Simultaneous determination of newly developed antiviral agents in pharmaceutical formulations by HPLC-DAD
© The Author(s) 2017
Received: 27 October 2016
Accepted: 14 December 2016
Published: 3 January 2017
Ombitasvir/paritaprevir/ritonavir/dasabuvir (Viekira Pak®) are the newest medicines approved for use in the treatment of hepatitis C virus (HCV) and are available in tablet form as an oral combination. Specifically, these agents are indicated in the treatment of HCV in patients with genotype 1 infection. Due to the therapeutic importance and increased use of Viekira Pak, proper methods for its determination in bulk and pharmaceutical formulations must be developed.
The present study describes the development and validation of a simple, rapid, selective and economical reverse phase high performance liquid chromatography-diode array detection (HPLC-DAD) method for the simultaneous determination of paritaprevir (PAR), ombitasvir (OMB), dasabuvir(DAS) and ritonavir (RIT) in bulk and pharmaceutical preparations. The proposed method was carried out using an RPC18 column (150 × 4.5 mm, 3.5 μ), with a mobile phase consisting of 10 mM phosphate buffer (pH 7)and acetonitrile (35:65, v/v) at a flow rate of 1 ml/min and a detection wavelength of 254 nm. Sorafenib (SOR) was selected as the internal standard to ensure that the quantitative performance was high. The method was validated based on its specificity, linearity, limit of detection, limit of quantitation, accuracy, precision, robustness and stability. The calibration curves for PAR, DAS, RIT and OMB were linear at 2.5–60, 1.25–30, 1.7–40 and 0.42–10 μg/ml, respectively, and all of the correlation coefficients were >0.999.
KeywordsAntiviral agents Viekira HPLC-DAD Ombitasvir Paritaprevir Ritonavir
Approximately 180 million individuals worldwide are infected with chronic hepatitis C virus (HCV), and 500,000 patients die each year from liver disease associated with hepatitis C, making it the most common blood borne pathogen [1–3]. HCV, which belongs to the genus Hepacivirus within the family of Flaviviridae, is an enveloped virus with a single positive-stranded RNA genome . In total, six different genotypes of HCV and multiple subtypes are known, and their distribution varies by region. In Saudi Arabia, HCV-genotype 4, followed by genotype 1, are the most prevalent [3, 5]. Increasing protective immune responses in human beings is difficult using classic approaches for virus control. As a result, an efficient vaccine for the prevention of HCV infection has not yet been developed, and the use of antiviral medications has been the only alternative considered for controlling the HCV epidemic . In the past, a combination of peg-interferon (alfa-2a or alfa-2b) and ribavirin was the only available treatment regimen for HCV. However, these drugs have major disadvantages, such as long treatment courses, suboptimal efficacy, and/or harmful side effects. Therefore, the development of a new category of more potent and safer antiviral agents was required. Direct-acting antiviral (DAA) therapies, which were recently discovered and approved, offer good tolerability, short treatment duration, fewer side effects, and high cure rates. DAAs work by targeting a variety of stages in the HCV life cycle [6–11].
A review of the literature revealed that CE [15, 16], HPLC [17–21], UPLC–MS/MS [22–24], LC–MS/MS [25, 26] and HPTLC [27, 28] methods have been reported for the analysis of RIT, individually or in combination with other drugs. However, a method for the simultaneous determination of OMB, PAR, RIT and DAS has not yet been reported. Therefore, the purpose of the present work was to develop a new method for the simultaneous determination of OMB, DAS, PAR and RIT in their bulk and pharmaceutical dosage forms. In this report, a simple, rapid, precise, accurate and selective RP-HPLC method was developed and validated in accordance with the international conference on harmonization (ICH) guidelines .
Chemicals and reagents
OMB, DAS, PAR, RIT and internal standard SOR were purchased from Haoyuan Chemexpress Co., Ltd. (Shanghai, China). Samples of Viekirax® and Exviera®tablets were obtained as gifts from King Faisal Specialist Hospital and Research Center (Riyadh, Saudi Arabia) and were manufactured by AbbVie Ltd. Acetonitrile (HPLC gradient-grade) was supplied by Panreac Quimica S.A. (Barcelona, Spain). Potassium dihydrogen orthophosphate anhydrous was obtained from WINLAB (Leicestershire, UK) and sodium hydroxide pellets were supplied by BDH Chemicals Ltd. (Poole, UK). Deionized water was used in all experiments.
Instrumentation and chromatographic conditions
The HPLC system (Waters, Milford, MA, USA) consisted of a waters 1525 binary HPLC pump, a Waters 2998 Photodiode Array Detector, and a Waters 2707 Autosampler. The data were acquired and processed using Windows XP-based Waters Breeze 2 software. Ultrapure water (18 MΩ/cm) was produced by a Milli-Q® Advantage A10® Water Purification System (Billerica, MA, USA).
The chromatographic separations were carried out on a reverse phase Waters Symmetry®C18 column (150 × 4.5 mm i.d., particle size 3.5 μm). The mobile phase was a mixture of acetonitrile and 10 mM potassium dihydrogen orthophosphate (65:35, v/v; pH adjusted to 7 with sodium hydroxide) delivered at a flow rate of 1 ml/min. The mobile phase was filtered through 0.45-µm Whatman®filterpaper and sonicated for 20 min. Analysis was performed at ambient temperature, and the elution of the compounds was monitored by diode array detection (DAD) from 190 to 400 nm. The chromatograms were recorded at 254 nm, and the injection volume was 20 µl.
Preparation of standard and sample solutions
Preparation of stock solutions
Accurate aliquots of 10 mg of PAR, RIT, OMB and internal standard SOR were each separately transferred into 10-ml volumetric flasks, dissolved using acetonitrile and diluted up to the mark with the same solvent to obtain primary stock solutions (concentration 1000 µg/ml) of each drug. The stock solution of DAS was prepared by weighing 10 mg of DAS and dissolving it in a very small amount of DMSO (10 drops); then, the final volume was achieved using acetonitrile to obtain a final concentration of 100 µg/ml.
Primary stock solutions of PAR, RIT, OM Band DAS were further diluted with the mobile phase to obtain working standards in the concentration range of 2.5–60, 1.25–30, 1.7–40 and 0.42–10 μg/ml for PAR, DAS, RIT and OMB, respectively. A standard concentration of 5 µg/ml SOR (internal standard) was added to the solutions.
Preparation of sample solutions
Ten tablets of Viekirax® (containing 75 mg PAR, 50 mg RIT and 12.5 mg OMB) were weighed and finely powdered. A quantity of the powder equivalent to 10 mg of PAR was weighed and transferred to a 10-ml volumetric flask. A small amount of acetonitrile was added to the flask, and the resulting mixture was sonicated for 20 min. The final volume was achieved using acetonitrile to obtain a final concentration of 1000 µg/ml of PAR. The solution was filtered through 0.45-µm filter paper (stock solution A).
Ten tablets of Exviera®tablet (containing 250 mg DAS) were powdered, and an amount equivalent to 10 mg of DAS was accurately weighed into a 10-ml volumetric flask and mixed with 10 drops of DMSO. A small amount of acetonitrile was then added to this flask. The solution was ultra sonicated for 20 min and filled with acetonitrile to obtain a final concentration of 1000 µg/ml of DAS. The solution was filtered through a 0.45-μm membrane filter (stock solution B).
Aliquots of sample stock solutions. A and B were further diluted with the mobile phase, and a constant amount of 5 μg/ml of SOR was added to each solution to obtain final concentrations of 40 μg/ml of PAR, 26.7 μg/ml of RIT, 6.7 μg/ml of OMB and 15 µg/ml of DAS. The resulting solutions were then subjected to analysis by the proposed HPLC method.
Results and discussion
Method development and optimization of chromatographic conditions
Validation of the optimized method was performed according to ICH Q2 (R1) guidelines . The following validation characteristics were addressed: specificity, detection limit, quantitation limit, linearity, precision, accuracy and robustness.
System suitability parameters
System suitability parameters for the determination of paritaprevir (PAR), dasabuvir (DAS), ritonavir (RIT), and ombitasvir (OMB) using the proposed HPLC method
Retention time (min) (Rt)
Capacity factor (k)
Efficiency (no. of theoretical plates)
The specificity of the proposed HPLC method was assessed by comparing the spectrum of each drug in the sample with the reference drug spectrum using the diode array detector. Chromatograms obtained from standard solutions were also compared to those from the sample solutions, and interference was not observed in the presence of formulation excipients, indicating that the specificity of the method was satisfactory Table 5.
Limit of detection (LOD) and limit of quantitation (LOQ)
Regression and statistical parameters for the determination of DAAs using the proposed HPLC method
Linearity range (µg/ml)
Correlation coefficient (r)
S a c
S b d
S y/x e
The linearity of the response of the detector for each drug was determined by plotting the response ratio (ratio of the peak area of the drug to that of the internal standard) versus the drug concentration and calculating the corresponding regression equation. The calibration curve was linear at concentrations of 2.5–60 µg/ml for PAR, 1.25–30 µg/ml for DAS, 1.7–40 µg/ml for RIT and 0.42–10 µg/ml for OMB. For all of the standard solutions, each concentration was injected in triplicate to obtain reproducible responses. According to the results of the regression analysis, which are given in Table 2, the method was linear, showing a correlation coefficient of >0.999.
The high values of the correlation coefficients (r) and negligible intercepts (a) indicated that the linearity of the calibration graphs was acceptable. Sy/x is a measure of the extent of deviation between the observed (measured) y-values and calculated y-values. For example, low values of Sy/x indicate that the point lies close to the regression line. The standard deviation (SD) of the intercept (Sa) and slope (Sb) were also calculated.
Precision and accuracy
Intra-day and inter-day precision and accuracy for the determination of paritaprevir (PAR), dasabuvir (DAS), ritonavir (RIT), and ombitasvir (OMB) using the proposed HPLC method
Intraday precision and accuracy (n = 3)
Interday precision and accuracy (n = 9)
Standard concentration (μg/ml)
Mean % recovery ± SD
Mean % recovery ± SD
100.53 ± 0.599
99.17 ± 1.227
99.11 ± 0.133
99.22 ± 0.396
101.39 ± 0.250
101.29 ± 0.232
99.72 ± 0.324
98.78 ± 0.567
100.75 ± 0.143
100.51 ± 0.271
100.38 ± 0.17
100.45 ± 0.352
101.19 ± 1.183
101.32 ± 0.996
100.67 ± 0.069
101.54 ± 0.466
100.92 ± 0.053
101.32 ± 1.164
99.23 ± 0.002
99.71 ± 1.612
100.85 ± 0.001
100.99 ± 0.312
101.64 ± 0.006
101.66 ± 0.386
Robustness of the proposed HPLC method
RSD % of peak areas
k ± SD
RSD % of peak areas
k ± SD
RSD % of peak areas
k ± SD
RSD % of peak areas
k ± SD
Percentage of acetonitrile in the mobile phase [64, 65 and 66 ml]
0.48 ± 0.007
2.08 ± 0.005
2.81 ± 0.001
6.11 ± 0.002
pH of the aqueous phase [6.9, 7 and 7.1]
0.53 ± 0.011
2.03 ± 0.010
2.73 ± 0.001
5.85 ± 0.003
Flow rate [0.9, 1, and 1.1 ml/min]
0.53 ± 0.005
2.12 ± 0.005
2.87 ± 0.000
6.25 ± 0.003
Injection volume [15, 20, 25 µl]
0.53 ± 0.024
1.97 ± 0.026
2.68 ± 0.01
5.66 ± 0.007
Detection wavelength [253, 254 and 255 nm]
0.55 ± 0.007
1.99 ± 0.003
2.69 ± 0.001
5.75 ± 0.002
The stabilities of both standard and sample solutions were examined, and changes in the corresponding chromatographs were not observed after 24 h at room temperature and over 3 weeks in a refrigerator (4 °C).
Analysis of pharmaceutical formulations
Assay of paritaprevir (PAR), dasabuvir (DAS), ritonavir (RIT) and ombitasvir (OMB) tablets by the proposed HPLC methods
Mean % recovery ± RSDa
101.89 ± 0.264
99.61 ± 0.498
101.93 ± 0.862
102.28 ± 0.011
A new method based on isocratic RP-HPLC with DAD was developed and validated for the simultaneous determination of paritaprevir, ombitasvir, ritonavir and dasabuvir in bulk and pharmaceutical formulations. The reliability assessment showed that the proposed method was linear, accurate, precise, reproducible, specific and robust. Moreover, all four drugs were successfully resolved and quantified within a single analytical run with a short operating time (the elution time of the last peak was 6.3 min). Therefore, the developed method can be used in quality control studies, in which cost and time are concerning factors.
high performance liquid chromatography-diode array detection ion
NZ designed the study, participated in discussing the results, and revised the manuscript. HM conducted the optimization and assay validation studies. AS performed the assays and prepared the manuscript. All authors read and approved the final manuscript.
This research project was supported by a grant from the “ResearchCenter of the Center for Female Scientific and MedicalColleges,” Deanship of Scientific Research, King Saud University.
The authors declare that they have no competing interests.
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