A new analytical method development and validation for the simultaneus estimation of ledipasvir and sofosbuvir using RP-HPLC

Kranthi K K et al, ICJPIR 2017, 4(1), 142-165
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Available online at www.icjpir.com ISSN: 2349-5448
Intercontinental journal of pharmaceutical
Investigations and Research
ICJPIR |Volume 4 | Issue 1 | Jan – Mar- 2017 Research Article
A new analytical method development and validation for the simultaneus
estimation of ledipasvir and sofosbuvir using RP-HPLC
K.Kranthi Kiran M.Pharm (Ph.D),Assoc. M.Saisri, M.Priyanka, M.Subhashini,
M.Manikanta
Assoc Professor & Jogaiah Institute of Technology & Sciences College of Pharmacy,Kalagampudi.
A.P India
Corresponding Author: K.Kranthi Kiran
Email Id: Kothapallikranthikiran@gmail.com
ABSTRACT
A simple and selective LC method is described for the determination of LEDIPASVIR and SOFOSBUVIR in
tablet dosage forms. Chromatographic separation was achieved on a c18 column using mobile phase consisting
of a mixture of Mixed Phosphate Buffer:ACN (55:45) with detection of 213 nm. Linearity was observed in the
range 60-140 µg/ml for LEDIPASVIR oxalate (r2
=0.999) and 6-14 µg /ml for SOFOSBUVIR (r2
=0.996) for
the amount of drugs estimated by the proposed methods was in good agreement with the label claim.
The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three
different levels. Recovery experiments indicated the absence of interference from commonly encountered
pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis,
showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine
analysis of pharmaceutical dosage form.
Keywords: Liquid chromatography(LC),RSD Relative standard deviation,r2
correlation coefficient.
INTRODUCTION
A drug includes all medicines intended for
internal or external use for or in the diagnosis,
treatment, mitigation or prevention of disease or
disorder in human beings or animals, and
manufactured exclusively in accordance with the
formulae mentioned in authoritative books [1].
Pharmaceutical analysis is a branch of
chemistry involving a process of identification,
determination, quantification, purification and
separation of components in a mixture or
determination of chemical structure of compounds.
There are two main types of analysis – Qualitative
and Quantitative analysis.Qualitative analysis is
performed to establish composition of a substance.

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It is done to determine the presence of a compound
or substance in a given sample or not. The various
qualitative tests are detection of evolved gas, limit
tests, color change reactions, determination of
melting point and boiling point, mass spectroscopy,
determination of nuclear half life etc.
AIM AND PLAN OF WORK
Aim
To develop new RP HPLC method for the
simultaneous estimation of Ledipasvir And
Sofosbuvir pharmaceutical dosage form [2].
Plan of Work
Solubility determination of Ledipasvir and
Sofosbuvir various solvents and buffers [3].
 Determine the absorption maxima of both the
drugs in UV–Visible region in different
solvents/buffers and selecting the solvents for
HPLC method development.
 Optimize the mobile phase and flow rates for
proper resolution and retention times.
 Validate the developed method as per ICH
guidelines.
METHODOLOGY
Mobile Phase
A mixture of Mixed Phosphate Buffer:ACN
were prepared [10]. The mobile phase was
sonicated for 10min to remove gases and filtered
through 0.45µ membrane filter for degassing of
mobile phase.
Determination of Working Wavelength
(λmax)
In estimation of drug wavelength maxima is
used.. So this wavelength is used in estimation to
estimate drug accurately [5].
Preparation of standard stock solution of
LEDIPASVIR
5 mg of LEDIPASVIR was weighed and
transferred in to 100ml volumetric flask and
dissolved in water and then make up to the mark
with water and prepare 100 µg /ml of solution by
diluting 1ml to 10ml with water [7-9].
Preparation of standard stock solution of
SOFOSBUVIR
10 mg of SOFOSBUVIR was weighed in to
100ml volumetric flask and dissolved in water and
then dilute up to the mark with water and prepare
100 µg /ml of solution by diluting 1ml to 10ml with
water [4].
RESULTS AND DISCUSSIONS
Solubility Studies
These studies are carried out at 25 0
C
Ledipasvir
Freely soluble in ethanol and methanol, and slightly
soluble in acetone and isopropanol and very
slightly soluble in water [6].
Sofosbuvir
Freely soluble in methanol and water.

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RESULTS
The wavelength of maximum absorption (λmax)
of the drug, 10 μg/ml solution of the drugs in
methanol were scanned using UV-Visible
spectrophotometer within the wavelength region of
200–400 nm against methanol as blank. The
resulting spectra and the absorption curve shows
the isobestic point was found to be 239 nm for the
combination.
METHOD DEVELOPMENT OF
LEDIPASVIR AND SOFOSBUVIR
Trial- 1
Chromatographic conditions
Mobile phase : Mixed phosphate buffer:ACN
pH : 6.8
Ratio : 55:45
Column : Inertsil ODS , (250×4.6× 5µ)
Wavelength : 213 nm
Flow rate : 1ml/min
Preparation of mixed standard solution
Weigh accurately 10 mg of LEDIPASVIR and
SOFOSBUVIR in 100 ml of volumetric flask and
dissolve in 10ml of mobile phase and make up the
volume with mobile phase.From above stock
solution 10µg/ml of LEDIPASVIR and
SOFOSBUVIR is prepared by diluting 1ml to 10ml
with mobile phase. This solution is used for
recording chromatogram.
Fig. 8.3.5: Chromatogram of SOFOSBUVIR and LEDIPASVIR by using mobile phase
Observation
 All the system suitability requirements were
met.
 The peak Asymmetry factor was less than 2 for
both SOFOSBUVIR and LEDIPASVIR.
 The efficiency was more than 2000
SOFOSBUVIR and LEDIPASVIR.
 Resolution between two peaks >1.5.
 Thedetails are given in the figure 8.3.8, hence
this method was for optimized.

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Table: Optimized chromatographic conditions
Mobile phase Mixed phosphate buffer:ACN(55:45)
Ph 6.8
Column Inertsil ODS 3V column,C18(150x4.6 ID) 5µm
Flow rate 1.0 ml/min
Column temperature Room temperature(20-25o
C)
Sample temperature Room temperature(20-25o
C)
Wavelength 213
Injection volume 20 µl
Run time 10min
Retention time About 3.060 min for LEDIPASVIR and 7.453 min for SOFOSBUVIR.
Assay
weigh accurately 10mg of LEDIPASVIR and 10
mg of SOFOSBUVIR in 100 ml of volumetric
flask and dissolve in 10ml of mobile phase and
make up the volume with mobile phase. From
above stock solution 10 µg/ml of LEDIPASVIR
and SOFOSBUVIR is prepared by diluting 1ml to
10ml with mobile phase. This solution is used for
Tablet sample
10 tablets (each tablet contains SOFOSBUVIR-
400 mg LEDIPASVIR-90 mg) were weighed and
taken into a mortar and crushed to fine powder and
uniformly mixed. Tablet stock solutions of
SOFOSBUVIR and LEDIPASVIR (μg/ml) were
prepared by dissolving weight equivalent to 10 mg
of SOFOSBUVIR and LEDIPASVIR and dissolved
in sufficient mobile phase. After that filtered the
solution using 0.45-micron syringe filter and
Sonicated for 5 min and dilute to 10ml with mobile
phase. Further dilutions are prepared in 5 replicates
of 10μg/ml of SOFOSBUVIR and
LEDIPASVIRwas made by adding 1 ml of stock
solution to 10 ml of mobile phase.
Calculation
The amount of SOFOSBUVIR and
LEDIPASVIRpresent in the formulation by using
the formula given below, and results shown in
above table:
Fig: Chromatogram of Assay standard preparation-1

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Fig: Chromatogram of Assay sample preparation-1

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Table : Assay Results
LEDIPASVIR SOFOSBUVIR
Standard Area Sample Area Standard Area Sample Area
Injection-1 9610.218 9618.037 929.107 933.278
Injection-2 9596.321 9610.218 898.860 902.356
Injection-3 9610.218 9545.801 909.64 928.769
Injection-4 9596.321 9394.586 898.86 934.043
Injection-5 9578.389 9612.063 891.613 918.958
Average Area 9598.293 9556.141 578.6002 923.4808
Assay(%purity) 99.5608344 101.97266
Observation
The amount of LEDIPASVIR and
SOFOSBUVIR present in the taken dosage form
was found to be 99.56 % and 101.97 %
respectively.
VALIDATION
Specificity by Direct comparison method
There is no interference of mobile phase,
solvent and placebo with the analyte peak and also
the peak purity of analyte peak which indicate that
the method is specific for the analysis of analytes in
their dosage form.
weigh accurately 10mg of LEDIPASVIR and 10
mg of SOFOSBUVIR in 100 ml of volumetric flask
and dissolve in 10ml of mobile phase and make up
the volume with mobile phase. From above stock
solution 10µg/ml of LEDIPASVIR and
SOFOSBUVIR is prepared by diluting 1ml to 10ml
with mobile phase. This solution is used for
Tablet sample
10 tablets (each tablet contains SOFOSBUVIR–
90 mg LEDIPASVIR -400 mg) were weighed and
taken into a mortar and crushed to fine powder and
uniformly mixed. Tablet stock solutions of
SOFOSBUVIR and LEDIPASVIR (100μg/ml)
were prepared by dissolving weight equivalent to
10 mg of SOFOSBUVIR and LEDIPASVIR and
dissolved in sufficient mobile phase. After that
filtered the solution using 0.45-micron syringe
filter and Sonicated for 5 min and dilute to 10ml
with mobile phase. Further dilutions are prepared in
5 replicates of 10μg/ml of SOFOSBUVIRand
LEDIPASVIR was made by adding 1 ml of stock
solution to 10 ml of mobile phase.
Fig: Chromatogram for specificity of LEDIPASVIR and SOFOSBUVIR sample

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Fig: Chromatogram for Specificity of LEDIPASVIR and SOFOSBUVIR standard
Observation
It is observed from the above data, diluent or
excipient peaks are not interfering with the
LEDIPASVIR and SOFOSBUVIR peaks.
Linearity and range
Preparation of standard stock solution
Preparation of standard stock solution
Standard stock solutions of LEDIPASVIR and
SOFOSBUVIR (microgram/ml) were prepared by
dissolving 10 mg of LEDIPASVIR and
SOFOSBUVIR dissolved in sufficient mobile phase
and dilute to 100 ml with mobile phase.
Table: Linearity Preparations
Preparations Volume from standard
stock transferred in ml
Volume made up in ml
(with mobile phase)
Concentration of solution(µg
/ml)
Preparation 1 0.5 0.5 10 50 50
Preparation 2 0.75 0.75 10 75 75
Preparation 3 1.0 1.0 10 100 100
Preparation 4 1.5 1.5 10 150 150
Preparation 5 1.75 1.75 10 175 175

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Fig. : Chromatogram of LEDIPASVIR and SOFOSBUVIR preparation-1
Fig. Chromatogram of LEDIPASVIR and SOFOSBUVIR preparation-2
Fig. Chromatogram of LEDIPASVIR and SOFOSBUVIR preparation-3

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Fig: Chromatogram of LEDIPASVIR and SOFOSBUVIR preparation-4
Fig. Chromatogram of LEDIPASVIR and SOFOSBUVIR for preparation-5
Table linearity of LEDIPASVIR
S .No. Conc.(µg/ml ) Area
1 50 8118.069
2 75 8859.874
3 100 9690.218
4 150 10999.32
5 175 11897.59
Table: linearity of SOFOSBUVIR
S.No. Conc.(µg/ml ) Area
1 50 774.04
2 75 876.449
3 100 999.107
4 150 1199.86
5 175 1298.846

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Fig. 9.3.9: Linearity graph of LEDIPASVIR Fig. 9.3.9.1: Linearity graph of SOFOSBUVIR
Acceptance criteria
The relationship between the concentration of
LEDIPASVIR and SOFOSBUVIR and area of
LEDIPASVIR and SOFOSBUVIR should be linear
in the specified range and the correlation should
not be less than 0.99.
Observation
The correlation coefficient for linear curve
obtained between concentration vs. Area for
standard preparations of LEDIPASVIR and
SOFOSBUVIR is 0.999 and 0.996. The relationship
between the concentration of LEDIPASVIR and
SOFOSBUVIR and area of LEDIPASVIR and
SOFOSBUVIR is linear in the range examined
since all points lie in a straight line and the
correlation coefficient is well within limits.
ACCURACY
Accuracy of the method was determined by
Recovery studies. To the formulation (pre analyzed
sample), the reference standards of the drugs were
added at the level of 100%, 120%, 140%. The
recovery studies were carried out three times and
the percentage recovery and percentage mean
recovery were calculated for drug is shown in table.
To check the accuracy of the method, recovery
studies were carried out by addition of standard
drug solution to pre-analyzed sample solution at
three different levels 100%, 120%, 140%.
Fig. 9.4.1: Chromatogram of 50% recovery (injection 1)
y = 29.696x + 6646.4
R² = 0.998
0
2000
4000
6000
8000
10000
12000
14000
0 50 100 150 200
Conc
Linearity of Ledipasvir y = 4.2149x + 566.02
R² = 0.999
0
200
400
600
800
1000
1200
1400
0 50 100 150 200
Conc
Linearity of Sofosbuvir

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Fig: Chromatogram of 150% recovery (injection 3)
Table 9.4.9.1 : Recovery results for LEDIPASVIR
Recovery level Accuracy LEDIPASVIR Average % Recovery
Amount taken(mcg/ml) Area %Recovery
100 75 9603.072
99.38428878
99.41098976
99.15405317
75 9605.652
75 9533.752

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Table 9.4.9.2 : Recovery results for SOFOSBUVIR
Observation
The percentage mean recovery of LEDIPASVIR
and SOFOSBUVIR is 99.59 % and 100.43%
respectively.
Precision
Method precision
Method precision
Prepared sample preparations of
SOFOSBUVIRand LEDIPASVIR as per test
method and injected 6 times in to the column.
Acceptance criteria
The % Relative standard deviation of Assay
preparations of SOFOSBUVIR and LEDIPASVIR
should be not more than 2.0%.
98.66688096 99.02230106
100.4409523
120 100 10641.469 99.11779744
98.83130831
99.11779744
100 10610.711
100 10641.469
140 150 11814.282
97.81486708
97.78136045
105.7266292
150 11810.235
150 12769.881
Recovery level Accuracy SOFOSBUVIR Average % Recovery
Amount taken(mcg/ml) Area %Recovery
100 75 913.199
191.8314016
189.2721763
195.3025203
102.83
99.36
99.103
75 901.016
75 929.723
120 100 921.96
96.83589175
96.16326273
97.79263406
100 915.556
100 931.069
140 150 1370.413
86.36285626
83.89274067
84.24678469
150 1331.217
150 1336.835

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Fig. 9.5. 1: Chromatogram of precision injection 1
Fig. 9.5.2: Chromatogram of precision injection 2

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Table: Results for Method precision of LEDIPASVIR and SOFOSBUVIR
S.No. Rt Area S.No. Rt Area
1 2.397 3898.811 1 5.630 591.352
2 2.403 3954.691 2 5.650 605.193
3 2.393 3996.327 3 5.603 610.259
4 2.387 3941.128 4 5.597 620.470
5 2.347 3902.778 5 5.523 610.067
6 2.403 3948.922 6 5.650 614.702
avg 2.3883 3940.443 avg 5.609 608.674
stdev 0.0212 36.195 stdev 0.048 9.921
%RSD 0.89 0.92 %RSD 0.85 1.63
Observation
Test results for SOFOSBUVIR and
LEDIPASVIR are showing that the %RSD of
Assay results are within limits.
Robustness
Chromatographic conditions variation
To demonstrate the robustness of the method,
prepared solution as per test method and injected at
different variable conditions like using different
conditions like flow rate and wavelength. System
suitability parameters were compared with that of
method precision.
Acceptance criteria
The system suitability should pass as per the
test method at variable conditions.

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Fig. : Chromatogram of LEDIPASVIR and SOFOSBUVIR Robustness (0.8 ml/min)
Fig. : Chromatogram of LEDIPASVIR and SOFOSBUVIR Robustness (1.2 ml/min)
Fig: Chromatogram of LEDIPASVIR and SOFOSBUVIR for Robustness (211nm)

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Fig: Chromatogram of LEDIPASVIR and SOFOSBUVIR for Robustness (215nm)
Ruggedness
The ruggedness of the method was studied by
the determining the analyst to analyst variation by
performing the Assay by two different analysts
Acceptance criteria
The % Relative standard deviation of Assay
values between two analysts should be not more
than 2.0%.
Fig. 9.9.1: Chromatogram of Analyst 01 standard preparation

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Fig. 9.9.2: Chromatogram of Analyst 01 sample preparation
Fig. 9.9.3: Chromatogram of Analyst 02 standard preparation

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Fig. 9.9.4: Chromatogram of Analyst 02 sample preparation
Observation
From the observation the between two analysts Assay values not greater than 2.0%, hence the method was
rugged.
BIBLIOGRAPHY
[1]. "Ledipasvir" (PDF). United States Adopted Name.
[2]. "Ledipasvir-submitted-to-FDA".
[3]. "GS-5885". Gilead Sciences.
[4]. ELECTRON: 100% Suppression of Viral Load through 4 Weeks’ Post-treatment for Sofosbuvir +
Ledipasvir (GS-5885) + Ribavirin for 12 Weeks in Treatment-naïve and -experienced Hepatitis C Virus
GT 1 Patients. Gane, Edward et al. 20th Conference on Retroviruses and Opportunistic Infections. March
3–6, 2013. Abstract 41LB.
[5]. CROI Sofosbuvir + Ledipasvir + Ribavirin Combo for HCV Produces 100% Sustained Response.
Highleyman, Liz. HIVandHepatitis.com. 2013.
[6]. "Gilead Files for U.S. Approval of Ledipasvir/Sofosbuvir Fixed-Dose Combination Tablet for Genotype 1
Hepatitis C". Gilead Sciences. 2014.
[7]. "U.S. Food and Drug Administration Approves Gilead's Harvoni (Ledipasvir/Sofosbuvir), the First Once-
Daily Single Tablet Regimen for the Treatment of Genotype 1 Chronic Hepatitis C". 2014.
[8]. Afdhal, N; Zeuzem, S; Kwo, P; Chojkier, M; Gitlin, N; Puoti, M; Romero-Gomez, M; Zarski, J. P.;
Agarwal, K; Buggisch, P; Foster, G. R.; Bräu, N; Buti, M; Jacobson, I. M.; Subramanian, G. M.; Ding, X;
Mo, H; Yang, J. C.; Pang, P. S.; Symonds, W. T.; McHutchison, J. G.; Muir, A. J.; Mangia, A; Marcellin,
P; Ion-1, Investigators. "Ledipasvir and sofosbuvir for untreated HCV genotype 1 infection". New
England Journal of Medicine. 370 (20): 1889–98. doi:10.1056/NEJMoa1402454. PMID 24725239. 2014.
[9]. http://www.gilead.com/~/media/Files/pdfs/medicines/liver-disease/harvoni/harvoni_pi.pdf
[10]. http://www.hepatitisc.uw.edu/page/treatment/drugs/ledipasvir-sofosbuvir

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