Development and validation of stability indicating RP-HPLC method for estimation of sofosbuvir in bulk and tablet dosage form

Jeyabaskaran M et al / Int. J. of Farmacia, 2016; Vol-(2) 4: 257-269
257
International Journal of Farmacia
Journal Home page: www.ijfjournal.com
Development and validation of stability indicating RP-HPLC method for
estimation of sofosbuvir in bulk and tablet dosage form
Jeyabaskaran M,1*
Prof.Rambabu C,2
Yasmin Sulthana3
1
Research Scholar,Department of Chemistry, Acharya Nagarjuna University Nagarjuna Nagar,
Guntur(Dist), Andhra Pradesh, India.
2
Professor and Head, Department of Chemistry, Acharya Nagarjuna University Nagarjuna Nagar,
Guntur(Dist), Andhra Pradesh, India.
3
Browns College of Pharmacy, Ammapalem (V), Konijerla (M) Wyra road, Khammam (Dist),
Telangana State, India.
Corresponding Author: Jeyabaskaran M
ABSTRACT
A simple, precise and accurate stability indicating RP-HPLC method has been developed and subsequently validated
for estimation of Sofosbuvir (SFB) in bulk and from their combination dosage form using a Kromasil C18 (250 mm ×
4.6 mm, 5µm) at 250
C. Mobile phase consisted mixture of 0.1% Ortho phosphoric acid buffer and acetonitrile in the
ratio 55:45 (v/v) with flow rate was 1 ml/min and detection was carried out by photodiode array detector at 259nm.
The retention time for SFB was found to be 2.069min. SFB and their dosage form were exposed to thermal,
photolytic, oxidative, acid-base hydrolytic stress conditions, the stressed samples were analyzed by the proposed
method. Peak purity results suggested no other co-eluting, interfering peaks from excipients, impurities, or
degradation products due to variable stress condition, and the method is specific for the estimation of SFB in presence
of their degradation products and impurities. The proposed method has permitted the quantification of SOF over
linearity in the range of 0.1– 0.6 mg/ml and its percentage recovery was found to be 100.39 %. The % RSD of
intraday and inter day precision were found 0.34% and 0.6% according to International Conference on Harmonization
(ICH) Q2B guidelines.
Keywords: Sofosbuvir, Rp-Hplc, Stability Indicates, Stress Conditions
INTRODUCTION
SOF is a prodrug of 2’-deoxy-2’-fluoro-2’-C-
methyluridine monophosphate that is phosphorylated
intra cellularly to the active triphosphate form. Used
for the treatment of chronic Hepatitis C [1].
Chemically, SFB is Propan-2-yl (2S)-2-{[(S)-
{[(3R,4R,5R)-5-(2,4-dioxo-1,2,3,4-tetrahydro
pyrimidin-1-yl) -4-fluoro-3-hydroxy-4-methyloxolan-
2-yl]methoxy(phenoxy)phosphoryl]amino}
propanoate [2] with empirical formula of
C22H29FN3O9P and molecular weight
529.4525 g/mol. It is a White to Off-white non-
hygroscopic crystalline solids [3]. Slightly soluble in
water (pH 1.2-7.7), freely soluble in ethanol and
acetone, soluble in 2-propanol and insoluble in
heptanes [4]. The chemical structure of SOF was
shown in Fig.01. SFB is a prodrug nucleotide analog
used as part of combination therapy to treat hepatitis
C virus (HCV) infection or to treat co-infection of
HIV and HCV. After metabolism to the active
antiviral agent 2'-deoxy-2'-α-fluoro-β-C-
methyluridine-5'-triphosphate (also known as GS-
461203), the triphosphate serves as a defective

258
substrate for the NS5B protein, an RNA-dependent
RNA polymerase required for replication of viral
RNA. SFB is used in combination therapy to treat
chronic hepatitis C virus (HCV) infected patients with
HCV genotype 1,2,3, or 4, and to treat HCV and HIV
co-infected patients. The combination therapy
includes either ribavirin alone or ribavirin and peg-
interferon alfa [5]. Sofosbuvir is nucleotide analog
inhibitor, which specifically inhibits HCV NS5B
polymerase. Sofosbuvir prevents HCV viral
replication by binding to the two Mg2+ ions present
in HCV NS5B polymerase's GDD active site motif
[6].
Stability testing is an important part of the process
of drug product development. The purpose of stability
testing is to provide evidence on how the quality of a
drug substance or drug product varies with time under
the influence of a variety of environmental factors,
such as temperature, humidity, and light, and enables
recommendation of storage conditions, retest periods,
and shelf lives to be established. The two main
aspects of a drug product that play an important role
in shelf life determination are assay of the active drug
and degradation products generated during the
stability study. The drug product in a stability test
sample needs to be determined using a stability
indicating method, as recommended by the
International Conference on Harmonization (ICH)
guidelines [7] and U.S. Pharmacopoeia (USP) 26 [8].
Although stability indicating methods have been
reported for assay of various drugs in drug products,
most of them describe assay procedures for drug
products containing only one active drug substance.
Only few stability indicating methods are reported for
assay of drug products containing two or more active
drug substances. The objective of this work was to
develop a simple, precise, and rapid column liquid
chromatography (LC) procedure that would serve as
stability indicating assay method for drug product of
SOF.
An extreme literature survey revealed that very
few analytical methods have been reported such as
HPLC [9-12] for SOF in individual and combination
with other drugs. In order to minimize the batch –to-
batch variation, it is very important to develop
suitable analytical methods for day –to- day analysis
of drugs. It was found that one attempt has been made
to develop stability indicating studies and estimation
of SOF by RP-HPLC at the starting of my work.
Therefore, it was thought of interest in development
and validating an advanced new sensitive, specific,
precise, accurate stability indicating RP-HPLC
method for estimation of SOF in bulk drug and in
pharmaceutical dosage form. We here in report a
simple, rapid and reliable HPLC for the estimation of
SOF in bulk and pharmaceutical dosage forms as per
ICH guidelines [13-16].
Fig 01: Structure of Sofosbuvir.

259
EXPERIMENTAL
REAGENTS AND MATERIALS
Pure standard of SFB was obtained as gift sample
from Lupin Pharmaceuticals, Mumbai. Acetonitrile,
Water HPLC grade (Merck Specialties Pvt Ltd,
Mumbai, India), Ortho phosphoric acid HPLC (merck
specialities pvt ltd, mumbai), All solvents used in this
work are HPLC grade. Sovaldi Tablets (Mylan
Pharmaceuticals Private Limited) containing
Sofosbuvir Marketed formulation was purchased from
local market, High precision weighing balance
(wensar instruments, hyderabad), micro pipette (in
labs,10-100 µl) were employed in the study. All the
glassware employed in the work cleaned with hot
water followed acetic anhydride then acetone and
dried in hot air oven whenever required. Working
environment was maintained in 25o
C. However, the
chemical structure and purity of the sample obtained
were confirmed by melting point, TLC Studies.
HPLC AND CHROMATOGRAPHIC
CONDITIONS
The analysis was performed on A Waters 2695
RP-HPLC separation module (Waters Corporation,
Milford, USA) equipped with PDA detector having
back pressure 5000psi, automatic injector and
Kromasil C18 (250mm × 4.6 mm, 5µ). Data
acquisition was performed by using Empower 2
software. Single pan Balance (shimadzu, AUX-220),
Control Dynamics pH meter (Mettler Toledo),
Sonicator (Powersonic 405, Lab india). Different
mobile phases were tested in order of their polarity to
find out the best conditions for the separation of
sofosbuvir. An isocratic RP-HPLC system was used
for analysis of samples at 25o
C column oven
temperature. The chromatographic separation was
achieved on Kromasil 250 mm x 4.6 mm, 5μ column
using 0.1% Ortho phosphoric acid buffer and
acetonitrile 55:45 % v/v as mobile phase at a flow rate
of 1 ml/min. The mobile phase was filtered through
0.45 µm nylon membrane filter and degassed before
use. The injection volume was 10 µl and the total
runtime was set at 5 minutes. The determination of
analytes was carried out at 259 nm using PDA
detector.
PROCEDURE RECOMMENDED
PREPARATION OF MOBILE PHASE
1 ml of Ortho phosphoric acid solution in a
1000ml of Volumetric flask adds about 100ml of
milli-Q water and final volume make up to 1000ml
with milli-Q water and 100% acetonitrile taken in the
ratio 55:45 (v/v) were employed as a mobile phase.
PREPARATION OF STOCK
SOLUTION
Accurately Weighed and transferred 40mg
Sofosbuvir working Standard into a 10ml clean dry
volumetric flask, add 5 ml of diluents (first dissolved
in methanol and make up Acetonitrile: Water
(50:50)), sonicated for 30 minutes and make up to the
final volume with diluents. From the above stock
solution, 1 ml was pipette out in to a 10ml volumetric
flask and then make up to the final volume with
diluents.
Table.01: Chromatographic Condition of SOF in API
Parameters Condition
Column Kromasil 250 mm x 4.6 mm, 5 µm.
Column Temperature 250
C
Wavelength 259nm
Diluent First dissolved in methanol and make up quantity
with Acetonitrile: Water in the ratio of (50:50)
Injector volume 10 µl
Flow rate 1 ml/min
Runtime 5 min
Retention time 2.069 min

260
Fig.02; A typical chromatogram of SFB
PREPARATION OF SAMPLE FOR
FORCED DEGRADATION STUDIES
Preparation Of Acid Induced Degradation
Product
4 ml (4mg) of SFB stock solution was taken into
10 ml volumetric flask and refluxed with 4 ml 2N
Hydrochloric acid at 800
C for 30 minutes. The
resultant solution was collected, diluted with diluent
to get the concentration of 400 µg/ml and 10 µl
solution was injected into HPLC system and
chromatograms were recorded.
Preparation Of Alkali Induced Degradation
Product
4 ml of SFB stock solution was taken into 10 ml
volumetric flask and refluxed with 4 ml of 0.1N
Sodium hydroxide at 800
C. After 30 minutes the
resultant solutions was diluted with diluent to get the
concentration of 400 µg/ml and 10 µl solution was
injected into HPLC system and chromatograms were
recorded.
Preparation Of Hydrogen Peroxide Induced
Degradation Product
volumetric flask and 4 ml of freshly prepared 10%
H2O2 solution was added into volumetric flask and
solution were kept at 800
C for 30 minutes. Then the
resultant solution was injected into HPLC system to
get the chromatograms.
Photochemical Stability Induced Product
The photochemical stability study of the drug was
studied by exposing the sample concentration of 4000
µg/ml to UV light in UV chamber for 7 days or 200
Watt hours/m2.
Then the resultant solution was diluted
and 10 µl solution was injected into the HPLC
system.
Dry Heat Induced Degradation Product
To study the dry heat degradation studies, the
standard drug solution of Sofosbuvir was placed in
oven for 6 hours at 1050
C. The resultant solutions
were diluted to get the concentration of 400 µg/ml and
10 µl solution was injected into HPLC system and
chromatograms were recorded to assess the stability
studies.
Preparation of water induced degradation
product
volumetric flask and heated with 4 ml of water at
80ºC for 30 .minutes. Then the resultant solution was
injected into HPLC system and chromatograms were
recorded.
Construction of Linearity
The concentrations of analyte were prepared from
the stock solution by taking suitable volume (0.25 –
1.5 ml) and diluted up to 10 ml to get the desired
concentrations for linearity in the range of 100 –
600µg/ml. The prepared solutions were filtered
through 0.45µm nylon membrane filter and each of
the dilutions was injected three times into the column.
The calibration curve for SFB was constructed by

261
plotting the mean peak area (Y-axis) against the
concentration (X-axis).
ESTIMATION OF SFB
Analysis of marketed formulation was purchased
from local market. 10 tablets were weighed and
average weight was calculated. Then from the
transferred the equivalent to one tablet to 100ml
volumetric flask, 70ml of diluent was added and the
mixture was allowed to stand with intermittent
sonication for 5 mins to ensure complete solubility of
drug. Further the volume made up with diluent and
the resulting solution was passed through 0.45µm
membrane filtered. From the filtered solution, 1ml
was pipette out into 10ml volumetric flask and made
upto 10ml with diluent. From the solution, 10µl was
injected into HPLC system and peak area was
recorded (Fig.3) with detector at 259nm. The % assay
was calculated with obtained peak area of detector
response. The % assay was found to be 100.39% for
Sofosbuvir. This indicates that developed method can
be used for routine analysis.
Table.02: % Assay results of Sofosbuvir in formulation
Tablets Drug Dosage
(mg)
Sample concentration
(µg/ml)
Amount found
(µg/ml)
% Assay
1 Sofosbuvir 400 400 401.56 100.39
Fig 03: Chromatogram showing assay of Sofosbuvir marketed dosage form (Sovaldi tablet)
METHOD VALIDATION
As per the International Conference on
Harmonization (ICH) guidelines, the method
validation parameters such as Specificity, Linearity,
Precision, Accuracy, Limit of Detection/
Quantification and Robustness were optimized.
RESULTS
The present RP-HPLC method for the
quantification of SFB in bulk and pharmaceutical
dosage forms, revealed as simple, rapid, accurate and
precise method with significant shorter retention time
of 2.069.min.
Accuracy
The accuracy of the proposed method was
determined by the standard addition method. It is the

262
closeness of the analytical results obtained by the
analysis of the true value. A known amount of
standard drug was added to the fixed amount of
injection solution. Accuracy was expressed as
percentage recovery. Recovery test was performed
with three different concentrations, i.e. 200 µg/ml,
400µg/ml and 600 µg/ml for Sofosbuvir. The %
recovery results were calculated and given in
Table.03.
Table.03: % Recovery results of Sofosbuvir
Conc. Sofosbuvir
Amount
added (µg/ml)
Amount recovered
(µg/ml)
%
Recovery
50% 200 198.21 99.10
200 199.77 99.88
200 198.47 99.24
100% 400 406.98 101.74
400 406.49 101.62
400 406.57 101.64
150% 600 599.28 99.88
600 603.15 100.53
600 598.95 99.82
Linearity
A series of six concentrations in the range of 100
to 600µg/ml of Sofosbuvir has been prepared and
peak areas were recorded at 259nm. A calibration
curve was plotted between peak area versus
concentration of respective Sofosbuvir and the
response of the drug was found to be linear. The
linear regression equation (y = mx + c) was found to
be y = 2452.2x + 22728. (Fig.4) for Sofosbuvir. The
linearity results were given in Table.04 & 05.
Table.04: Linearity results of Sofosbuvir
Concentration
(µg/ml)
Area
Average
area
% RSD
100
291315
291440 0.09
291748
291256
200
514225
516267 1.10
512112
522464
300
763943
767713 0.43
769903
769294
400
983383
988458 0.44
990845
991145
500
1262003
1257597 0.84
1245602
1265185
600
1492919
1487202 0.36
1482395
1486292

263
Fig .04: Calibration curve of Sofosbuvir
Table.05: Slope and intercept value of SFB Precision
Linearity curve Sofosbuvir
Slope Intercept
Value 2452.2 22728
Correlation
coefficient (r2
)
0.9991
Repeatability or Precision of the method was
determined by injecting six replicates of standard
solution at 400µg/ml of SFB into HPLC system. From
the results obtained it was found that the proposed
method was precise given in Table 06.
Table.06: Precision data Robustness
Injection Sofosbuvir concentration Area
1
400µg/ml
988616
2 991620
3 983591
4 984602
5 989132
6 980856
Mean 986403
STDV 4036.9
%RSD 0.4
Robustness study of the method was determined
by changing the parameters such as flow rate, mobile
phase ratio and temperature. Drug samples were
analyzed under small changed conditions and
chromatogram was recorded. It was found that these
deliberate changes were not affected the
chromatograms of both drug samples and given in
Table 07.

264
Table.07: Actual conditions and proposed conditions of the method
Effect of Flow rate
Parameters Actual conditions Proposed variations
Flow rate 1ml/min 0.9, and 1.1ml/min
Mobile phase ration 55:45 % v/v ±5%
Temperature 25 0
C 20 0
C, 30 0
C
By changing the flow rate (1ml/min ±0.1ml) no drastic changes were seen in chromatographic parameters and
were given in Table 08 & 09.
Table.08: Robustness data at flow rate 0.9ml/min of SFB
Parameters RT Area Average area % RSD
Sofosbuvir 2.260 971995 975116 0.35
2.264 978519
2.266 974440
2.268 976791
2.275 978479
2.281 970471
Table.09: Robustness data at 1.1ml/min of Sofosbuvir
Sofosbuvir 1.869 965678 955559 0.97
1.875 943208
1.879 944178
1.881 962880
1.881 960063
1.883 957347
Effect of Mobile phase
In mobile phase, organic phase was changed to
±5%. It was found that change in mobile phase was
not affected the chromatogram parameters and were
given in Table 10 & 11.
Table.10: Robustness data at mobile phase ration 55:45% v/v
Sofosbuvir 1.887 923741 931686 0.6
1.890 929021
1.892 937085
1.892 927732
1.897 936415
1.903 936119

265
Table.11: Robustness data at mobile phase ration 65:35% v/v
Effect of Temperature
Sofosbuvir
2.228 961352
964502 0.64
2.228 960588
2.229 955685
2.230 968539
2.232 967730
2.242 973119
Temperature of the column was changed to ±50
C
and chromatogram was recorded. From the results, it
was found that change in temperature also not
affected the chromatogram parameters and were given
in Table 12 & 13.
Table.12: Robustness data at temperature 20 0
C
Sofosbuvir 2.021 928242 925434 0.44
2.025 919000
2.025 921372
2.036 923694
2.036 931974
2.043 928319
Table.13: Robustness data at temperature 35 0
C
Sofosbuvir 2.031 937150 939030 0.51
2.037 940122
2.037 941376
2.037 932061
2.038 946158
2.045 937314
Limit of Detection (LOD)
Limit of detection is the known concentration of
SFB and establishing minimum concentration at
which the SFB can be reliably detected. It was
calculated based on the standard deviation of the
response and the slope of the standard calibration
curve. The LOD was found to be 0.762 µg/ml of SFB.
Limit of Quantification (LOQ)
Limit of quantification is the known concentration
of SFB and establishing minimum level at which the
SFB can be quantified with acceptable accuracy and
precision. The LOQ was found to be 2.308 µg/ml of
SFB. The LOD and LOQ results were given in
Table.14.

266
Table.14: LOD and LOQ results of SFB
Sample LOD LOQ
Sofosbuvir 0.762 µg/ml 2.308 µg/ml
FORCED DEGRADATION STUDIES
Forced degradation studies (FDS) or stability
indicating studies were carried out in presence of
degradation products of the samples. These studies
were performed to evaluate the specificity of the
proposed method. According to the ICH guidelines
Q1A (R2) various stresses induced degradation
conditions such as acid, alkali, oxidation, photolytic,
dry heat and water were used for the stability
indicating studies. All the degradation products were
resolved from sample peaks. Under each condition the
chromatograms were recorded and studied using PDA
detector. The sample peaks were tested for its purity
using chromatographic software.
EFFECT OF ACID AND ALKALINE
HYDROLYSIS
In acid and alkaline hydrolysis, sample solutions
were subjected to acid and alkali hydrolysis (2N HCl
and 0.1N NaOH respectively for 30 minutes at 800
C).
In acid induced degradation drug found to undergo
minor degradation with degradation product retention
time of 2.055 minutes (Fig.05) and in base induced
conditions also retention time 2.049min (Fig.06).
Fig.05: Chromatogram of Acid degradation
Fig.06: Chromatogram of base degradation

267
EFFECT OF OXIDATIVE
DEGRADATION
In oxidative stress conditions sample solutions
were added to the hydrogen peroxide solution (10 %
H2O2 for 30 minutes at 80 0
C) and it was found that
sample get degraded with the degradation product
retention time of Sofosbuvir 2.058 minutes (Fig.07).
Fig.07: Chromatogram of oxidative degradation
EFFECT OF PHOTOLYTIC, DRY
HEAT AND WATER DEGRADATION
When samples were subjected to photo and
thermal degradation and water, no degradation was
found in the samples which is indicated that the
samples were very stable in photolytic conditions
(Fig.08), dry heat (Fig.09) and water (Fig.10).
Fig.08: Chromatogram of photolytic degradation

268
Fig.09: Chromatogram of dry heat degradation
Fig.10: Chromatogram of water degradation
CONCLUSION
In the present study, a stability indicating RP-
HPLC method was developed and successfully
validated according to ICH guidelines for the
estimation of SFB. The method was validated for
various parameters like specificity, linearity,
accuracy, precision, robustness, LOD and LOQ. The
forced degradation studies were carried out and
degradation peaks were separated with developed
stability indicating RP-HPLC method. The validated
method was applied for the assay of the commercial
tablets of SFB in formulation. All the results obtained
of various parameters were found to be within the
acceptance limits. Thus the developed method in the
present work is simple, sensitive, accurate, rapid,
precise and robust. Hence the above method can be
successfully applied for estimation of SFB in both
bulk and tablet dosage form.
ACKNOWLEGDEMENT
The authors thank to lupin pharmaceticals, mumbai for providing gift sample of sofosbuvir for this research work.

269
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Development and validation of stability indicating RP-HPLC method for estimation of sofosbuvir in bulk and tablet dosage form

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Development and validation of stability indicating RP-HPLC method for estimation of sofosbuvir in bulk and tablet dosage form