INTRODUCTION:

Sofosbuvir N- [[P(S), 2′R] -2′-Deoxy -2′-fluoro -2′-methyl – P– phenyl - 5′-uridylyl] -l-alanine (fig.1). Sofosbuvir a recently approved nucleotide analog, is a highly potent inhibitor of the NS5B polymerase in the Hepatitis C virus (HCV), and has shown high efficacy in combination with several other drugs, with and without PEG-INF, against HCV.

Sofosbuvir is used for treatment of chronic HCV genotype 1, 2, 3, or 4 infection in treatment-naive (previously untreated) or previously treated adults without cirrhosis or with compensated cirrhosis, including those with HIV infection and those with hepatocellular carcinoma awaiting liver transplantation[1,2].

Fig. 1: Chemical structure of Sofosbuvir

Velpatasvir Methyl { (1R) -2 - [ ( 2S, 4S) - 2 -( 5 - { 2 - [ ( 2S , 5S ) – 1 - { ( 2S ) – 2 - [ (methoxy carbonyl) amino] – 3– methyl butanoyl } – 5–metl pyrrolidin – 2 – yl ] - 1,11–dihydro [2] benzo pyrano [4',3':6,7] naphtha [1, 2 - imidazol -9 –yl} (methoxy methyl) pyrrolidin-1-yl –2 –oxo -1-phenyl ethyl} carbamate (fig.2) is a white to tan or yellow hygroscopic solid. Only one solid form is known to date. Velpatasvir 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 1 being the most common in the United States, and affecting 72% of all chronic HCV patients[3]. Velpatasvir acts as a defective substrate for NS5A (Non-Structural Protein 5A), a non-enzymatic viral protein that plays a key role in Hepatitis C Virus replication, assembly, and modulation of host immune responses[4]. Velpatasvir belongs to Biopharmaceutics Classification System (BCS) Class 4 (low solubility relative to dose and low permeability) and exhibits pH-dependent solubility; it is soluble at pH 1.2, sparingly soluble at pH 2 and practically insoluble at pH > 5.

Fig. 2: Structure of Velpatasvir

The combination of Sofosbuvir and Velpatasvir as a tablet had high overall cure rates of 99% for genotype 2 (GT2) patientsand 95% for genotype 3 (GT3) patients and velpatasvir was also found to be highly effective in patients with GT1, GT4, GT5, and GT6, with overall cure rates ranging from 97–100^[5]. Literature survey reveals that many analytical methods are reported for determination of sofosbuvirindividuallyand sofosbuvir with other combination drugs [5,6,7,8,9]. However, few methods have been reported for simultaneous estimation of these two drugs by reverse phase HPLC where no method has been reported about stability data.

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. An ideal stability-indicating method is one that resolves the drug and its degradation products efficiently. Consequently, the implementation of an analytical methodology to determine sofosbuvir and velpatasvir simultaneously, in presence of its degradation products is rather a challenge for pharmaceutical analyst. Therefore, it was thought necessary to study the stability of sofosbuvir and velpatasvir under acidic, alkaline, oxidative, UV and photolytic conditions. The developed method is simple, accurate, precise, reproducible and repeatable stability-indicating method and suitable for routine analysis of sofosbuvir and velpatasvir in combined dosage form. The method was validated and complies with ICH guidelines [10,11,12,13,14].

MATERIALS AND METHODS:

Distilled water (HPLC grade), Acetonitrile(HPLC grade), Methanol(HPLC grade), Phosphate buffer, Potassium dihydrogen ortho phosphate buffer, Ortho-phosphoric acid used were of Rankem, Hyderabad, India. The tablet formulation containing 400mg of sofosbuvir and 100mg of velpatasvir was procured from local market and used for analysis of marketed formulation. The liquid chromatographic system used was WATERS HPLC 2695 SYSTEM equipped with quaternary pumps, Photo Diode Array detector and Auto sampler integrated with Empower 2 Software was used for LC peak integration and Data processing. UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2 mm and 10 mm and matched quartz cells integrated with UV-win 6 was used for measuring absorbance of sofosbuvir and velpatasvir solutions. Electronics Balance-Denver, pH meter-BVK enterprises, Ultrasonicator-BVK enterprises were used in this study.

Preparation of Mobile Phase and Stock Solutions:

Buffer (0.1% OPA) and Acetonitrile were mixed in the ratio of 50:50. This mixture was sonicated for 10 min and filtered through 0.22 µm membrane filter and used as mobile phase. Stock solutions were prepared by weighing 40 mg of sofosbuvir and 10 mg velpatasvir. and were transferred to two separate 10 ml volumetric flasks and the volume was made up to the mark with diluent to obtain a solution containing required concentrations of sofosbuvir and velpatasvir. The solutions were further diluted with the same solvent to obtain final concentrations of sofosbuvir and velpatasvir. The HPLC analysis was performed on the above mentioned chromatographic system by using isocratic elution mode using a mobile phase of 0.1% OPA Buffer:Acetonitrile (50:50, v/v) on BDS C18 column (250×4.6 mm, 5 µm particle size) with 1 ml/min flow rate at 240 nm using photodiode array detector by maintain ambient temperature.

Linearity profile Sofosbuvir and Velpatasvir:

Tablet formulation containing sofosbuvir and velpatasvir in a ratio of 4:1 was taken and suitable aliquots of sofosbuvir and velpatasvir were prepared to obtain the final concentrations of 100-600 μg/ml and 25-150 μg/ml respectively. Calibration curves were constructed by plotting average peak areas versus concentrations and regression equations were computed for both the drugs (Table 1).

Analysis of Marketed Formulations:

Twenty tablets were weighed accurately and the average weight of tablets was calculated, then the weight equivalent to one tablet was transferred into a 100 ml volumetric flask, 50 ml of diluent was added and sonicated for 25 min, further the volume was made up to the mark with diluent and filtered by HPLC filters and volume was made upto the mark with diluent to get the required concentrations. The above solution was filtered using Whatman filter paper No1. Above sample solution was injected into HPLC and peak areas were measured under optimized chromatographic conditions.

METHOD VALIDATION:

The method of analysis was validated as per the recommendations of ICH [21]for the parameters like accuracy, linearity, precision, detection limit, quantization limit and robustness. The accuracy of the method was determined by calculating percentage recovery of sofosbuvir and velpatasvir. For both the drugs, recovery studies were carried out by applying the method to drug sample to which known amount of sofosbuvir and velpatasvir corresponding to 50, 100 and 150% of label claim had been added (standard addition method). At each level of the amount six replicates were performed and the results obtained were compared.

Intraday and Interday precision study of sofosbuvir and velpatasvir was carried out by estimating the corresponding responses of three replicates on the same day and on three different days by the using 400 μg/ml and 100 μg/ml concentrations of sofosbuvir and velpatasvir respectively.

The limit of detection (LOD) and limit of quantification (LOQ) were calculated the formulae

LOD = 3.3(SD)/S and

LOQ = 10 (SD)/S,

where SD = standard deviation of response (peak area) and S = average of the slope of the calibration curve.

System suitability tests are an integral part of chromatographic method which is used to verify reproducibility of the chromatographic system. To ascertain its effectiveness, certain system suitability parameters were checked by injecting the drug solution at the concentration level 400 μg/ml and 100 μg/ml for sofosbuvir and velpatasvir respectively to check the reproducibility of the system and the results are shown in Table 2.

Robustness evaluation studies were carried out by doing deliberate changes in flow rate, ratio of the organic mobile phase and temperature. One factor was changed at one time to estimate the effect. Each factor selected was changed at three levels (-1, 0, +1) with respect to optimized parameters. Robustness of the method was done at the concentration levels of 400 μg/ml and 100 μg/ml for sofosbuvir and velpatasvir respectively.

Forced degradation studies:

Forced degradation studies of both the drugs were carried out under conditions of hydrolysis, dry heat, oxidation, UV light and photolysis.

Preparation of Solutions:

Sofosbuvir and velpatasvir were weighed (400 mg and 100 mg each) and transferred into two 100 ml volumetric flasks and diluted up to the mark with diluents to give 4000 μg/ml of sofosbuvir and 1000 μg/ml of velpatasvir. These stock solutions were used for forced degradation studies.

Basic degradation:

Forced degradation in basic media was performed by taking 10ml of stock solution of sofosbuvir and velpatasvir each in separate round bottom flasks. Then 1ml of 2N NaOH was added and these mixtures were heated for up to 30 min at 60° C in dark, in order to exclude the possible degradation effect of light.

Acid degradation:

Forced degradation in acidic media was performed by keeping the drug in contact with 2N Hydrochloric acid for up to 30 min at ambient temperature as well as heating for up to 30 min at 60°in dark.

Peroxide degradation:

Degradation with hydrogen peroxide was performed by taking 1 ml of stock solution of sofosbuvir and velpatasvir in two different flasks and add 1 ml of 20% (w/v) hydrogen peroxide in each of the flasks. These mixtures were kept for up to one day in the dark.

Neutral degradation:

To study neutral degradation, 1ml of stock solution of sofosbuvir and velpatasvir taken in two different flasks, then 1 ml of HPLC grade water was added in each flask, these mixtures were heated for 30 min at 60°in the dark.

Dry heat degradation:

For dry heat degradation, solid drugs were kept in Petri dish in oven at 105°for 1hr. Thereafter, 400 mg of sofosbuvir and 100 mg velpatasvir were weighed and transferred to two separate 10 ml volumetric flasks and diluted up to the mark with diluents.

UV degradation:

For UV degradation study, the stock solutions of both drugs (4000 µg/ml of sofosbuvir and 1000 µg/ml of velpatasvir) were exposed to UV Chamber for 1day or 200 Watt hours/m²in photo stability chamber.

For HPLC analysis, all the degraded sample solutions were diluted with mobile phase to obtain final concentration of 400 μg/ml of sofosbuvir and 100 μg/ml of velpatasvir. Similarly mixture of both drugs in a concentration of 400 μg/ml of sofosbuvir and 100 μg/ml velpatasvir each was prepared prior to analysis by HPLC. Besides, solutions containing 400 μg/ml of sofosbuvir and 100 mg of velpatasvir drug separately were also prepared without being performing the degradation of both the drugs. Then 10μl solution of above solutions were injected into HPLC system and analyzed under the chromatographic condition described earlier.

RESULTS AND DISCUSSION:

The mobile phase consisting of 0.1% OPA Buffer: Acetonitrile (50:50, v/v) pH 3.0 adjusted with o-phosphoric acid, at 1ml/min flow rate was optimized which gave two sharp, well-resolved peaks with minimum tailing factor for sofosbuvir and velpatasvir (fig.3). The retention times for sofosbuvir and velpatasvir were 2.267 min and 2.983 min, respectively. UV overlain spectra of both sofosbuvir and velpatasvir showed that both drugs absorbed appreciably at 240 nm, so this wavelength was selected as the detection wavelength. The calibration curve for sofosbuvir and velpatasvir was found to be linear over the range of 100- 600 μg/ml and 20-150 μg/ml, respectively. The data of regression analysis of the calibration curves is shown in Table 1. The proposed method was successfully applied for the determination of sofosbuvir and velpatasvir in their combined tablet dosage form. The results for the combination were comparable with the corresponding labeled amounts. The developed method was also found to be specific, since it was able to separate other excipients present in tablet from the two drugs (fig.3).

The LOD and LOQ values were found to be 0.20 μg/ml and 0.61 μg/ml for sofosbuvir while 0.21 μg/ml and 0.65 μg/ml for velpatasvir respectively. The results for validation and system suitability parameters are summarized in Table 2. Results for robustness for both the drugs are presented in Table 2.

Insignificant differences in peak areas and less variability in retention times were observed. The degradation study indicated that sofosbuvir and velpatasvir were stable to neutral hydrolysis, acid, base, H₂O₂, UV radiation, and dry heat under experimental conditions. In all conditions the drug degrades very small as observed by the decreased area in the peak of the drug when compared with peak area of the same concentration of the no degraded drug, without giving any additional degradation peaks. Summary of degradation studies of both the drugs is given in Table 3.

In the proposed study, stability-indicating HPLC method was developed for the simultaneous determination of sofosbuvir and velpatasvir and validated as per ICH guidelines. Statistical analysis proved that method was accurate, precise, and repeatable. The developed method was found to be simple, sensitive and selective for analysis of sofosbuvir and velpatasvir in combination without any interference from the excipients. The method was successfully used for determination of drugs in a pharmaceutical formulation. Assay results for combined dosage form using proposed method showed 99.86 % of sofosbuvir and 99.61 % of velpatasvir. The results indicated the suitability of the method to study stability of sofosbuvir and velpatasvir under various forced degradation conditions like acid, base, dry heat, neutral, photolytic and UV degradation. It can be concluded that the method separates the drugs from their degradation products; it may be employed for route analysis of sofosbuvir and velpatasvir.

Fig. 3: Typical Chromatogram Sofosbuvir and Velpatasvir

Table 1: Linear regression data for calibration curves

Parameters (Units)	Sofosbuvir	Velpatasvir
Linearity range (µg/ml)	100-600µg/ml	25-150µg/ml
r²±SD	0.999	0.999
Slope±SD	5618±16.04	3546±3.464 3.464 3.464 ±0.0104 ±0.053
Intercept±SD	2324±340.6	558±229.8
Average of SE of estimation	3.8	2.6

Table 2: Summary of validation and system suitability parameters

Parameter (Units)	Sofosbuvir	Velpatasvir
Linearity range (µg/ml)	100-600	25-150
Correlation coefficient	0.99989±1.195	0.99995±1.067
LOD (μg/ml)	0.20	0.21
LOQ (μg/ml)	0.61	0.65
Recovery (%)	100.34	101.37
Precision (%RSD)
Interday (n=3)	0.1	0.236
Intraday (n=3)	0.3	0.30
Robustness	Robust	Robust
Retention Time ± allowable time (min.)	2.66±0.01	2.98±0.01
Resolution		3.7
Theoretical Plates	8486	8453
Tailing Factor (asymmetry factor)	1.25	1.12

Table 3: Summary of degradation data

Condition	% Degradation
Condition	Sofosbuvir	Velpatasvir
Acid, 2 N HCL (heated, at 60°)	3.49	3.64
Base, 2N NAOH (heated at 60°)	3.49	3.64
Oxidative, 20% w/v H₂O₂(ambient, in dark)	3.61	2.90
Neutral hydrolysis (heated, at 60°)	0.84	0.90
Dry Heat (105°)	4.57	4.11
UV Radiation at 256 nm	1.79	2.44

Table 4 Comparison Table

Validation Parameters	Method developed		Reported method [16]		Reported method [17]
Validation Parameters	Sofosbuvir	Velpatasvir	Sofosbuvir	Velpatasvir	Sofosbuvir	Velpatasvir
Retention time (min)	3.44	4.68	2.843	3.875	4.25	6.05
Linearity (µg/ml)	100-600	25-150	80-240	20-60	100-600	25-150
Regression equation(r²)	0.999	0.999	0.9997	0.9997	0.999	0.997
LOD (µg/ml)	0.20	0.21	0.005	0.001	7.71	2.74
LOQ (µg/ml)	0.61	0.65	0.02	0.003	23.37	8.31
Accuracy (%)	100.34	101.37	99.5-99.7	99-100.5	99.40	99.60
Precision (%RSD)	0.1	0.236	0.25	0.42	0.92	0.6
Mobile phase	Buffer: Acetonitrile (50:50)		0.1%v/v Trifloro acetic acid in Water: Methanol (42:58).		Acetonitrile: 0.1%perchloric acid (50:50 v/v)

CONCLUSIONS:

Rapid reproducible RP-HPLC method for simultaneous estimation of sofosbuvir and velpatasvir was developed and compared with the developed methods and reported in the above table 4. In the developed method retention time was very less with simple mobile phase when compared with the developed methods. As per the ICH guidelines the validation parameters were performed and found to be within the limits. Degradation studies were performed by exposing the drugs to different stress conditions such as acid, base, peroxide, heat, UV and neutral and found very less degradation where no other method have been reported about the degradation studies. Based on all the above parameters we can conclude that the developed method is simple reproducible and stability indicating method for routine simultaneous analysis of these drugs in bulk and pharmaceutical dosage forms.

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Received on 28.07.2018 Modified on 31.08.2018

Research J. Pharm. and Tech 2018; 11(12): 5425-5430.

DOI: 10.5958/0974-360X.2018.00990.3