RP-HPLC Method Development and Validation for

Asian J. Pharm. Tech. 2017; Vol. 7: Issue 3

ISSN- 2231–5705 (Print) ISSN- 2231–5713 (Online)

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www.asianpharmaonline.org www.ajptonline.com

RESEARCH ARTICLE

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)-5dihydropyrimidin-1(2H)-yl)-4fluoro-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 chainterminating 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-fluoro3-methyltetrahydrofuran-2-yl)methoxy) phosphorylamino) propanoate (Fig.No.1).It is a White to off-white non-hygroscopic crystalline

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

: Figure 1. Structure of Sofosbuvir

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Slightly soluble in water (pH 1.2-7.7), freely soluble in MATERIALS AND METHODS: ethanol and acetone, soluble in 2-propanol, and insoluble Chemicals: in heptanes [3] Sofosbuvir was obtained from HETERO Pharmaceuticals. And was used as such without further Literature survey reveals a few HPLC methods, LC purification [7]. method have been used. The objective of the present work was to develop simple, rapid, accurate, specific and Reagents: economic RP-HPLC stability indicating method. Methanol (HPLC grade), Water (HPLC grade), Potassium dihydrogen phosphate (GR grade), The aim of the present work was to develop and validate Orthophosphoric acid (GR grade) a simple, fast and reliable isocratic RP method with UV detection for the determination of Sofosbuvir in bulk Instruments and Equipments: form. The important features and novelty of the High Performance Liquid Chromatography (Waters proposed method included simple sample treatment with 2695 HPLC, Class) with 2487 pumps, auto injector with sonicator of small amount of powder sample at ambient loop volume of 10 μl (Rheodyne), programmable temperature, shot elution time(less than 5 min)SFS, good variable wavelength PDA detector[8]. precision (R.S.D. less than 2%). Preparation of mobile phase: The aim of the present work was to develop and validate Mix a mixture of above buffer 100mL of Methanol a simple, fast and reliable isocratic RP Method with UV HPLC (100%) and degas in ultrasonic water bath for detection for the determination of Sofosbuvir in bulk 5minutes.Filter through 0.45μ filter under vacuum form. The important features and novelty of the filtration. proposed method included simple sample treatment with sonicator of small amount of powder sample at ambient Preparation of standard and sample solutions: temperature, shot elution time(less than 5 min) SFS, Stock solution of SFS (1mg/mL) was prepared by good precision (R.S.D. less than 2%). weighing 10mg and dissolving in the mobile phase methanol (100%v/v). Standard solutions of SFS were Conformation of the applicability of developed method prepared in the range of 20 μg/mL to 100 μg/mL by validated according to the international conference on diluting the stock solution with mobile phase. The eluate Harmonization (ICH) [6] was monitored at 260nm. Each solution was then injected into the column and chromatograms were recorded.

Fig 2. Chromatogram showing blank (mobile phase preparation)

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Fig .3 Chromatogram of standard sofosbuvir

Table 1.Results of system suitability for Sofosbuvir S.No 1 2 3 4 5 Mean Std. Dev. % RSD

Peak Name Sofosbuvir Sofosbuvir Sofosbuvir Sofosbuvir Sofosbuvir

RT 3.513 3.516 3.515 3.517 3.512

Area (µV*sec) 2947505 2958475 2965847 2952642 2951645 2955223 7114.704 0.24075

USP Plate Count 7462 7462 6472 7183 7428

USP Tailing 1.1 1.1 1.1 1.1 1.1

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 Average Peak Area Concentration g/ml 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 1 Sofosbuvir 2 Sofosbuvir 3 Sofosbuvir 4 Sofosbuvir 5 Sofosbuvir Mean Std.dev %RSD

Retention time 3.528 3.516 3.514 3.519 3.512

Area (µV*sec) 2958333 2951049 2959294 2953391 2950744 2954562 4028.083 0.136334

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USP Plate Count 7583 7593 8674 7958 9745

USP Tailing 1.1 1.1 1.1 1.1 1.1


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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

10. 11. 12.

13.

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