INTRODUCTION:

The chemical name of Ombitasvir (OMB) is Dimethyl ([(2S,5S)-1-(4-tert-butylphenyl) pyrrolidine-2,5- diyl]bis{benzene-4,1-diylcarbamoyl(2S)pyrrolidine-2,1-diyl[(2S)-3-methyl-1-oxobutane-1,2- diyl]})biscarbamate hydrate. The molecular formula is C₅₀H₆₇N₇O8•4.5H₂O (hydrate) and the molecular weight for the drug substance is 975.20 (hydrate). The drug substance is white to light yellow to light pink powder, and is practically insoluble in aqueous buffers but is soluble in ethanol.

Paritaprevir (PAR) chemically is (2R,6S,12Z,13aS,14aR,16aS)-N-(cyclopropylsulfonyl)-6- {[(5-methylpyrazin-2-yl)carbonyl]amino}-5,16-dioxo-2-(phenanthridin-6-yloxy)-1,2,3,6,7,8,9,10,11,13a,14,15,16,16a-tetradecahydrocyclopropa[e]pyrrolo[1,2-a][1,4] diazacyclopentadecine-14a(5H)-carboxamide dihydrate. The molecular formula is C₄₀H₄₃N₇O₇S•2H₂O (dihydrate) and the molecular weight for the drug substance is 801.91 (dihydrate). The drug substance is white to off-white powder with very low water solubility.

The chemical name of Ritonavir (RIT) is [5S-(5R*,8R*,10R*,11R*)]10-Hydroxy-2-methyl-5-(1- methyethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oicacid,5-thiazolylmethyl ester. The molecular formula is C₃₇H₄₈N₆O₅S₂ and the molecular weight for the drug substance is 720.95. The drug substance is white to off white to light tan powder practically insoluble in water and freely soluble in methanol and ethanol³. The molecular structures of Ombitasvir, Paritaprevir and Ritonavir were shown in Figure 1.

Figure 1: Molecular Structures of Paritaprevir (A), Ombitasvir (B) and Ritonavir (C).

Several analytical methods have been described in the literature for the estimation of OMB, PAR and RIT alone and/or in combination with other drugs in bulk and pharmaceutical formulations by using RP HPLC^1-11. To the best of our knowledge there is no UPLC method available. UPLC was chosen for present study as analysis can be completed in short time^12-14. Hence, an attempt was made to develop a stability indicating method by UPLC for simultaneous quantification of OMB, PAR and RIT in pharmaceutical formulation.

MATERIALS AND METHODS:

Reagents and chemicals:

Reference standards of Ombitasvir, Paritaprevir and Ritonavir were obtained as gift samples from Aurobindo Pharma ltd, Hyderabad. The fixed dose generic product of Ombitasvir/Paritaprevir/Ritonavir Technivie(12.5mg/75mg/50mg) was procured from commercial source. Acetonitrile (UPLC Lichrosolv) of chromatographic grade, AR grade of Potassium dihydrogen orthophosphate (Merck, Mumbai), Orthophosphoric acid (Merck, Mumbai), Sulphuric acid (Qualigens), Sodium Hydroxide (Merck, Mumbai) and Hydrogen Peroxide were used for present study.

Instrumentation and Chromatographic conditions:

The liquid chromatographic analysis for simultaneous estimation of Ombitasvir, Paritaprevir and Ritonavir was carried out using Waters Acquity UPLC system equipped with quaternary pump, an inbuilt auto injector and PDA 2996 detector controlled by Empower 2 software. An electronic balance (Shimadzu), pH meter, Ultra bath sonicator, Hot air oven, UV chamber were also used during study.

Chromatographic separations of OMB, PAR and RIT were performed in a SB C8column (100×2.1mm,1.8µm) at ambient temperature, with pH4 phosphate buffer and Acetonitrile in the ratio of 50:50v/v as mobile phase at a flow rate of 0.5ml/min with an injection volume of 0.50µl. Detection of separated analytes was done at 250 nm with a runtime of 3min.

Preparation of standard solutions:

Accurately Weigh and transfer 18.75mg of Paritaprevir, 3.125mg of Ombitasvir and 12.5mg of Ritonavir working Standards into a 25ml clean dry volumetric flasks, add 10ml of diluent (water and acetonitrile, 50:50v/v) and sonicated for 10 minutes. Make up the final volume with diluents to obtain the concentrations of 750µg/ml of Paritaprevir, 125µg/ml of Ombitasvir and 500µg/ml of Ritonavir. Further dilutions were made to obtain standard solutions of known concentrations in the range of 18.75-112.5µg/ml of PAR, 3.125-18.75 µg/ml of OMB and 12.5-75µg/ml of RIT.

Preparation of sample solutions:

An Accurately weighed equivalent weight of the combination powder (Technivie) sample transferred into a 100ml volumetric flask, 75ml of diluent was added and sonicated for 25 min. Further the volume was made up with diluent and filtered through 0.45µ filter. 1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluents which contains 75µg/ml Paritaprevir, 12.5µg/ml Ombitasvir and 50µg/ml of Ritonavir.

Method validation^15-17:

The developed analytical method was validated according to the ICH guidelines¹⁸ for the parameters Specificity, system suitability, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantification (LOQ).

System Suitability:

The standard solutions were injected into UPLC system and the system suitability parameters such as theoretical plates, tailing factor and resolution were evaluated.

Specificity:

Method specificity is unequivocally assessed for the presence of interfering peaks by injecting blank and placebo solutions. Absence of peaks at the retention times of analytes indicates absence of interference and specificity of the method.

Linearity:

Series of six standard solutions of known concentrations in triplicate were used to assess linearity range from peak area versus concentration. Calibration curves are plotted and correlation coefficient, slope and intercept are calculated by using straight line equation.

Precision and Accuracy:

The method precision was determined by injecting six solutions into UPLC system and calculating the % RSD values. The accuracy of the method was determined by recovery studies. The sample solutions of 3 concentrations (n = 3) at 50%, 100% and 150% levels of target assay were analyzed by standard addition method. The % recovery and RSD were evaluated.

LOD and LOQ:

Limit of Detection and Limit of Quantification were calculated based on calibration data from the equations LOD = 3.3σ/S and LOQ = 10 σ/S respectively. σ denotes standard deviation of intercept and S denotes slope.

Robustness:

Robustness of the method was determined by deliberately changing the optimized analytical conditions such as mobile phase composition by ± 5%, flow rate by ± 0.1ml/min, and column temperature by ± 5ᵒC.

Forced degradation studies:

Stability indicating nature of the method and identification of possible degradants can be achieved by performing the degradation studies by exposing the analytes to various stress conditions such as acid hydrolysis, base hydrolysis, water hydrolysis, oxidation, exposure to UV light and high temperature¹⁹.

Acid, Alkali, Neutral and Oxidative degradation were performed by reflexing 1ml of standard solution and 1ml of 2N HCl, 1ml of 2N NaOH, 1ml of water and 1ml of 20%H₂O₂at 60ᵒC for 30mins respectively. The resultant solution was neutralized and diluted to obtain concentrations of 75ppm of PAR, 12.5ppm of OMB and 50ppm of RIT and injected into UPLC system. The chromatograms obtained were assessed for the sample stability.

Dry heat degradation was studied by placing standard solution in oven at 105ᵒC for 6hrs. For UPLC analysis the resultant solution was diluted to 75ppm of PAR, 12.5ppm of OMB and 50ppm of RIT and injected to obtain the chromatograms which were then assessed to indicate sample stability.

Photochemical stability of analyte was assessed by exposing the solution containing 750ppm of PAR, 125ppm of OMB and 500ppm of RIT to UV light in UV chamber for 7days. The resultant solution was diluted and injected into UPLC system to record and assess the chromatograms.

RESULTS AND DISCUSSION:

Method Development:

For the simultaneous estimation of PAR, OMB and RIT, a specific, accurate and suitable UPLC method was developed by applying different set of conditions to achieve system suitability parameters within acceptable limits. A variety of mobile phase combinations in different proportions at different flow rates over different stationary phases were used to optimize the method.

The mobile phase composing of Phosphate buffer of pH 4 and acetonitrile in the ratio of 50:50v/v at a flow rate of 0.5ml/min over SB C8 column were selected as they provided better resolution and separation with an elution time of 3min. detection of analytes was done at 250nm by PDA detector and the retention times were found to be 0.908, 1.190 and 1.739 minutes for PAR, OMB and RIT respectively.

Method validation:

The optimized set of chromatographic conditions were validated in compliance with ICH guidelines to assure the intended use of proposed method.

Specificity:

The blank, placebo and standard chromatograms were compared to check the presence of any interfering peaks at the retention times of analytes which were shown in Figure 2. Specificity of method is indicated by the absence of interfering peaks.

System Suitability:

Chromatographic parameters such as plate count, tailing factor, resolution, peak area and retention times were evaluated and found to be in compliance with acceptable limits. The results of system suitability and validation parameters were given in Table 1.

Table 1: System Suitability and Validation results

Parameter	Paritaprevir	Ombitasvir	Ritonavir
USP Plate count	7987	2286.5	4814.83
USP tailing	1.346	1.063	1.151
Resolution	___	2.96	5.35
Retention time (Min)	0.908	1.190	1.739
Linearity range (µg/ml)	18.75 – 112.5	3.125 – 18.75	12.5 – 75
Correlation coefficient	0.9999	0.9999	0.9994
Slope	8392.1	6584	9063.9
Intercept	1995.3	479.83	5150.4
LOD (µg/ml)	0.99	0.06	0.18
LOQ (µg/ml)	2.99	0.17	0.53
Flow rate Minus	0.6	0.4	0.7
Flow rate plus	0.8	0.3	0.8
Mobile Phase Minus	0.6	0.7	0.5
Mobile phase Plus	0.2	0.3	0.9
Column temp Minus	0.3	0.2	0.6
Column temp Plus	1.0	0.6	0.5
Solution Stability – 0hrs	0.5	0.9	0.6
Solution Stability - 24hrs % RSD	0.7	0.3	0.5
Assay	99.71%	99.04%	99.26%

Linearity:

The standard solutions in the concentration range of 18.75 – 112.5ppm of PAR, 3,125 – 18.75ppm of OMB and 12.5 – 75ppm of RIT showed linear relationship with correlation coefficient of more than 0.999. The calibration curves were shown in Figure 3.

Figure 3: Calibration curves of PAR, OMB and RIT.

Precision:

The proposed method was precise for estimation of PAR, OMB and RIT as the %RSD were less than 2.0% as given in table 2.

Accuracy:

The percentage recovery of spiked sample solutions at 50%, 100% and 150% of target levels were evaluated thrice by the proposed method. The results in the Table 3 confirm the adequate accuracy of developed method.

Table 2: Precision Data

Table 2.1: Results of method precision

Sample No	Paritaprevir		Ombitasvir		Ritonavir
Sample No	Peak area	% Assay	Peak area	% Assay	Peak area	% Assay
1.	633401	99.72	83245	99.54	459341	99.45
2.	630206	99.22	83375	99.69	461810	99.99
3.	631046	99.35	83111	99.38	460260	99.65
4.	628320	98.92	83287	99.59	460521	99.71
5.	631735	99.45	83055	99.31	461976	100.02
6.	631758	99.46	83873	100.29	460151	99.63
Avg.		99.35		99.63		99.74
S.D.		0.269		0.35		0.221
% RSD		0.3		0.4		0.2

STDEV – Standard deviation, %RSD – Percentage Relative Standard Deviation

Table 2.2: Results of Inter day precision

Injection No.	Paritaprevir peak areas		Ombitasvir peak areas		Ritonavir peak areas
Injection No.	Day - 1	Day -2	Day - 1	Day -2	Day - 1	Day -2
Inj - 1	633401	633245	83245	81995	459341	440562
Inj - 2	630266	626709	83375	81975	461810	441527
Inj - 3	631046	621732	83111	82519	460260	439979
Inj - 4	628320	623732	83287	82369	460521	437859
Inj - 5	631735	627772	83055	82356	461976	436465
Inj - 6	631758	623996	83873	82458	460151	436951
Avg.	631088	626198	83324	82279	460677	438891
S.D.	1706.3	4081.0	293.0	235.3	1022.8	2080.3
% RSD	0.3	0.7	0.4	0.3	0.2	0.5

STDEV – Standard deviation, %RSD – Percentage Relative Standard Deviation

Table 3: Results of % recovery studies

	Paritaprevir			Ombitasvir			Ritonavir
% level	PPM Spiked	PPM found	% recovery	PPM Spiked	PPM found	% recovery	PPM Spiked	PPM found	% recovery
50	37.5	37.803	100.81	6.25	6.246	99.95	25	25.085	100.34
	37.5	37.185	99.16	6.25	6.264	100.222	25	24.980	99.92
	37.5	37.256	99.35	6.25	6.245	99.93	25	24.902	99.61
100	75	74.250	99.00	12.5	12.497	99.98	50	49.621	99.25
	75	74.449	99.27	12.5	12.507	100.06	50	49.303	98.61
	75	75.270	100.36	12.5	12.378	99.03	50	49.328	98.66
150	112.5	111.236	98.88	18.75	18.596	99.18	75	74.904	99.87
	112.5	112.569	100.06	18.75	18.571	99.05	75	74.718	99.62
	112.5	112.187	99.72	18.75	18.741	99.96	75	75.310	100.41
Mean			99.62			99.71			99.59
STDEV			0.66			0.48			0.65
%RSD			0.67			0.48			0.65

STDEV – Standard deviation, %RSD – Percentage Relative Standard Deviation

Figure 4: UPLC chromatograms of Forced Degradation studies

Table 4: Results of Forced degradation studies

Stress Condition	Paritaprevir		Ombitasvir		Ritonavir
	% Assay	% Degradation	% Assay	% Degradation	% Assay	% Degradation
Acid	93.41	6.59	95.86	4.14	94.19	5.81
Alkali	95.73	4.27	95.96	4.04	94.17	5.83
Oxidation	96.79	3.21	96.08	3.92	95.97	4.03
Thermal	97.12	2.88	96.80	3.20	96.46	3.54
UV	98.57	1.43	98.65	1.35	98.01	1.99
Neutral	99.13	0.87	99.22	0.78	99.44	0.56

Robustness:

The chromatograms obtained by deliberate variation in chromatographic conditions were assessed for system suitability parameters. No changes were observed indicating that the method is robust. The results were shown in Table 1.

Forced degradation studies:

The percent degradation of PAR, OMB and RIT was within limits. The degradation products were resolved from the analytes indicating method specificity. The degradation chromatograms were shown in Figure 4 and the results were given in Table 4.

Assay:

The developed method was applied for the assay of Technivie tablets. The assay values were found to be 99.71%, 99.04% and 99.26% for PAR, OMB and RIT respectively. The standard and sample chromatogram were shown in Figure 5.

Figure 5: UPLC chromatograms of Standard and Sample.

CONCLUSION:

The proposed stability indicating UHPLC method allows precise, linear, rapid and stable quantification of Paritaprevir, Ombitasvir and Ritonavir in bulk and tablet dosage forms. The results of parameters validated were complying the acceptance criteria given in ICH guidelines. The developed method can be choice for rapid quantification of samples in routine and quality control analysis of formulation.

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Received on 18.01.2021 Modified on 13.04.2021

Research J. Pharm.and Tech 2022; 15(3):1307-1312.

DOI: 10.52711/0974-360X.2022.00218