2793578

274499.8

5

Voxilaprevir

3.437

2778483

276713.0

6

Voxilaprevir

3.438

2778485

246712.0

mean

 

 

27854628

 

Std.dev

 

 

758206.4

 

%RSD

 

 

0.82

 

 

 

 

 

Intermediate precision:

Table.No.10. Showing results for intermediate precision of Velpatasvir

 

Peak name

Rt

Area

Height

1

Velpatasvir

2.756

5698542

539568.1

2

Velpatasvir

2.688

5682534

536985.4

3

Velpatasvir

2.633

5695846

539584.1

4

Velpatasvir

2.613

5689452

534569.8

5

Velpatasvir

2.617

5636591

534985.5

6

Velpatasvir

3.439

5634595

535875.6

mean

 

 

5600593

 

Std.dev

 

 

203577.3

 

%RSD

 

 

0.44

 

 

Table.No.11 Showing results for Intermediate precision of Voxilaprevir

 

Peak name

Rt

Area

Height

1

Voxilaprevir

3.617

2624315

231325.6

2

Voxilaprevir

3.635

2623598

231315.4

3

Voxilaprevir

3.461

2623541

231250.1

4

Voxilaprevir

3.447

2624987

231342.6

5

Voxilaprevir

3.438

2635698

231765.2

6

Voxilaprevir

3.439

2635699

264818.2

mean

 

 

2626428

 

Std.dev

 

 

5215.58

 

%RSD

 

 

0.19

 

 

System suitability:

Table.12 Results for system suitability of  Velpatasvir

Injection

RT (min)

Peak area

TP

TF

1

2.526

124652

1554.31

1.28

2

2.767

127376

1634.55

1.31

3

2.764

122803

1623.37

1.31

4

2.808

125382

1622.73

1.23

5

2.789

122153

1460.39

1.32

6

2.799

122155

1634.88

1.27

Mean

123634

-

-

SD

631.0

-

-

%RSD

0.6

-

-

 

Table.13 Results for system suitability of Voxilaprevir

Injection

RT(min)

Peak area

TP

TF

1

3.901

434308

4315.31

1.17

2

3.904

436736

4232.73

1.17

3

3.905

436821

4372.54

1.17

4

 3.907

435350

4354.17

1.17

5

3.908

425462

4322.22

1.17

6

3.909

435465

4328.19

1.18

Mean

 

44531.3

-

-

SD

 

1257.3

-

-

%RSD

 

0.3

-

-

 

Detection limit LOD:

Table .No.14 Showing results for Limit of Detection

Drug name

Standard deviation (σ)

Slope (s)

LOD (µg)

Velpatasvir

373625.50

581075863

3.17

Voxilaprevir

5772.40

476579210

0.0172

 

Quantitation limit LOQ

Table.No.15 Showing results for Limit of Quantitation

Drug name

Standard deviation (σ)

Slope (s)

LOQ (µg)

Velpatasvir

372727.80

574265980

5.80

Voxilaprevir

5761.30

478828490

0.212

 

Robustness:

Table.No.16 Showing system suitability results for Velpatasvir

S. No

Flow rate (ml/min)

System suitability results

USP Plate Count

USP Tailing

1

0.8

5339

1.4

2

1

4668

1.3

3

1.2

5216

1.4

 

Table.No.17 Showing system suitability results for Voxilaprevir

S. No

Flow rate (ml/min)

System suitability results

USP Plate Count

USP Tailing

1

0.8

7036

1.3

2

1

6089

1.2

3

1.2

6998

1. 3

 

Fig.No.4 Chromatogram showing less flow rate 0.8ml/min

 

 

Fig.No.5 Chromatogram showing less flow rate 1.2 ml/min

 

 

Fig.No.6 Chromatogram showing more organic phase ratio

 

 

 

 

 

Fig.No.7 Chromatogram showing less organic phase ratio 

 

Table.No.18 Showing system suitability results for Velpatasvir

S. No

Change in organic composition in the mobile phase

System suitability results

USP Plate Count

USP Tailing

1

5 % less

6232

1.4

2

*Actual

4668

1.3

3

5 % more

6387

1.4

 

Table.No.19 Showing system suitability results for Voxilaprevir

S. No

Change in organic composition in the mobile phase

System suitability results

USP Plate Count

USP Tailing

1

5 % less

5437

1.3

2

*Actual

6089

1.2

3

5 % more

4817

1.2

 

SUMMARY AND CONCLUSION:

A new method was established for simultaneous estimation of Velpatasvir  and Voxilaprevir by RP-HPLC method. The chromatographic conditions were success fully developed for the separation of Velpatasvir  and Voxilaprevir by using Thermosil C18 column (4.0×125mm) 5µ, flow rate was 1ml/min, mobile phase ratio was (70:30 v/v) methanol: Sodium acetate buffer pH 3 (pH was adjusted with orthophosphoricacid), detection wavelength was 252nm. The instrument used was Waters HPLC Auto Sampler, Separation module 2690, photo diode array detector 996, Empower-software version-2. The retention times were found to be 2.566 mins and 3.417 mins. The % purity of Velpatasvir  and Voxilaprevir was found to be 101.27% and 99.24% respectively. The system suitability parameters for Velpatasvir  and Voxilaprevir such as theoretical plates and tailing factor were found to be 4668, 1.3 and 6089 and 1.2, the resolution was found to be 6.0. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study n Velpatasvir  and Voxilaprevir was found in concentration range of 5µg-25µg and 50µg-250µg and correlation coefficient (r2) was found to be 0.999 and 0.999, % recovery was found to be 99.56% and 99.47%, % RSD for repeatability was 0.86 and 0.82, % RSD for intermediate precision was 0.44 and 0.19 respectively. The precision study was precise, robust, and repeatable.LOD value was 3.17 and 5.80, and LOQ value was 0.0172 and 0.212 respectively.Hence the suggested RP-HPLC method can be used for routine analysis of Velpatasvir  and Voxilaprevir in API and Pharmaceutical dosage form.

 

REFERENCES:

1.     J. Sandya rani et.al, A New RP-HPLC Method Development and Validation for Simultaneous Estimation of Sofosbuvir and Velpatasvir in Pharmaceutical Dosage Form. International Journal of Engineering Technology Science and Research. 2017, 4(11), 145-152.

2.     Sarath Nalla et al., A Stability Indicating RP-HPLC Method for Simultaneous Estimation Of Velpatasvir and Sofosbuvir in Combined Tablet Dosage Forms. Journal of Pharmacy and Pharmaceutical Sciences. 2017,6(9),159-161.

3.     Hyock Joo Kwon et al,  Method Development and Validation of Stability Indicating RP-HPLC Method for Simultaneous Estimation of  Sofosbuvir  and Velpatasvir  In Bulk  and Its Pharmaceutical Formulations. Eurasian Journal of Analytical chemistry.  2016, 11(4),197-210.

4.     Anusha Tiyyagura et al, Method Development and Validation for the Simultaneous Estimation Of Velpatasvir  and Sofosbuvir in Pharmaceutical Dosage Form by RP-HPLC. IJPCBS. 2012, 3(1), 44-54.

5.     Ganta Srinivas et al, Method Development and Validation of Stability Indicating RP-HPLC Method for Simultaneous Estimation of Atazanavir and Velpatasvir  In Bulk and Its Pharmaceutical Formulations. Asian Journal Pharm Clin Res,2014,7(2),105-113.

 

 

 

 

 

Received on 22.05.2018          Modified on 20.06.2018

Accepted on 16.07.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(2):477-482.

DOI: 10.5958/0974-360X.2019.00084.2