Stability indicating RP-HPLC Method development and Validation for the Estimation of Sofosbuvir, Velpatasvir and Voxilaprevir in Bulk and Pharmaceutical dosage form

 

Tej Kumar Kokkirala*, Duvvuri Suryakala

Department of Chemistry, GITAM Institute of Science,

GITAM, Visakhapatnam - 530045, Andhra Pradesh, India.

 

ABSTRACT:

A simple, accurate, precise RP-HPLC method was developed for the simultaneous estimation of the Sofosbuvir, Velpatasvir and Voxilaprevir Tablet [Vosevi^®] dosage form. The method was carried through Agilent C18 150 x 4.6mm, 5m, with mobile phase containing buffer 0.01N Na₂HPO₄ and acetonitrile in the ratio of 60:40, pumped through column at 1ml/min flow rate, pH was adjusted to 4.5 with dilute orthophosphoric acid solution at 220 nm wavelength. Retention time of Sofosbuvir, Velpatasvir and Voxilaprevir were 2.229 min, 2.957 min and 3.568 min respectively. The proposed triple combination method can be used for estimation of these drugs in combined dosage forms. All the validation study was found statistically significant because all the statistical parameters were within the acceptance range for both accuracy and precision. The %RSD of Sofosbuvir, Velpatasvir and Voxilaprevir were observed as 0.8, 1.0 and 0.7 respectively. The % recovery was obtained as 100. 27%, 100.78% and 100.10% for Sofosbuvir, Velpatasvir and Voxilaprevir respectively. The high recovery and low coefficient of variation % revealed the reliability of the method for quantitative study of three drugs in Vosevi^® tablets. The method is a rapid and cost-effective quality-control tool for routine quantitative analysis of Sofosbuvir, Velpatasvir and Voxilaprevir in tablet dosage form.

 

 

 

INTRODUCTION:

Vosevi^® is an oral tablet, a fixed dose combination containing the combination of Sofosbuvir, Velpatasvir and Voxilaprevir [400mg: 100mg: 100mg] used for the treatment of adult patients with chronic HCV infection without cirrhosis or with compensated cirrhosis (Child-Pugh A).

 

Sofosbuvir is chemically known as (S)-isopropyl-2-((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2­-yl) methoxy) (phenoxy) phosphorylamino) propanoate.

 

Sofosbuvir has been approved to Gilead with the brand name Sovaldi^®, with the dosage strength of 400 mg oral tablet. Sovaldi^® is a hepatitis C virus (HCV) nucleotide analog NS5B polymerase inhibitor indicated for the treatment of chronic hepatitis C (CHC) infection as a component of a combination antiviral treatment regimen. Sofosbuvir is a white to off-white crystalline solid with a solubility of ≥ 2 mg/ml across the pH range of 2 – 7.7 at 37 °C and is slightly soluble in water^1,2.

 

Velpatasvir is chemically known as methyl-{(1R)-2-[(2S,4S)-2-(5-{2[(2S,5S)-1-{(2S)-2-[(methoxy carbonyl) amino]-3-methylbutanoyl}-5-methylpyrrolidin-2-yl]-1,11-dihydro[2]benzopyrano[4',3':6,7] naphtho[1,2-d] imidazol-9-yl}-1H-imidazol-2-yl)-4(methoxymethyl) pyrrolidin-1-yl]-2-oxo-1-phenylethyl} carbamate. Velpatasvir is not approved as such alone in US. Gilead has got the first approval for Velpatasvir in combination with Sofosbuvir with the brand name Epclusa® as an oral tablet with the strength of 400 mg [Sofosbuvir]; 100mg [Velpatasvir]³.

 

Voxilaprevir is chemically identified as (1aR,5S,8S,9S,10R,22aR)-5-tert-butylN-{(1R,2R)-2-(difluoromethyl)-1-[(1-methyl cyclopropanesulfonyl) carbamoyl] cyclopropyl}-9-ethyl-18,18-difluoro-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methano cyclopropa [18,19] [1,10,3,6] dioxadiaza cyclononadecino [11,12-b] quinoxaline-8-carboxamide. Voxilaprevir has not yet been approved as alone in US, whereas it is approved to Gilead in combination with Sofosbuvir and Velpatasvir. Voxilaprevir is a white to light brown solid and is slightly hygroscopic to hygroscopic. Voxilaprevir is practically insoluble (less than 0.1 mg/ml) below pH 6.8⁴. The Chemical structures of Sofosbuvir, Velpatasvir and Voxilaprevir are as depicted in Figure – 1.

 

Figure 1: Chemical structures of Sofosbuvir; Velpatasvir and Voxilaprevir

 

Stability indicating RP-HPLC method development and validation for the simultaneous determination of Sofosbuvir and Velpatasvir in tablet dosage forms reported by Sarath et al in-World Journal of Pharmacy and pharmaceutical sciences⁵. A new RP-HPLC method for the simultaneous assay of Sofosbuvir and Ledipasvir in combined dosage form has been reported by Nagaraju et al in International Journal of Chemtech research⁶. RP-HPLC method development and validation for Velpatasvir and Voxilaprevir by simultaneous determination in bulk and their pharmaceutical dosage forms was reported by Abdul et al in IJCPS⁷.

 

The previously reported prior-art HPLC method for the simultaneous estimation of Sofosbuvir, Velpatasvir and Voxilaprevir reported by Sridevi et al is time consuming and tedious⁸. The present work authors have developed a specific improved RP-HPLC method, wherein the retention time is very less and the method is linear, precise. In the current study to develop a simple stability indicating method selected RP-HPLC technique. The HPL is a powerful analytical tool for development of simple analytical methods with less run time, most of the stability indicating methods were published, Dongala et al 2019 explained the importance of HPLC in pharmaceutical industry^9-14. The current optimized HPLC method have high sensitivity and getting close to 100% recovery, assay values. Hence, the HPLC method being developed by present work authors is more cost-effective, time saving and more stable. The detailed comparison of current improved HPLC method with respect to the previously published method has been depicted in Results and discussion at Table 9.

 

MATERIALS AND METHODS:

Instruments:

Waters (2695) PDA detector HPLC using the software Empower 2. Used Metler Toledo analytical balance from the range of 1mg to 200g. To extract the API from sample matrix used Bransonic ultra sonicator.

 

Source(s) of Chemicals and Reagents:

The material and the corresponding sources (s) as given below

 

Reference sample is from Spectrum Pharmalabs, Hyderabad, Telangana, India; Test sample (Vosevi^® formulation) is from Local Pharmacy, Hyderabad, Telangana, India; HPLC grade: Acetonitrile, Methanol and water from Merck chemical division, Mumbai, Maharashtra, India; AR grade: Potassium dihydrogen ortho phosphate, ortho-phosphoric acid and sodium dihyrogen ortho phosphate from Rankem, Mumbai, Maharashtra, India.

 

Sample Processing:

Diluents:

Based up on the solubility of the drug, diluents were selected, initially dissolved in methanol and diluted with Acetonitrile: 0.01N Na₂HPO₄ (50/50 v/v).

 

Preparation of Standard stock solutions:

Accurately weighed 40mg of Sofosbuvir, 10mg of Velpatasvir and 10mg of Voxilaprevir were transferred into three 25ml volumetric flasks distinctly. 10ml of methanol was added to flasks and sonicated for 15 minutes. Flasks were made up with diluents and labelled as Standard stock solution 1, 2 and 3. From the each stock solution 1 ml was pipetted out and taken into a 10 ml volumetric flask and made up with diluents.

 

Preparation of Sample stock solutions:

5 tablets of Vosevi^® were weighed and the average weight of each tablet was calculated and the weight equivalent to 1 tablet was transferred into a 100ml volumetric flask. 25ml of diluent was added to the volumetric flask and sonicated for 50 minutes; further the volume made up with diluent and filtered. From the filtered solution 0.4ml was pipetted out into a 10ml of another volumetric flask and made up to 10ml with diluents (160ppm; 40ppm and 40ppm).

 

Preparation of buffer:

0.01 N Na₂HPO₄ Buffer:

1.41grams of accurately weighed sodium dihyrogen ortho phosphate and about 900ml of milli-Q water were added to a 1000ml volumetric flask, degas to sonicate and finally make up the volume with water followed by adjusting PH to 4.5 with dilute orthophosphoric acid solution.

 

Method development:

Method development was carried out by using different columns, different type of solvent with varying strengths, pH of the buffer solution varied to determine the chromatographic conditions to give the best separation. Optimization of the mobile-phase conditions were done, so that solvent and excipients did not interfere with the components.

 

Other parameters including assay sensitivity, solvent noise, and time taken for analysis, proper range for eluted peaks, and use of the unchanged solvent system for drug extraction from formulation matrices are considered. After trying different columns, the final choice of the stationary phase that gave a satisfactory resolution and run time was the reversed-phase column Agilent C18 150 x 4.6mm, 5m).

 

Mobile phases containing buffer solutions series with different pH values in combination and different volume ratios of acetonitrile and methanol are used as modifiers for testing. The best results were obtained by use of a mixture of buffer (1.41gm of sodium dihydrogen orthophosphate in a 1000ml of Volumetric flask, add about 900ml of milli-Q water, with pH adjusted to 4.5 using dilute orthophosphoric acid. By testing the effect of different flow rate on the peak area and resolution the flow rate was determined; a flow rate of 1.0ml/min is found to be optimum. All experiments were carried out at ambient temperature.

 

To determine the appropriate wavelength for simultaneous determination of solutions of Sofosbuvir, Velpatasvir and Voxilaprevir in the mobile phase are scanned by a UV-vis spectrophotometer in the range 200–400nm. From the overlaid UV spectra, suitable wavelength considered for monitoring the drugs was 220 nm. Solutions of each substance in the mobile phase were also injected directly for HPLC analysis, and the responses (peak area) were recorded at 220nm. It is observed that, there is no interference from the mobile phase or baseline disturbance, and these three drugs absorbed well at 220nm. It is, therefore, concluded that 220nm is the most suitable wavelength for analysis of both the drugs with suitable sensitivity.

 

Method Validation:

The developed method was validated in accordance with ICH guidelines^15-18.

 

System suitability parameters:

The system suitability parameters were determined by preparing standard solutions of Sofosbuvir, Velpatasvir and Voxilaprevir and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined. The results were incorporated in Table – 1.

 

The % RSD for the area of six standard injections results should not be more than 2%.

 

Specificity:

Checking of the interference in the optimized method. We should not found interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

 

Linearity:

The linearity study was done by injecting the different concentrations (50% - 150%) of the drugs. From the linearity curve the correlation coefficient was found to be less than 1.0. The results were incorporated in Tables – 2 and 3.

 

 

Accuracy:

The recovery study was done by standard addition method for three drugs at 50%, 75% and 150% level. The results were given in Table 4.

 

Precision:

Six different sample solutions were prepared from the commercial tablets and injected into HPLC. The %RSD was beneath 1% for all drugs.

 

Intermediate precision:

The %RSD of the area and RT values were analysed for two different analysts. The results were incorporated in Table 5.

 

LOD and LOQ:

The LOD and LOQ were calculated from the linearity curve method and depicted in Table 6.

 

Robustness:

No changes were observed in chromatograms when changing the flow rate, the concentration of mobile phase and temperature. Hence the method was robust. The results were shown in Table 7.

 

Degradation studies:

Forced degradation studies play in important role for identification of degradation pathways and will prove the stability indicating nature^19-22

 

Oxidation:

To 1ml of stock solutions of Sofosbuvir, Velpatasvir and Voxilaprevir, 1ml of 20% of hydrogen peroxide (H₂O₂) was added separately. The solutions were kept for 30 minutes at 60^°C. For HPLC study, the resultant solution was diluted to obtain 160µg/ml, 40µg/ml and 40µg/ml of all components and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Acid Degradation Studies:

To 1ml of stock solution Sofosbuvir, Velpatasvir and Voxilaprevir, 1ml of 2N Hydrochloric acid was added and refluxed for 30 minutes at 60°C, further neutralized with 2N sodium hydroxide. The resultant solution was diluted to obtain 160µg/ml, 40µg/ml and 40µg/ml of all components and 10µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Alkali Degradation Studies:

To 1ml stock solution of Sofosbuvir, Velpatasvir and Voxilaprevir, 1ml of 2N sodium hydroxide was added and refluxed for 30 minutes at 60°C, further neutralized with 2N Hydrochloric acid. The resultant solution was diluted to obtain 160µg/ml, 40µg/ml and 40µg/ml of all components and 10µl was injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Dry Heat Degradation Studies:

The standard drug solution was placed in an oven at 105°C for 1 h to study dry heat degradation. For HPLC study, the resultant solution was diluted obtain 160 µg/ml, 40µg/ml and 40µg/ml of all components and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

 

Photo Stability studies:

The photochemical stability of the drug was also studied by exposing the 1600µg/ml, 400µg/ml and 400 µg/ml solutions to UV Light by keeping the beaker in UV Chamber for 1 day or 200 Watt hours/m² in photo stability chamber^. For HPLC study, the resultant solution was diluted to obtain 160µg/ml, 40µg/ml and 40µg/ml of all components and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Neutral Degradation Studies:

Stress testing under neutral conditions was studied by refluxing the drug in water for 6 hours at a temperature of 60°C. For HPLC study, the resultant solution was diluted to obtain 160µg/ml, 40µg/ml and 40µg/ml of all components and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

 

Results for stability studies of Sofosbuvir, Velpatasvir and Voxilaprevir were depicted in Table 8.

 

RESULTS AND DISCUSSION:

Method development:

Method development was carried out by using different columns, different type of solvent with varying strengths, pH of the buffer solution varied to determine the chromatographic conditions to give the best separation. Optimization of the mobile-phase conditions were done, so that solvent and excipients did not interfere with the components.

 

Other parameters including assay sensitivity, solvent noise, and time taken for analysis, proper range for eluted peaks, and use of the unchanged solvent system for drug extraction from formulation matrices are considered. After trying different columns, the final choice of the stationary phase that gave a satisfactory resolution and run time was the reversed-phase column Agilent C18 150 x 4.6mm, 5m).

 

Mobile phases containing buffer solutions series with different pH values in combination and different volume ratios of acetonitrile and methanol are used as modifiers for testing. The best results were obtained by use of a mixture of buffer (1.41gm of sodium dihydrogen orthophosphate in a 1000ml of Volumetric flask, add about 900ml of milli-Q water, with pH adjusted to 4.5 using dilute orthophosphoric acid. By testing the effect of different flow rate on the peak area and resolution the flow rate was determined; a flow rate of 1.0ml/min is found to be optimum. All experiments were carried out at ambient temperature.

 

To determine the appropriate wavelength for simultaneous determination of solutions of Sofosbuvir, Velpatasvir and Voxilaprevir in the mobile phase are scanned by a UV-vis spectrophotometer in the range 200–400nm. From the overlaid UV spectra, suitable wavelength considered for monitoring the drugs was 220 nm. Solutions of each substance in the mobile phase were also injected directly for HPLC analysis, and the responses (peak area) were recorded at 220nm. It is observed that, there is no interference from the mobile phase or baseline disturbance, and these three drugs absorbed well at 220nm. It is, therefore, concluded that 220nm is the most suitable wavelength for analysis of both the drugs with suitable sensitivity. Optimized chromatogram of Sofosbuvir, Velpatasvir, and Voxilaprevir has been depicted in Figure 2.

 

Figure 2: Optimized Chromatogram of Sofosbuvir, Velpatasvir and Voxilaprevir

 

System Suitability:

According to ICH guidelines, plate count should be more than 2000, tailing factor should be less than 2 and resolution must be more than 2. All the system suitable parameters were passed and were within the limits. System suitability was shown in Table 1.

 

Table 1: System Suitability Parameters

 

Table 2: Linearity Parameters

 

Table 3: Results for Linearity

 

Linearity:

Concentration range of 40-240 (μg/mL), Velpatasvir range of 10-60 μg/ml and Voxilaprevir range of 10-60 μg/ml were found to be linear with correlation coefficients 0.999 were within limits. The results were shown in Tables 2 and 3.

 

Accuracy:

The Percentage accuracy was a relative standard deviation for accuracy at each level is well within the limit. Over all the percentage recovery of the relative standard deviation was found to be 100.27 %, 100.78 % and 100.10 % for all the levels was within the limit. The results were shown in Table 4.

Precision:

Percentage relative standard deviation of six results was within the limit. Results were as depicted in Table – 5.

 

Linearity Graph for Sofosbuvir, Velpatasvir, Voxilaprevir

 

Table 4: Accuracy tables of Sofosbuvir, Velpatasvir and Voxilaprevir

Table 4.1: Accuracy table of Sofosbuvir

 

Table 4.2 Accuracy table of Velpatasvir

 

Table 4.3: Accuracy table of Voxilaprevir

 

 

 

 

Table 5: Results of Precision

 

Limit of Detection (LOD):

Limit of detection of target assay concentration of Sofosbuvir, Velpatasvir and Voxilaprevir by using linearity curve method. The results were incorporated in Table – 6.

 

Limit of Quantification (LOQ):

Limit of quantification of the target assay concentration of Sofosbuvir, Velpatasvir and Voxilaprevir by using linearity curve method. The results were incorporated in Table – 6.

 

Table 6: Results for LOD and LOQ

 

Robustness:

No changes were observed in chromatograms when changing the flow rate, the concentration of mobile phase and temperature. Hence the method was robust. The results were shown in Table – 7.

 

 

 

 

 

Table 7: Robustness

 

In the above conditions, the parameters like % RSD of peak area, tailing factor and theoretical plates showed were within the limit.

 

Forced Degradation Study:

The forced degradation studies were conducted and all the parameters for Sofosbuvir, Velpatasvir and Voxilaprevir were within the limits. Degradation studies demonstrated the specificity of the developed method in the presence of degradation products. The purity of drug peaks was confirmed by purity angles. Sofosbuvir, Velpatasvir and Voxilaprevir were subjected to the various stress conditions. Significant degradation was observed in acidic, basic, oxidative studies and no degradation was observed in thermal, photolytic and neutral degradation studies. Degradation study was shown in Table – 8 and Figures – 3 to 5, wherein Figure 3 depicts Acid degradation Chromatogram, Figure 4 depicts Base degradation Chromatogram, Figure 5 depicts Peroxide degradation Chromatogram.

 

Figure – 3: Acid degradation chromatogram

 

Figure – 4: Base degradation chromatogram

 

Figure – 5: Peroxide degradation chromatogram

 

Table 8: Results for stability studies of Sofosbuvir, Velpatasvir and Voxilaprevir

Table 8.1: Degradation Data of Sofosbuvir, Velpatasvir and Voxilaprevir under acidic conditions

 

Table 8.2: Degradation Data of Sofosbuvir, Velpatasvir and Voxilaprevir under basic conditions

 

Table 8.3 Degradation Data of Sofosbuvir, Velpatasvir and Voxilaprevir under peroxide conditions

 

Table – 9: The in-detail comparison of current developed RP-HPLC method w.r.t. previously published prior-art method

	Mobile phase ratio		Retention times	Run time	LOD ppm	LOQ ppm	Recovery%	Assay %
Prior-art method8	0.3M KH2PO4: ACN(80:20)	Sofosbuvir	4.6min	15 min	1.33	4.44	100.1%	99.6
		Velpatasvir	8.1min		2.72	9.07	99.9%	99.2
		Voxilaprevir	10.6min		1.16	3.92	99.7%	100.1
Current method	0.01N Na2HPO4: ACN(60:40)	Sofosbuvir	2.2min	6 min	0.38	1.14	100.2%	100.19
		Velpatasvir	2.9min		0.32	0.96	100.78%	99.69
		Voxilaprevir	3.5min		0.08	0.24	100.10%	99.96

 

CONCLUSION:

The proposed stability indicating RP-HPLC developed method allows a simple, linear, precise, rapid and stable quantitative determination of Sofosbuvir, Velpatasvir and Voxilaprevir in bulk and tablet dosage forms compared to the published prior-art method. All the validation parameters were found to be among the boundaries in keeping with ICH guidelines. The projected current technique was found to be cost-effective, rapid, simple, specific for the drugs of interest regardless of the excipients comprising and also the less LOD and LOQ concentration, short retention times permits the analyst for the analysis of number of samples in a quick span of time with high recovery and assay. Further, the current technique was found to be easy, accurate, precise, rugged, robust and stable under forced degradation stress conditions.

 

Hence in our view, the established technique appears to be applicable for the routine HPLC analysis of the marketed formulations successfully.

 

ACKNOWLEDGEMENT:

The authors are thankful to the Spectrum labs, Hyderabad for providing the gift samples of Sofosbuvir, Velpatasvir and Voxilaprevir.

 

CONFLICT OF INTEREST:

Nil.

 

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Received on 21.01.2020           Modified on 19.02.2020

Accepted on 26.03.2020         © RJPT All right reserved