INTRODUCTION:

Vosevi^TM drug (sofosbuvir, velpatasvir and voxilaprevir) is a consolidation emolument that Contains Sofosbuvir (SOF) is a once-daily pangenotypic HCV NS5B nucleotide polymerase inhibitor official in the United States and abroad for the prescription of chronic HCV infection [1]. It is an oral pro drug that subjected to hepatic metabolism into a dynamic nucleotide analog that effectively inhibits the NS5B polymerase, thereby anticipating viral replication [2].

The structure of SOF showed below and it is chemically (S)-Isopropyl2-((S)-(((2R, 3R, 4R, 5R)-5-(2, 4-dioxo-3, 4dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy (phenoxy) phosphorylamino) propanoate [3]. Velpatasvir (VEL) is a once-daily pangenotypic HCV NS5A protein inhibitor, including equivalent potency in vitro against genotype 3 compared with other genotypes, approved in the United States and abroad for the treatment of HCV infection in combination with SOF [4-5]. Inhibition of the NS5A protein disrupts HCV replication, assembly and possibly egress [Ross-T]. The structure of VEL showed below and chemically it is Methyl{(1R)-2-[(2S,4S)-2-(5-{2[(2S,5S)-1-{(2S)-2-[(methoxycarbonyl)amino]-3-methylbutanoyl}-5- methylpyrrolidin-2-yl]1,11dihydro[2]benzopyrano[4',3':6,7 ]naphtha [1,2-d] imidazol-9-yl} -1H-imidazol-2-yl)-4(methoxymethyl)pyrrolidin-1-yl]-2-oxo-1phenylethyl}carbamate[6]. Voxilaprevir (VOX) is a novel macro cyclic NS3/4A protease inhibitor that has recently completed phase III clinical development in combination with SOF/VEL[7]. It has admirable activity across all HCV genotypes and against most of the RASs associated with first-generation protease inhibitors. The structure of VOX showed below and chemically it is (1aR, 5S, 8S, 9S, 10R, 22aR)-5-tert-butylN-{(1R, 2R) -2- (difluoromethyl)-1-[(1methylcyclopropanesulfonyl) 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-methanocyclopropa[18, 19] [1, 10, 3, 6] dioxadiazacyclononadecino[11, 12-b] quinoxaline-8-carboxamide [8]. Now, SOF, VEL and VOX are official in US pharmacopeia [9]. Literature survey divulge that a few analytical methods have been reported for the quantitative estimation of SOF, VEL some of them were being: RP-HPLC method development and validation for estimation sofosbuvir in pure and tablet dosage form [10], Development and validation of RP-HPLC method for quantitative analysis of sofosbuvir in pure and pharmaceutical formulation [11-12], a stability indicating RP-HPLC method for simultaneous estimation of velpatasvir and sofosbuvir in combined tablet dosage forms [13-14], and a new RP-HPLC Method for the simultaneous assay of sofosbuvir and ledipasvir in Combined Dosage Form [15] also Evolution of sofosbuvir, velpatasvir plus voxilaprevir as fixed dose co formulation for treating hepatitis C in [8], besides that several methods reported for the determination of SOF, VEL and VOX like Quantitative detection methods applying mass spectrometric detection are limited to four UPLC-MS [16-19] and one UPLC-ESI-MS/MS [20] two LC–MS/MS methods [21-22]. There is no official method for this combination so far according to the best of our knowledge. As per literature survey, several methods have been reported for estimation of sofosbuvir, velpatasvir and voxilaprevir individually or with the combination of some other drugs. With this current proposed method sofosbuvir, velpatasvir and voxilaprevir determines simple and economical dosage formulations.

The main aim of this method was to determine and validate the SOF, VEL and VOX based on International Conference on Harmonization [23] guidelines. This method was made use of a reproducible procedure for the quantitative analysis of drug samples as the bulk drug and in tablet dosage forms. The designed method was considered as an advisable to develop precise, accurate, simple RP-HPLC method for stability indicating and simultaneous estimation of SOF, VEL and VOX in tablets.

MATERIAL AND METHODS:

INSTRUMENTATION:

The analysis was performed on a HPLC instrument used was of WATERS HPLC 2965 SYSTEM with Auto Injector and PDA Detector and it was controlled by using software Empower 2. and it has UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz was be used for measuring absorbance for SOF, VEL and VOX solutions. The separation and quantization were made on column (Waters C18 250 x 4.6 mm, 5m.m.)

MATERIALS:

HPLC grade acetonitrile, water, ortho phosphoric acid AR grade were purchased from SD Fine Chem. Mumbai, India were used in this method development and validation study. The drug samples were kindheartedly provided by Spectrum pharmaceuticals Pvt Ltd, Kukutpally, Hyderabad, India, and the formulation samples analytical grade of sodium hydroxide, hydrochloric acid, hydrogen peroxide and high purity distilled water were used. The standards preparation of three drugs were made use of Vosevi^TM tablets labeled claim was 400 mg of sofosbuvir, 100 mg of velpatasvir and 100 mg of voxilaprevir.

GENERAL PROCEDURE:

CHROMATOGRAPHIC CONDITIONS:

A mobile phase system consisting of 0.01M ortho-phosphoric acid and acetonitrile was used in the ratio of 50:50% v/v at a P^H 2.5 adjusted with ortho phosphoric acid and it is also used as diluents for preparing the working solution of drugs. The separation was achieved with the elution method. The flow rate was 1.0 ml/min. The injection volume was 10mL. The eluant was monitored by the photo diode array detector (PDA) from 200 to 400 nm, and chromatograms were extracted at the wavelengths of 220 nm. The total run time was 6 minutes and all establishments were performed at 30°C.

STANDARD SOLUTIONS:

Accurately Weighed and transferred 40 mg, 10 mg and 10 mg of sofosbuvir, velpatasvir and voxilaprevir working Standards into a 10 ml, 10 ml and 10 ml clean dry volumetric flask, add 5ml of diluents, sonicated for 30 minutes and made up to the final volume with diluents to obtain the concentrations of 4000 µg/ml SOF, 1000 µg/ml VEL and 1000µg/ml VOX. From the above stock solutions 1 ml was pipette out in to a 10 ml volumetric flask and then made up to the final volume with diluents and achieved concentrations 400 ppm, 100 ppm and 100 ppm. Three injections were finished for each concentration and chromatographs were gained under the previously described liquid chromatographic conditions. The peak areas were plotted in opposition to the consequent concentrations to erect the calibration graphs.

ASSAY OF SAMPLE PREPARATION:

Five Vosevi^TM tablets were weighed and calculated the average weight of each tablet then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask and then 60 ml of diluents were added. This solution was sonicated for 25 min and additional volume made up to the mark with diluents and filtered. From the resultant solution 1ml was pipette out into a 10 ml volumetric flask and made up to10 ml with diluents and acquired final concentrations were 400 ppm, 100 ppm and 100 ppm for SOF, VEL and VOX respectively. These specified concentrations were used for general procedure and recovered concentrations were calculated from the consequent calibration graphs. For regular addition assay, test solutions were spiked with aliquots of regular solutions of the three compounds to gain total concentrations contained by the earlier specified ranges then treated as under common process. Recovered concentrations were deliberated by assimilating the analyte response with the growth response achieved after addition of the standard.

STABILITY-INDICATING AND FORCED DEGRADATION STUDIES:

Forced degradation studies were executed on SOF, VEL and VOX standards based on the following conditions:

(i) Oxidation:

1 ml stock solution of SOF, VEL and VOX was taken and added 1 ml of 20% hydrogen peroxide (H₂O₂) separately. This solution was set aside for 30 min at 60°C temperature. The consequential solution was diluted to get 400 ppm, 100 ppm and 100 ppm concentrations to study HPLC. From the resultant solution, 10 µl was injected into the system and the chromatograms were recorded to evaluate the stability of drug samples.

(ii) Acid degradation studies:

1 ml of stock solution and 1 ml of 2N Hydrochloric acid was added and refluxed for 30 mins at 60⁰C. To get 400 ppm, 100 ppm and 100 ppm concentrations for HPLC study, the resultant solution was diluted. For the study of acid degradation 10 µl injected into the system and corresponding chromatograms are achieved. These chromatograms were assessed for stability of samples.

(iii) Alkali degradation studies:

To 1 ml of stock solution of SOF, VEL and VOX and 1 ml of 2N sodium hydroxide was added and refluxed for 30 mins at 60°C. The resultant solution was diluted to obtain concentrations 400 ppm, 100 ppm and 100 ppm. From the above solution, 10 µl was injected into the system and the chromatograms were recorded to evaluate the stability of sample.

(iv) Dry heat degradation studies:

The standard stock solution was placed in oven at 105⁰c for 6 hours to study dry heat degradation. For HPLC study, the resultant solution was diluted to obtain concentrations 400 ppm, 100 ppm and 100 ppm for SOF, VEL and VOX. From the tested solution, 10µl was injected into the system and the chromatograms were achieved to analyze the dry heat stabilities of the samples.

(v) Photo stability studies:

The sample solutions possessing 400 ppm, 100 ppm and 100 ppm concentrations was kept in beaker and exposed to UV light in the UV chamber for seven days or 200 Watt hours/m² for photo stability studies of HPLC. After the above condition, 10 µl were injected into the system and the chromatograms were recorded to review the stability of sample drugs.

(vi) Neutral degradation studies:

Stress testing under neutral conditions was studied by refluxing the drugs in water for 6 hours at a temperature of 60ºC. The resultant solution was diluted for required concentrations of 400 ppm, 100 ppm and 100 ppm for SOF, VEL and VOX respectively. The 10 µl solution from the above was injected into the system and the chromatograms were registered to determine the stability of the sample.

RESULTS AND DISCUSSION:

The stability-indicating method was developed into more clearly and obligatory due to advancement of International Conference on Harmonization (ICH) guidelines. These guidelines essential for the treatment of forced degradation studies based on different conditions, like photo, oxidation, dry heat and others followed by separation of drugs from degradation products [23]. HPLC has specificity, sensitivity and high resolution capability. Therefore it has incredible reputation in stability studies along with separation of several components. The planned research liable towards the study of the chromatographic behavior of stress degradation studies along with the simultaneous estimation of the SOF, VEL and VOX in their combined formulation. The degradation studies of this drug combination has not reported so far in the literature as per our knowledge and motivated us to develop an RP-HPLC- PAD stability indicating test where the degradation products were resolved from the integral drugs. Stability studies must be considered over development of chromatographic methods especially when degraded products are unknown or not available [24]. Hence, stability indicating studies were performed for SOF, VEL and VOX in order to effectuate the possible proportional degradants and test their chromatographic actions using the developed RP-HPLC method. Oxidative, acidic, alkali, and neutral degradation studies were executed at room temperature or with the help of heating besides that photolytic and dry heat degradation studies were applied.

STABILITY INDICATING ASPECTS:

The degradation studies were eluted for each of the SOF, VEL and VOX in this drug combination with regard to generate the probable admissible degradants and checked their chromatographic action using the developed method. The degradation studies like Oxidation, acidic, alkali, dry heat and photolytic tests were executed with various conditions of temperature and time. Oxidation (with H₂O₂) degradation was performed for 30 min at 60°C showed 96.16% from the peak area analyzed with standard of the same concentration (Fig.1A). There was only one degradation peak observed for SOF conducted at a retention time of 2.119 min in oxidative degradation. SOF is exposed to stress studies in both acidic and alkali media. In strong acidic medium, degradation of SOF was observed from the reduction of its peak area which attained 4.5% of the expected area with 2N Hcl refluxed for 30 min at 60°C. The degradation peak of acid media observed at a retention time 2.099(Fig. 1B), correspondingly, SOF refluxed for 30 min at 60°C with 2N NaOH caused degradation of peak area which attained 5.4% of the expected area with a retention time 2.122 min (Fig.1C). The solution of SOF was kept in the beaker in UV Chamber for 3days or 200 Watt hours/m² in photo stability chamber. RP-HPLC chromatogram showed the degradation up to 3% after being exposed to the UV light in UV chamber for 7 days or 200 Watt hours/m². Finally, the peak was observed at a retention time 2.105 min (Fig. 1D). The degradation under dry heat conditions which carried out in an oven at 105^oC for 6 hours and 4.4% degradation were observed at a retention time 2.117 min (Fig.1E). Finally, stress testing under neutral conditions was studied by refluxing the SOF in water for 6 hours at a temperature of 60^oC and observed the stress of 0.9% and the peak was achieved at a retention time 2.106 min (Fig.1F). The peak areas were mostly identical to that of standards of the similar concentration; moreover, the chromatograms did not showed any extra peaks. Correspondingly, the same degradation studies were applied to analyze the stability indicating behavior of VEL. Oxidation (with H₂O₂) degradation was conducted by refluxing for 30 min at 60^oC showed 97.16% from the peak area analyzed with standard of the same concentration (Fig. 1A). There was only one degradation peak observed for VEL conducted at a retention time of 2.711 min in oxidative degradation. VEL was exhibited to stress studies in acidic as well as alkali media. In strong acidic medium, degradation of VEL was observed from the reduction of its peak area which attained 3.77% of the expected area at 2N Hcl refluxed for 30 min at 60^oC. The degradation peak of acid media perceived at a retention time 2.671 min (Fig. 1B), correspondingly, VEL refluxed for 30 min at 60^oC With 2N NaOH caused degradation of peak area which procured 4.08% of the expected area with a retention time 2.739 min (Fig. 1C). ). The solution of VEL was kept in the beaker in UV Chamber for 3days or 200 Watt hours/m² in photo stability chamber. RP-HPLC chromatogram showed the degradation up to 1.24% after being naked to the UV light in UV chamber for 7 days or 200 Watt hours/m². Finally, the peak was observed at a retention time 2.695 min (Fig. 1D). The degradation under dry heat conditions which carried out in oven at 105^oC for 6 hours and 1.88% degradation were observed at a retention time 2.725 min for VEL (Fig. 1E). And also, stress testing under neutral conditions was studied by refluxing the VEL in water for 6 hours at a temperature of 60^oC and observed the stress of 0.48% and the peak was achieved at a retention time 2.688 min (Fig. 1F). The peak areas were mostly identical to that of standards of the similar concentration; besides, the chromatograms did not showed any extra peaks for VEL. In the same way, the same degradation studies were performed to analyze the stability indicating behavior of VOX. Oxidation (with H₂O₂) degradation was performed by refluxing the solution for 30 min at 60^oC and showed 96.57% from the peak area analyzed with standard of the same concentration (Fig. 1A). There was only one degradation peak detected for VOX conducted at a retention time of 3.449 min in oxidative degradation. VOX is reveled to stress studies in acidic and alkali media. In strong acidic medium, degradation of VOX was detected from the reduction of its peak area which obtained 4.5% of the anticipated area with 2N Hcl and solution was refluxed for 30 min at 60^oC. The degradation peak of acid media found at a retention time 3.404 min (Fig. 1B), correspondingly, VOX refluxed for 30 min at 60^oC with 2N NaOH caused degradation of peak area which acquired 5.4% of the expected area with a retention time 3.507 min (Fig. 1C). ). The solution of VOX was kept in the beaker in UV Chamber for 3days or 200 Watt hours/m² in photo stability chamber. RP-HPLC chromatogram showed the degradation up to 3.0% after being uncovered to the UV light in UV compartment for 7 days or 200 Watt hours/m². Finally, the peak was observed at a retention time 3.435 min (Fig. 1D). The degradation under dry heat conditions which carried out in oven at 105^oC for 6 hours and 4.4% degradation were detected at a retention time 3.474 min for VOX (Fig. 1E). And also, stress testing under neutral conditions was studied by refluxing the VOX in water for 6 hours at a temperature of 60^oC and observed the stress of 0.9% and the peak was achieved at a retention time 3.437 min (Fig. 1F). The peak areas were mostly identical to that of standards of the analogous concentration; furthermore, the chromatograms did not showed any extra peaks for VOX in all the conditions. Resolution was computed between any of the three drugs and the nearest degradation resultant peaks. Resolution was observed not <3.99; this suggest a tolerable baseline separation between the sample drugs and the degradation compounds. It is significant to mention that peak purity analysis results acquired from the photo diode array detector (PDA) proved that SOF, VEL and VOX peaks are homogenous and uncontaminated in all the assay samples subjected to stress degradation studies.

METHOD DEVELOPMENTS:

Six trials were performed for the method development and the best peaks with least fronting factor was elevated for SOF, velpatasvir and voxilaprevir to be with retention times were 2.098, 2.650, 3.353 minutes respectively. The resultant chromatogram revealed in the Figure-2.

Figure 2: Chromatogram of sofosbuvir, velpatasvir and voxilaprevir

METHOD VALIDATION:

The method was validated as per ICH guidelines [23]. The different validation parameters which were performed are following: linearity, precision, accuracy, specificity, and limit of detection, limit of quantification, robustness, degradation studies and the stability indicating capability.

SYSTEM SUITABILITY TEST:

Six replicate injections of standard solutions were injected and the chromatograms were recorded. The system was suitable for analysis of sofosbuvir,

velpatasvir and voxilaprevir if the % relative standard deviation (%RSD) of area counts in six repeated injections should be not more than 2.0%. USP tailing factor for sofosbuvir, velpatasvir and voxilaprevir peak should be not more than 2.0. USP resolution factor between the peaks corresponding to sofosbuvir, velpatasvir and voxilaprevir should be more than 2.0. The results are shown in the Table-1

Table-1: system suitability results

Parameter	sofosbuvir	velpatasvir	voxilaprevir
USP tailing Factor	1.09	1.08	1.00
USP plate count	4744	5367	6804
USP resolution	--	4.0	4.5
LOD(µg/ml)	0.08	0.29	0.10
LOQ(µg/ml)	0.25	0.87	0.31
% RSD	0.6	0.6	0.8

LINEARITY AND CONCENTRATION RANGES:

Working dilutions of sofosbuvir was 100 to 600, velpatasvir and voxilaprevir within the diverge of 25 to 150 ppm was prepared by taking suitable aliquots of solutions of drug in several 10 ml volumetric flask and made up to the mark with mobile phase and those are depicted in table-2. 10μl amount of every dilution was injected in to the column with a flow rate of 1ml/min. The samples in elute were monitored at 220 nm and the subsequent chromatograms were recorded. From these, calculated the mean peak areas and showed in table-3 for sofosbuvir, and table-4 for velpatasvir and voxilaprevir a plot of concentration verses peak areas was produced and drawn in the figure-3, 4 and 5. The regression of the plot was calculated by least square regression method. Regression equation of sofosbuvir was y = 4738.x + 2618(R²=0.999), velpatasvir was y = 9819.x + 1192(R²=0.999) voxilaprevir was y = 11255.x + 661.5(R²=0.999).

Table-3: linearity peak area values of sofosbuvir

S.No	concentration	Peak areas of sofosbuvir
1	0	0
2	100	494136
3	200	976995
4	300	1390358
5	400	1852799
6	500	2384378
7	600	2870497

*Each value is a mean of three readings

Table-4: linearity peak area values of velpatasvir and voxilaprevir

S.No	concentration	Peak areas of velpatasvir	Peak areas of voxilaprevir
1	0	0	0
2	25	242857	283385
3	50	490421	565515
4	75	743036	846166
5	100	994828	1115505
6	125	1222287	1413699
7	150	1470104	1689340

*Each value is a mean of three readings

Figure-3: Linearity graph for sofosbuvir

Figure-4: Linearity graph for velpatasvir

Figure-5: Linearity graph for voxilaprevir

X-Axis = Concentration Y-Axis = Peak area

PRECISION:

The standard sofosbuvir, velpatasvir and voxilaprevir solutions were injected for six times and measured the area for all six trails in RP- HPLC. The %RSD for the area of six repeat injections was founded to be within the specific limits. The data of system precision was shown in the Table-5, repeatability was presented in table-6, and inter day precision was showed in table-7.

Acceptance Criteria:

The % RSD should not be more than 2%

Table-5: Precision results of system

S.No	Area of sofosbuvir	Area of velpatasvir	Area of voxilaprevir
1	1833838	993751	1138804
2	1825026	997928	1139022
3	1810161	995179	1136675
4	1812970	986105	1122704
5	1821630	994635	1117374
6	1802937	981580	1134727
Mean	1817760	991530	1131551
S.D	11195.4	6281.0	9208.9
%RSD	0.6	0.6	0.8

Table-6: Repeatability results

S.No	Area of sofosbuvir	Area of velpatasvir	Area of voxilaprevir
1	1828854	991589	1122592
2	1803830	992751	1125614
3	1826611	996806	1133485
4	1815364	993116	1127449
5	1808059	991356	1126220
6	1821711	984304	1127453
Mean	1817405	991654	1127136
S.D	10101.1	4098.8	3586.4
%RSD	0.6	0.4	0.3

Table-7: Inter day precision results

S.No	Area of sofosbuvir	Area of velpatasvir	Area of voxilaprevir
1	1823838	993751	1118804
2	1825026	991928	1119022
3	1810161	995179	1116675
4	1812970	986105	1122704
5	1821630	994635	1117374
6	1802937	981580	1134727
Mean	1816094	990530	1121551
S.D	8814.4	5485.9	6783.8
%RSD	0.5	0.6	0.6

ACCURACY:

Accuracy of the readings was computed by % recovery of six different concentrations of sofosbuvir, velpatasvir and voxilaprevir at 50%, 100% and 150% and also standard addition technique was carried out for same samples. The results acquired including the means of the recovery and standard deviations were displayed in Table-8.

Acceptance Criteria:

The % Recovery for sofosbuvir, velpatasvir and voxilaprevir at each stage should be between 99 to 101%.

Table-8: Accuracy

	Sofosbuvir			Velpatasvir			Voxilaprevir
%Concentration	50	100	150	50	100	150	50	100	150
Trail-I	100.94	100.22	100.75	99.54	101.26	99.55	99.69	99.99	100.18
Trail-II	100.97	100.66	99.23	101.31	99.67	100.39	101.67	100.50	99.66
Trail-III	100.34	99.52	100.18	99.60	101.81	99.90	101.18	99.09	99.39
AVG (%Recovery)	101.42	100.13	100.05	100.15	100.91	99.95	100.85	99.86	99.74
SD	1.36	0.57246	0.7684	1.00	1.1084	0.4205	1.03	0.7162	0.3999
%RSD	1.34	0.5717	0.7680	1.00	1.0984	0.4207	1.03	0.7172	0.4009

Table-9: Specificity

S.No.	Name of the sample	No. of injections	Area of sofosbuvir	Area of velpatasvir	Area of voxilaprevir
1	Blank	1	-	-	-
2	Placebo	1	-	-	-
3	Standard	1	1825026	986105	1122704
4	Water sample	1	1813262	988782	1123617

RECOVERY STUDIES:

To estimate the accuracy and precision of the proposed method recovery studies were carried out. A fixed amount of sample was taken and reference drugs were added at 50%, 100% and 150% levels. The results were analyzed and the results were within the limits.

SPECIFICITY:

The specificity of the RP-HPLC method is furnished, where complete separations of sofosbuvir, velpatasvir and voxilaprevir were distinguished in presence of other inert excipients used in tablets. In addition, there was no deterrence at the retention time of in the chromatogram of placebo solution. In the case of peak purity analysis with PDA, purity gradient was always not greater than purity threshold for the analytes. The results of peaks of analyte were pure. The excipient in the formulation does not obstruct the analyte. The data was listed in the Table-9.

LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION:

Limit of Detection (LOD) is the least concentration of an analyte in a sample that can be identified but not quantified. LOD is indicated as a concentration at a précised signal to noise ratio. The LOD will depend on the procedure of analysis along with type of instrument. In the chromatography, detection limit is the injected quantity that consequences in a peak with a height at least thrice or twice as high as baseline noise level. LOD was computed by using formula LOD=3.3SD/S. The chromatogram was showed in (fig.6). The LOD was found to be 0.08, 0.29 and 0.10 for SOF, VEL and VOX respectively.

Limit of quantification (LOQ) is defined as least concentration of analyte in a sample that can be estimated with tolerable precision, accuracy and reliability by a specified method under affirmed experimental conditions. LOQ is uttered as a concentration at a specified signal to noise ratio. In the chromatography, limit of quantification is the injected quantity that consequences in a peak with a height, ten times as high as base line noise level. LOQ is calculated by using the formula LOQ=10(SD/S). The chromatogram was showed in (fig.7) and The LOQ was found to be 0.25, 0.87 and 0.31 for SOF, VEL and VOX accordingly.

Figure-6: LOD chromatogram of SOF, VEL and VOX

Figure-7: LOQ chromatogram of SOF, VEL and VOX

ROBUSTNESS:

Robustness is denoted by making speculate changes in the chromatographic conditions like change in temperature, mobile phase composition and flow rate were assessed for the impact on the present method. It was founded from the chromatograms that the results were not exceeding the limits. This represents that the method developed is robust and shown in the Table-10.

Table-10: Robustness of sofosbuvir, velpatasvir and voxilaprevir

Parameter		Sofosbuvir	Velpatasvir	Voxilaprevir
Temp, ±5	25	1223118	656477	748858
Temp, ±5	35	1218887	655711	748001
Flow rate ±0.1	0.9	1886048	1029022	1175362
Flow rate ±0.1	1.1	1817916	993517	1132466
Mobile Phase change ±5	45:55	1210521	653102	742508
Mobile Phase change ±5	55:45	1221549	656403	747569

CONCLUSION:

This paper describes a new simple, reliable, economic elution of stability-indicating RP-HPLC-PDA method for the simultaneous estimation of SOF, VEL and VOX. The simultaneous estimation of three drugs was conducted with various parameters like system suitability, linearity, accuracy, precision, LOD, and LOQ etc. The results were in within the limit. The forced degradation studies also executed for the same drugs by considering several degradation conditions like oxidation, acidic, alkali, thermal, neutral and photolytic conditions. The executed method was effectively employed from the resolution of employed samples peaks. No such detailed stability indicating method has been reported for the analysis of this triplicate drug mixture. The photo diode array detector (PDA) was used as tool for peak integrity and purity confirmation to finish the present developed method. Therefore the proposed study method can be used for quantification of SOF, VEL and VOX in bulk and pharmaceutical dosage form. Finally, this method was carefully validated; as a result, it can be suggested for routine analysis and for testing quality through stability studies of the drugs.

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Received on 19.04.2018 Modified on 15.06.2018

Research J. Pharm. and Tech 2018; 11(9): 4147-4156.

DOI: 10.5958/0974-360X.2018.00762.X


(A)	(B)

(C)	(D)

(E)	(F)

S.No	Pipetted from stock (ml)	Volume of flask (ml)	Concentration in ppm(SOF)	Concentration in ppm(VEL)	Concentration in ppm(VOX)	%Linearity Level
1	0.25	10	100	25	25	25
2	0.5	10	200	50	50	50
3	0.75	10	300	75	75	75
4	1	10	400	100	100	100
5	1.25	10	500	125	125	125
6	1.5	10	600	150	150	150