RP HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF GRAZOPREVIR AND ELBASVIR IN BULK AND ITS DOSAGE FORM WITH STRESS STUDIES

RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR

SIMULTANEOUS ESTIMATION OF GRAZOPREVIR AND ELBASVIR

IN BULK AND ITS DOSAGE FORM WITH STRESS STUDIES

Rupa Lavyanya S.*, Bangaruthalli J.1, M. L. N. Renuka2, P. Akhila3 and

N. Arunkumar4

A.U. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India.

ABSTRACT

Objective: The objective of this study was to develop and validate

rapid, specific, sensitive, and precise reverse-phase high performance

liquid chromatography (RP-HPLC) method for the quantitative

determination of Grazoprevir and Elbasvir. No interference from any

component of pharmaceutical dosage form was observed. Methods:

The chromatographic separation was performed on Inertsil ODS

column discovery (4.6 x 250mm, 5 μ particle size) the optimized

mobile phase consists of 0.1% OPA: Methanol (50:50% v/v) with a

flow rate of 1.0 ml/min and ultraviolet (UV) detection at 225 nm.

Results: The retention times were 1.867 and 2.812 min for

Grazoprevir and Elbasvir, respectively. Quantification is achieved with PDA detection at 254

nm over the concentration range of 50–150 μg/ml for Grazoprevir and for Elbasvir 25–125

μg/ml, with a correlation coefficient of 0.999 and 0.999, respectively. The developed method

was validated according to the International Conference on Harmonization (ICH) guidelines

with respect to linearity, accuracy, precision, specificity, and robustness. Forced degradation

study was extended out under acidic, alkaline, oxidative, photolytic, and thermal conditions

to demonstrate the stability-indicating capability of the developed HPLC method. The

degradation products were well resolved from the main peak, thus proved the

stability-indicating power of the method. The results of the analysis were validated statistically.

Conclusion: The method is precise, accurate, linear, robust, and fast. The short retention time

allows the analysis of a large number of samples in a short period of time and, therefore,

should be cost-effective for routine analysis in the pharmaceutical industry.

Volume 8, Issue 8, 546-558. Research Article ISSN 2277– 7105

Article Received on 28 April 2019,

Revised on 18 May 2019, Accepted on 07 June 2019,

DOI: 10.20959/wjpr20198-15190

*Corresponding Author

Rupa Lavyanya S.

A.U. College of

Pharmaceutical Sciences,

Andhra University,

KEYWORDS: Grazoprevir, Elbasvir, High performance liquid chromatography,

Development, Validation, Forced degradation.

INTRODUCTION

Hepatitis C virus (HCV) is a single-stranded RNA virus that is estimated to affect 130–150

million people worldwide. HCV is a single-stranded RNA virus that is categorized into nine

distinct genotypes. HCV infection has been associated with both hepatic and extrahepatic

manifestations that increase graft loss and mortality after transplantation. The combination of

antiviral drugs grazoprevir (Fig. 1), and elbasvir (Fig. 2), is more effective for the treatment

of patients chronically infected with HCV. Elbasvir/grazoprevir (Zepatier) is a combination

product with an FDA-approved indication for the treatment of chronic HCV genotypes (GTs)

1 and 4 in adults.

Grazoprevir

Grazoprevir chemically known as (1R,18R,20R,24S,27S) -N-{(1R,2S) -1

[(cyclopropylsulfonyl) carbamoyl] -2-vinylcyclopropyl} -7-methoxy- 24-(2-methyl-2

propanyl)-22,25-dioxo-2,21-dioxa-4,11,23,26-tetraazapentacyclo[24.2.1.03,12.05,10.0,18,20]

nonacosa-3,5,7,9,11- Benton-27-carboxamide is a direct acting antiviral medication used as a

part of combination therapy to treat chronic hepatitis C, an infectious liver disease caused by

infection with HCV. It is a protease inhibitor of HCV NS3/4A that prevents cleavage of the

poly protein necessary for replication.

Elbasvir

Elbasvir chemically known as dimethyl N,N’-([(6S)-6H-indolo[1,2-c]

[1,3]benzoxazine-3,10-diyl]bis{1H-imidazole-5,2-diyl-(2S)-pyrrolidine-2,1-diyl[(2S)-1-oxo-3-methylbutane-1,2

diyl]}) biscarbamate is a NS5A inhibitor, preventing hepatitis C viral RNA replication and

virion assembly. Elbasvir is a drug approved by the FDA for the treatment of Hepatitis C.

Fig 2: Elbasvir structure.

METHODS

Instruments and apparatus

The Acquity waters HPLC system equipped with PDA detector and autosampler integrated

with Empower Software was used to perform development and validation of the method. An

Inertsil ODS 4.6 x 250mm column with particle size of 5 μm was used as a stationary phase

for chromatographic separation and determination of grazoprevir and elbasvir. Sartorius

analytical balance was used for all weighings, Metsar pH meter was used for the pH

measurement of buffer solution, and Labman Sonicator was used to dissolve the standard and

samples.

MATERIALS

Working standards of grazoprevir and elbasvir were gifted by spectrum laboratories

(Hyderabad, India). Dosage form ZEPATIER tablets purchased from the market.

HPLC-grade acetonitrile, methanol, and orthophosphoric acid (OPA) purchased from Merck Ltd,

Mumbai, India, were used in the study.

Preparation of solutions

Preparation of 0.1% OPA buffer

1 mL of OPA is taken in 1000 mL of HPLC water; pH was adjusted with 0.1M NaOH upto

Preparation of mobile phase

Accurately measured 100 mL (50%) of above buffer and 100 mL of Methanol HPLC (50%)

were mixed and degassed in an ultrasonic water bath for 10 minutes and then filtered through

0.45 µ filter under vacuum filtration.

Diluent Preparation

The Mobile phase was used as the diluent.

PREPARATION OF THE GRAZOPREVIR & ELBASVIR STANDARD & SAMPLE

SOLUTION

Standard Solution Preparation

Accurately weigh and transfer 100 mg of Grazoprevir and 50 mg of Elbasvir working

standard into a 100 mL clean dry volumetric flask, add about 70 mL of Diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent. (Stock

solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

up to the mark with diluent.

Sample Solution Preparation

Accurately weigh and transfer 100 mg of Grazoprevir and 50 mg of Elbasvir working

standard into a 100 mL clean dry volumetric flask add about 70 mL of Diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent. (Stock

solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

up to the mark with diluent.

METHOD VALIDATION

PRECISION

Preparation of stock solution

Accurately weigh and transfer 100 mg of Grazoprevir and 50 mg of Elbasvir working

standard into a 100 mL clean dry volumetric flask add about 70 mL of Diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent. (Stock

solution). Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask

INTERMEDIATE PRECISION/RUGGEDNESS

To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision

was performed on different day.

Preparation of stock solution

Accurately weigh and transfer 100 mg of Grazoprevir and 50 mg of Elbasvir working

standard into a 100 mL clean dry volumetric flask add about 70 mL of Diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent. (Stock

solution). Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask

and dilute up to the mark with diluent.

SPECIFICITY

For Specificity Blank and Standard are injected into system. There is no interference of any

peak in blank with the retention time of the analytical peaks.

ACCURACY

Preparation of Standard stock solution

Accurately weigh and transfer 100 mg of Grazoprevir and 50 mg of Elbasvir working

standard into a 100 mL clean dry volumetric flask add about 70 mL of Diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent. (Stock

solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

up to the mark with diluent.

Preparation Sample solutions

For preparation of 50% solution (With respect to target Assay concentration)

Accurately weigh and transfer 50 mg of Grazoprevir and 25 mg of Elbasvir working standard

into a 100 mL clean dry volumetric flask add about 70 mL of Diluent and sonicate to dissolve

it completely and make volume up to the mark with the same solvent. (Stock solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

For preparation of 100% solution (With respect to target Assay concentration)

Accurately weigh and transfer 100 mg of Grazoprevir and 50 mg of Elbasvir working

standard into a 100 mL clean dry volumetric flask add about 70 mL of Diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent. (Stock

solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

up to the mark with diluent.

For preparation of 150% solution (With respect to target Assay concentration)

Accurately weigh and transfer 150 mg of Grazoprevir and 75 mg of Elbasvir working

standard into a 100 mL clean dry volumetric flask add about 70 mL of Diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent. (Stock

solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

up to the mark with diluent.

LINEARITY

Preparation of stock solution

Accurately weigh and transfer 100 mg of Grazoprevir and 50 mg of Elbasvir working

standard into a 100 mL clean dry volumetric flask add about 70 mL of Diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent. (Stock

solution).

Preparation of Level – I

0.5 mL of above stock solutions has taken in 10mL of volumetric flask, dilute up to the mark

with diluent.

Preparation of Level – II

1 mL of above stock solutions has taken in 10mL of volumetric flask, dilute up to the mark

with diluent.

Preparation of Level – III

1.5 mL of above stock solutions has taken in 10mL of volumetric flask, dilute up to the mark

Preparation of Level – IV

2 mL of above stock solutions has taken in 10mL of volumetric flask, dilute up to the mark

with diluent.

Preparation of Level – V

2.5 mL of above stock solutions has taken in 10mL of volumetric flask, dilute up to the mark

with diluent.

DETECTION LIMIT

LIMIT OF DETECTION: (for Grazoprevir)

Preparation of 1.05µg/mL solution

Accurately weigh and transfer 100 mg of Grazoprevir working standard into a 100 mL clean

dry volumetric flask add about 70 mL of Diluent and sonicate to dissolve it completely and

make volume up to the mark with the same solvent. (Stock solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

up to the mark with diluent.

Further pipette 0.07mL of the above stock solution into a 10mL volumetric flask and dilute

up to the mark with diluent.

LIMIT OF QUANTIFICATION

Preparation of 3.60 µg/mL solution

Accurately weigh and transfer 100 mg of Grazoprevir working standard into a 100 mL clean

dry volumetric flask add about 70 mL of Diluent and sonicate to dissolve it completely and

make volume up to the mark with the same solvent. (Stock solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

up to the mark with diluent.

Further pipette 0.24 mL of the above stock solution into a 10mL volumetric flask and dilute

LIMIT OF DETECTION: (for Elbasvir)

Preparation of 2.1 µg/mL solution

Accurately weigh and transfer 50 mg of Elbasvir working standard into a 100 mL clean dry

volumetric flask add about 70 mL of Diluent and sonicate to dissolve it completely and make

volume up to the mark with the same solvent. (Stock solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

up to the mark with diluent.

Further pipette 0.28 mL of the above stock solution into a 10mL volumetric flask and dilute

up to the mark with diluent.

LIMIT OF QUANTIFICATION

Preparation of 7.05 µg/mL solution

Accurately weigh and transfer 50 mg of Elbasvir working standard into a 100 mL clean dry

volumetric flask add about 70 mL of Diluent and sonicate to dissolve it completely and make

volume up to the mark with the same solvent. (Stock solution).

Further pipette 1.5 mL of the above stock solutions into a 10mL volumetric flask and dilute

up to the mark with diluent.

Further pipette 0.94mL of the above stock solution into a 10mL volumetric flask and dilute

up to the mark with diluent.

DEGRADATION STUDIES

The International Conference on Harmonization (ICH) guideline entitled stability testing of

new drug substances and products requires that stress testing be carried out to elucidate the

inherent stability characteristics of the active substance. The aim of this work was to perform

the stress degradation studies on the Grazoprevir and Elbasvir using the proposed method.

Preparation of stock

Accurately weigh and transfer 100 mg of Grazoprevir and 50 mg of Elbasvir working

standard into a 100 mL clean dry volumetric flask add about 70 mL of Diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent. (Stock

Hydrolytic degradation under acidic condition

Pipette 1.5 mL of above solution into a 10mL volumetric flask and 3 mL of 0.1N HCl was

added. Then, the volumetric flask was kept at 60ºC for 24 hours and then neutralized with 0.1

N NaOH and make up to 10mL with diluent. Filter the solution with 0.44µ syringe filters and

place in vials.

Hydrolytic degradation under alkaline condition

Pipette 1.5 mL of above solution into a 10mL volumetric and add 3mL of 0.1N NaOH was

added in 10mL of volumetric flask. Then, the volumetric flask was kept at 60ºC for 24 hours

and then neutralized with 0.1N HCl and make up to 10mL with diluent. Filter the solution

with 0.44 µ syringe filters and place in vials.

Thermal induced degradation

Grazoprevir and Elbasvir sample was taken in Petri dish and kept in Hot air oven at 1100C for

3 hours. Then the sample was taken and diluted with diluents and injected into UPLC and

analyzed.

Oxidative degradation

Pipette 1.5 mL above stock solution into a 10mL volumetric flask and 1mL of 30% w/v of

hydrogen peroxide added in 10 mL of volumetric flask and the volume was made up to the

mark with diluent. The volumetric flask was then kept at room temperature for 15 min filter

the solution with 0.45 µ syringe filters and place in vials.

Photo degradation: Pipette 0.3 mL above stock solution into a 10mL volumetric flask and

expose to sunlight for 24hrs and the volume was made up to the mark with diluent. Filter the

solution with 0.45 µ syringe filters and place in vials.

RESULTS

S. No. Name RT (min)

Area (µV sec)

Height (µV)

USP resolution

USP tailing

USP plate count 1 Grazoprevir 1.867 191234 24816 1.18 3828.18 2 Elbasvir 2.812 107339 6206 4.96 1.12 3115.92

SYSTEM SUITABILITY

Figure 4: Chromatogram for Blank.

Table 1: Results of system suitability parameters.

S. No. Name RT(min) Area

(µV sec)

Height (µV)

USP resolution

USP tailing

USP plate count

1 Grazoprevir 1.867 191234 24816 1.18 3828.18

2 Elbasvir 2.798 107339 6206 4.96 1.12 3115.92

LINEARITY

The linearity range was found to lie from 50µg/ml to 250µg/ml of Grazoprevir, 25µg/ml to

125µg/ml 0f Elbasvir and chromatograms are shown below.

Table 2: Area of different concentration of Grazoprevir and Elbasvir.

S. No

Grazoprevir Elbasvir

Concentration (µg/ml) Area Concentration (µg/ml) Area

1 50 65787 25 32441

2 100 131783 50 67728

3 150 194311 75 100630

4 200 256245 100 134448

5 250 317748 125 172463

PRECISION

Precision of the method was carried out for both sample solutions as described under

Table 3: Results of Precision for Grazoprevir and Elbasvir.

Injection Area

Grazoprevir Elbasvir

Injection-1 191345 107339

Injection-2 191232 107232

Injection-3 191671 107131

Injection-4 191999 107399

Injection-5 192898 107018

Injection-6 194679 107089

Average 192304.0 107201.3

Standard Deviation 1308.1 148.4

%RSD 0.7 0.1

ACCURACY

Sample solutions at different concentrations (50%, 100%, and 150%) were prepared and the

% recovery was calculated.

Table 4: Accuracy (recovery) data for Grazoprevir and Elbasvir

%

Concen-tration

Grazoprevir Elbasvir

Area Amount Added (mg) Amount Found (mg) % Recovery Mean

Recovery Area

Amount Added (mg) Amount Found (mg) % Recovery Mean Recovery

50% 95105 40 39.79 99.47

99.40

53846 5 25.06 100.24

99.74

100% 191399 80 79.74 99.67 107344 10 49.96 99.91

150% 285309 120 118.86 99.05 159676 15 74.31 99.08

LIMIT OF DETECTION FOR GRAZOPREVIR AND ELBASVIR

The lowest concentration of the sample was prepared with respect to the base line noise and

measured the signal to noise ratio.

Table 5: Results of LOD.

Drug name Baseline

noise (µV)

Signal obtained (µV)

S/N ratio

Grazoprevir 58 174 3.00

Elbasvir 58 173 2.98

LIMIT OF QUANTIFICATION FOR GRAZOPREVIR AND ELBASVIR

The lowest concentration of the sample was prepared with respect to the base line noise and

Table 6: Results of LOQ.

Drug name Baseline

noise (µV)

Signal obtained (µV)

S/N ratio

Grazoprevir 58 579 9.98

Elbasvir 58 580 10.00

ASSAY

Standard and sample solution injected as described under experimental work. The

corresponding chromatograms and results are shown below.

Table 7: Results of Assay for Grazoprevir and Elbasvir.

Label Claim (mg) % Assay

Grazoprevir 100 100.14

Elbasvir 50 99.59

Table 8: Degradation results for Grazoprevir and Elbasvir.

Sample Name

Grazoprevir Elbasvir

Area % Degraded Area % Degraded

Standard 191642 107223

Acid 183252 4.38 98959 7.71

Base 183532 4.23 98921 7.74

Peroxide 183253 4.38 98978 7.69

Thermal 187552 2.13 98851 7.81

Photo 186452 2.71 98789 7.87

CONCLUSION

A rapid and robust method for the analysis of grazoprevir and elbasvir was developed and its

applicability as a method for analyzing stability was checked. The method was found to be

specific, accurate, precise, and reproducible. Force degradation studies confirmed its ability

to determine stability because no interference from degradation products was observed.

Moreover, no influence from excipients was found, allowing it to be used in the final drug

product analysis. The method was also validated in accordance with ICH requirements.

REFERENCES

1. Vander Wal S, Snyder LR. J. Chromatogr, 1983; 225: 463.

2. A Practical Guide to HPLC Detection, Academic Press, San Diego, CA, 1983.

3. Poole CF, Schutte SA. Contemporary Practice of Chromatography, Elsevier, Amsterdam,

1984; 375.

4. Krull IS. In Chromatography and Separation Chemistry: Advances and Developments,

5. Li G, Szulc ME, Fischer DH, Krull IS. In Electrochemical Detection in Liquid

Chromatography and Capillary Electrophoresis, Kissinger PT. edn., Chromatography

Science Series, Marcel Dekker, New York, 1997.

6. Kissinger PT, Heineman WR. eds., Laboratory Techniques in Electroanalytical

Chemistry, Chaptor 20, Marcel Dekker, New York, 1984.

7. Swarbrick JC, Boylan James, Encyclopedia of pharmaceutical technology, 1998;

217-224.

8. AOAC Peer-Verified Methods Program, Manual on policies and procedures, Arlington,

Va., USA (1998). http://www.aoac.org/vmeth/PVM.pdf.

9. Winslow PA, Meyer RF. Defining a master plan for the validation of analytical methods,

J Validation Technology, 1997; 361–367.

10. Breaux J, Jones K, Boulas P. Pharmaceutical Technology, Analytical Technology and

Testing, 2003; 6-13.

11. Huber L, George S. Diode-array detection in high-performance liquid chromatography,

New York, Marcel Dekker, ISBN 0-8247-4, 1993.

12. Sumalatha Nallagundla, Nallagundla H S Reddy2, Vishal Vemula, Bharath Kumar,

Analytical Method Development and Validation of Elbasvir and Grazoprevir in Bulk and

Tablet Formulations by Rp- HPLC, International Journal of Pharmaceutical Science

Invention ISSN (Online): 2319 – 6718, ISSN (Print): 2319 – 670X www.ijpsi.org, August

2017; 6(8): 01-05.

13. Padmavathi Sakinala, Mrs.G. Sai Sri Lakshmi, Sravanthi. Gali, T Varalakshmi, RP-HPLC

Method development and validation for the simultaneous estimation of Grazoprevir and

Elbasvir in bulk and pharmaceutical dosage, International Journal of Pharmaceutical

Science Invention ISSN (Online): 2319 – 6718, ISSN (Print): 2319 – 670X

www.ijpsi.org, August 2017; 6(8): 01-05.

14. Syeda Saniya Fatima, R. Vani, Stability indicating analytical method development and

validation for estimation of Ceftazidime and Avibactam in bulk and pharmaceutical