RP-HPLC Method for Estimation of Daclatasvir in Bulk and Tablet Dosage Form

INTRODUCTION

The quality of a drug plays an important role in ensuring the safety and efficacy of the drugs. Quality assurance and control of pharmaceutical and chemical formulations is essential for ensuring the availability of safe and effective drug formulations to consumers. Hence Analysis of pure drug substances and their pharmaceutical dosage forms occupies a pivotal role in assessing the suitability to use in patients. The quality of the analytical data depends on the quality of the methods employed in generation of the data.¹

CHROMATOGRAPHY

Chromatography (Chroma means ‘colour’ and graphene means to ‘write’) is the collective term for a set of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a “mobile phase” through a stationary phase2 , which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)^3-6

Liquid chromatography is an analytical chromatographic technique that is useful for separating ions or molecules that are dissolved in a solvent. If the sample solution is in contact with a second solid or liquid phase to differing degrees due to differences in adsorption, ion exchange, partitioning or size. These differences will allow the mixture components to be separated from each other by using these differences to determine the timeof the solutes through a column. During 1970’s, most chemical separations were carried outusing a variety of techniques including opencolumn chromatography, paper chromatography and thin layer chromatography (TLC). However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time pressure liquid chromatography began to be used to decreased flow through time, thus reducing separation time of compounds being isolated by column chromatography. However, flow rates were inconsistent, and the question of whether it was better to have constant flow rate or constant pressure debated. High pressure liquid chromatography quickly improved with the development of column packing materials. Additional convenience of on-line detectors became rapidly a powerful separation technique and is today called as High Performance Liquid Chromatography (HPLC).

CLASSIFICATION OF HPLC

Based on modes of chromatography

Normal phase chromatography

Reverse phase chromatography

Based on principle of separation:

Adsorption chromatography

Partition chromatography

Ion exchange chromatography

Size exclusion chromatography

Affinity chromatography

Chiral phase chromatography

Based on elution technique:

Isocratic separation

Gradient separation

Based on the scale of operation:

Analytical HPLC

Preparative HPLC

DRUG PROFILE^7-11

Daclatasvir is a direct-acting antiviral agent against Hepatitis C Virus (HCV) used for the treatment of chronic HCV genotype 1 and 3 infection. It is marketed under the name DAKLINZA and is contained in daily oral tablets as the hydrochloride salt form. Hepatitis C is an infectious liver disease caused by infection with Hepatitis C Virus (HCV). HCV is a singlestranded RNA virus that is categorized into nine distinct genotypes, with genotype 1 being the most common in the United States, and affecting 72% of all chronic HCV patients . It was the first drug with demonstrated safety and therapeutic efficacy in treating HCV genotype 3 without the need for co-administration of interferon or Ribavirin and stops HCV viral RNA replication and protein translation by directly inhibiting HCV protein NS5A which is critical for HCV viral transcription and translation. It reaches steady state in human subjects after about 4 days of once-daily 60 mg oral administration, with a peak dose in concentration occurring about 2 hours after administration which comes in the form of an oral tablet, with a bioavailability of 67%. It is predominantly metabolized by the liver enzyme CYP3A4, and is also a P-glycoprotein substrate. It is highly protein bound which was measured to be around 99% in people dosed multiple times with Daclatasvir independent of dose strength. It has a volume of distribution of 47L following an oral dose of 60 mg and an IV dose of 100 µg. It has a molecular weight of 738.9 g/mol, the other medications used in combination include sofosbuvir, ribavirin, and interferon vary depending on the virus type and whether the person has cirrhosis. Common side effects with Sofusbivir and Daclatasvir include headache, feeling tired, and nausea. With daclatasvir, sofusbivir, and ribavirin the most common side effects are headache, feeling tired, nausea, and red blood cell breakdown. It should not be used with St. John’s wort, rifampin, or carbamazepine. It was approved for use in Europe in 2014 and the United States and India in 2015. It is on the World Health Organization’s List of Essential Medicines, the most effective and safe medicines needed in a health system. It is methyl N-[(2S)-1-[(2S)-2-[5-[4-[4-[2-[(2S)-1-[(2S)- 2-(methoxycarbonylamino)-3-methylbutanoyl] pyrrolidin-2-yl]-1H-imidazol-5-yl]phenyl] phenyl]-1H-imidazol-2-yl]pyrrolidin-1- yl]-3-methyl-1-oxobutan-2-yl]carbamate; dihydrochloride having a molecular weight of 738.9 g/mol.

K.Sumathi, K. Thamizhvanan and S.Vijayraj¹² developed and validated a novel, simple precise method for the estimation of Daclatasvir in single dosage form HPLC – WATERS Model No.2690 series Compact System Consisting of Inertsil-C18 ODS column with a mobile phase constituting of Acetonitrile and Methanol (70:30) Flow rate 1ml/ min and detection was carried out at 230nm. The selected chromatographic conditions were found to effectively separate Daclatasvir (Rt: 2.658 m). The developed method was validated for linearity, accuracy, precision, LOD, LOQ, robustness, ruggedness and for system suitability parameters as per ICH guidelines. Linearity for Daclatasvir was found in the range of 20-80μg/ml, respectively. The method was found to be robust. The proposed method could be used for routine analysis of Daclatasvir in single dosage forms. The bulk drug was subjected to forced degradation studies like acid, alkali, oxidative, thermal conditions.

HanaaSaleh, Gamal H. Ragab, Mohammed A. Othman¹³ developed and validated a sensitive, simple, selective and accurate method for analysis of antiviral drug Daclatasvir (BMS-790052, DCV) in pure form and in tablet dosage form in the presence of its degradation products. The chromatographic separation achieved by isocratic elution on Hypersil BDS C18, 4.6×150 mm, 5 µm column at 25 °C. The mobile phase was a mixture of 0.05M Potassium dihydrogen phosphate (pH - 4.5) and acetonitrile in ratio of 50:50 (v/v). The injection volume was 10 µl. The flow rate was 1 ml/ minute. The detection wavelength was 320 nm. The developed method was validated as per ICH guidelines; it was precise, accurate and robust. The calibration curve of Daclatasvir was linear in range 0.5- 100 µg/ml with a correlation coefficient ≥ 0.999. Also the validated method was helpful for rapid routine analysis as the run time was less than 3 minute; the retention time for Daclatasvir was about 2.33 minute. The method was successfully applied to analysis of Daclatasvir in tablet form and the recovery was from 99.71% to 100.86%.

MagdyAtef Wadie, Samia Mahmoud Mostafa, Sobhy MohamedEl.Adl, Mohamed SalehElgawish¹⁴ developed and validated a sensitive,simple,selective method for simultaneous analysis of antiviral drugs Sofosbuvir, Daclatasvir and Ribavirin that allow reduction in treatment duration for HCV patients that in turn decrease the cost of the treatment.

Amira S. Eldin, Shereen M. Azab, Abdalla Shalaby, Magda El-Maamly¹⁵ developed and validated a simple, rapid and reproducible reversed-phase high performance liquid chromatography (RP-HPLC) and ultraviolet derivative spectrophotometric (UVDS) methods for the simultaneous determination of daclatasvir (DAC) and sofosbuvir (SOF) in pure and in pharmaceutical dosage forms .

The present research work describes a HPLC and UV spectrophotometric method for estimation of Daclatasvir, in API and aims at developing a simple, accurate and precise RP-HPLC method for its estimation in bulk and Pharmaceutical dosage forms.

MATERIALS AND METHODS

Instrumentation: An Alliance module equipped with a UV spectrophotometer for finding out the λmax values of the drug used throughout this study. A Denali C8 (250mm*4.6mm 5μ) column was employed for the method development. The chromatographic system was monitored by EMPOWER2 software. Analytes were monitored by UV detection at 304 nm using an isocratic mode with 0.01N KH2 PO4 : Acetonitrile in the ratio of 50:50 V/V was used as mobile phase. The flow rate was set at 1.0 ml/min and effluent was monitored at 304 nm. The temperature and run time were maintained at 30 °C and 5-10 m respectively. Solubility of the compounds was enhanced by sonication on an ultrasonicator (BVK Enterprise).

Chemicals and solvents: The reference sample of Daclatasvir was obtained from Abbott India Ltd. HPLC grade water (prepared by using 0.45 Millipore Milli –Q) was procured from Standard Reagents, Hyderabad. HPLC grade Acetonitrile, HPLC grade water, Potassium dihydrogen phosphate, Glacial acetic acid, Methanol were bought from Rankem.

Selection of mobile phase: The objective of this experiment was to optimize the method for estimation of Daclatasvir based on the literature survey. Various mobile phases were tested to select the best possible system. The various mobile phases used included Water: methanol (50:50A),Water:Acetonitrile (50:50), 0.01N KH₂ PO₄ buffer: Acetonitrile (50:50A), 0.1% OPA: Acetonitrile (60:40). Better peak resolution and adequate retention time were obtained with the ratio of 0.01N KH2PO4: Acetonitrile (50:50).

Preparation of the Buffer:

0.01N Potassium dihyrogen orthophosphate: 1.36 g of Potassium dihyrogen Ortho phosphate was taken in a 1000 ml of volumetric flask , about 900 ml of milli-Q water was added and degassed to sonicate and finally made up to the volume with water. 1 ml of Triethylamine was added to adjust the pH to 3.0 with dilute Orthophosphoric acid solution.

Preparation of Mobile Phase: The mobile phase was prepared by mixing 500 ml of phosphate buffer and 500 ml of Acetonitrile in a 1000 ml clean and dry flask. The mobile phase was then degassed using Ultra-Sonicator to remove dissolved gases and the resultant mobile phase was filtered through a 0.45 μm membrane filter under vacuum.

Preparation of the standard solution: 15 mg of Daclatasvir was accuaretly weighed and transferred into 25 ml volumetric flasks, 3/4^th of diluent was added and sonicated for 10 minutes. Flasks were made up with diluent and labelled as Standard stock solution which contains 600 µg/ml of Daclatasvir.

Preparation of Standard working solutions (100% solution): 1 ml of Daclatasvir from each stock solution was pipetted out and taken into a 10 ml volumetric flask and made up with diluents which contains 60 µg/ml of Daclatasvir.

Preparation of Sample solution: 5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask, 5ml of diluent was added and sonicated for 25 m, further the volume was made up with diluent and filtered.

METHOD DEVELOPMENT: The standard solution was subjected to UV Spectrophotometer to find its absorption maxima.

The absorbance maxima for the drug Daclatasvir was found to be 304 nm.

VALIDATION

Prior to validation studies blank solution was injected and chromatograms were recorded. Optimized conditions were maintained, the drug was eluted with good retention time and peak area which was shown in the fig. 3.

The system suitability parameters were determined by preparing standard solutions of Daclatasvir (60 µg/ml) and the solutions were injected six times.

Parameters like peak tailing, resolution and USP plate count were determined. The % RSD for the area of six standard injections results should not be more than 2%.The system suitability data was represented in figure 4 and table 1.

Discussion: The number of theoretical plates of Daclatasvir were found to be greater than 2000. The %RSD was found to be 1.2.

Linearity : The linearity of the method was established by determining the absorbance of different concentrations of Daclatasvir over a range of 15-90 µg/ml respectively. The linearity data was given in table 2 and chromatograms were figured as 5,6,7,8,9,10.

Discussion: The response was found to be linear in the concentration range from 15 μg/ml to 90 μg/ml and the correlation coefficient was found to be 0.999.

Accuracy: To determine the accuracy of the proposed method, recovery studies were carried out by analyzing the samples with the measured concentration and the added concentration of the drug. Each sample was injected thrice .The percent recoveries of the drugs were estimated. The accuracy data was represented in table 3 along with the chromatograms in figures 12,13,14.

Discussion: The percentage recovery for Daclatasvir with 50%, 100% and 150% was found to be 99.87%.

Precision: Precision is one of the important factors which determine the reliability of an analytical method that was tested and was found to be suitable. Both system and method precision were performed and results were given in table 4 with the chromatogram figured 15.

Discussion: The percentage RSD for Daclatasvir was found to be 0.6%.

Repeatability: Six working sample solutions of 60ppm were injected and the % Amount and % RSD was calculated . The results were tabulated in the table 5 with the chromatogram given in the figure 16.

Discussion: The percentage RSD for Daclatasvir was found to be 0.9%.

Robustness: The robustness of the proposed method was determined by analysis of aliquots from homogenous lots by differing physical parameters like volume of injection, wavelength, mobile phase composition, flow rate ,pH, temperature which may differ but the responses were still within the limits of the assay.

The results were tabulated in the table 6 with the chromatograms represented in the figures 17,18,19,20,21,22.

Discussion: The percentage RSD for Daclatasvir was not more than 2% for variation in flow rate, mobile phase, temperature conditions

Limit of Detection (LOD): Limit of detection (LOD) is the lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental condition. It was given as figure 23.

3.3σ

LOD =   -------------

S

Where, σ = the standard deviation of the response

S = the slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte

Discussion: From the chromatogram the LOD value for Daclatasvir was found to be 0.364 μg/ml.

Limit of Quantification (LOQ): Limit of quantitation (LOQ) is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. It is given as figure 24.

10σ

LOQ =  --------------

S

Where,

σ = the standard deviation of the response

S = the slope of theS calibration curve

The slope S may be estimated from the calibration curve of the analyte

Discussion: From the chromatogram the LOQ value for Daclatasvir was found to be 1.103 μg/ml.

Assay: Assay of marketed formulations of Daclatasvir was carried by injecting sample corresponding to equivalent weight into HPLC system and recovery studies were carried out. The results were represented in the table 7 and was given as figure 25.

The % Assay of marketed formulations of Daclatasvir were found to be 100.64 %.

SUMMARY AND CONCLUSION

In the present work, an attempt was made to provide a newer, sensitive, simple, accurate and economical RP-HPLC method. It was successfully applied for the determination of Daclatasvir in pharmaceutical dosage forms without the interferences of other constituents in the formulations. The standard solution was subjected to UV spectrophotometer to find the absorption maxima. Different mobile phase compositions were tried, to get good optimum results. Mobile phase and flow rate selection was done based on peak parameters (height, tailing, theoretical plates, capacity factor), run time etc. The system with 0.01N KH₂ PO₄ buffer: Acetonitrile in the ratio of 50:50 with 1.0 ml/m flow rate was quite robust. The optimum wavelength for detection was 304 nm at which better detector response for drug was obtained. The average retention time of Daclatasvir was found to be 2.28 minutes .The calibration curve was linear in concentration range of 15-90 μg/ml. The low values of % RSD indicate the method was precise and accurate. Sample to sample precision and accuracy were evaluated using, three samples of five and three different concentrations respectively, which were prepared and analyzed on same day. These results show the accuracy of the assay. The LOD and LOQ values were also in limits. By using above method assay of marketed formulation was carried out which was found to be 100.64 %. The proposed method was validated in accordance with ICH parameters and the results of all methods were very close to each other as well as to the label value of commercial pharmaceutical formulation. There was no significant difference in the results achieved by the proposed method. The proposed method for the assay of the Daclatasvir in the commercially available dosage formulation was simple, accurate, economical, and rapid. It can be easily adopted for routine quality control for monitoring the assay in the API, in-process samples, and the finished tablet formulation.

ACKNOWLEDGEMENT

I am very much thankful to the Director, Principal, Staff, Nonteaching Staff, Karnataka College of Pharmacy, Bangalore for providing me the facilities to carry out the research work.

CONFLICTS OF INTEREST

Certify that we have no conflict of interest in the subject matter.