ESTIMATION OF DACLATASVIR IN PHARMACEUTICAL DOSAGE FORM BY ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY

ESTIMATION OF DACLATASVIR IN PHARMACEUTICAL DOSAGE FORM BY ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY

A. Viswanath ¹, A. Lakshmanrao ¹, Aziz Unnisa ², B. Anupama ^{* 3} and Kondepati Haritha ³

Acharya Nagarjuna University ¹, Guntur – 522510, Andhra Pradesh, India.

University of Hail ², Saudi Arabia.

KVSR Siddhartha College of Pharmaceutical Sciences ³, Vijayawada - 520008, Andhra Pradesh, India.

ABSTRACT: Daclatasvir is an antiviral drug used in combination with other drugs includes sofosbuvir, ribavirin, and interferon, depending on the virus type to treat cirrhosis caused by hepatitis C (HCV). Several methods have been found for quantification, but those are not cost-effective, and they are time-consuming. The present study developed a simple, precise, accurate and cost-effective UPLC method to determine daclatasvir quantity in tablet dosage forms. A simple and selective UPLC method is described for the determination of Daclatasvir Chromatographic separation was achieved on a Acquity BEH C18 (50 × 3.0mm. 1.7 µm) using a mobile phase consisting 0.1% of Orthophosphoric acid: Acetonitrile in a ratio of 60:40 v/v with detection of 248 nm. Linearity was observed in the range 50-150 µg/ml for Daclatasvir (r² = 1.000). The amount of drugs estimated by the proposed method was in good agreement with the label claim. The proposed method was validated as per ICH guidelines and applied for the determination of the cited drug in the dosage form.

UPLC, Daclatasvir, Orthophosphoric acid, Acetonitrile

INTRODUCTION: Daclatasvir is chemically dimethyl N, N’-([1,1’-biphenyl]-4, 4’-diylbis{1H- imidazole-5,2-diyl-[(2S)-pyrrolidine-2,1-diyl][(2S)-3- methyl- 1- oxobutane-1, 2-diyl]}) dicarbamate. Daclatasvir has molecular weight: 738.89 g/mol and molecular formula: C₄₀H₅₀N₈O₆. It is an antiviral drug used in combination with other medicaments to treat hepatitis C (HCV). The other medicines used in combination include interferon, sofosbuvir, and ribavirin, depending on the virus type 1. The dose of daclatasvir present in the formulation was determined by using the Ultra Performance Liquid Chromatography method. UPLC has greater sensitivity, resolution, and speed of analysis.

UPLC operates at high pressure than HPLC, and fine particles, i.e., less than 2.5 µm are used, and mobile phases at high linear velocities decrease the length of the column, reduces solvent consumption, and save time ².

The UPLC is based on the use of a stationary phase consisting of particles less than 2.5 µm whereas the HPLC column is typically filled with 3-5 µm particles. The principle of this evolution is governed by the Van Deemeter equation, which is an empirical formula that describes the relationship between the linear velocity of flow rate and plate height ^{3, 4}.

H = A + B / v + Cv

Where; A, B and C are constants, v is the linear velocity, the carrier gas flow rate.

*The A term is independent of velocity and represents "eddy" mixing. It is the smallest when the packed column particles are small and uniform.

The B term represents axial diffusion or the natural diffusion tendency of molecules. This effect is diminished at high flow rates, and so this term is divided by v.

*The C term is due to kinetic resistance to equilibrium in the separation process. The kinetic resistance is the time lag involved in moving from the gas phase to the stationary packing phase and back again. The greater the flow of gas, the more a molecule on the packing tends to lag behind molecules in the mobile phase. Thus the term is proportional to v.

Therefore it is possible to increase throughput and thus the speed of analysis without affecting the chromatographic performance. The advent of UPLC has demanded the development of a new instrumental system for liquid chromatography, which can take advantage of the separation performance (by reducing dead volumes) and consistent with the pressures (about 8000 to 15,000 PSI, compared with 2500 to 5000 PSI in HPLC). Efficiency is proportional to column length and inversely proportional to the particle size ^{5, 6}.

This technology has the advantage of chromatographic principles to run separations using a packed column with similar particle sizes less than 2.5 µm are used with high flow rates speed gives superior resolution and sensitivity.

MATERIALS AND METHODS:

Chemicals and Reagents: The drug standard of Daclatasvir was kindly supplied by Madras Pharmaceuticals, Chennai, with certified purity of 99.97 ± 0.512. Daklinza 10 mg Tablets were purchased from apollo pharmacy, Hyderabad. HPLC grade acetonitrile, water, and methanol were obtained from Rankem. Analytical grade Potassium Dihydrogen orthophosphate, Dipotassium hydrogen orthophosphate and O-Phosphoric acid were obtained from Merck.

A Shimadzu (UV-1800) double beam UV-Vis spectrophotometer with 1cm quartz cuvette connected to a personal computer loaded with UV probe 2.21 software was used.

Chromatographic Method: ^{7, 8} Chromatographic separations were achieved by UPLC-agilent 1290 infinity with a quaternary solvent manager, with autosampler injector and photodiode array detector, coupled with Empower software for data acquisition. Acquity BEH C18 (50 × 3.0 mm. 1.7 µm) was used as the stationary phase for the development of the chromatographic separation, optimization, and method validation. Isocratic elution was conducted using a mobile phase 0.1% Orthophosphoric acid: Acetonitrile (60:40) v/v.

The flow rate was set at 0.5 mL/min. Column temperature was adjusted at 30 °C, and samples were injected at 10 µL injection volume with a run time of 5 min at a temperature 10 °C and determined at a wavelength of 248 nm. Daclatasvir 1 mg/mL stock solution was prepared for the UPLC method by dissolving 100mg of daclatasvir in 100 mL of the mobile phase.

Preparation of 0.1% Ortho Phosphoric Acid: Taken 1 mL of orthophosphoric acid and transferred in to a 1000 mL of water & filtered through 0.45 µm filters to remove all fine particles and gases.

RESULTS AND DISCUSSION:

UPLC Method Development: The main target of the proposed UPLC method was to achieve separation of daclatasvir within short runtime. To determine the stationary phase (Acquity BEH C18 (50 × 3.0 mm. 1.7 µm)) column was chosen because it provided better peak symmetry. For organic modifier, a different ratio of orthophosphoric acid and acetonitrile were checked. It was found that orthophosphoric acid found better resolution. Mobile phase ratio was found to be a mixture of orthophosphoric acid: acetonitrile 60:40 v/v.

Flow rate at 0.5 mL/min was selected as the optimum flow rate. The optimum wavelength for detection was 248 nm. The retention time was 1.190 min, respectively. According to the ICH guidelines, the system sustainability tests should be carried out prior to analysis. Several parameters were studied, including tailing factor, retention time, height equivalent to theoretical plates, and RSD% of peak area for repetitive injections were studied. In all deliberately varied chromatographic conditions, the chromatogram of solution showed satisfactory resolution, as shown in Fig. 1 and results are shown in Table 1.

FIG. 1: UPLC CHROMATOGRAM OF DACLATASVIR

TABLE 1: UPLC CHROMATOGRAM OF DACLATASVIR

Validation of Proposed Methods: The developed method was validated as per ICH guidelines.

Linearity and Concentration Range: ⁹ Aliquots equivalent to 50-150 µg/mL of working solution (1mg/mL) of daclatasvir were transferred into a 10 mL volumetric flask, and the volume was diluted with the mobile phase. The linearity values were summarized in Table 2.

TABLE 2: LINEARITY DATA OF DACLATASVIR

The correlation coefficient R² was determined and was found to be 1.00 for DACLATASVIR were given in Table 3. The linearity graph shown in Fig. 2 and the chromatograms are shown in Fig. 3-7.

FIG.2: GRAPH FOR LINEARITY DATA OF DACLATASVIR

TABLE 3: LINEARITY RESULTS OF DACLATASVIR

FIG. 3: CHROMATOGRAM OF LINEARITY FOR PREPARATION 1

FIG. 4: CHROMATOGRAM OF LINEARITY FOR PREPARATION 2

FIG. 5: CHROMATOGRAM OF LINEARITY FOR PREPARATION 3

FIG. 6: CHROMATOGRAM OF LINEARITY FOR PREPARATION 4

FIG. 7: CHROMATOGRAM OF LINEARITY FOR PREPARATION 5

System Suitability and Method Precision: ¹⁰ The system suitability was evaluated by giving daclatasvir injection six times, and the chromatograms were recorded.

The results were summarised in Table 4. The plate count and tailing factor results were found to be within limits.

The method precision chromatograms were recorded, and the results were summarized in Table 5.

TABLE 4: RESULTS FOR SYSTEM SUITABILITY OF DACLATASVIR

FIG. 8: CHROMATOGRAM OF METHOD PRECISION-01

FIG. 9: CHROMATOGRAM OF METHOD PRECISION-02

FIG. 10: CHROMATOGRAM OF METHOD PRECISION-03

FIG. 11: CHROMATOGRAM OF METHOD PRECISION-04

FIG. 12: CHROMATOGRAM OF METHOD PRECISION-05

FIG. 13: CHROMATOGRAM OF METHOD PRECISION-06

TABLE 5: METHOD PRECISION RESULTS FOR DACLATASVIR

Specificity: A chromatogram of blank and placebo solutions had shown no peaks at the retention times of daclatasvir. It indicates that diluent or excipient peaks do not interfere with the daclatasvir peak. The chromatograms are shown in Fig. 14 and Fig. 15.

Accuracy: The accuracy of the proposed method were determined by analyzing three different laboratory preparations of daclatasvir in different ratios within the linearity range. The values of mean percentage recoveries were shown in Table 6. The chromatograms are shown in Fig. 16-24 ¹¹.

TABLE 6: RESULTS FOR RECOVERY OF DACLATASVIR

Limit of Detection (LOD) & Quantitation (LOQ): ^{12, 13} According to ICH guidelines LOD and LOQ can be calculated using the standard deviation of the response and the slope. LOD = 3.3 × σ/S and LOQ = 10 × σ/S. Where, σ = the standard deviation of the response and S = the slope of the calibration curve. LOD and LOQ of the drug were found to be 35.14µg/mL and 106.48 µg/mL, respectively.

Robustness: ¹⁴ The Robustness of the method was determined. The chromatograms are shown in Fig. 25-28. The results obtained by deliberate variation in method parameters are summarized below in Table 7.

TABLE 7: RESULTS FOR ROBUSTNESS OF DACLATASVIR

Ruggedness: The ruggedness of the method was studied by determining the analyst-to-analyst variation by performing the Assay by two different analysts. The chromatograms are shown in Fig. 29- 32. The method is rugged, and the results are summarized in Table 8.

TABLE 8: RUGGEDNESS RESULTS OF DACLATASVIR

Analysis of Pharmaceutical Dosage Forms: ¹⁵ The proposed method was applied for the determination of daclatasvir in pharmaceutical dosage form Daklinza 10 mg tablets, without interference from the excipients, and good recoveries were obtained by applying the standard addition technique. The results were summarized in Table 9 and 10.

TABLE 9: RESULTS OF DAKLINZA DOSAGE FORM

TABLE 10: RESULTS OF ASSAY

CONCLUSION: A new precise, accurate, rapid method has been developed for the estimation of Daclatasvir pharmaceutical dosage form by UPLC. The cited drug was analyzed without any interference from excipients indicates the selectivity of the method. The proposed method is highly sensitive, precise, and accurate, as indicated by % recovery, % mean recovery and % RSD values.

From the results, it indicates that the UPLC method is applicable to assay of this antiviral drug with minimum sample preparation, cost, and time effectiveness with a satisfactory level of accuracy and precision. Hence, it is successfully applied for the quantification of API content in the commercial formulations of Daclatasvir in educational institutions and Quality control laboratories. UPLC method is economical, faster, consumes less mobile phase than HPLC; it indicates it is faster and eco-friendly.

ACKNOWLEDGEMENT: The authors are very grateful to Acharya Nagarjuna University, KVSR Siddhartha College of Pharmaceutical Sciences, and SAGTE for providing the facilities to carry out the work.

CONFLICTS OF INTEREST: The authors declare that there are no conflicts of interest. All authors contributed the work equally and worked together in the preparation of abstracts, experimental work and writing the paper.

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    How to cite this article:

    Viswanath A, Lakshmanrao A, Unnisa A, Anupama B and Haritha K: Estimation of daclatasvir in pharmaceutical dosage form by ultra performance liquid chromatography. Int J Pharm Sci & Res 2021; 12(2): 973-83. doi: 10.13040/IJPSR.0975-8232.12(2).973-83.

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