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A validated gradient stability-indicating LC method for the analysis of

valsartan in pharmaceutical dosage form

Article in International Journal of Pharmacy and Pharmaceutical Sciences · September 2016

DOI: 10.22159/ijpps.2016v8i9.12581

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 International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 8, Issue 9, 2016

Original Article
A VALIDATED GRADIENT STABILITY-INDICATING LC METHOD FOR THE ANALYSIS OF
VALSARTAN IN PHARMACEUTICAL DOSAGE FORM

TRIPTI SHARMA*1a, SUDAM CHANDRA SIa

aDepartment of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Siksha 'O' Anusandhan University, Bhubaneswar 751003, India

Email: [email protected]
Received: 04 May 2016 Revised and Accepted: 22 Jul 2016
ABSTRACT
Objective: The objective of this research work was to develop a sensitive, precise, specific, linear and stability-indicating gradient HPLC method for
the estimation of valsartan in bulk drug and in pharmaceutical preparations.
Methods: Chromatographic separation was achieved on C-18 stationary phase with a gradient mobile phase consisting of orthophosphoric acid
buffer (the pH of the solution was adjusted to 4.2±0.05 with triethylamine) and methanol. The eluent was monitored with PDA detector at 225 nm
with a flow rate of 1.0 ml/min, run time of 65 min.
Results: The method was linear over the range of 20-120μg/ml. The correlation coefficient was found to be 0.9994±0.02. In order to check the
selectivity of the method for pharmaceutical preparations, forced degradation studies were carried out. Valsartan was found to be stable at light and
oxidation experiments. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit
of quantification, linearity, accuracy, precision and robustness. .The LOQ was found to be 0.26µg/ml and the LOD was found to be 0.79µg/ml.
Valsartan showed good correlation coefficient in the concentration range of 20-120μg/ml. The developed method was compared statistically by
applying two-way anova and student's t-test to correlate with an isocratic method and was applied to bulk drug and tablet dosage form. There was
no significant difference between the two methods.
Conclusion: The proposed method was found to be accurate, precise, sensitive and robust. Hence, it can be used successfully for the routine
analysis of valsartan in pharmaceutical formulation and for analysis of stability samples obtained during accelerated stability study.
Keywords: RP-HPLC, Valsartan, Degradation products, Pharmaceutical dosage forms, Two-way ANOVA and student's t-test
© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/)
DOI: http://dx.doi.org/10.22159/ijpps.2016v8i9.12581

INTRODUCTION MATERIALS AND METHODS

Valsartan (VAL) is chemically N-(1-Oxopentyl)-N-[2'-(1H-tetrazol-5- Chemicals and reagents
yl) [1,1'-biphenyl]-4-yl] methyl]-L-valine.[1] It is a new potent,
highly selective and orally active antihypertensive drug belonging to A reference standard sample of VAL was obtained as a gift from
the family of Angiotensin II type I receptor antagonist. Angiotensin II Macleod’s Pharmaceuticals Ltd and commercial dosage form
receptor type I antagonists have widely used in the treatment of containing the studied drug were purchased from local market.
hypertension, heart failure, myocardial infarction and diabetic HPLC grade methanol, orthophosphoric acid, triethylamine, and
nephropathy.[2] Literature survey revealed that HPLC, LC-MS, protein water were HPLC grade purchased from E. Merck, Mumbai, India. All
precipitation, capillary electrophoresis and simultaneous UV- the other chemicals and reagents used were of analytical grade and
spectrophotometric methods [3-15] are reported for estimation of purchased from SD Fine Chemicals, Mumbai, India.
VAL alone or on combination with other agents. As the published Instrumentation and chromatographic system
literature and knowledge of the molecule suggest, reversed phase
liquid chromatography (RP-HPLC) is suitable for analysis of VAL. The chromatographic system consisted of a JASCO (Japan)
various methods are available for estimation of VAL in isocratic mode, chromatograph equipped with an LC–Net II/ADC, an MU–2010 plus
but as the main aim was to resolve the compound from degraded PDA detector, a PU–2089 plus quaternary pump, an online degasser
products and impurities if any, the gradient method was chosen. and a rheodyne model 7725 injector valve with 20 µl sample loop.
The chromatograph is coupled with “Chrompass” software (version
1.7.403.1).
The chromatographic separations were performed at ambient
temperature using Symmetry C18 column (4.6 mm×150 mm,
particle size 5.0 µm). Separation was achieved using a gradient
method. Mobile phase A comprised of orthophosphoric acid buffer
(the pH of the solution was adjusted to 4.2±0.05 with triethylamine).
Mobile phase B was methanol. orthophoshoric acid buffer (pH 4.2)
was prepared by adding 5.5 ml of orthophosphoric acid in 1000 ml
of HPLC grade water, adjusting the pH to 4.2±0.05 with
triethylamine, diluting to 1000 ml with HPLC-grade water, and
filtering through 0.45 m membrane filter. For the preparation of
diluent, a mixture of buffer pH 4.2 and methanol (50:50v/v) filtered
and degassed for 30 min prior to use. The eluent was monitored
with PDA detector at 225 nm with a flow rate of 1.0 ml/min, run
time of 65 min; sample size of 20 µl was carried out at room
Fig. 1: Chemical structure of Valsartan temperature all over the study.
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Int J Pharm Pharm Sci, Vol 8, Issue 9, 128-133

Table 1: Gradient composition for analysis of VAL

Time (min) Mobile phase A (% v/v) Mobile phase B (% v/v) Comment
0→40 55 45 Isocratic
40→55 55→20 45→80 Linear gradient
55→57 20 80 Isocratic
57→58 20→55 80→45 Linear gradient
58→65 55 45 Re-equilibration

Preparation of stock solution Method validation

Stock solutions were prepared by accurately weighing 10 mg of VAL The method was validated according to International Conference on
and transferring to 10 ml volumetric flasks containing 6 ml of Harmonization [17] guidelines for validation of analytical
methanol. The flasks were sonicated for 10 min to dissolve the procedures.
solids. Volumes were made up to the mark with diluents, which gave
1000μg/ml the drugs. Aliquots from the stock solutions were RESULTS AND DISCUSSION
appropriately diluted with diluents to obtain working standards of HPLC method development and optimization
100μg/ml of VAL.
Some important parameters, like pH of the mobile phase,
Calibration standards and quality control sample concentration of buffer solution, percentage and type of organic
Different calibration standards ranging from 20, 40, 60, 80, 100 and modifiers (acetonitrile and methanol), different columns (CN
120μg/ml were prepared by appropriate dilution of standard column, C8 and C18), flow rates (0.5 to 2.0 ml/min) were attempted
solution (1000μg/ml) with mobile phase. Three quality control to resolve the good chromatographic separation of VAL. VAL and
samples at concentrations 60, 80 and 100μg/ml representing 50, degraded products could not be separated without pH adjustment of
100 and 150% respectively of assay concentration (40μg/ml) were the mobile phase. Various pH ranges (3 to 6) for mobile phase were
prepared from the standard solution. An aliquot of 20μL of the tried, and the best resolution was obtained with pH 4.2.
solution was injected into HPLC system. The method was optimized to provide a good separation of the
Preparation of assay solution components (acceptable theoretical plates and resolution between
peaks) with a sufficient sensitivity and suitable peak symmetry
To determine the VAL content of tablet formulations, twenty (peak tailing factor<2). Symmetry C18 column (4.6 mm×150 mm,
tablets were weighed, to determine the average weight of the 5.0 µm particles were preferred over the other columns. The mobile
tablets, and then crushed and mixed using a mortar and pestle. A phase consisted of 55% phosphate buffer and 45% methanol to 80%
portion of powder equivalent to 1000μg/ml was accurately buffer and 20% methanol within 40 min with a 1 ml/min flow rate.
weighed into each of three 10 ml volumetric flasks and 5 ml Retention times for VAL under these conditions were 27.04 (fig. 2).
methanol was added. Each solution was sonicated for 20 min to The quantitative analysis of the drug and its degradation products
achieve complete dissolution of the VAL and the solutions were were performed at 225 nm.
then diluted to volume with diluents, to yield concentrations of
1000μg/ml, and filtered through a 0.22μm nylon membrane filter. Gonzalez et al. [5] developed a HPLC method coupled to
The solution obtained was analyzed by HPLC. fluorescence detection using gradient elution mode and was applied
to plasma samples obtained from hypertensive patients under
Forced degradation study clinical studies after oral administration of a therapeutic dose of
some of these angiotensin II receptor antagonists. The objective of
Acidic degradation
this research work reported was to develop a simple, precise,
50 mg of VAL was accurately weighed and dissolved in 10 ml of accurate gradient stability-indicating LC method for analysis of VAL
methanol, then 5 ml of 0.1N HCl were added and kept at 80 °C about in bulk and its formulations in the presence of degradation products
2 h in a water bath, the solution was allowed to attend ambient and impurities.
temperature then the solution was neutralized by 0.1N NaOH to pH
7 and the volume made up to 50 ml with methanol.
Alkali degradation
50 mg of VAL was accurately weighed and dissolved in 10 ml of
methanol, then 5 ml of 0.1N NaOH was added and kept at 80 °C
about 2 h in a water bath. Then the solution was neutralized by
0.1 N HCl to pH 7 and the volume made up to 50 ml with
methanol.
Oxidative degradation
50 mg of VAL was accurately weighed and dissolved in 10 ml of
methanol, then 5 ml of 10% H2O2 solution were added and kept at 80
°C about 2 h in a water bath then volume was made up to 50 ml with
methanol.
Fig. 2: HPLC chromatogram of valsartan (pure drug) in gradient
Thermal degradation mode
50 mg of VAL was spread in a borosilicate glass Petri dish and placed
in a hot air oven maintained at 80 °C for 24 h, and then the solution
was prepared to achieve a final concentration of 80μg/ml with Method validation
methanol.
System suitability
Photodegradation
System suitability test is an integral part of method development
50 mg of VAL was (covered with aluminum foil) and exposed in the and is used to ensure adequate performance of the chromatographic
UV chamber for 24 h; then the solution was prepared to achieve a system. Retention time, a number of theoretical plates, asymmetrical
final concentration of 80μg/ml with methanol. factor, and peak area were evaluated for five replicate injections of

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Int J Pharm Pharm Sci, Vol 8, Issue 9, 128-133

the drug at a concentration of 80μg/ml. The results given in table 2

were within acceptable limits.

Table 2: System suitability of HPLC method for VAL

Parameters (Mean*±%RSD)
Retention time 27.04±0.59
No. of theoretical plates 9154.2±0.72
Asymmetrical factor 1.05±0.47
Peak area 7050360±0.68
*Mean of five determinations

Linearity
VAL showed good correlation coefficient in the concentration range
of 20-120μg/ml (fig. 3). Linearity was calculated by determining six
standard working solutions containing VAL in triplicate (table 3). Fig. 3: Calibration curve of valsartan in gradient mode
For the method, the linearity of calibration graph was validated by
the high value of correlation of coefficient and the RSD for slope and
intercept value was less than 2%. Accuracy
The accuracy of the method was checked by recovery study using
Table 3: Linear regression data for calibration curve of VAL standard addition method known the amount of standard VAL was
Parameters VAL added into the pre-analyzed sample and subjected it to the proposed
Linearity range(μg/ml) 20-120 high performance liquid chromatographic method.
r *±RSD% 0.9994±0.02 These studies were carried out at three levels i. e, (50, 100 and 150%).
Slope*±RSD% 94265±0.26 The recovery studies were carried out and the % recovery and
Intercept*±RSD%. 453560±1.60
standard deviation of the % recovery were calculated and presented in
*Mean of three determinations table 4.

Table 4: Recovery study by standard addition method for VAL

Sample Amount taken (μg/ml) Amount % Amount added Mean amount present (μg/ml) Recovery mean*
(μg/ml) (%)±SD
40 50 20 60 99.98±0.25
Valzaar-80 40 100 40 80 99.90±0.35
40 150 60 100 99.82±0.55
40 50 20 60 100.21±0.47
Diavon-80 40 100 40 80 99.94±0.48
40 150 60 100 100.26±0.45
*Mean of five determinations

Precision concentration levels on the same day. The inter-day precision

was calculated by assaying three samples of each at three
The precision of the method was determined by repeatability different concentration levels on three different days. The
(intraday precision) and intermediate precision (inter-day results are expressed as a relative standard deviation. The
precision) of VAL standard solutions. Repeatability was relative standard deviations were below 2%, which signifies the
calculated by assaying three samples of each three different precision of both the methods (table 5).

Table 5: Precision of the method

Name of the formulation Intra-day precision (n =6) Inter-day precision (n = 6)
Mean*(%)±RSD Mean*(%)±RSD
Valzaar-80 99.97±0.35 99.91±0.72
Diavon-80 100.11±0.33 100.13±0.61
*Mean of three determinations injected six times at each concentration level

Limits of detection (LOD) and quantification (LOQ) parameters unchanged, and in parallel, the chromatographic
profile was observed and recorded. The chromatographic
The LOQ and LOD were determined based on the 10 and 3.3 times parameters were interchanged within the range of 1-10% of the
the standard deviation of the response, respectively, divided by the optimum recommended conditions. The studied parameters were:
slope of the calibration curve. The LOQ was found to be 0.26µg/ml, mobile phase pH, flow rate, and detector wavelengths. The results
and the LOD was found to be 0.79µg/ml. indicated that the small change in the conditions did not
Robustness significantly affect the determination of VAL. Under all
deliberately varied conditions, the %RSD for the assay values
In order to measure the extent of the method robustness, the most (n=3) for VAL was found to be well within the acceptance limit of
critical parameters were interchanged while keeping the other 2%. The results are reported in table 6.

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Int J Pharm Pharm Sci, Vol 8, Issue 9, 128-133

Table 6: Results from testing the robustness of the method

Condition Modification Mean area* ±SD RSD Mean RT*
(%) (min)±SD
Mobile phase flow 0.8 7078494±16792 0.23 29.95±0.05
rate (mL/min) 1 7062968±54957 0.77 27.08±0.04
1.2 7097530±57017 0.8 26.95±0.15
Mobile Phase pH 4 7033714±34615 0.49 27.04±0.10
4.2 7070556±17718 0.25 27.08±0.04
4.4 7074327±26629 0.37 27.05±0.08
Detector wavelength (nm) 223 7072746±32695 0.46 26.94±0.06
225 7067339±20404 0.28 26.94±0.11
227 7077785±13108 0.18 27.13±0.10
*Mean of three determinations

Specificity and degradation studies 12.2% degradation was observed for VAL. The results from forced
degradation studies are given table 7.
When establishing the stability-indicating properties of analytical
methods, the intermediate degradation products should not Chromatograms obtained from after degradation under different
interfere with any stage of drug analysis. VAL was found to be stable stress conditions are shown in–fig. 4, respectively. No peaks co-
at light and oxidation experiments. In acidic condition VAL degraded eluted with the drug peak, suggesting the method enabled specific
up to 9.2%, in basic condition up to 6.5% and in thermal condition analysis of VAL in the presence of its degradation products.

Table 7: Summary of forced degradation results of VAL

Condition Time % Assay of active substance % Degradation
Treated with 5 ml of 0.1 N HCl solution and kept at 80 °C on water bath 2h 90.8 9.2
Treated with 5 ml of 0.1 N NaOH solution and kept at 80 °C on water bath 2h 93.5 6.5
Treated with 5 ml of 10.0 % H2O2solution 2h 98.2 1.8
and kept at 80 °C on a water bath
Heated at 80 °C in oven 24 h 87.8 12.2
Exposed in the UV chamber 24 h 98.8 1.2

Fig. 4: LC chromatograms of Valsartan (a) after acidic

degradation (b) after basic degradation (c) after oxidative
degradation (d) after thermal degradation (e) after
photodegradation (f) formulation I (g) Formulation II

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Int J Pharm Pharm Sci, Vol 8, Issue 9, 128-133

Use of the method for analysis of marketed formulations Statistical comparison of gradient HPLC method and Isocratic HPLC
Two marketed samples have been analyzed to see the performance To test the difference between the developed gradient liquid
of the method. The first formulation taken was Valzaar-80 which chromatographic method and our previous work isocratic HPLC
contains 80 mg of VAL; the second formulation taken was Diavon-80 method for estimation of VAL [16] statistical tests were performed
contains 80 mg of VAL. Results obtained have been summarized in for the level of confidence 95% (P = 0.05). Two-way ANOVA and
the table 8. One-way ANOVA was applied to test both methods–sample
interaction and differences in method precision. Two-way ANOVA
was used for comparison of two formulations by two
Table 8: Results from assay of VAL in marketed formulation chromatographic methods (table 9). One way ANOVA was used for
Formulation Mean*±SD RSD (%) Recovery (%) comparison of each formulation by chromatographic methods (table
Valzaar-80 80.04±0.33 0.41 100.05 9). To test means between different chromatographic methods
Diavon-80 79.93±0.28 0.35 99.91 paired student’s t–test was applied. The test removes any variation
between samples. For VAL, the two formulations were compared for
*Mean of six determinations the chromatographic methods (table 10).

Table 9: Comparison of chromatographic methods between valzaar-80 and diavon-80

ANOVA-Two Way with replication
Source of Variation SS df MS F stat P-value F crit
Sample 0.0082 1 0.0082 0.0868 0.7713 4.3512
Columns 0.0031 1 0.0031 0.0331 0.8573 4.3512
Interaction 0.0455 1 0.0455 0.4786 0.4969 4.3512
Within 1.9010 20 0.0950
Fstat<Fcrit
ANOVA: Single Factor (VALZAAR-80)
Source of Variation SS df MS F stat P-value F crit
Between Groups 0.0363 1 0.0363 0.3221 0.5828 4.9646
Within Groups 1.1269 10 0.1126
Fstat<Fcrit
ANOVA: Single Factor(DIAVON-80)
Source of Variation SS df MS F stat P-value F crit
Between Groups 0.0123 1 0.0123 0.1595 0.6979 4.9646
Within Groups 0.7741 10 0.0774
Fstat<Fcrit

Table 10: Student’s t–test for different chromatographic methods

t-Test: paired two sample for means
Parameters Valzaar-80 Diavon-80
Pearson correlation -0.5097 -0.4082
t Stat 0.5053 0.3460
t Critical two-tail = 2.5705 t stat<t crit t stat<t crit

CONCLUSION 2. Kumari B, Garg R. Drug profile of valsartan: a review. World J

Pharm Sci 2015;3:1598-606.
A gradient stability-indicating reversed phase high performance 3. Koçyiğit KB, Unsalan S, Rollas S. Determination and validation
liquid chromatographic method for valsartan in bulk and of ketoprofen, pantoprazole and valsartan together in human
pharmaceutical dosage form was undertaken in the present research plasma by high-performance liquid chromatography.
work. The method was found to be specific, as there was no Pharmazie 2006;61:586-9.
interference of any co-eluting impurities after stress degradation 4. Daneshtalab N, Lewanczuk RZ, Jamali F. High performance
study. The method was validated according to ICH guidelines and liquid chromatographic analysis of angiotensin II receptor
correlated by statistical analysis. The proposed method is found to antagonist valsartan using a liquid extraction method. J
be accurate, precise, sensitive and robust. Hence, it can be used Chromatogr B: Anal Technol Biomed Life Sci 2002;766:345-9.
successfully for the routine analysis of valsartan in pharmaceutical 5. González L, López JA, Alonso RM, Jiménez RM. Fast screening
formulation and for analysis of stability samples obtained during method for the determination of angiotensin II receptor
accelerated stability study. antagonists in human plasma by high-performance liquid
chromatography with fluorimetric detection. J Chromatogr A
ACKNOWLEDGMENT 2002;949:49-60.
The authors express their sincere thanks to Macleod’s 6. Koseki N, Kawashita H, Haraa H, Niina M, Tanaka M, Kawai R, et
Pharmaceuticals Ltd., India for providing gift sample of valsartan. al. Development and validation of a method for quantitative
Thanks are also due to the Professor and Chairman Siksha ‘O’ determination of valsartan in human plasma by liquid
Anusandhan University, Bhubaneswar for providing laboratory chromatography-tandem mass spectrometry. J Pharm Biomed
Anal 2007;43:1769-74.
facility for carrying out the present research work.
7. Hao L, Yingwu W, Yao J, Yunbiao T, Jiang W, Limei Z, et al. A
CONFLICTS OF INTERESTS liquid chromatography/tandem mass spectrometry method for
the simultaneous quantification of valsartan and
Declared none hydrochlorothiazide in human plasma. J Chromatogr B: Anal
REFERENCES Technol Biomed Life Sci 2007;852:436-42.
8. Selvan PS, Gowda KV, Mandal U, Solomon WDS, Pal TK.
1. Budavari S. The Merck index, Merck and Co. Press. Edn 12. Simultaneous determination of fixed dose combination of
Whitehouse Station, New Jersey; 1997. nebivolol and valsartan in human plasma by liquid

132
 Sharma et al.
Int J Pharm Pharm Sci, Vol 8, Issue 9, 128-133

chromatographic-tandem mass spectrometry and its valsartan in a pharmaceutical formulation. J Pharm Biomed
application to pharmacokinetic study. J Chromatogr B: Anal Anal 2002;30:371-5.
Technol Biomed Life Sci 2007;858:143-50. 14. Agrahari V, Kabra V, Gupta S, Nema RK, Nagar M, Karthikeya C,
9. Jing N, Bingren X, Yuqi F, Danhua W. Isolation and identification et al. Determination of inherent stability of valsartan by stress
of process impurities in crude valsartan by HPLC, mass degradation and its validation by HPLC. Int J Pharm Clin Res
spectrometry, and nuclear magnetic resonance spectroscopy. 2009;1:77-81.
J Liq Chromatogr Relat Technol 2006;29:553-68. 15. Bhatia M Sudesh, Kokil S Uttamrao. Determination and
10. Macek J, Klíma J, Ptácek P. Rapid determination of valsartan in validation of valsartan and its degradation products by
human plasma by protein precipitation and high-performance isocratic HPLC. J Chem Metal 2009;3:1-12.
liquid chromatography. J Chromatogr B: Anal Technol Biomed 16. Sharma T, Moitra SK, Si SC, Sankar DG. Development and
Life Sci 2006;832:169-72. validation of a HPLC method for the determination of valsartan
11. Hillaert S, Bossche VW. Simultaneous determination of and its degradation products in a pharmaceutical formulation.
hydrochlorothiazide and several angiotensin-II-receptor Int J Pharm Pharm Sci 2012;4:299-303.
antagonists by capillary electrophoresis. J Pharm Biomed Anal 17. ICH. Stability Testing of New Drug Substances and Products.
2003;31:329-39. International Conference on Harmonization, IFPMA, Geneva;
12. Satana E, Altmay S, Göger NG, Özkan SA, Sentürk Z. 2003.
Simultaneous determination of valsartan and
How to cite this article
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13. Tatar S, Sağlík S. Comparison of UV-and second derivative- pharmaceutical dosage form. Int J Pharm Pharm Sci
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