RP-HPLC Method Development and Validation for Estimation of Sofosbuvir in Pure and Tablet Dosage Form

 

P. Swathi*, K. Rajeswar Dutt, K. N. V Rao, M. Alagar Raja

Nalanda College of Pharmacy, Charlapally, Nalgonda, Telangana.

*Corresponding Author E-mail:

 

ABSTRACT:

A rapid and precise Reverse Phase High Performance Liquid Chromatographic method has been developed for the validation of Sofosbuvir, in its pure form as well as in tablet dosage form. Chromatography was carried out on a Hypersil C18 (4.6×150mm, 5µ) column using a mixture of Methanol (100% v/v) as the mobile phase at a flow rate of 1.0 mL/min, the detection was carried out at 265 nm. The retention time of the Sofosbuvir was 3.515 ±0.02min. The method produce linear responses in the concentration range of 20-100µg/mL of Sofosbuvir. The method precision for the determination of assay was below 2.0% RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

 

KEY WORDS: Sofosbuvir; RP-HPLC; Validation; Tablet dosage forms.

 

 


INTRODUCTION:

Sofosbuvir is a prodrug of 2’-deoxy methyluridine monophosphate that is phosphorylated

Intra cellularly to the active triphosphate the treatment of Chronic Hepatitis C[1] triphosphate is a non-obligate chain-terminating analogue of UTP that competes for incorporation at the HCV NS5B polymerase active site. Viral RNA synthesis is inhibited secondary to incorporation of the phosphorlated metabolite into nascent viral RNA by the HCV RNA-dependent RNA polymerase. Chemically, It is (S)-isopropyl-2-((S)-(((2R,3R,4R,5R)-5- dihydropyrimidin-1(2H)-yl)-4-fluoro-3-methyl tetrahydrofuran-2-yl) methoxy) phosphorylamino) propanoate (Fig.No.1).

 

It is a White to off-white non-hygroscopic crystalline Sofosbuvir is a prodrug of 2’-deoxy methyluridine monophosphate that is phosphorylated intra cellularly to the active triphosphate the treatment of Chronic Hepatitis C[1] triphosphate is a non-obligate chain-terminating analogue of UTP that competes for incorporation at the HCV NS5B polymerase active site. Viral RNA synthesis is inhibited secondary to incorporation of the phosphorlated metabolite into nascent viral RNA by the HCV RNA-dependent RNA polymerase. Chemically, It is (S)-isopropyl-2-((S)-(((2R,3R,4R,5R)-5-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-methyltetrahydrofuran-2-yl)methoxy) phosphorylamino) propanoate (Fig.No.1).It is a White to off-white non-hygroscopic crystalline

:

Figure 1. Structure of Sofosbuvir

Slightly soluble in water (pH 1.2-7.7), freely soluble in ethanol and acetone, soluble in 2-propanol, and insoluble in heptanes [3]

 

Literature survey reveals a few HPLC methods, LC method have been used. The objective of the present work was to develop simple, rapid, accurate, specific and economic RP-HPLC stability indicating method.

 

The aim of the present work was to develop and validate a simple, fast and reliable isocratic RP method with UV detection for the determination of Sofosbuvir in bulk form. The important features and novelty of the proposed method included simple sample treatment with sonicator of small amount of powder sample at ambient temperature, shot elution time(less than 5 min)SFS, good precision (R.S.D. less than 2%).

 

The aim of the present work was to develop and validate a simple, fast and reliable isocratic RP Method with UV detection for the determination of Sofosbuvir in bulk form. The important features and novelty of the proposed method included simple sample treatment with sonicator of small amount of powder sample at ambient temperature, shot elution time(less than 5 min) SFS, good precision (R.S.D. less than 2%).

 

Conformation of the applicability of developed method validated according to the international conference on Harmonization (ICH) [6]

 

MATERIALS AND METHODS:

Chemicals:

Sofosbuvir was obtained from HETERO Pharmaceuticals. And was used as such without further purification [7].

 

Reagents:

Methanol (HPLC grade), Water (HPLC grade), Potassium dihydrogen phosphate (GR grade), Orthophosphoric acid (GR grade)

 

Instruments and Equipments:

High Performance Liquid Chromatography (Waters 2695  HPLC, Class) with 2487 pumps, auto injector with loop volume of 10 μl (Rheodyne), programmable variable wavelength PDA detector[8].

 

Preparation of mobile phase:

Mix a mixture of above buffer 100mL of Methanol HPLC (100%) and degas in ultrasonic water bath for 5minutes.Filter through 0.45μ filter under vacuum filtration.

 

Preparation of standard and sample solutions:

Stock solution of SFS (1mg/mL) was prepared by weighing 10mg and dissolving in the mobile phase methanol (100%v/v). Standard solutions of SFS were prepared in the range of 20 μg/mL to 100 μg/mL by diluting the stock solution with mobile phase. The eluate was monitored at 260nm. Each solution was then injected into the column and chromatograms were recorded.


 

Fig 2. Chromatogram showing blank (mobile phase preparation)

 

Fig .3 Chromatogram of standard sofosbuvir


 


Table 1.Results of system suitability for Sofosbuvir

S.No

Peak Name

RT

Area (µV*sec)

USP Plate Count

USP Tailing

1

Sofosbuvir

3.513

2947505

7462

1.1

2

Sofosbuvir

3.516

2958475

7462

1.1

3

Sofosbuvir

3.515

2965847

6472

1.1

4

Sofosbuvir

3.517

2952642

7183

1.1

5

Sofosbuvir

3.512

2951645

7428

1.1

Mean

 

 

2955223

 

 

Std. Dev.

 

 

7114.704

 

 

% RSD

 

 

0.24075

 

 


Table 2. Optimized method of parameters

Column C18

Hypersil C18 (4.6×150mm) 5µ

Mobile Phase

Methanol (100%)

Flow Rate

1.0 mL /min.

Run Time

6 min.

Column Temp.

Ambient

Volume Of Injection Loop

10µL

Detection  Wave Length

265 nm

Linearity Range

20-100 µg/mL

 

Table 3. Calibration data of Sofosbuvir

Concentration mg/ml

Average Peak Area

20

1083048

40

1973321

60

2955166

80

4063921

100

5006038

Slope (m)

49935

Intercept (C)

16821

r2

0.9997

 

Fig .03 Calibration graph of Sofosbuvir

 


Table 4. Precision studies of Sofosbuvir

S. No

Peak name

Retention time

Area (µV*sec)

USP Plate Count

USP  Tailing

1

Sofosbuvir

3.528

2958333

7583

1.1

2

Sofosbuvir

3.516

2951049

7593

1.1

3

Sofosbuvir

3.514

2959294

8674

1.1

4

Sofosbuvir

3.519

2953391

7958

1.1

5

Sofosbuvir

3.512

2950744

9745

1.1

Mean

 

 

2954562

 

 

Std.dev

 

 

4028.083

 

 

%RSD

 

 

0.136334

 

 


RESULTS AND DISCUSSION:

In this paper we developing the reverse phased column procedure for a suitable method for the pharmaceutical analysis of Sofsobuvir drug. Atypical Chromatogram obtained by using the mobile phase (Figure No 2). The precision and Accuracy of the method was determined. Sofosbuvir dosage forms inter and intraday studies were performed in two consecutive days. The method was validated for linearity, precision and accuracy parameters [9]. Linearity of the method was studied by injecting six concentrations of drug prepared in the mobile phase in the range 20-100 µg/mL and solutions are analyzed through the high pressure liquid chromatographic technique (Figure No. 3). The peak area were plotted against concentration was subjected to linear plot and the results present in table (Table no.3). Precision of this method was studied in inter day and intraday variation [12]. The precision of intraday studies was repeated on two consecutive days. The developed method was found to be precise as the percentage of

 

RSD values for inter-day and intra –day precision studies were found to be less than 2%.

 

CONCLUSION:

The proposed method was found to be simple, precise, accurate, rapid and specific for determination of Sofosbuvir from pure and its dosage forms. The mobile phase is simple to prepare and economical. The developed method is accurate, precise and reliable for the analysis of Sofosbuvir in Pharmaceutical formulations. This method was validated for linearity, accuracy and precision of sofosbuvir drug. The RSD values for all parameters were found to be <2, which indicates the validity of method and results obtained by this method is with fair agreement. Hence, this method can be easily and conveniently adopted for routine analysis of Sofosbuvir in pure form and also can be used for dissolution or similar studies.

 

REFERENCES:

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13.     Code Q2B, Validation of Analytical Procedures; Methodology. ICH Harmonized  Tripartite Guidelines, Geneva, Switzerland, (1996), PP 1- 8.

 

 

 

 

 

 

Received on 22.05.2017       Accepted on 16.06.2016     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2017; 7 (3): 153-156.

DOI: 10.5958/2231-5713.2017.00025.3