INTRODUCTION:

Hepatitis C is a comprehensive liver disease produced by the hepatitis C virus (HCV) and can increase liver cirrhosis, liver failure, liver cancer and liver transplantation. Daclatasvir, is a nucleotide analogue NS5A polymerase inhibitor. Sofosbuvir, is a nucleotide analogue HCV NS5B polymerase inhibitor that is used in the treatment of chronic hepatitis C genotypes 1,2,3 or 4.


Fig 1: Chemical Structure of Sofosbuvir	Fig 2: Chemical Structure of Daclatasvir

High performance liquid chromatography is defined as a separation of mixtures of compounds due to differences in their distribution equilibrium between two phases, the stationary phase packed inside columns and the mobile phase, delivered through the columns by high pressure pumps. Components whose distribution into the stationary phase is higher, are retained longer, and get separated from those with lower distribution into the stationary phase. HPLC is one of the most widely used analytical instruments for quantification of various analytes.^1-5 Novel methods will service as tools in quality control laboratories prior to the best method inclusion in pharmacopeias.^14-18The developed methods are validated as per ICH guidelines. The study was analytically validated according to the ICH guidelines.^6-10The sofosbuvir and daclatasvir combination is associated with a high rate of SVR4 in difficult-to-treat patients infected with genotype 1 or 4. Various methods were reported for estimation of sofosbuvir and daclatasvir in individual and other drug combinations.^11-15The present study describes a validated RP-HPLC method for the simultaneous quantitative detection of sofosbuvir and daclatasvir in its pure and commercially available tablet form. The developed method was more sensitive than the previously reported HPLC methods.

MATERIALS AND METHODS:

Sofosbuvir and Daclatasvir pure drugs (API), Combination Sofosbuvir and Daclatasvir (Darvoni) tablets, Distilled water, Acetonitrile, Phosphate buffer, Methanol, Potassium dihydrogen ortho phosphate buffer, Ortho-phosphoric acid. All the above chemicals and solvents are from Rankem.

Diluent:

Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in the ratio of 50:50

Preparation of Standard stock solutions:

Accurately weighed 40 mg of Sofosbuvir, 6mg of Daclatasvir and transferred to 25ml volumetric flask and 3/4^th of diluents was added to these flask and sonicated for 10 minutes. Flask were made up with diluents and labeled as Standard stock solution. (1600µg/ml of Sofosbuvir and 240µg/ml of Daclatasvir)

Preparation of Sample stock solutions:

5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to tablet was transferred into a 100 ml volumetric flask, 50ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (4000µg/ml of Sofosbuvir and 600µg/ml of Daclatasvir)

Preparation of Sample working solutions (100% solution):

0.4ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (160µg/ml of Sofosbuvir and 24µg/ml of Daclatasvir).

System suitability parameters:

The system suitability parameters were determined by preparing standard solutions of Sofosbuvir (160ppm) and Daclatasvir (24ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined. The %RSD for the area of six standard injections results should not be more than 2%.

Validation:

The proposed method was validated as per ICH guidelines. The parameters studied for validation were system suitability, specificity, linearity, precision, accuracy (recovery), ruggedness and robustness, limit of detection and limit of quantification.

RESULT AND DISCUSSION:

Optimized wavelength selected was 279 nm.

Table 1: Method development was done by changing various mobile phase ratios, buffers etc.

Chromatographic Conditions	Trail 1	Trail 2	Trail 3	Trail 4	Trail 5	Optimized
Mobile phase	Methanol: Water (50:50)	Water: Acetonitrile (50:50)	Acetonitrile: Water (45:55)	Acetonitrile: Water (55:45)	Acetonitrile: Water (60:40)	Acetonitrile: Water (60:40)
Flow rate	1 ml/min	1.0 ml/min	1.0 ml/min	1.0 ml/min	0.8 ml/min	0.7ml/min
Column	Kromosil C18 (4.6 x 250mm, 5µm)	Ascentis C18 (4.6 x 150mm, 5µm)	Kromosil C18 (4.6 x 150mm, 5µm)	Discovery C18 (4.6 x 150mm, 3.5µm)	Ascentis C18 (4.6 x 150mm, 3.5µm)	Ascentis C18 (4.6 x 150mm, 5µm)
Detector wave length	279nm	279nm	279nm	279nm	279nm	279nm
Column temperature	30°C	30°C	30°C	30°C	30°C	30°C
Injection volume	10mL	10mL	10mL	10mL	10mL	10mL
Run time	10 min	10 min	10 min	10.0 min	5.0 min	5.0 min
Diluent	Water and Acetonitrile in the ratio 50:50	Water and Acetonitrile in the ratio (50:50)	Water and Acetonitrile in the ratio 50:50	Water and Acetonitrile in the ratio 50:50	Water and Acetonitrile in the ratio 50:50	Water and Acetonitrile in the ratio 50:50
Results	Peak eluted with void volume, Tailing is not good, so, Further trial is carried out.	Retention time is more so, Further trial is carried out.	Retention times of the peaks were more than the literature, hence further trial is carried out.	Peaks were eluted at void time, hence further trial is carried out.	Peaks were eluted at void time, hence further trial is carried out.	Both peaks have good resolution, tailing factor, theoretical plate count and resolution.

Fig.3. Optimized Chromatogram

Daclatasvir and Sofosbuvir were eluted at 2.186 min and 2.747 min respectively with good resolution. Plate count and tailing factor was very satisfactory, so this method was optimized and to be validated. (Figure-3).

Specificity:

Retention times of Sofosbuvir and Daclatasvir were 2.186 min and 2.747 min respectively. We did not found and interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Linearity:

Six linear concentrations of Sofosbuvir (40-240µg/ml) and Daclatasvir (6- 36µg/ml) were injected in a duplicate manner. Average areas were mentioned above and linearity equations obtained for Sofosbuvir was y = 9010x + 21702 and of Daclatasvir was y = 10136x + 1757 Correlation coefficient obtained was 0.999 .

Table 2: Linearity for Sofosbuvir and Daclatasvir

Sofosbuvir		Daclatasvir
Conc (μg/mL)	Peak area	Conc (μg/mL)	Peak area
0	0	0	0
40	383540	6	61890
80	758279	12	123758
120	1112232	18	185457
160	1474488	24	247506
200	1833235	30	309513
240	2158526	36	361259

Precision:

Table 3: System precision of Sofosbuvir and Daclatasvir

S. No	Area of Sofosbuvir	Area of Daclatasvir
1.	1042454	247496
2.	1061619	248687
3.	1050784	251651
4.	1062308	247323
5.	1059490	244217
6.	1049332	245659
Mean	1054331	247506
S.D	8024.9	2562.7
%RSD	0.8	1.0

From a single volumetric flask of working standard solution six injections were given and the obtained areas were mentioned above. Average area, standard deviation and %RSD were calculated for two drugs. %RSD obtained as 0.8% and 1.0% respectively for Sofosbuvir and Daclatasvir. As the limit of Precision was less than “2” the system precision was passed in this method.

Repeatability:

Multiple sampling from a sample stock solution was done and six working sample solutions of same concentrations were prepared, each injection from each working sample solution was given and obtained areas were mentioned in the above table. Average area, standard deviation and % RSD were calculated for two drugs and obtained as 0.4% and 0.3% respectively for Sofosbuvir and Daclatasvir As the limit of Precision was less than “2” the system precision was passed in this method.

Accuracy:

Table 4: Accuracy table of Sofosbuvir

% Level	Amount Spiked (μg/mL)	Amount recovered (μg/mL)	% Recovery	Mean %Recovery
50%	80	79.5216426	99.40	99.88%
	80	79.6751387	99.59
	80	80.5498335	100.69
100%	160	158.416426	99.01
	160	160.972586	100.61
	160	159.77636	99.86
150%	240	238.616648	99.42
	240	242.066593	100.86
	240	238.677802	99.45

Table 5: Accuracy table of Daclatasvir

% Level	Amount Spiked (μg/mL)	Amount recovered (μg/mL)	% Recovery	Mean % Recovery
50%	12	11.906472	99.22	99.80%
	12	12.0067088	100.06
	12	12.0667916	100.56
100%	24	24.1822218	100.76
	24	23.8091949	99.20
	24	23.7585833	98.99
150%	36	36.1834057	100.51
	36	35.9342936	99.82
	36	35.667719	99.08

Three levels of Accuracy samples were prepared by standard addition method. Triplicate injections were given for each level of accuracy and mean %Recovery was obtained as 99.88% and 99.80% for Sofosbuvir and Daclatasvir respectively.

Robustness:

Robustness conditions like Flow minus (0.55ml/min), Flow plus (0.65ml/min), mobile phase minus (55B:45A), mobile phase plus (45B:55A), temperature minus (25°C) and temperature plus (35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

CONCLUSION:

A simple, Accurate, precise method was developed for the simultaneous estimation of the Sofosbuvir and Daclatasvir in Tablet dosage form. Retention time of Sofosbuvir and Daclatasvir were found to be 2.198 min and 2.765 min. %RSD of the Sofosbuvir and Daclatasvir were found to be 0.4 and 0.3 respectively. %Recovery was obtained as 99.88% and 99.80% for Sofosbuvir and Daclatasvir respectively. LOD, LOQ values obtained from regression equations of Sofosbuvir and Daclatasvir were 1.73, 5.23 and 0.12, 0.36 respectively. Regression equation of Sofosbuvir is y = 9010x+21702, and y = 10136x+1757 of Daclatasvir Retention times were decreased and that run time was decreased, so the method developed was simple and economical that can be adopted in regular Quality control test in Industries.

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Received on 07.03.2021 Modified on 15.07.2021

Research J. Pharm. and Tech. 2022; 15(6):2447-2450.

DOI: 10.52711/0974-360X.2022.00408