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
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:
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.
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.
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
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
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).
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
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).
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
% Assay= x PR
AR x Standard D.F x WT×
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
Sample D.F = Sample
PR = Purity of working
LA = Labeled amount of
The method was
validated according to ICH guidelines in order to determine the linearity,
precision, accuracy and ruggedness of the method7.8.
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 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 studies were
carried out to ascertain the reproducibility of the proposed method.
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.
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 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 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 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.
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,
σ = the standard
deviation of the response,S = slope of the calibration curve.
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.
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.
spectrum of Sofosbuvir
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
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.
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.
Repeatability studies of Sofosbuvir
Absorbance of Mean
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%.
The % RSD forsofosbuvir
should be < 2.0 %.
The percentage recovery
for sofosbuvir was found to be 100.02 %. The results for assay are within
Table 5 Assay
studies of sofosbuvir
Mean ± S.D
Percentage(%) purity forsofosbuvir should be 98-102 %.
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:
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.
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
λ max (nm)
y = 0.034x
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