Development and Validation of UV Spectroscopic Method for the Estimation of Sofosbuvir in Bulk and Pharmaceutical Dosage form

 

N. Jagruthi, Shalem K., Vijaya Sri*, M.A.Madhuri

Department of Pharmaceutical Analysis, Malla Reddy College of Pharmacy, (Affiliated to Osmania University) Maisammaguda, Secunderabad-500 014

*Corresponding Author E-mail:

 

ABSTRACT:

The main objective was to develop and validate the UV-spectrophotometric method for the estimation of sofosbuvir in bulk and pharmaceutical formulations as per ICH guidelines. A simple, sensitive and accurate UV- spectrophotometric method has been developedusing acetonitrile: water (45:55 v/v)a solventfor the estimation of sofosbuvir from bulk and pharmaceutical formulation. The λmax of sofosbuvir was found to be 260 nmit was provedlinearity in the concentration range 4–24 μg/ml with a correlation coefficient value of 0.999. The % recovery was found to be in the range of99.6-101.2%. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.495and 1.502µg/ml, respectively. The % RSD low values are indicates the accuracy and precise of the method. The above method was a rapid toolforroutine analysis of sofosbuvir the bulk and in the pharmaceutical dosage form.

 

KEYWORDS:Sofosbuvir, ICH, Method development, UV-spectrophotometric and pharmaceutical formulation.

 

 


INTRODUCTION:

Sofosbuvirchemicallyis Isopropyl (2S)-2-[[[(2R, 3R, 4R, 5R) - 5-(2, 4-dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl] methoxy-phenoxy-phosphoryl] amino] propanoate1-2. Sofosbuvir is a nucleotide analogue used in combination with other drugs for the treatment of HCV infection. It has been marketed since 2013. Compared to previous treatments, Sofosbuvir-based regimens provide a higher cure rate, fewer side effects, and a 2- to 4-fold reduced duration of therapy.

 

 

 

The chemical structure of Sofosbuvir shown in Fig. 1

 

Fig .1 Structure of sofosbuvir

 

A thorough literature survey has revealed that UV spectroscopy3, HPLC (4-5)UPLC(6-7)and LC-MS/Ms(8)method for Sofosbuvir estimation in bulk, pharmaceutical dosage and biological samples in single or combined dosage form. For the assay of Sofosbuvir in tablet dosage form, a simple and precise method was developed and validated by using a mobile phase comprising a mixture of Acetonitrile: water in the volume ratio of (45:55) v/v.

 

The aim of present research work for the development and validation of UV method, an attempt was made. To develop a simple, accurate, precise and rapid method for the estimation of Sofosbuvir in bulk and pharmaceutical dosage form.

 

MATERIALS AND METHODS:

Chemicals:

Sofosbuvir was gifted from Hetero labs, Hyderabad. Methanol was purchased from Merck Chemical Company. Acetic acid purchased from SDFCL chemicals. Whatman no 5 filter paper was obtained from Modern Science lab, (Nasik, India). All the glassware used were class A grade.

 

Instrumentation:

An electronic balance (Shimadzu TX223L), a sonicator (spectral lab, model UCB 40) and UV- Visible Spectrophotometer (Shimadzu model-2203) and the high pressure liquid chromatographic system used was an Agilent High Pressure Liquid Chromatography 1260 series. Pump, Eclipse C18 column (5 µm particle size x 4.6 × 250 mm) (made in USA) and diode array detector G1315D was used for data acquisition. Digital pH meter (Systronics model - 802), were used in this study.

 

SELECTION OF SOLVENT:

Ideal properties of a solvent are a)Drug should be soluble in the solvent used b) Drug should show stability in the solvent used c) The drug should not react with the solvent used.

 

 Solutions of Sofosbuvir was prepared in different solvents like Water, Methanol and Acetonitrile. UV spectrum of each was recorded by scanning between 200-400 nm. Among these solvents, Acetonitrile: water (45:55 v/v) gave good response. Hence, Acetonitrile: water (45:55 v/v) was selected as solvent for further studies.

 

PREPARATION OF STANDARD STOCK SOLUTION OF SOFOSBUVIR BY USING ACETONITRILE: WATER (45:55 V/V):

Standard stock solution of sofosbuvir was prepared by transferring 20 mg of sofosbuvir into a 10 ml volumetric flask containing 4mL of (45:55v/v) Acetonitrile and water. It was then sonicated for 15 minutes and solution was diluted up to the volume by methanol and water. From these, further dilutions were made using Acetonitrile: water (45:55 v/v) to produce solution of sofosbuvir (200µg/ml).

 

 

 

SELECTION OF WAVELENGTH FOR ANALYSIS OF SOFOSBUVIR:

 0.1 ml of standard stock solution of sofosbuvir was transferred into a 10 ml volumetric flask and diluted to a mark with Acetonitrile: water (45:55 v/v) to give concentration of 2μg/ml. The resulting solution was scanned in the UV range (200–400 nm) and wavelength corresponding to maximum absorbance (λmax) was found at 260nm.

 

PREPARATION OF SAMPLE SOLUTION:

Twenty tablets are analysed for their drug content by UV spectrophotometric methods. The tablet contents were crushed into a fine powder and suitably diluted in Acetonitrile: water (45:55 v/v) to yield a concentration of 2.0 µg/ml for sofosbuvir. Further dilutions were made using Acetonitrile: water (45:55 v/v) to obtain a final concentration of 2.0 µg/ml. The spectrum was recorded at 260 nmagainst blank solution of Acetonitrile: water (45:55 v/v).

 

ASSAY:

Series of sample solutions i.e. 50μg/mL and 100μg/mL of Sofosbuvir was scanned and the absorbance of sample solutions were measured. The amount of Sofosbuvir in tablet dosage form (Sofovir 400mg) was determined and the results obtained were comparable with the corresponding label claim to obtain % recoveries.

Determine the amount of % Sofosbuvir in tablets according to the following formula

 

AT x WS × Sample D.F x Average Weight

 

% Assay=                              x PR

 

AR x Standard D.F x WT× LA

 

Where,

WS = weight of standard; WT = weight of sample

AT = Absorbance of Sofosbuvirin the test solution

AR = Absorbance of Sofosbuvirin the standard solution

Std. D.F = Standard dilution factor

Sample D.F = Sample dilution factor

PR = Purity of working standard [%]

LA = Labeled amount of Sofosbuvir

 

VALIDATION OF PROPOSED METHOD:

The method was validated according to ICH guidelines in order to determine the linearity, precision, accuracy and ruggedness of the method7.8.

 

SPECIFICITY:

Specificity of the method was done by comparing the chromatogram of drug with the chromatogram of blank (mobile phase) and found that there was no interaction with the Analyte.

 

 

LINEARITY:

Linearity was evaluated by eight point standard curve in concentration range of 4- 24 µg/ml (4,8,12,16,20 and 24 µg/ml) of Sofosbuvir. The calibration curve was obtained by plotting absorbance against concentration (μg/ml). Each set was analyzed to plot a calibration curve. Standard deviation (SD), slope, intercept, and correlation coefficient of determination (r2) of the calibration curves were calculated to ascertain the linearity of the method.

 

PRECISION:

Precision studies were carried out to ascertain the reproducibility of the proposed method.

 

Repeatability:

Repeatability was determined by preparing six replicates of 8 µg/ml of Sofosbuvir and the absorbance was measured at 260 nm without changing the parameter of the proposed UV method. The %RSD was calculated.

 

Intermediate Precision(reproducibility):

The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses on the same day and next day for three different concentration of standard solution of Sofosbuvir (12, 16 and 18µg/ml). The result was reported in terms of relative standard deviation (%RSD).

 

ACCURACY:

Accuracy of the proposed method was determined using recovery studies by standard addition method. The recovery studies were carried out by adding different amounts (50, 100 and 150%) of the pure drug to the pre-analysed formulation. The solutions were prepared in triplicates and the % recovery was calculated.

 

ROBUSTNESS:

Robustness studies were carried by changing the flow rate of mobile phase from 0.8 to 1.2 mL/min, and wavelength from 259 to 263.Sofosbuvir made in triplicates and were analyzed.

 

RUGGEDNESS STUDIES:

Ruggedness studies were performed by preparing three replicates of 4µg/ml, analysing by two different analyst and on two different instruments and the results are reported as %RSD.

 

LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION:

The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines.

 

LOD = 3.3 × σ/S,

LOQ = 10 × σ/S,                                                                                                                                

 

Where,

 σ = the standard deviation of the response,S = slope of the calibration curve.

 

RESULTS:

The goal of the present study is to develop and validate a simple, accurate and precise Ultraviolet spectrophotometric method for the estimation of sofosbuvir in bulk and pharmaceutical dosage forms.

 

SELECTION OF WAVELENGTH:

The standard stock solution of sofosbuvir of 2µg/ml concentration was prepared in acetonitrile : water (45:55 v/v) and scanned from 200-400 nm. Maximum absorption spectrum was recorded at 261 nm.

 

 

Fig2Absorption spectrum of Sofosbuvir

 

 

 

 

LINEARITY:

The linearity was found in the concentration range of 4- 24 µg/ml for the developed UV spectroscopy method. The X-axis is concentration and the Y-axis is absorbance. The correlation coefficient was found to be 0.999 and the regression equation was found to be Y=0.034x.

 

 

 Fig 3 Linearity graph for Sofosbuvir at 261nm

 

 

PRECISION:

Repeatability studies were carried out by taking test concentration and repeating it six times. Interday and intraday precision were done by taking three concentrations and repeating it three times and the values for repeatability, intraday precision and inter day precision in terms of %RSD.

 

 

 

 

Acceptance criteria:

A method is said to be precise if the %RSD value is <2.0%. The results show that the %RSD value for repeatability, intraday and inter day precision is <2.0% which indicate that they meet the acceptance criteria and hence the method is said to be precise.

 

 

 

 

Table 1. Repeatability studies of Sofosbuvir

Concentration

[µg/ml]

Absorbance

at 261nm

Absorbance of Mean

 ± S.D(n=6)

 

%RSD

8

0.256

 

 

 

 0.257±0.00187

 

 

 

 0.806

8

0.261

8

0.257

8

0.258

8

0.256

8

0.258

 

ACCURACY:

Accuracy studies were carried out at 50%, 100% and 150% by adding known amount of standard drug solution, i.e. (20, 40, 60 µg/ml) to the sample solution whose concentration is maintained constant, i.e. 40µg/ml and the accuracy of the method was confirmed by recovery studies and the % recovery for the marketed formulation was determined and were found to be in the range of 100.1-100.6%.


Table 2 Intermediate Precision of sofosbuvir

 

Intraday precision of Sofosbuvir

Inter day precision of Sofosbuvir

Conc

(µg/ml)

Absorbance

Absorbance Mean± S.D(n=3)

 

%RSD

Absorbance

Absorbance Mean±S.D(n=3)

 

%RSD

 

12

 0.423

 

0.424±0.001

 

0.27

 0.415

 

0.415±0.003

 

0.73

 0.425

 0.419

 0.425

 0.413

 

16

 0.543

 

 0.54±0.002

 

 0.48

 0.526

 

0.524±0.005

 

1.00

 0.538

 0.528

 0.539

 0.518

 

18

 0.756

 

0.754±0.001

 

0.76

 0.756

 

 0.758±0.003

 

 0.50

 0.753

 0.763

 0.753

 0.757

 


Acceptance criteria:

A method is said to be accurate if the % recovery studies is in the range of 98-102%. The results for accuracy indicate that the % recovery values is within the range of 98-102% which indicate that the method is accurate as it meets the necessary criteria.


 

 

 

 

Table 3Determination of accuracy results for sofosbuvir at 261nm

Spiked level (%)

Formulation

Conc (µg/ml)

Pure

Drug Conc (µg/ml)

Amount recovered (µg/ml)

% Recovery

%Mean recovery

±SD

 

%RSD

 

50

4

2

6.2

100.6

 

100.4 ± 0.24

 

0.243

4

2

6.2

100.5

4

2

6.0

100.1

 

100

4

4

8.5

101.2

 

100.3 ± 0.78

 

0.786

4

4

7.9

99.7

4

4

8.0

100.0

 

150

4

6

10.1

100.3

 

100.0 ± 0.36

 

0.366

4

6

10.0

100.2

4

6

9.8

99.6

 


LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION:

The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation. LOD and LOQ values are 0.495,1.502 µg/ml.

 

LOD = 3.3 (σ/S)

 

LOQ = 10 (σ/S)

 

Where,

S: Slope of the calibration curve, σ: Average standard deviation of the response.

 

LOD = 0.495µg/ml

 

LOQ = 1.502µg/ml

 

RUGGEDNESS STUDIES:

This study was performed by analyzing 4 µg/ml of sofosbuvir by two different analysts and on two instruments, results of the % RSD obtained was less than two which is within the acceptance limits. (Table-4).

 


 

 

 

Table.4Ruggedness of sofosbuvir

 

Different analyst

 

Concentration  (µg/ml)

Absorbance mean± S.D

%RSD

Analyst -1

4

0.15±0.000707107

 0.471

Analyst -2

4

0.1505±0.000707

0.469

Different instrument

Instrument-1

4

0.148±0.002121

1.433

Instrument -2

4

0.148±0.002828

1.91

 


Acceptance Criteria:

The % RSD forsofosbuvir should be < 2.0 %.

 

ASSAY:

The percentage recovery for sofosbuvir was found to be 100.02 %. The results for assay are within acceptable limits.

 

Table 5 Assay studies of sofosbuvir

Labelled claim

(mg)

Amount found

(mg)

%purity

 

 

40

4.89

99.56

5.01

100.04

5.00

100.00

5.09

100.36

4.98

99.92

5.06

100.24

Mean ± S.D

5.01±0.069

100.02±0.277

 %RSD

0.507

0.507

 

Acceptance Criteria:

The Percentage(%) purity forsofosbuvir should be 98-102 %.

 

CONCLUSION:

The proposed UV spectrophotometric methods were found to be simple and rapid and mentioned summarised results table in 6 .The method requires measurement of absorbance of the drug at 260 nm.The proposed UV method are very simple, precise, accurate, rapid and cost effective for the estimation of sofosbuvir from its pharmaceutical dosage forms by the spectrophotometric method. Hence it can be utilized for routine analysis in bulk and pharmaceutical dosage forms.

 

RECOMMEND FUTURE RESEARCH:

This method was successfully applied to the determination of sofosbuvir content in marketed formulation. The method will be apply to routine analysis of pharmaceutical dosage form and pharmacokinetic studies.

 

ACKNOWLEDGEMENTS:

Authors are grateful to authorities of Malla Reddy College of Pharmacy, for providing the facilities to conduct this research work.

 

Table 6 Summary of Validated parameters

Parameters

Method

λ max (nm)

260nm

Correlation coefficient (r2)

0.999

Regression equation (y=mx+c)

y = 0.034x

Slope (m)

0.033

Intercept(c)

0.002

Accuracy

99.67-101.25

Precision (%RSD)

0.3-1.3

LOD (µg/ml)

0.495

LOQ (µg/ml)

1.502

 

REFERENCES:

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Received on 17.01.2018         Modified on 28.02.2018

Accepted on 20.04.2018         © AJRC All right reserved

Asian J. Research Chem. 2018; 11(3):563-568.

DOI:10.5958/0974-4150.2018.00101.3