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.

 

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.

 

 

 

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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Received on 22.05.2017       Accepted on 16.06.2016     

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