1. INTRODUCTION:

Sofosbuvir, L-Alanine, N-[[P(S),2'R]-2'-deoxy-2'-fluoro-2'-methyl-P-phenyl-5'-uridylyl]-, 1-methylethyl ester is a new drug candidate for treating hepatitis C has molecular formula C22H29FN3O9P and molecular weight of 529.5 g/mol¹ It acts against six major Hepatitis C Virus (HCV) genotypes and stops HCV from multiplying and infecting new cells.² Sofosbuvir offers benefits like less side effects, high potency, oral administration, well tolerated and high barrier to resistance. The efficiency and safety were proven in many well-designed clinical trials.^3,4,5,6

The uridine nucleotide analog of sofosbuvir is a phosphoramidate pro-drug that must be triphosphorylated inside the cells to produce its activity. The necessary enzymes for its activation are available in the human hepatic cells, therefore it is changed over to its active metabolite during the first pass metabolism, directly at the desired site of action i.e. the liver.⁷. From the literature survey, it was noted that few methods like UV, RP-HPLC, TLC, HPTLC methods have been developed for simultaneous estimation of sofosbuvir in combination with another drug in marketed preparation and as stability indicating methods.^8-16. Hence, in this study an effort has been taken to develop UV and HPLC methods for sofosbuvir estimation in bulk and pharmaceutical preparation and also the force degradation study was accomplished by utilizing the same optimized HPLC method with Cosmosil C18 (250mm×4.6ID, Particle size: 5µ) column, methanol: water (70:30) as mobile phase and detection wavelength of 260nm.

2. MATERIAL AND METHODS:

Chemicals and Reagents:

A pure standard reference of sofosbuvir was acquired as a gift sample from Dr. Reddy’s Lab Ltd. Hyderabad, India. Methanol and water of HPLC grade from Thermo Fisher Scientific and Sofosbuvir tablets manufactured by Mylan pharmaceuticals private Ltd. were bought from the local marketplace.

Instruments:

HPLC:

HPLC analysis was executed on HPLC-3000 series (Binary gradient framework, Analytical technologies Ltd.) manufactured by Analytical Technologies Ltd., comprising of a siphon with the loop limit of 20µl, UV-Vis (model no. UV2012) detector, waters column (250 mm x 4.6mm, 5μ). The Borosilicate 25μl glass syringe was utilized for testing. Mdi 0.2μm membrane filter paper was utilized.

UV-Visible spectrophotometer:

The absorbance were recorded on UV-Visible spectrophotometer (UV-3000-M) manufactured by Analytical Technologies Ltd., with a spectral band width 2cm furnished with quartz cell of 1mm at medium scan speed.

Identification of Drug:

Melting Point:¹⁷

The melting point of sofosbuvir was resolved by utilizing a capillary method on a melting point device

FTIR:^18,19

Fourier transform infrared (FT-IR Alpha Brucker, Germany) was utilized to recognize the characteristic functional group, blank reading was taken by Potassium Bromate (KBr). A small quantity of the sofosbuvir was combined with KBr at the time of analysis from 4000-400cmˉ ˡ

Solubility:^20,21

The solubility study of sofosbuvir was investigated by utilizing various solvents, like methanol, ethanol and water.

Selection and optimization of UV-visible Spectrophotometric method:^22,23,24

Selection of mobile phase:

Mobile phase was selected by utilizing different solvents in different concentrations, from the solvents tested, the one which gave the precise results in UV spectrophotometer was selected.

Preparation of standard stock solution:

10mg of the sofosbuvir pure drug was precisely weighed and moved into 10ml volumetric flask and to it adequate volume of diluents was combined to dissolve the drug, later the volume was then made up till the mark utilizing the mobile phase to acquire standard solutions of 1000μg/ml.

Selection of detection wavelength:

So as to establish the wavelength of maximum absorption (λmax) for sofosbuvir, 1 ml working solution was diluted till 10ml with the mobile phase and was scanned for the entire UV region i.e. 200-400nm in UV spectrophotometer on spectrum mode.

Establishment of calibration curve:

From the working standard solution 0.6, 1.2, 1.8, 2.4, 3 ml were pipette out and moved to 10ml volumetric flask separately. To each of the flask mobile phase was combined to make the volume up till the mark to get solution of strength 6, 12, 18, 24, 30μg/ml. Absorbance of each solution was recorded at the selected analytical wavelength, to determine whether the span of concentration is obeying Beer-Lamberts law.

Assay:

The average weight of the sofosbuvir tablets, was calculated by recording weight of 10 tablets independently. The equivalent weight of 12.02mg of sofosbuvir tablet powder (which is equal to 10mg of pure drug) was combined with 5ml of the mobile phase in a volumetric flask of 10ml. Sonication was carried out for 10mins so as to dissolve the contents of the powdered tablet, then with the mobile phase the volume was made till the mark. This solution was transferred to falcon tubes and centrifuged for 30 minutes to settle down the undissolved contents. Supernatant was then sifted through 0.22µ membrane filter, 0.3ml of this solution was diluted up till 10ml with mobile phase to acquire 30µg/ml of solution which was scanned in UV spectrophotometer in triplicate. % Assay was calculated by utilizing following equation:

% Assay=Concentration of drug in μg/ml X Dilution Factor X 100 X Average weight of sample / Label Claim X Weight of the sample taken.

Validation of method as per guidelines of ICH:^25,26

Accuracy:

The drug recovery study was completed with the method of standard addition in which a known amount of standard drug in three levels, i.e. 50%, 100% and 150% was combined with pre-analyzed sample. Absorbance of each level were recorded three times and the results obtained were compared to estimate the outcomes

Precision:

Intermediate precision:

Intermediate precision was executed by utilizing the optimized method for 18µg/ml of solution in triplicates for 2 times a day and for 2 consequent days. Estimation of absorbance was used to calculate % relative standard deviation (%RSD).

Linearity:

Preparation of sample solution:

The sample solution of sofosbuvir tablets, (1mg/ml) was made by dissolving equivalent weight of 12.02mg tablet powder in 10ml of volumetric flask by sufficient amount of mobile phase to obtain concentration of 1000µg/ml. This solution is further diluted to detect the linearity. From the above solution 1ml was withdrawn and diluted with mobile phase till 10ml in a volumetric flask to get a solution strength of 100µg/ml.

Preparation of linearity sample solution:

From the above sample solution various concentrations of analyte in ascending order was prepared. From the sample solution 0.6, 1.2, 1.8, 2.4, 3 ml were moved to 10 ml volumetric flask separately and diluted with mobile phase to get solution strength ranging from 6 to 30 µg/ml. The calibration curve’s linearity was determined through regression coefficient by plotting a graph of absorbance versus concentration.

Robustness:

To assess the optimized method’s robustness some of the parameters were purposely fluctuated. These parameters fluctuated in this study are flow rate (±1), the wavelength of determination (±1). Robustness of the technique was done at the constant concentration of 18 μg/ml and the factor changed was detection wavelength to measure its impact on the optimized method.

Selection and Optimization of HPLC method^27,28

Selection of mobile phase:

Methanol and water indifferent combinations were run and from the chromatograms obtained mobile phase which gave sharp peaks and minimum tailing was selected.

Preparation of standard stock solution:

The solution of sofosbuvir (1mg/ml) was made by dissolving 10mg of pure drug in 10ml of volumetric flask in it an adequate amount of diluent was combined to make up the volume till the mark to acquire concentration of 1000µg/ml.

Preparation of working standard solution:

From the standard stock solution 1ml was pipette out and transferred to 10ml volumetric flask, volume was made up till the mark with the diluent to acquire a solution of concentration 100µg/ml

Selection of detection wavelength:

The working standard solution of 100µg/ml was scanned for an entire UV range (200-400nm) in a 1cm quartz cell utilizing methanol: water (70:30 v/v) as blank.

Establishment of calibration curve:

From the working standard stocks of sofosbuvir drug, 0.1, 0.2, 0.3, 0.4, and 0.5ml was pipette out and was combined up to 10ml with mobile phase to acquire 10, 20, 30, 40 and 50(µg/ml) separately in a volumetric flask. The absorbance of each concentration was taken at 260nm, and the estimation of regression coefficient was determined from the calibration curve.

Assay:

The average weight of the sofosbuvir tablets was determined by weighing 10 tablets independently. The assay of sofosbuvir tablet was determined by dissolving equivalent weight of 12.02mg tablet powder (which is equal to 10mg of unadulterated medications) in 10ml of mobile phases in a volumetric flask to get a concentration of 1000µg/ml. The solution was sonicated for 10mins to dissolve the tablet contents. 0.3ml of this solution was moved to the 10ml volumetric flask and diluted with the mobile phase up to the mark to get a solution of 30µg/ml. Then the solution was sifted through 0.22µ membrane filter. After chromatographic advancement peak region was estimated at 260nm and drug amount available in the test sample was evaluated from the region of peak. % Assay was calculated by using following formula:

% Assay=Concentration of drug in μg/ml X Dilution Factor X 100 X Average weight of sample/Label Claim X Weight of the sample taken

HPLC Method validation:^29,30

The optimized HPLC technique was validated by the following parameters given by ICH and in USP:

Accuracy (Recovery):

A method of standard addition was utilized to additionally assess the accuracy of the method. Standard drug was combined with 3 levels (i.e. 50, 100, 150%) to the pre-analyzed drug sample and from the chromatograms; region under the curve was assessed to determine amount recovered and the accuracy of the method.

The solutions were set up by the method of standard addition as follows.

Table No. 2: Composition of sample solution for accuracy by HPLC method

Sr. No.	% of standard Drug Added	Concentration drug from Tablet	Concentration of Standard added	Final Concentration
1.	50%	20ppm	10ppm	30ppm
2.	100%	20ppm	20ppm	40ppm
3.	150%	20ppm	30ppm	50ppm

Specificity:

The specificity of the technique was controlled by taking chromatogram of mobile phase, and test sample solution to see any hindrance of the mobile phase and excipients in the test sample.

Precision:

Analytical method’s precision is the level of agreement among single sample outcomes when the method is applied over a range of different concentration of a homogeneous sample. Precision is normally determined by standard deviation or the relative standard deviation. Precision is the proportion of the level of repeatability of an analytical technique under typical activity.

Intermediate Precision:

Intraday Precision:

The method’s intraday precision was measured by testing the sample solution of 30µg/ml,2 times a day and its relative standard deviation was determined.

Inter day Precision:

The method’s inter day precision was detected by examining the sample solution 2 times each on 2 consequents.

Linearity:

The linearity of an optimized method was controlled by its ability to demonstrate that test outcomes are directly proportional to the analyte’s concentration in a given range.

Preparation of linearity sample solution:

The sample solution of sofosbuvir (1mg/ml) was set up by dissolving 12.02mg, tablet powder in 10ml of volumetric flask in adequate diluent and further volume was made up till 10ml to acquire a concentration of 1000µg/ml. The working sample solution was set up by stepwise dilution of stock solution. From the above solution, various concentrations ranging from 10-50 µg/ml were prepared.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ were determined utilizing following equations as stated in ICH rules.

LOD= 3.3 × σ/S

LOQ= 10 × σ/S

Where σ is the standard deviation of the response and S is the slope of the calibration curve.

Robustness:

It is the measure of method’s ability to stay unaffected by little yet intentional variety in strategy parameter and gives a sign of its dependability under typical utilization. To check the Robustness of this method parameters that were varied are flow rate and wavelength and results were recorded in triplicate.

3. RESULTS AND DISCUSSION:

Validation of UV method:

Accuracy:

Optimized method was utilized for the estimation of drug in bulk and pharmaceutical products in the way of spiking extra pure sofosbuvir to the pre-analyzed sample, RSD of accuracy study determined was below 2% for each level and the recovery observed was 99.97% with 0.0057% of RSD.

Precision:

Intermediate precision anticipated were performed by utilizing the analysisthe18μg/ml of solution in triplicate. The RSD was seen to be 0.08754% for intraday precision and 0.06745% for inter-day precision. Therefore, from the results obtained it can be said that the proposed UV method was seen to be precise.

Linearity:

For assessing linearity, calibration graph, i.e. concentration versus absorbance was plotted. It was observed that optimized method, obey beer- lambert’s law as the graph of calibration obtained was linear with the 0.999 regression coefficient.

Robustness:

For the assurance of technique's robustness, parameter, i.e. wavelength was varied within a practical range and the quantitative impact of the factor was resolved. Robustness of the strategy was assessed at a concentration level of 18μg/ml in triplicate (n=3).

Selection and Optimization of HPLC method:

Selection and optimization of mobile phase:

Different combination of solvents was run with different ratio, a minimum tailing with a sharp peak of sofosbuvir was observed with methanol: water in the 70:30 ratio.

Figure No.1: Sofosbuvir: Methanol: Water (70:30) (4.3 minutes of retention time)

Validation of HPLC method:

Accuracy:

The accuracy study of sofosbuvir tablets was performed by adding standard drug in 3 levels, i.e. 50%, 100%, 150% and the amount recovered after analysis is determined. Each level was analyzed in triplicate. RSD of accuracy study was noticed to be below 2% for all the 3 levels whereas, in recovery study, 99.75% of sofosbuvir was recovered with the relative standard deviation of 0.17%.

Specificity:

Specificity was performed to determine of the interference mobile phase and excipients of the tablets in the optimized method. From specificity study, we got that no peaks were observed for any of the tablet excipients and also of the mobile phase. Hence there is no interference of the mobile phase and excipients in the optimized method.

Precision:

Intermediate precision (Intra-day and Inter-day)

The intraday and inter-day precision study for sofosbuvir was done by analyzing the sample twice around the same day and also on two consequent days and comparing their results by keeping the concentration 30μg/ml constant. The RSD obtained was 0.19% for intra-day and 0.11% for inter day precision. Hence it can be said that the proposed HPLC method is précised.

Linearity:

The graph obtained of calibration curve plotted based on concentration versus peak area was noticed to be linear with 0.999 regression coefficient and 70552x slope. From this we get that concentration of sofosbuvir in the span of 10-50μg/ml obeys the Beer-Lambert’s law.

LOD/LOQ:

LOD and LOQ were resolved dependent on standard deviation obtained from the calibration curve. The values obtained for sofosbuvir are given in table no.3.

Table No. 3: LOD and LOQ of Sofosbuvir

Parameters	Results
LOD (μg/ml)	0.5764
LOQ (μg/ml)	1.7468

Robustness:

For the assurance of method's robustness, parameters like flow rate and detection wavelength was varied in a practical range and the quantitative impact of the factors was resolved. Method’s strength was assessed in triplicate at a constant concentration of 20 μg/ml of sofosbuvir sample solution.

Table No. 4: Result of robustness for Sofosbuvir at different flow rate

Sr. No.	Concentration μg/ml	flow rate	Area
1	20	0.8 ml/min	1460254
2	20	0.9 ml/min	1461256
3	20	1.0 ml/min	1465874
		Mean	1462461
		SD	2997.62
		%RSD	0.204971

Table No. 5: Result of robustness for Sofosbuvir at different wavelength

Sr. No.	Concentration μg/ml	Wavelength	Area
1	20	258 nm	1461257
2	20	260 nm	1461256
3	20	262 nm	1461444
		Mean	1461319
		SD	108.254
		%RSD	0.00740799

Forced degradation studies:^31,32

Forced degradation studies were performed on sofosbuvir, which reveals that the drug was decomposed when exposed to oxidizing agents, acid, alkaline, photolytic and heat separately. From this study, we can conclude that this method (i.e. Mobile phase- methanol: water {70:30}, detection wavelength- 260nm, flow rate- 0.9ml/min, injection volume 20µl) can be utilized for observing the stability of sofosbuvir in bulk and pharmaceutical product.

Oxidative decomposition:

Stock solution and hydrogen peroxide mixture was injected into the HPLC system to evaluate the drug degraded in the presence of hydrogen peroxide (oxidizing agent) from the degraded sample’s peak zone, it was noted that 6.64% of the sofosbuvir was degraded due to oxidation.

Figure No.2: Chromatogram representing oxidative degradation of sofosbuvir

Acid decomposition:

Adding 0.1N HCl in the stock solution, it was injected into the system and from the chromatogram produced, it was recorded that due to the presence of acid 16.60 % of the sofosbuvir got degraded. The chromatogram of acid degradation is represented below.

Figure No. 3: Chromatogram representing acid degradation of Sofosbuvir

Alkaline decomposition:

Combination of stock with 0.1N NaOH solution was injected in the system for assessing the alkaline degradation. From the peak zone of chromatograms, it was evaluated that 10.45% of the drug was degraded in the presence of sodium hydroxide.

Figure No. 4: Chromatogram representing alkaline degradation of Sofosbuvir

Photolytic decomposition:

A stock solution was exposed to UV light in stability chamber at room temperature for 24 hours. From the chromatogram of this solution, it was noticed that 1.55 % of the sofosbuvir was degraded when exposed to UV light.

Figure No. 5: Chromatogram representing photolytic degradation of Sofosbuvir

Thermal decomposition:

Thermal degradation was evaluated by exposing a stock solution to 60°C by keeping in the oven. The resultant solution was injected into the system, from the results of a chromatogram, it was detected that 2.89% of the drug was degraded due to increase in temperature.

Figure No.6: Chromatogram representing thermal degradation of sofosbuvir

The table given below summarizes the outcomes of force degradation study.

Table No 6: Degradation study results of Sofosbuvir

Sr. No.	Condition	Area of degraded sample	% Degraded up to	Actual % degradation
1	Oxidative degradation	3325495	93.35653097	6.643469033
2	Acidic degradation	2970645	83.39483654	16.60516346
3	Alkaline degradation	3189547	89.54006645	10.45993355
4	Photolytic degradation	3506870	98.44826642	1.551733576
5	Thermal degradation	3458965	97.10343066	2.896569342

4. CONCLUSION:

In this research work a UV spectroscopic method and a HPLC method were developed to determine the sofosbuvir in marketed preparation. Parameters of the optimized methods are mobile phase-methanol: water (70:30) detection wavelength 260nm, flow rate 0.9ml/min, injection volume-20µl. Assay value of sofosbuvir was observed to be 99.36% by UV spectroscopy and 99.77% by HPLC method. These optimized methods were validated as stated by ICH in Q1R2 guidelines. Linearity was obtained in the span of concentration from 6-30µg/ml in UV spectroscopy and from 10-50µg/ml in HPLC method with 0.999 regression coefficient.% RSD of accuracy, precision and robustness of the optimized method was below 2 %. LOD and LOQ were calculated by HPLC method and was noticed to be 0.57μg/ml and 1.74μg/ml. The optimized HPLC method was also utilized to study force degradation and was noticed that sofosbuvir was deteriorated in presence of oxidizing agent, acidic agent, alkaline agent, heat and UV light. Hence the proposed methods can be employed successfully for the sofosbuvir estimation in bulk and marketed preparation and also degradation evaluation for regular and routine analysis.

5. ACKNOWLEDGEMENT:

The authors extend their gratitude to the Department of Pharmacy and Research Section of Mewar University, Rajasthan, India for providing essential facilities and supportive encouragement for research work.

6. REFERENCES:

1. Bhatia HK, Singh H, Grewal N, Natt NK. Sofosbuvir: A novel treatment option for chronic hepatitis C infection. Journal of Pharmacology and Pharmacotherapeutics. 2014 Oct; 5(4): 278.

2. Safety of Sofosbuvir, Daclatasvir in HCV Patients and RAVS in Resistent and Relapsed Cases. https://clinicaltrials.gov/ct2/show/ NCT03572140 [Last accessed on 2020 Feb 24].

3. Sofosbuvir, Ledipasvir, Ribavirin for Hepatitis C Cirrhotics, Genotype 1 [cited 2020 Feb 24]. Available from: https://clinicaltrials.gov/ct2/show/ NCT02705534[Last accessed on 2020 Feb 24]

4. Sofosbuvir, Daclatasvir, Ribavirin for Hepatitis C Virus (HCV) Cirrhotics. https://clinicaltrials.gov/ct2/ show/NCT02596880 [Last accessed on 2020 Feb 24]

5. Treating Hepatitis C in CRF Using Sofosbuvir and Daclatasvir. https://clinicaltrials.gov/ct2/show/ NCT03063879 [Last accessed on 2020 Feb 24]

6. Randomized, Open-Label, Study to Evaluate the Safety and Efficacy of Sofosbuvir Tablet Plus Ribavirin Tablet (Part A) Versus Single Dose (2 Tablets) of EHCV Containing Sofosbuvir, Ribavirin, and Natural Anti-hemolytic (B) in Egyptian Adults with Chronic Genotype 4 HCV Infection. https:// clinicaltrials.gov/ct2/show/NCT02483156[Last Accessed on 2020 Feb 24]

7. Herbst Jr DA, Reddy KR. Sofosbuvir, a nucleotide polymerase inhibitor, for the treatment of chronic hepatitis C virus infection. Expert Opinion on Investigational Drugs. 2013 Apr 1; 22(4): 527-36.

8. El Hamd MA, Ali R, Marzouk AA, Abdelmageed OH. Validated Ultraviolet-Spectrometric Method for Determination of Sofosbuvir in Tablets Formulation. Journal of Applied Pharmaceutical Science. 2017 Feb; 7(02): 114-9.

9. Mansour FR. A new innovative spectrophotometric method for the simultaneous determination of sofosbuvir and ledipasvir. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2018 Jan 5; 188: 626-32.

10. Rani JS, Devanna N. A new RP-HPLC method development and validation for simultaneous estimation of sofosbuvir and velpatasvir in pharmaceutical dosage form. Int. J. Eng. Technol. Sci. Res. 2017 Nov; 4: 145-52.

11. Zaman B, Siddique F, Hassan W. RP-HPLC method for simultaneous determination of sofosbuvir and ledipasvir in tablet dosage form and its application to in vitro dissolution studies. Chromatographia. 2016 Dec 1; 79(23-24): 1605-13.

12. Salama FM, Attia KA, Abouserie AA, El-Olemy A, Abolmagd E. Application of TLC densitometric method for simultaneous estimation of the newly co-formulated antiviral agents ledipasvir and sofosbuvir in their tablet dosage form. Analytical Chemistry Letters. 2017 Mar 4; 7(2): 241-7.

13. El-Gizawy SM, El-Shaboury SR, Atia NN, Abo-Zeid MN. New, simple and sensitive HPTLC method for simultaneous determination of anti-hepatitis C sofosbuvir and ledipasvir in rabbit plasma. Journal of Chromatography B. 2018 Aug 15; 1092: 432-9.

14. El-Shaboury SR, El-Gizawy SM, Atia NN, Abo-Zeid MN. Validated spectrodensitometric method for simultaneous estimation of sofosbuvir, ribavirin and saxagliptin in their pure and pharmaceutical dosage forms. Current Pharmaceutical Analysis. 2018 May 1; 14(3): 212-8.

15. Abo-Zeid MN, El-Gizawy SM, Atia NN, El-Shaboury SR. Efficient HPTLC-dual wavelength spectrodensitometric method for simultaneous determination of sofosbuvir and daclatasvir: Biological and pharmaceutical analysis. Journal of Pharmaceutical and Biomedical Analysis. 2018 Jul 15; 156: 358-65.

16. Nebsen M, Elzanfaly ES. Stability-indicating method and LC–MS-MS characterization of forced degradation products of sofosbuvir. Journal of Chromatographic Science. 2016 Oct 17; 54(9): 1631-40.

17. Reddy CS, Khan KA, Nagaraja C. A Review on the Determination of Melting Point Measurement System. International Journal of Advanced Research in Electrical, Electronics and Instrumentation Engineering. 2016; 5(2): 975-9.

18. Bunaciu AA, Aboul-Enein HY, Fleschin S. Recent applications of fourier transform infrared spectrophotometry in herbal medicine analysis. Applied Spectroscopy Reviews. 2011 Apr 19; 46(4): 251-60.

19. Lim L, Yan F, Bach S, Pihakari K, Klein D. Fourier transform mass spectrometry: The transformation of modern environmental analyses. International Journal of Molecular Sciences. 2016 Jan; 17(1): 104.

20. Larsson J. Methods for measurement of solubility and dissolution rate of sparingly soluble drugs. [Last accessed on 2020 Feb 25]

21. Savjani KT, Gajjar AK, Savjani JK. Drug solubility: importance and enhancement techniques. ISRN Pharmaceutics. 2012 Jul 5; 2012.

22. Eldin AS, Azab SM, Shalaby A, El-Maamly M. The development of a new validated HPLC and spectrophotometric methods for the simultaneous determination of daclatasvir and sofosbuvir: antiviral drugs. Journal Pharmacy and Pharmacology Research. 2017; 1(1): 28-42.

23. Hassouna ME, Mohamed MA. Novel and facile spectrophotometric techniques for the determination of sofosbuvir and ledipasvir in their tablet dosage form. J Anal Pharm Res. 2018; 7(2): 92-9.

24. Abo-Talib NF, El-Ghobashy MR, Tammam MH. Spectrophotometric methods for simultaneous determination of sofosbuvir and ledipasvir (HARVONI tablet): comparative study with two generic products. Journal of AOAC International. 2017 Jul 1; 100(4): 976-84.

25. Baker MM, El-Kafrawy DS, Mahrous MS, Belal TS. Validated spectrophotometric and chromatographic methods for analysis of the recently approved hepatitis C antiviral combination ledipasvir and sofosbuvir. In Annales Pharmaceutiques Francaises 2018 Jan 1 (Vol. 76, No. 1, pp. 16-31). Elsevier Masson.

26. Lalitha KV, Reddy JR, Devanna N. Stability indicating RP-HPLC method development and validation for estimation of sofosbuvir in pharmaceutical dosage form. The Pharma Innovation. 2018 May 1; 7(5, Part J): 656.

27. Vanaja B, Vageesh NM, Kistayya C, Urukundu V. RP-HPLC method development and validation for simultaneous estimation of sofosbuvir and velpatasvir in pure and pharmaceutical dosage form. Innovat International Journal of Medical and Pharmaceutical Sciences. 2018 Jan 1; 3(1).

28. Bandla J, Ganapaty S. Stability indicating RP-HPLC method development and validation for the simultaneous determination of Sofosbuvir and Velpatasvir in tablet dosage forms. Indian Journal of Pharmaceutical and Biological Research. 2017 Sep 30; 5(04): 10-6.

29. Rote AP, Alhat J, Kulkarni AA. Development and validation of RP-HPLC method for the simultaneous estimation of ledipasvir and sofosbuvir in bulk and pharmaceutical dosage form. Int J Pharm Sci Drug Res. 2017; 9(6): 291-8.

30. Miraghaei S, Mohammadi B, Babaei A, Keshavarz S, Bahrami G. Development and validation of a new HPLC-DAD method for quantification of sofosbuvir in human serum and its comparison with LC–MS/MS technique: Application to a bioequivalence study. Journal of Chromatography B. 2017 Sep 15; 1063: 118-22.

31. Rote AP, Alhat J, Kulkarni AA. Development and validation of RP-HPLC method for the simultaneous estimation of ledipasvir and sofosbuvir in bulk and pharmaceutical dosage form. Int J Pharm Sci Drug Res. 2017; 9(6): 291-8.

32. Mastanamma SK, Chandini SK, Reehana SK, Saidulu P. Development and validation of stability indicating RP-HPLC method for the simultaneous estimation of Sofosbuvir and Ledipasvir in bulk and their combined dosage form. Future Journal of Pharmaceutical Sciences. 2018 Dec 1; 4(2): 116-23.

Received on 07.09.2020 Modified on 11.01.2021

Research J. Pharm. and Tech. 2021; 14(8):4165-4172.

DOI: 10.52711/0974-360X.2021.00721

Sr. No.	% Recovery	Concentration of drug in Tablet	Concentration of Standard drug added	Final Concentration
1.	50%	20ppm	10ppm	30ppm
2.	100%	20ppm	20ppm	40ppm
3.	150%	20ppm	30ppm	50ppm