Hindawi Publishing CorporationISRN ChromatographyVolume 2013 Article ID 461461 6 pageshttpdxdoiorg1011552013461461

Research ArticleStability Indicating Simultaneous Validation ofTelmisartan and Cilnidipine with Forced DegradationBehavior Study by RP-HPLC in Tablet Dosage Form

Reema H Rupareliya and Hitendra S Joshi

Department of Chemistry Saurashtra University Rajkot Gujarat-360 005 India

Correspondence should be addressed to Hitendra S Joshi drhsjoshi49gmailcom

Received 26 October 2013 Accepted 25 November 2013

Academic Editors B K Mandal and M L Trehy

Copyright copy 2013 R H Rupareliya and H S Joshi This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A simple precise and accurate RP-HPLCmethod has been developed and validated for the simultaneous assay of Telmisartan andCilnidipine in tablets Isocratic RP-HPLC method was developed on Waters C18 250 times 46mm 5120583m column using mobile phaseas acetonitrile (ACN) buffer pH 30 with orthophosphoric acid (68 32) at a flow rate of 10mLmin and the detection was carriedout at 245 nm using photodiode array detector Forced degradation study was carried out by oxidation hydrolysis photolysis andheating the drug The method was validated for specificity linearity precision accuracy robustness and solution stability Themethod was found to be linear in the concentration range of 40ndash160 120583gmL with correlation coefficient of 09990 for Telmisartanand 10ndash40120583gmL with correlation coefficient of 09989 for Cilnidipine Degradation products produced as a result of stress studiesdid not interfere with the detection of agomelatine therefore the assay can be considered to be stability indicating

1 Introduction

Telmisartan is chemically nominated as 41015840-[(141015840-dimethyl-21015840-propyl[261015840-bi-1H-benzimidazole]-11015840-yl) methyl] [111015840-biphenyl]-2-carboxylic acid (Figure 1) Its molecular formulais C33H30N4O2andmolecular weight is 51462 It is a diabetes

angiotensin receptor blocker that shows high affinity for theangiotensin II type 1 (AT1) receptors has a long durationof action and has the longest half-life of any angiotensin IIreceptor blocker (ARB) [1] In clinical studies Telmisartanshows comparable antihypertensive activity to other majorantihypertensive classes such as angiotensin convertingenzyme (ACE) inhibitors beta-blockers and calciumantagonists [2]

Cilnidipine is chemically nominated as 14-dihydro-26-dimethyl-4-(3-nitrophenyl)-35-pyridine carboxylic acid 2-methoxyethyl(2E)-3-phenyl-propenyl ester (Figure 2) It isa dual blocker of L-type voltage-gated calcium channels invascular smooth muscle and N-type calcium channels insympathetic nerve terminals that supply blood vessels [3]

The parent drug stability test guideline Q1A (R2) issuedby the International Conference on Harmonization (ICH)

suggests that stress testing is an essential part of developmentstrategy and is carried out under more serve condition thanaccelerated conditions These studies provide informationto establish its inherent stability characteristics leading toidentification of degradation products and hence supportingthe suitability of the proposed analytical methods [4ndash6]According to ICH guidelines stress testing should includethe effect of temperature light oxidizing agents and suscep-tibility across a wide range of pH values and separation ofdrugs from degradation products [7] It is also suggested thatanalysis of stability sample should be done by using validatedstability testing methods

There are many reported methods for analysis of Telmis-artan [8ndash13] and Cilnidipine [3 14ndash16] either alone or incombination with other drugs in pharmaceutical dosageforms or individually in biological fluids Simultaneous RP-HPLC andHPTLC estimation of Telmisartan andCilnidipinein combined tablet dosage form has been reported [17 18]To our knowledge there has been no stability indicatingRP-HPLC method reported for Telmisartan and Cilnidip-ine combination in which ICH recommended that stress

2 ISRN Chromatography

N

N

N

N HOO

Figure 1 Chemical structure of Telmisartan

N+

O

OO

O OO

NH

Ominus

O3-(2-Methoxyethyl) O5-[(E)-3-phenylprop-2-enyl]26-dimethyl-4-(3-nitrophenyl)-14-dihydropyridine-35-

dicarboxylate

Figure 2 Chemical structure of Cilnidipine

conditions be applied Therefore the stability indicatingmethodwas developed by applying different stress conditionslike acidic alkali H

2O2 thermal and photodegradation

2 Experimental

21 Instrumentation The chromatographic system used toperform development and validation of this assay methodwas comprised of a LC-10ATvp binary pump a SPD-M10Avpphotodiode array detector and a Rheodyne manual injectormodel 7725i with 20120583L loop (Shimadzu Kyoto Japan)connected to a multi-instrument data acquisition and dataprocessing system (LC solution Shimadzu)

22 Reagents and Reference Substance Telmisartan and Cil-nidipine standards were provided by Unique ChemicalsPanoli India and CPL Chemicals Ankleshvar India respec-tively Cilacar T tablets containing 40mg Telmisartan and10mg Cilnidipine were obtained from market HPLC gradeACN was obtained from Spectrochem Pvt Ltd MumbaiIndia HPLC grade water was produced in-house by usingMilli Q (Millipore Milford USA) system Membrane filtersof 045120583m (Millipore) were used Analytical grade sodiumphosphate monobasic dihydrate was obtained from SiscoResearch Laboratories Mumbai India Hydrochloric acidglacial acetic acid sodium hydroxide pellets and 30 vvhydrogen peroxide solution were obtained from RanbaxyFine Chemicals New Delhi India

23 Chromatographic Conditions Chromatographic analysiswas performed on Waters C 18 (250mm times 46mm id5 120583m particle size) column at ambient temperature Themobile phase consisted of ACN 001M sodium phosphates

monobasic dehydrate buffer pH 30 with phosphoric acid(68 32 vv) Buffer solution was filtered through a 045120583mnylon membrane (Millipore Pvt Ltd Bangalore India) andmobile phase was degassed in an ultrasonic bath (SpincotechPvt Ltd Mumbai) The flow rate of the mobile phase wasadjusted to 10mLmin and the injection volume was 20120583LDetection was performed at 245 nm

24 Standard Preparation Standard solution containingTelmisartan (100 120583gmL) and Cilnidipine (25 120583gmL) wasprepared by dissolving accurately about 1000mgTelmisartanand 250mgCilnidipine in 100mL volumetric flask by diluent(methanol) (stock standard solution) 10mL of stock solutionwas pipetted out into 100mL volumetric flask and dilutedup to mark with diluent to get concentration 100120583gmL forTelmisartan and 25 120583gmL for Cilnidipine

25 Test Preparation Twenty tablets were weighed and theaverage weight of tablet was determined From these fivetablets were weighed and transferred into a 500mL volumet-ric flask About 50mL diluent was added and sonicated for aminimum of 30min with intermittent shaking

Then content was brought back to room temperatureand diluted to volume with diluent The sample was filteredthrough 045 120583m nylon syringe filter 25mL of filtrate stocksolution was pipetted out into 100mL volumetric flask anddiluted up to mark with diluent The concentration obtainedwas 100 120583gmL of Telmisartan and 25 120583gmL of Cilnidipine

26 Degradation Study The degradation samples were pre-pared by transferring powdered tablets which had beenequivalent to 400mg Telmisartan and 100mg Cilnidipineinto a 250mL round bottomed flask Then drug contentwas employed for acidic alkaline and oxidant media andalso for thermal and photolytic stress conditions After thedegradation treatments were completed the stress contentsolutions were allowed to equilibrate to room temperatureand diluted with diluent to attain 100 120583gmL Telmisartan and25 120583gmL Cilnidipine concentration Specific degradationconditions were described as follows

27 Acidic Degradation Condition Acidic degradation studywas performed by heating the drug content in 1NHCl at 60∘Cfor 30min and mixture was neutralized

28 Alkali Degradation Condition Alkaline degradationstudy was performed by ambient temperature in 1 N NaOHfor 30min and mixture was neutralized

29 Oxidative Degradation Condition Oxidation degrada-tion study was performed by heating the drug content in 30vv H2O2at 80∘C for 1 hour Here 80∘C temperature was used

because at 60∘C degradation was not obtained

210 Thermal Degradation Condition Thermal degradationwas performed by exposing solid drug to dry heat of 80∘C

ISRN Chromatography 3

in a conventional oven for 72 hr Here 80∘C temperature wasused because at 60∘C degradation was not obtained

211 Photolytic Degradation Condition Photolytic degrada-tion study was performed by exposing the drug content inUV-light for 72 hr

3 Method Validation

31 Specificity Study The specificity of the method wasdetermined by checking the interference of placebo withanalyte and the proposed method was eluted by checkingthe peak purity of Telmisartan and Cilnidipine during theforce degradation study The peak purity of the Telmisartanand Cilnidipine was found to be satisfactory (09999) and(09998) under different stress conditionsTherewas no inter-ference of any peak of degradation product with drug peak

32 Linearity Linearity test solutions for the assay methodwere prepared at seven concentration levels from 40 to 160of assay analyte concentration (40 60 80 100 120 140and 160 120583gmL) with correlation coefficient of 09990 fortelmisartan and (10 15 20 25 30 35 and 40 120583gmL) withcorrelation coefficient of 09989 for Cilnidipine The peakareas versus concentration data were evaluated by linearregression analysis

33 LOD and LOQ The limit of detection and limit of quan-tificationwere evaluated by serial dilutions of telmisartan andCilnidipine stock solution in order to obtain signal-to-noiseratio of 3 1 for LOD and 10 1 for LOQ

34 Precision The precision of the assay method was evalu-ated in terms of repeatability by carrying out six independentassays of telmisartan and Cilnidipine test sample preparationand calculated the RSD of assay (intraday) Intermediateprecision of the method was checked by performing sameprocedure on a different day (interday) by another personunder experimental condition

35 Accuracy An accuracy study was performed by addingknown amounts of Telmisartan and Cilnidipine to theplacebo preparationThe actual andmeasured concentrationswere compared Recovery of the method was evaluated atthree different concentration levels (corresponding to 50100 and 150 of test preparation concentration) For eachconcentration level three sets were prepared and injected induplicate

36 Robustness The robustness of study was carried outto evaluate the influence of small but deliberate variationson the chromatographic conditions The factors chosen forthis study were the flow rate (plusmn01mLmin) mobile phasecomposition [ACN-buffer (70 30 and 66 34 vv)] andusing different lot of LC column

37 Solution Stability The stability study of solution for testpreparation was carried out The solution was stored at

7006005004003002001000

minus100

00 10 20 30 40 50 60 70 80 90 100 110

Telmisartan

Cilnidipine(mAU

)

245nm 4nm(100)

(min)

Figure 3 Chromatogram of standard preparation

ambient temperature 2ndash5∘C and tested at intervals of 1224 36 and 48 hr The responses for the aged solution wereevaluated using a freshly prepared standard solution

38 Result and Discussion An analytical method based onLC with UV detection was developed and validated for assayand determination of Telmisartan and Cilnidipine in tabletdosage forms The analytical conditions were selected aftertesting the different parameters such as diluents buffer bufferconcentration organic solvent for mobile phase and mobilephase composition and other chromatographic conditionsOur preliminary trials using different compositions ofmobilephases consisting of water with methanol or acetonitrile didnot give good peak shape Then different pH of water wasused with methanol and acetonitrile but good peak shapewas not obtained By keeping mobile phase compositionCAN-001M sodium phosphate monobasic dehydrate buffer(68 32 vv) pH 3 best peak shape was obtained For theselection of organic constituent of mobile phase acetonitrilewas chosen to reduce the longer retention time and to attaingood peak shape A detection wavelength of 245 nm wasselected after scanning the standard solution over the range190ndash370 nm by use of the PDA detector Detection at 245 nmresulted in good response and good linearity Chromatogramof standard preparation is represented in Figure 3

The drug substance was easily extracted from the phar-maceutical dosage form usingmethanolThe tablet dispersedreadily in water and the drug substance was freely solublein methanol Solutions of standard and test preparation werefound to be stable in methanol which was used as a diluent

After development of the analytical method it was vali-dated in accordance with ICH and USP guidelines This fur-nished evidence that the method was suitable for its intendedpurpose The intensive approach described in this paperwas used to develop and validate a liquid chromatographicanalytical method that can be used for assay validation oftelmisartan andCilnidipine in a pharmaceutical dosage formDegradation product produced as a result of stress did notinterfere with detection of Telmisartan and Cilnidipine andthe assay method can thus be regarded as stability indicating

The specificity of the method was evaluated by checkingthe interference of placebo with analyte and the proposedmethod was evaluated by checking the peak purity of Telmis-artan and Cilnidipine during the force degradation studyThere was no interference of any peak of degradation productwith drug peak

4 ISRN Chromatography

500

400

300

200

100

0

00 25 50 75 100 125 150 175 200 225 250 275

Telmisartan

Cilnidipine(mAU

)

245nm

(100)4nm

(min)

Figure 4 Chromatogram of alkali forced degradation study

R2 = 09990

12000000

10000000

8000000

6000000

4000000

2000000

00 005 01 015 02

y = 699636125000x + 1214036071

Figure 5 Linearity curve for Telmisartan

Major degradation was found in alkali condition thatproduct was degraded up to 574 for Telmisartan wheretwo degradation peaks were found at 3460 and 5786 whilefor Cilnidipine the product was degraded up to 3299 at12096min (Figure 4) There was no degradation found inacid 3 H

2O2 30 H

2O2 thermal and photo degradation

for both Telmisartan and CilnidipineTo determine linearity a calibration graph was obtained

by plotting Telmisartan and Cilnidipine concentration (mgmL) against peak area Linearity was good in the concen-tration range 40minus160 120583gmL for Telmisartan (Figure 5) and10ndash40 120583gmL for Cilnidipine (Figure 6) The response ofthe drug was found to be linear The regression equationfor Telmisartan was 119910 = 6996361250119909 + 12140361 withcorrelation coefficient 09990 and for Cilnidipine was 119910 =10029430714 + 2139704 with correlation coefficient 09989where 119909 is the concentration in 120583gmL and 119910 is the peak areain absorbance units (AU)

The LOD values for telmisartan and Cilnidipine werefound to be 001 ppm and 009 ppm respectively The LOQvalues for telmisartan and Cilnidipine were 006 ppm and04 ppm

For assay of Telmisartan (119899 = 6) RSD of system precisionwas 020 on the same day (intraday) and 036 on differentdays (interday) The mean values of assay and RSD formethod precision (repeatability) were 10035 and 043respectively for assay on same day (intraday) while 9972and 056 respectively for assay on different days (interday)

For assay of Cilnidipine (119899 = 6) RSD of system precisionwas 014 on the same day (intraday) and 022 on differentdays (interday) The mean values of assay and RSD

5000000

4000000

3000000

2000000

1000000

0

0 001 002 003 004 005

R2 = 09989

y = 1002943071429x + 213970357

Figure 6 Linearity curve for Cilnidipine

for method precision (repeatability) were 9983 and 086respectively for assay on the same day (intraday) while9993 and 045 respectively for assay on different days(interday)

Intermediate precision was established by determiningthe overall (intraday and interday) method precision Forintermediate precision (119899 = 12) overall assay and RSDvalue was 10004 and 058 respectively for Telmisartanwhile 9988 and 066 for CilnidipineThe precise result forcontent uniformity was indicative of uniform distribution ofthe drug in the tablets without significant variation this is inaccordance with the USP which stipulates acceptance limitsfor drug content uniformity and RSD as 85ndash115 and lt6respectively [19]

The accuracy of the method was assessed by determina-tion of recovery for three concentrations covering the rangeof the method Known amounts (50 100 and 150120583gmL) forTelmisartan and (125 250 and 375 120583gmL) for Cilnidipinewere added to a placebo preparation and the amount ofTelmisartan and Cilnidipine recovered in the presence ofplacebo interface was calculated The mean recovery ofTelmisartan was 9837 9809 and 9979 respectivelyand the mean recovery of Cilnidipine was 9968 9889and 9952 respectively (Table 1)

The robustness of the method was assessed by assayingtest solutions under different analytical conditions deliber-ately changed from the original conditions For each differentanalytical condition the standard solution and test solutionwere prepared separately The result obtained from assay ofthe test solution was not affected by varying the conditionsand was in accordance with the true value System suitabilitydata were also found to be satisfactory during variation ofthe analytical conditions (Table 2) The analytical methodtherefore remains unaffected by slight but deliberate changesin the analytical conditions

During the study of the stability of stored solutions ofstandards and test preparations for assay determination thesolutions were found to be stable for up to 48 hr Assay valuesobtained after 48 hr were statistically identical with the initialvalue without measurable loss (Table 3)

Before each measurement of validation data a systemsuitability test was performed by measurement of generalcharacteristics such as peak asymmetry number of the-oretical plates and RSD () of peak area observed for

ISRN Chromatography 5

Table 1 Evaluation data of accuracy study

Level () Theoretical concentrationa

(mgmL)Observed concentrationa

(mgmL) Recovery RSD

Telmisartan50 004998 004917 9837 168100 009995 009804 9809 058150 014993 014963 9979 120

Cilnidipine50 001253 001246 9969 049100 002505 002477 9889 013150 003752 003734 9952 015

aEach value corresponds to the mean of six determinations

Table 2 Evaluation data of robustness study

Robust conditions Assay System suitability parametersTheoretical plates Asymmetry

Flow 09mLmin 10031 3448 103Flow 11mLmin 9976 3372 098Buffer-ACN(34 66 vv) 9977 3397 102

Buffer-ACN(30 70 vv) 9976 3362 107

Column change 10037 3290 103

Table 3 Evaluation data of stability study

Intervals Assay for test solution

stored at 2ndash8∘C

Assay for test solutionstored at ambient

temperatureTelmisartan Cilnidipine Telmisartan Cilnidipine

Initial 9956 9971 9999 996612 h 9961 9985 10011 1001724 h 9954 9984 10014 997736 h 9998 9923 10018 994548 h 9974 10015 9989 10020

a standard solutionThe values obtainedwere satisfactory andin accordance with in-house limits (Tables 4 and 5)

4 Conclusion

This LC method for simultaneous assay validation of Telmis-artan and Cilnidipine in a tablet formulation was successfullydeveloped and validated for its intended purpose In thisstudy stability of Telmisartan and Cilnidipine in presentdosage form was established through employment of ICHrecommended stress condition The developed procedurehas been evaluated over the specificity linearity accuracyprecision and robustness in order to ascertain the stability ofthe analytical method It has been proved that it was specificlinear precise accurate robust and stability indicatingHence the method is recommended for routine qualitycontrol analysis and also stability sample analysis

Table 4 Evaluation data of system suitability study for Telmisartan

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 102 3628 124Linearity 053 3290 146Precision 020 3799 134Intermediateprecision 036 3730 138

Accuracy 083 3528 129Solution stability 016 3026 120Robustness 071 3413 145bMeans not more than and cmeans not less than

Table 5 Evaluation data of system suitability study for cilnidipine

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 026 10214 135Linearity 069 9823 123Precision 014 10195 129Intermediateprecision 022 10166 140

Accuracy 055 10081 138Solution stability 029 10372 115Robustness 062 10224 126bMeans not more than and cmeans not less than

Acknowledgments

The authors are thankful to Unique Chemicals Panoli Indiaand CPL Chemicals Ankleshvar India for providing Telmis-artan and Cilnidipine standard and facilities and Grantsgiven under UGC-Special Assistance Programme Depart-ment Research Support (DRS) and Department of Scienceamp Technology (DST) New Delhi Fund For Improvementof Science amp Technology (FIST) National Facility for DrugDiscovery (NFDD) and Department of Chemistry Saurash-tra University Rajkot-360 005 india for providing analyticalfacilities

6 ISRN Chromatography

References

[1] M RamaMohana Reddy A Praveen Kumar V Krishna Reddyand S Wasimul Haque ldquoStability-indicating hplc method forsimultaneous estimation of low level impurities of telmisartanand hydrochlorothiazide in tablet dosage formsrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 1pp 497ndash504 2012

[2] A Gupta R M Charde and M S Charde ldquoDeterminationof telmisartan and forced degradation behavior by RP-HPLCin tablet dosage formrdquo International Journal of PharmaceuticalChemistry vol 2 no 3 pp 93ndash99 2012

[3] M S Mohammed ldquoSpctrophotometric method for the estima-tion of cilnidipine in bulk and pharmaceutical dosage formsrdquoOriental Journal of Chemistry vol 29 no 1 pp 131ndash134 2013

[4] A S Rathore L Sathiyanarayanan and K R MahadikldquoStability-indicating high-performance thin-layer chromato-graphic method for quantitative estimation of emtricitabine inbulk drug and pharmaceutical dosage formrdquo ISRN Chromatog-raphy vol 2012 Article ID 278583 7 pages 2012

[5] P P Vekariya and H S Joshi ldquoDevelopment and validation ofRP-HPLCmethod for azilsartan medoxomil potassium quanti-tation in human plasma by solid phase extraction procedurerdquoISRN Spectroscopy vol 2013 Article ID 572170 6 pages 2013

[6] S Singh B Singh R Bahuguna L Wadhwa and R SaxenaldquoStress degradation studies on ezetimibe and development of avalidated stability-indicating HPLC assayrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 41 no 3 pp 1037ndash10402006

[7] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[8] C D Niranjan S M Patil J K Sabaji and A N ChivateldquoDevelopment of UV spectrophotometric method for estima-tion and validation of telmisartan as a pure APIrdquo Journal ofPharmacy Research vol 5 no 6 pp 3331ndash3333 2012

[9] K K Pradhan U S Mishra A Sahoo K C Sahu DMishra and R Dash ldquoMethod development and validation ofTelmisartan in bulk and pharmaceutical dosage forms by UVSpectrophotometric methodrdquo International Journal of Researchin Pharmaceutical Sciences vol 2 no 4 pp 526ndash530 2011

[10] G Mahathi Y Ravindra Reddy and P Uttam Prasad ldquoDevel-opment and validation of telmisartan and atorvastatin calciumin combined dosage form by RP-HPLCrdquo International Journalof Pharmacy and Technology vol 3 no 3 pp 3370ndash3389 2011

[11] A M Raja N Spoorthy D Banji K N V Rao C Vanita andS D Kumar ldquoSimultaneous estimaton of metroprolol succinateand telmisartan in tablet dosage form by RP-HPLCrdquo Journal ofPharmacy Research vol 5 no 8 pp 4585ndash4587 2012

[12] P Vani and S K Kalyana ldquoA rapid stability-indicating simul-taneous determenation of hydrochlorothiazide ramipril andtelmisartan in combined dosage form by ultra performanceliquid chromatographyrdquo Pharmacia Letter vol 5 no 3 pp 81ndash89 2013

[13] K E Parmar R S Mehta N D Patel and K E ParmarldquoDevelopment and validation of HPTLC method for simul-taneous determination of telmisartan and chlorthalidone inbulk and pharmaceutical dosage formrdquo International Journal ofPharmacy and Pharmaceutical Sciences vol 5 no 2 pp 420ndash425 2013

[14] H S Karmalkar V V Vaidya N A Gomes M P Choukekarand M B Kekare ldquoDetermination of cilnidipine from pharma-ceutical formulation by high performance thin layer chromato-graphicmethodrdquoAnalytical Chemistry vol 7 no 8 pp 573ndash5762008

[15] K-R Lee Y-J Chae J-H Lee et al ldquoQuantification ofcilnidipine in human plasma by liquid chromatography-massspectrometryrdquo Journal of Liquid Chromatography and RelatedTechnologies vol 35 no 2 pp 308ndash320 2012

[16] P P Chaudhari and A V Bhalerao ldquoMethod validation forspectrophotometric estimation of cilnidipinerdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 5pp 96ndash98 2012

[17] P Pawar S V Gandhi P B Deshpande B Padmanabh SVanjari and S U Shelar ldquoSimultaneous RP-HPLC estimationof cilnidipine and telmisartan in combined table dosage formrdquoChemica Sinica vol 4 no 2 pp 6ndash10 2013

[18] P Pawar P B Deshpande S Gandhi and V Bhavnani ldquoHighperformance hin layer chromatographic determenation of cilni-dipine and telmisartan in combined dosage formrdquo InternationalResearch Journal of Pharmacy vol 3 no 6 pp 219ndash222 2012

[19] G J Kher V R Ram K L Dubal A H Bapodara and HS Joshi ldquoValidation of a stability-indicating LC method forassay of leflunomide in tablets and for determination of contentuniformityrdquo International Journal of ChemTech Research vol 3no 2 pp 523ndash530 2011

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CatalystsJournal of

2 ISRN Chromatography

N

N

N

N HOO

Figure 1 Chemical structure of Telmisartan

N+

O

OO

O OO

NH

Ominus

O3-(2-Methoxyethyl) O5-[(E)-3-phenylprop-2-enyl]26-dimethyl-4-(3-nitrophenyl)-14-dihydropyridine-35-

dicarboxylate

Figure 2 Chemical structure of Cilnidipine

conditions be applied Therefore the stability indicatingmethodwas developed by applying different stress conditionslike acidic alkali H

2O2 thermal and photodegradation

2 Experimental

21 Instrumentation The chromatographic system used toperform development and validation of this assay methodwas comprised of a LC-10ATvp binary pump a SPD-M10Avpphotodiode array detector and a Rheodyne manual injectormodel 7725i with 20120583L loop (Shimadzu Kyoto Japan)connected to a multi-instrument data acquisition and dataprocessing system (LC solution Shimadzu)

22 Reagents and Reference Substance Telmisartan and Cil-nidipine standards were provided by Unique ChemicalsPanoli India and CPL Chemicals Ankleshvar India respec-tively Cilacar T tablets containing 40mg Telmisartan and10mg Cilnidipine were obtained from market HPLC gradeACN was obtained from Spectrochem Pvt Ltd MumbaiIndia HPLC grade water was produced in-house by usingMilli Q (Millipore Milford USA) system Membrane filtersof 045120583m (Millipore) were used Analytical grade sodiumphosphate monobasic dihydrate was obtained from SiscoResearch Laboratories Mumbai India Hydrochloric acidglacial acetic acid sodium hydroxide pellets and 30 vvhydrogen peroxide solution were obtained from RanbaxyFine Chemicals New Delhi India

23 Chromatographic Conditions Chromatographic analysiswas performed on Waters C 18 (250mm times 46mm id5 120583m particle size) column at ambient temperature Themobile phase consisted of ACN 001M sodium phosphates

monobasic dehydrate buffer pH 30 with phosphoric acid(68 32 vv) Buffer solution was filtered through a 045120583mnylon membrane (Millipore Pvt Ltd Bangalore India) andmobile phase was degassed in an ultrasonic bath (SpincotechPvt Ltd Mumbai) The flow rate of the mobile phase wasadjusted to 10mLmin and the injection volume was 20120583LDetection was performed at 245 nm

24 Standard Preparation Standard solution containingTelmisartan (100 120583gmL) and Cilnidipine (25 120583gmL) wasprepared by dissolving accurately about 1000mgTelmisartanand 250mgCilnidipine in 100mL volumetric flask by diluent(methanol) (stock standard solution) 10mL of stock solutionwas pipetted out into 100mL volumetric flask and dilutedup to mark with diluent to get concentration 100120583gmL forTelmisartan and 25 120583gmL for Cilnidipine

25 Test Preparation Twenty tablets were weighed and theaverage weight of tablet was determined From these fivetablets were weighed and transferred into a 500mL volumet-ric flask About 50mL diluent was added and sonicated for aminimum of 30min with intermittent shaking

Then content was brought back to room temperatureand diluted to volume with diluent The sample was filteredthrough 045 120583m nylon syringe filter 25mL of filtrate stocksolution was pipetted out into 100mL volumetric flask anddiluted up to mark with diluent The concentration obtainedwas 100 120583gmL of Telmisartan and 25 120583gmL of Cilnidipine

26 Degradation Study The degradation samples were pre-pared by transferring powdered tablets which had beenequivalent to 400mg Telmisartan and 100mg Cilnidipineinto a 250mL round bottomed flask Then drug contentwas employed for acidic alkaline and oxidant media andalso for thermal and photolytic stress conditions After thedegradation treatments were completed the stress contentsolutions were allowed to equilibrate to room temperatureand diluted with diluent to attain 100 120583gmL Telmisartan and25 120583gmL Cilnidipine concentration Specific degradationconditions were described as follows

27 Acidic Degradation Condition Acidic degradation studywas performed by heating the drug content in 1NHCl at 60∘Cfor 30min and mixture was neutralized

28 Alkali Degradation Condition Alkaline degradationstudy was performed by ambient temperature in 1 N NaOHfor 30min and mixture was neutralized

29 Oxidative Degradation Condition Oxidation degrada-tion study was performed by heating the drug content in 30vv H2O2at 80∘C for 1 hour Here 80∘C temperature was used

because at 60∘C degradation was not obtained

210 Thermal Degradation Condition Thermal degradationwas performed by exposing solid drug to dry heat of 80∘C

ISRN Chromatography 3

in a conventional oven for 72 hr Here 80∘C temperature wasused because at 60∘C degradation was not obtained

211 Photolytic Degradation Condition Photolytic degrada-tion study was performed by exposing the drug content inUV-light for 72 hr

3 Method Validation

31 Specificity Study The specificity of the method wasdetermined by checking the interference of placebo withanalyte and the proposed method was eluted by checkingthe peak purity of Telmisartan and Cilnidipine during theforce degradation study The peak purity of the Telmisartanand Cilnidipine was found to be satisfactory (09999) and(09998) under different stress conditionsTherewas no inter-ference of any peak of degradation product with drug peak

32 Linearity Linearity test solutions for the assay methodwere prepared at seven concentration levels from 40 to 160of assay analyte concentration (40 60 80 100 120 140and 160 120583gmL) with correlation coefficient of 09990 fortelmisartan and (10 15 20 25 30 35 and 40 120583gmL) withcorrelation coefficient of 09989 for Cilnidipine The peakareas versus concentration data were evaluated by linearregression analysis

33 LOD and LOQ The limit of detection and limit of quan-tificationwere evaluated by serial dilutions of telmisartan andCilnidipine stock solution in order to obtain signal-to-noiseratio of 3 1 for LOD and 10 1 for LOQ

34 Precision The precision of the assay method was evalu-ated in terms of repeatability by carrying out six independentassays of telmisartan and Cilnidipine test sample preparationand calculated the RSD of assay (intraday) Intermediateprecision of the method was checked by performing sameprocedure on a different day (interday) by another personunder experimental condition

35 Accuracy An accuracy study was performed by addingknown amounts of Telmisartan and Cilnidipine to theplacebo preparationThe actual andmeasured concentrationswere compared Recovery of the method was evaluated atthree different concentration levels (corresponding to 50100 and 150 of test preparation concentration) For eachconcentration level three sets were prepared and injected induplicate

36 Robustness The robustness of study was carried outto evaluate the influence of small but deliberate variationson the chromatographic conditions The factors chosen forthis study were the flow rate (plusmn01mLmin) mobile phasecomposition [ACN-buffer (70 30 and 66 34 vv)] andusing different lot of LC column

37 Solution Stability The stability study of solution for testpreparation was carried out The solution was stored at

7006005004003002001000

minus100

00 10 20 30 40 50 60 70 80 90 100 110

Telmisartan

Cilnidipine(mAU

)

245nm 4nm(100)

(min)

Figure 3 Chromatogram of standard preparation

ambient temperature 2ndash5∘C and tested at intervals of 1224 36 and 48 hr The responses for the aged solution wereevaluated using a freshly prepared standard solution

38 Result and Discussion An analytical method based onLC with UV detection was developed and validated for assayand determination of Telmisartan and Cilnidipine in tabletdosage forms The analytical conditions were selected aftertesting the different parameters such as diluents buffer bufferconcentration organic solvent for mobile phase and mobilephase composition and other chromatographic conditionsOur preliminary trials using different compositions ofmobilephases consisting of water with methanol or acetonitrile didnot give good peak shape Then different pH of water wasused with methanol and acetonitrile but good peak shapewas not obtained By keeping mobile phase compositionCAN-001M sodium phosphate monobasic dehydrate buffer(68 32 vv) pH 3 best peak shape was obtained For theselection of organic constituent of mobile phase acetonitrilewas chosen to reduce the longer retention time and to attaingood peak shape A detection wavelength of 245 nm wasselected after scanning the standard solution over the range190ndash370 nm by use of the PDA detector Detection at 245 nmresulted in good response and good linearity Chromatogramof standard preparation is represented in Figure 3

The drug substance was easily extracted from the phar-maceutical dosage form usingmethanolThe tablet dispersedreadily in water and the drug substance was freely solublein methanol Solutions of standard and test preparation werefound to be stable in methanol which was used as a diluent

After development of the analytical method it was vali-dated in accordance with ICH and USP guidelines This fur-nished evidence that the method was suitable for its intendedpurpose The intensive approach described in this paperwas used to develop and validate a liquid chromatographicanalytical method that can be used for assay validation oftelmisartan andCilnidipine in a pharmaceutical dosage formDegradation product produced as a result of stress did notinterfere with detection of Telmisartan and Cilnidipine andthe assay method can thus be regarded as stability indicating

The specificity of the method was evaluated by checkingthe interference of placebo with analyte and the proposedmethod was evaluated by checking the peak purity of Telmis-artan and Cilnidipine during the force degradation studyThere was no interference of any peak of degradation productwith drug peak

4 ISRN Chromatography

500

400

300

200

100

0

00 25 50 75 100 125 150 175 200 225 250 275

Telmisartan

Cilnidipine(mAU

)

245nm

(100)4nm

(min)

Figure 4 Chromatogram of alkali forced degradation study

R2 = 09990

12000000

10000000

8000000

6000000

4000000

2000000

00 005 01 015 02

y = 699636125000x + 1214036071

Figure 5 Linearity curve for Telmisartan

Major degradation was found in alkali condition thatproduct was degraded up to 574 for Telmisartan wheretwo degradation peaks were found at 3460 and 5786 whilefor Cilnidipine the product was degraded up to 3299 at12096min (Figure 4) There was no degradation found inacid 3 H

2O2 30 H

2O2 thermal and photo degradation

for both Telmisartan and CilnidipineTo determine linearity a calibration graph was obtained

by plotting Telmisartan and Cilnidipine concentration (mgmL) against peak area Linearity was good in the concen-tration range 40minus160 120583gmL for Telmisartan (Figure 5) and10ndash40 120583gmL for Cilnidipine (Figure 6) The response ofthe drug was found to be linear The regression equationfor Telmisartan was 119910 = 6996361250119909 + 12140361 withcorrelation coefficient 09990 and for Cilnidipine was 119910 =10029430714 + 2139704 with correlation coefficient 09989where 119909 is the concentration in 120583gmL and 119910 is the peak areain absorbance units (AU)

The LOD values for telmisartan and Cilnidipine werefound to be 001 ppm and 009 ppm respectively The LOQvalues for telmisartan and Cilnidipine were 006 ppm and04 ppm

For assay of Telmisartan (119899 = 6) RSD of system precisionwas 020 on the same day (intraday) and 036 on differentdays (interday) The mean values of assay and RSD formethod precision (repeatability) were 10035 and 043respectively for assay on same day (intraday) while 9972and 056 respectively for assay on different days (interday)

For assay of Cilnidipine (119899 = 6) RSD of system precisionwas 014 on the same day (intraday) and 022 on differentdays (interday) The mean values of assay and RSD

5000000

4000000

3000000

2000000

1000000

0

0 001 002 003 004 005

R2 = 09989

y = 1002943071429x + 213970357

Figure 6 Linearity curve for Cilnidipine

for method precision (repeatability) were 9983 and 086respectively for assay on the same day (intraday) while9993 and 045 respectively for assay on different days(interday)

Intermediate precision was established by determiningthe overall (intraday and interday) method precision Forintermediate precision (119899 = 12) overall assay and RSDvalue was 10004 and 058 respectively for Telmisartanwhile 9988 and 066 for CilnidipineThe precise result forcontent uniformity was indicative of uniform distribution ofthe drug in the tablets without significant variation this is inaccordance with the USP which stipulates acceptance limitsfor drug content uniformity and RSD as 85ndash115 and lt6respectively [19]

The accuracy of the method was assessed by determina-tion of recovery for three concentrations covering the rangeof the method Known amounts (50 100 and 150120583gmL) forTelmisartan and (125 250 and 375 120583gmL) for Cilnidipinewere added to a placebo preparation and the amount ofTelmisartan and Cilnidipine recovered in the presence ofplacebo interface was calculated The mean recovery ofTelmisartan was 9837 9809 and 9979 respectivelyand the mean recovery of Cilnidipine was 9968 9889and 9952 respectively (Table 1)

The robustness of the method was assessed by assayingtest solutions under different analytical conditions deliber-ately changed from the original conditions For each differentanalytical condition the standard solution and test solutionwere prepared separately The result obtained from assay ofthe test solution was not affected by varying the conditionsand was in accordance with the true value System suitabilitydata were also found to be satisfactory during variation ofthe analytical conditions (Table 2) The analytical methodtherefore remains unaffected by slight but deliberate changesin the analytical conditions

During the study of the stability of stored solutions ofstandards and test preparations for assay determination thesolutions were found to be stable for up to 48 hr Assay valuesobtained after 48 hr were statistically identical with the initialvalue without measurable loss (Table 3)

Before each measurement of validation data a systemsuitability test was performed by measurement of generalcharacteristics such as peak asymmetry number of the-oretical plates and RSD () of peak area observed for

ISRN Chromatography 5

Table 1 Evaluation data of accuracy study

Level () Theoretical concentrationa

(mgmL)Observed concentrationa

(mgmL) Recovery RSD

Telmisartan50 004998 004917 9837 168100 009995 009804 9809 058150 014993 014963 9979 120

Cilnidipine50 001253 001246 9969 049100 002505 002477 9889 013150 003752 003734 9952 015

aEach value corresponds to the mean of six determinations

Table 2 Evaluation data of robustness study

Robust conditions Assay System suitability parametersTheoretical plates Asymmetry

Flow 09mLmin 10031 3448 103Flow 11mLmin 9976 3372 098Buffer-ACN(34 66 vv) 9977 3397 102

Buffer-ACN(30 70 vv) 9976 3362 107

Column change 10037 3290 103

Table 3 Evaluation data of stability study

Intervals Assay for test solution

stored at 2ndash8∘C

Assay for test solutionstored at ambient

temperatureTelmisartan Cilnidipine Telmisartan Cilnidipine

Initial 9956 9971 9999 996612 h 9961 9985 10011 1001724 h 9954 9984 10014 997736 h 9998 9923 10018 994548 h 9974 10015 9989 10020

a standard solutionThe values obtainedwere satisfactory andin accordance with in-house limits (Tables 4 and 5)

4 Conclusion

This LC method for simultaneous assay validation of Telmis-artan and Cilnidipine in a tablet formulation was successfullydeveloped and validated for its intended purpose In thisstudy stability of Telmisartan and Cilnidipine in presentdosage form was established through employment of ICHrecommended stress condition The developed procedurehas been evaluated over the specificity linearity accuracyprecision and robustness in order to ascertain the stability ofthe analytical method It has been proved that it was specificlinear precise accurate robust and stability indicatingHence the method is recommended for routine qualitycontrol analysis and also stability sample analysis

Table 4 Evaluation data of system suitability study for Telmisartan

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 102 3628 124Linearity 053 3290 146Precision 020 3799 134Intermediateprecision 036 3730 138

Accuracy 083 3528 129Solution stability 016 3026 120Robustness 071 3413 145bMeans not more than and cmeans not less than

Table 5 Evaluation data of system suitability study for cilnidipine

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 026 10214 135Linearity 069 9823 123Precision 014 10195 129Intermediateprecision 022 10166 140

Accuracy 055 10081 138Solution stability 029 10372 115Robustness 062 10224 126bMeans not more than and cmeans not less than

Acknowledgments

The authors are thankful to Unique Chemicals Panoli Indiaand CPL Chemicals Ankleshvar India for providing Telmis-artan and Cilnidipine standard and facilities and Grantsgiven under UGC-Special Assistance Programme Depart-ment Research Support (DRS) and Department of Scienceamp Technology (DST) New Delhi Fund For Improvementof Science amp Technology (FIST) National Facility for DrugDiscovery (NFDD) and Department of Chemistry Saurash-tra University Rajkot-360 005 india for providing analyticalfacilities

6 ISRN Chromatography

References

[1] M RamaMohana Reddy A Praveen Kumar V Krishna Reddyand S Wasimul Haque ldquoStability-indicating hplc method forsimultaneous estimation of low level impurities of telmisartanand hydrochlorothiazide in tablet dosage formsrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 1pp 497ndash504 2012

[2] A Gupta R M Charde and M S Charde ldquoDeterminationof telmisartan and forced degradation behavior by RP-HPLCin tablet dosage formrdquo International Journal of PharmaceuticalChemistry vol 2 no 3 pp 93ndash99 2012

[3] M S Mohammed ldquoSpctrophotometric method for the estima-tion of cilnidipine in bulk and pharmaceutical dosage formsrdquoOriental Journal of Chemistry vol 29 no 1 pp 131ndash134 2013

[4] A S Rathore L Sathiyanarayanan and K R MahadikldquoStability-indicating high-performance thin-layer chromato-graphic method for quantitative estimation of emtricitabine inbulk drug and pharmaceutical dosage formrdquo ISRN Chromatog-raphy vol 2012 Article ID 278583 7 pages 2012

[5] P P Vekariya and H S Joshi ldquoDevelopment and validation ofRP-HPLCmethod for azilsartan medoxomil potassium quanti-tation in human plasma by solid phase extraction procedurerdquoISRN Spectroscopy vol 2013 Article ID 572170 6 pages 2013

[6] S Singh B Singh R Bahuguna L Wadhwa and R SaxenaldquoStress degradation studies on ezetimibe and development of avalidated stability-indicating HPLC assayrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 41 no 3 pp 1037ndash10402006

[7] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[8] C D Niranjan S M Patil J K Sabaji and A N ChivateldquoDevelopment of UV spectrophotometric method for estima-tion and validation of telmisartan as a pure APIrdquo Journal ofPharmacy Research vol 5 no 6 pp 3331ndash3333 2012

[9] K K Pradhan U S Mishra A Sahoo K C Sahu DMishra and R Dash ldquoMethod development and validation ofTelmisartan in bulk and pharmaceutical dosage forms by UVSpectrophotometric methodrdquo International Journal of Researchin Pharmaceutical Sciences vol 2 no 4 pp 526ndash530 2011

[10] G Mahathi Y Ravindra Reddy and P Uttam Prasad ldquoDevel-opment and validation of telmisartan and atorvastatin calciumin combined dosage form by RP-HPLCrdquo International Journalof Pharmacy and Technology vol 3 no 3 pp 3370ndash3389 2011

[11] A M Raja N Spoorthy D Banji K N V Rao C Vanita andS D Kumar ldquoSimultaneous estimaton of metroprolol succinateand telmisartan in tablet dosage form by RP-HPLCrdquo Journal ofPharmacy Research vol 5 no 8 pp 4585ndash4587 2012

[12] P Vani and S K Kalyana ldquoA rapid stability-indicating simul-taneous determenation of hydrochlorothiazide ramipril andtelmisartan in combined dosage form by ultra performanceliquid chromatographyrdquo Pharmacia Letter vol 5 no 3 pp 81ndash89 2013

[13] K E Parmar R S Mehta N D Patel and K E ParmarldquoDevelopment and validation of HPTLC method for simul-taneous determination of telmisartan and chlorthalidone inbulk and pharmaceutical dosage formrdquo International Journal ofPharmacy and Pharmaceutical Sciences vol 5 no 2 pp 420ndash425 2013

[14] H S Karmalkar V V Vaidya N A Gomes M P Choukekarand M B Kekare ldquoDetermination of cilnidipine from pharma-ceutical formulation by high performance thin layer chromato-graphicmethodrdquoAnalytical Chemistry vol 7 no 8 pp 573ndash5762008

[15] K-R Lee Y-J Chae J-H Lee et al ldquoQuantification ofcilnidipine in human plasma by liquid chromatography-massspectrometryrdquo Journal of Liquid Chromatography and RelatedTechnologies vol 35 no 2 pp 308ndash320 2012

[16] P P Chaudhari and A V Bhalerao ldquoMethod validation forspectrophotometric estimation of cilnidipinerdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 5pp 96ndash98 2012

[17] P Pawar S V Gandhi P B Deshpande B Padmanabh SVanjari and S U Shelar ldquoSimultaneous RP-HPLC estimationof cilnidipine and telmisartan in combined table dosage formrdquoChemica Sinica vol 4 no 2 pp 6ndash10 2013

[18] P Pawar P B Deshpande S Gandhi and V Bhavnani ldquoHighperformance hin layer chromatographic determenation of cilni-dipine and telmisartan in combined dosage formrdquo InternationalResearch Journal of Pharmacy vol 3 no 6 pp 219ndash222 2012

[19] G J Kher V R Ram K L Dubal A H Bapodara and HS Joshi ldquoValidation of a stability-indicating LC method forassay of leflunomide in tablets and for determination of contentuniformityrdquo International Journal of ChemTech Research vol 3no 2 pp 523ndash530 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

ISRN Chromatography 3

in a conventional oven for 72 hr Here 80∘C temperature wasused because at 60∘C degradation was not obtained

211 Photolytic Degradation Condition Photolytic degrada-tion study was performed by exposing the drug content inUV-light for 72 hr

3 Method Validation

31 Specificity Study The specificity of the method wasdetermined by checking the interference of placebo withanalyte and the proposed method was eluted by checkingthe peak purity of Telmisartan and Cilnidipine during theforce degradation study The peak purity of the Telmisartanand Cilnidipine was found to be satisfactory (09999) and(09998) under different stress conditionsTherewas no inter-ference of any peak of degradation product with drug peak

32 Linearity Linearity test solutions for the assay methodwere prepared at seven concentration levels from 40 to 160of assay analyte concentration (40 60 80 100 120 140and 160 120583gmL) with correlation coefficient of 09990 fortelmisartan and (10 15 20 25 30 35 and 40 120583gmL) withcorrelation coefficient of 09989 for Cilnidipine The peakareas versus concentration data were evaluated by linearregression analysis

33 LOD and LOQ The limit of detection and limit of quan-tificationwere evaluated by serial dilutions of telmisartan andCilnidipine stock solution in order to obtain signal-to-noiseratio of 3 1 for LOD and 10 1 for LOQ

34 Precision The precision of the assay method was evalu-ated in terms of repeatability by carrying out six independentassays of telmisartan and Cilnidipine test sample preparationand calculated the RSD of assay (intraday) Intermediateprecision of the method was checked by performing sameprocedure on a different day (interday) by another personunder experimental condition

35 Accuracy An accuracy study was performed by addingknown amounts of Telmisartan and Cilnidipine to theplacebo preparationThe actual andmeasured concentrationswere compared Recovery of the method was evaluated atthree different concentration levels (corresponding to 50100 and 150 of test preparation concentration) For eachconcentration level three sets were prepared and injected induplicate

36 Robustness The robustness of study was carried outto evaluate the influence of small but deliberate variationson the chromatographic conditions The factors chosen forthis study were the flow rate (plusmn01mLmin) mobile phasecomposition [ACN-buffer (70 30 and 66 34 vv)] andusing different lot of LC column

37 Solution Stability The stability study of solution for testpreparation was carried out The solution was stored at

7006005004003002001000

minus100

00 10 20 30 40 50 60 70 80 90 100 110

Telmisartan

Cilnidipine(mAU

)

245nm 4nm(100)

(min)

Figure 3 Chromatogram of standard preparation

ambient temperature 2ndash5∘C and tested at intervals of 1224 36 and 48 hr The responses for the aged solution wereevaluated using a freshly prepared standard solution

38 Result and Discussion An analytical method based onLC with UV detection was developed and validated for assayand determination of Telmisartan and Cilnidipine in tabletdosage forms The analytical conditions were selected aftertesting the different parameters such as diluents buffer bufferconcentration organic solvent for mobile phase and mobilephase composition and other chromatographic conditionsOur preliminary trials using different compositions ofmobilephases consisting of water with methanol or acetonitrile didnot give good peak shape Then different pH of water wasused with methanol and acetonitrile but good peak shapewas not obtained By keeping mobile phase compositionCAN-001M sodium phosphate monobasic dehydrate buffer(68 32 vv) pH 3 best peak shape was obtained For theselection of organic constituent of mobile phase acetonitrilewas chosen to reduce the longer retention time and to attaingood peak shape A detection wavelength of 245 nm wasselected after scanning the standard solution over the range190ndash370 nm by use of the PDA detector Detection at 245 nmresulted in good response and good linearity Chromatogramof standard preparation is represented in Figure 3

The drug substance was easily extracted from the phar-maceutical dosage form usingmethanolThe tablet dispersedreadily in water and the drug substance was freely solublein methanol Solutions of standard and test preparation werefound to be stable in methanol which was used as a diluent

After development of the analytical method it was vali-dated in accordance with ICH and USP guidelines This fur-nished evidence that the method was suitable for its intendedpurpose The intensive approach described in this paperwas used to develop and validate a liquid chromatographicanalytical method that can be used for assay validation oftelmisartan andCilnidipine in a pharmaceutical dosage formDegradation product produced as a result of stress did notinterfere with detection of Telmisartan and Cilnidipine andthe assay method can thus be regarded as stability indicating

The specificity of the method was evaluated by checkingthe interference of placebo with analyte and the proposedmethod was evaluated by checking the peak purity of Telmis-artan and Cilnidipine during the force degradation studyThere was no interference of any peak of degradation productwith drug peak

4 ISRN Chromatography

500

400

300

200

100

0

00 25 50 75 100 125 150 175 200 225 250 275

Telmisartan

Cilnidipine(mAU

)

245nm

(100)4nm

(min)

Figure 4 Chromatogram of alkali forced degradation study

R2 = 09990

12000000

10000000

8000000

6000000

4000000

2000000

00 005 01 015 02

y = 699636125000x + 1214036071

Figure 5 Linearity curve for Telmisartan

Major degradation was found in alkali condition thatproduct was degraded up to 574 for Telmisartan wheretwo degradation peaks were found at 3460 and 5786 whilefor Cilnidipine the product was degraded up to 3299 at12096min (Figure 4) There was no degradation found inacid 3 H

2O2 30 H

2O2 thermal and photo degradation

for both Telmisartan and CilnidipineTo determine linearity a calibration graph was obtained

by plotting Telmisartan and Cilnidipine concentration (mgmL) against peak area Linearity was good in the concen-tration range 40minus160 120583gmL for Telmisartan (Figure 5) and10ndash40 120583gmL for Cilnidipine (Figure 6) The response ofthe drug was found to be linear The regression equationfor Telmisartan was 119910 = 6996361250119909 + 12140361 withcorrelation coefficient 09990 and for Cilnidipine was 119910 =10029430714 + 2139704 with correlation coefficient 09989where 119909 is the concentration in 120583gmL and 119910 is the peak areain absorbance units (AU)

The LOD values for telmisartan and Cilnidipine werefound to be 001 ppm and 009 ppm respectively The LOQvalues for telmisartan and Cilnidipine were 006 ppm and04 ppm

For assay of Telmisartan (119899 = 6) RSD of system precisionwas 020 on the same day (intraday) and 036 on differentdays (interday) The mean values of assay and RSD formethod precision (repeatability) were 10035 and 043respectively for assay on same day (intraday) while 9972and 056 respectively for assay on different days (interday)

For assay of Cilnidipine (119899 = 6) RSD of system precisionwas 014 on the same day (intraday) and 022 on differentdays (interday) The mean values of assay and RSD

5000000

4000000

3000000

2000000

1000000

0

0 001 002 003 004 005

R2 = 09989

y = 1002943071429x + 213970357

Figure 6 Linearity curve for Cilnidipine

for method precision (repeatability) were 9983 and 086respectively for assay on the same day (intraday) while9993 and 045 respectively for assay on different days(interday)

Intermediate precision was established by determiningthe overall (intraday and interday) method precision Forintermediate precision (119899 = 12) overall assay and RSDvalue was 10004 and 058 respectively for Telmisartanwhile 9988 and 066 for CilnidipineThe precise result forcontent uniformity was indicative of uniform distribution ofthe drug in the tablets without significant variation this is inaccordance with the USP which stipulates acceptance limitsfor drug content uniformity and RSD as 85ndash115 and lt6respectively [19]

The accuracy of the method was assessed by determina-tion of recovery for three concentrations covering the rangeof the method Known amounts (50 100 and 150120583gmL) forTelmisartan and (125 250 and 375 120583gmL) for Cilnidipinewere added to a placebo preparation and the amount ofTelmisartan and Cilnidipine recovered in the presence ofplacebo interface was calculated The mean recovery ofTelmisartan was 9837 9809 and 9979 respectivelyand the mean recovery of Cilnidipine was 9968 9889and 9952 respectively (Table 1)

The robustness of the method was assessed by assayingtest solutions under different analytical conditions deliber-ately changed from the original conditions For each differentanalytical condition the standard solution and test solutionwere prepared separately The result obtained from assay ofthe test solution was not affected by varying the conditionsand was in accordance with the true value System suitabilitydata were also found to be satisfactory during variation ofthe analytical conditions (Table 2) The analytical methodtherefore remains unaffected by slight but deliberate changesin the analytical conditions

During the study of the stability of stored solutions ofstandards and test preparations for assay determination thesolutions were found to be stable for up to 48 hr Assay valuesobtained after 48 hr were statistically identical with the initialvalue without measurable loss (Table 3)

Before each measurement of validation data a systemsuitability test was performed by measurement of generalcharacteristics such as peak asymmetry number of the-oretical plates and RSD () of peak area observed for

ISRN Chromatography 5

Table 1 Evaluation data of accuracy study

Level () Theoretical concentrationa

(mgmL)Observed concentrationa

(mgmL) Recovery RSD

Telmisartan50 004998 004917 9837 168100 009995 009804 9809 058150 014993 014963 9979 120

Cilnidipine50 001253 001246 9969 049100 002505 002477 9889 013150 003752 003734 9952 015

aEach value corresponds to the mean of six determinations

Table 2 Evaluation data of robustness study

Robust conditions Assay System suitability parametersTheoretical plates Asymmetry

Flow 09mLmin 10031 3448 103Flow 11mLmin 9976 3372 098Buffer-ACN(34 66 vv) 9977 3397 102

Buffer-ACN(30 70 vv) 9976 3362 107

Column change 10037 3290 103

Table 3 Evaluation data of stability study

Intervals Assay for test solution

stored at 2ndash8∘C

Assay for test solutionstored at ambient

temperatureTelmisartan Cilnidipine Telmisartan Cilnidipine

Initial 9956 9971 9999 996612 h 9961 9985 10011 1001724 h 9954 9984 10014 997736 h 9998 9923 10018 994548 h 9974 10015 9989 10020

a standard solutionThe values obtainedwere satisfactory andin accordance with in-house limits (Tables 4 and 5)

4 Conclusion

This LC method for simultaneous assay validation of Telmis-artan and Cilnidipine in a tablet formulation was successfullydeveloped and validated for its intended purpose In thisstudy stability of Telmisartan and Cilnidipine in presentdosage form was established through employment of ICHrecommended stress condition The developed procedurehas been evaluated over the specificity linearity accuracyprecision and robustness in order to ascertain the stability ofthe analytical method It has been proved that it was specificlinear precise accurate robust and stability indicatingHence the method is recommended for routine qualitycontrol analysis and also stability sample analysis

Table 4 Evaluation data of system suitability study for Telmisartan

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 102 3628 124Linearity 053 3290 146Precision 020 3799 134Intermediateprecision 036 3730 138

Accuracy 083 3528 129Solution stability 016 3026 120Robustness 071 3413 145bMeans not more than and cmeans not less than

Table 5 Evaluation data of system suitability study for cilnidipine

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 026 10214 135Linearity 069 9823 123Precision 014 10195 129Intermediateprecision 022 10166 140

Accuracy 055 10081 138Solution stability 029 10372 115Robustness 062 10224 126bMeans not more than and cmeans not less than

Acknowledgments

The authors are thankful to Unique Chemicals Panoli Indiaand CPL Chemicals Ankleshvar India for providing Telmis-artan and Cilnidipine standard and facilities and Grantsgiven under UGC-Special Assistance Programme Depart-ment Research Support (DRS) and Department of Scienceamp Technology (DST) New Delhi Fund For Improvementof Science amp Technology (FIST) National Facility for DrugDiscovery (NFDD) and Department of Chemistry Saurash-tra University Rajkot-360 005 india for providing analyticalfacilities

6 ISRN Chromatography

References

[1] M RamaMohana Reddy A Praveen Kumar V Krishna Reddyand S Wasimul Haque ldquoStability-indicating hplc method forsimultaneous estimation of low level impurities of telmisartanand hydrochlorothiazide in tablet dosage formsrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 1pp 497ndash504 2012

[2] A Gupta R M Charde and M S Charde ldquoDeterminationof telmisartan and forced degradation behavior by RP-HPLCin tablet dosage formrdquo International Journal of PharmaceuticalChemistry vol 2 no 3 pp 93ndash99 2012

[3] M S Mohammed ldquoSpctrophotometric method for the estima-tion of cilnidipine in bulk and pharmaceutical dosage formsrdquoOriental Journal of Chemistry vol 29 no 1 pp 131ndash134 2013

[4] A S Rathore L Sathiyanarayanan and K R MahadikldquoStability-indicating high-performance thin-layer chromato-graphic method for quantitative estimation of emtricitabine inbulk drug and pharmaceutical dosage formrdquo ISRN Chromatog-raphy vol 2012 Article ID 278583 7 pages 2012

[5] P P Vekariya and H S Joshi ldquoDevelopment and validation ofRP-HPLCmethod for azilsartan medoxomil potassium quanti-tation in human plasma by solid phase extraction procedurerdquoISRN Spectroscopy vol 2013 Article ID 572170 6 pages 2013

[6] S Singh B Singh R Bahuguna L Wadhwa and R SaxenaldquoStress degradation studies on ezetimibe and development of avalidated stability-indicating HPLC assayrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 41 no 3 pp 1037ndash10402006

[7] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[8] C D Niranjan S M Patil J K Sabaji and A N ChivateldquoDevelopment of UV spectrophotometric method for estima-tion and validation of telmisartan as a pure APIrdquo Journal ofPharmacy Research vol 5 no 6 pp 3331ndash3333 2012

[9] K K Pradhan U S Mishra A Sahoo K C Sahu DMishra and R Dash ldquoMethod development and validation ofTelmisartan in bulk and pharmaceutical dosage forms by UVSpectrophotometric methodrdquo International Journal of Researchin Pharmaceutical Sciences vol 2 no 4 pp 526ndash530 2011

[10] G Mahathi Y Ravindra Reddy and P Uttam Prasad ldquoDevel-opment and validation of telmisartan and atorvastatin calciumin combined dosage form by RP-HPLCrdquo International Journalof Pharmacy and Technology vol 3 no 3 pp 3370ndash3389 2011

[11] A M Raja N Spoorthy D Banji K N V Rao C Vanita andS D Kumar ldquoSimultaneous estimaton of metroprolol succinateand telmisartan in tablet dosage form by RP-HPLCrdquo Journal ofPharmacy Research vol 5 no 8 pp 4585ndash4587 2012

[12] P Vani and S K Kalyana ldquoA rapid stability-indicating simul-taneous determenation of hydrochlorothiazide ramipril andtelmisartan in combined dosage form by ultra performanceliquid chromatographyrdquo Pharmacia Letter vol 5 no 3 pp 81ndash89 2013

[13] K E Parmar R S Mehta N D Patel and K E ParmarldquoDevelopment and validation of HPTLC method for simul-taneous determination of telmisartan and chlorthalidone inbulk and pharmaceutical dosage formrdquo International Journal ofPharmacy and Pharmaceutical Sciences vol 5 no 2 pp 420ndash425 2013

[14] H S Karmalkar V V Vaidya N A Gomes M P Choukekarand M B Kekare ldquoDetermination of cilnidipine from pharma-ceutical formulation by high performance thin layer chromato-graphicmethodrdquoAnalytical Chemistry vol 7 no 8 pp 573ndash5762008

[15] K-R Lee Y-J Chae J-H Lee et al ldquoQuantification ofcilnidipine in human plasma by liquid chromatography-massspectrometryrdquo Journal of Liquid Chromatography and RelatedTechnologies vol 35 no 2 pp 308ndash320 2012

[16] P P Chaudhari and A V Bhalerao ldquoMethod validation forspectrophotometric estimation of cilnidipinerdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 5pp 96ndash98 2012

[17] P Pawar S V Gandhi P B Deshpande B Padmanabh SVanjari and S U Shelar ldquoSimultaneous RP-HPLC estimationof cilnidipine and telmisartan in combined table dosage formrdquoChemica Sinica vol 4 no 2 pp 6ndash10 2013

[18] P Pawar P B Deshpande S Gandhi and V Bhavnani ldquoHighperformance hin layer chromatographic determenation of cilni-dipine and telmisartan in combined dosage formrdquo InternationalResearch Journal of Pharmacy vol 3 no 6 pp 219ndash222 2012

[19] G J Kher V R Ram K L Dubal A H Bapodara and HS Joshi ldquoValidation of a stability-indicating LC method forassay of leflunomide in tablets and for determination of contentuniformityrdquo International Journal of ChemTech Research vol 3no 2 pp 523ndash530 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 ISRN Chromatography

500

400

300

200

100

0

00 25 50 75 100 125 150 175 200 225 250 275

Telmisartan

Cilnidipine(mAU

)

245nm

(100)4nm

(min)

Figure 4 Chromatogram of alkali forced degradation study

R2 = 09990

12000000

10000000

8000000

6000000

4000000

2000000

00 005 01 015 02

y = 699636125000x + 1214036071

Figure 5 Linearity curve for Telmisartan

Major degradation was found in alkali condition thatproduct was degraded up to 574 for Telmisartan wheretwo degradation peaks were found at 3460 and 5786 whilefor Cilnidipine the product was degraded up to 3299 at12096min (Figure 4) There was no degradation found inacid 3 H

2O2 30 H

2O2 thermal and photo degradation

for both Telmisartan and CilnidipineTo determine linearity a calibration graph was obtained

by plotting Telmisartan and Cilnidipine concentration (mgmL) against peak area Linearity was good in the concen-tration range 40minus160 120583gmL for Telmisartan (Figure 5) and10ndash40 120583gmL for Cilnidipine (Figure 6) The response ofthe drug was found to be linear The regression equationfor Telmisartan was 119910 = 6996361250119909 + 12140361 withcorrelation coefficient 09990 and for Cilnidipine was 119910 =10029430714 + 2139704 with correlation coefficient 09989where 119909 is the concentration in 120583gmL and 119910 is the peak areain absorbance units (AU)

The LOD values for telmisartan and Cilnidipine werefound to be 001 ppm and 009 ppm respectively The LOQvalues for telmisartan and Cilnidipine were 006 ppm and04 ppm

For assay of Telmisartan (119899 = 6) RSD of system precisionwas 020 on the same day (intraday) and 036 on differentdays (interday) The mean values of assay and RSD formethod precision (repeatability) were 10035 and 043respectively for assay on same day (intraday) while 9972and 056 respectively for assay on different days (interday)

For assay of Cilnidipine (119899 = 6) RSD of system precisionwas 014 on the same day (intraday) and 022 on differentdays (interday) The mean values of assay and RSD

5000000

4000000

3000000

2000000

1000000

0

0 001 002 003 004 005

R2 = 09989

y = 1002943071429x + 213970357

Figure 6 Linearity curve for Cilnidipine

for method precision (repeatability) were 9983 and 086respectively for assay on the same day (intraday) while9993 and 045 respectively for assay on different days(interday)

Intermediate precision was established by determiningthe overall (intraday and interday) method precision Forintermediate precision (119899 = 12) overall assay and RSDvalue was 10004 and 058 respectively for Telmisartanwhile 9988 and 066 for CilnidipineThe precise result forcontent uniformity was indicative of uniform distribution ofthe drug in the tablets without significant variation this is inaccordance with the USP which stipulates acceptance limitsfor drug content uniformity and RSD as 85ndash115 and lt6respectively [19]

The accuracy of the method was assessed by determina-tion of recovery for three concentrations covering the rangeof the method Known amounts (50 100 and 150120583gmL) forTelmisartan and (125 250 and 375 120583gmL) for Cilnidipinewere added to a placebo preparation and the amount ofTelmisartan and Cilnidipine recovered in the presence ofplacebo interface was calculated The mean recovery ofTelmisartan was 9837 9809 and 9979 respectivelyand the mean recovery of Cilnidipine was 9968 9889and 9952 respectively (Table 1)

The robustness of the method was assessed by assayingtest solutions under different analytical conditions deliber-ately changed from the original conditions For each differentanalytical condition the standard solution and test solutionwere prepared separately The result obtained from assay ofthe test solution was not affected by varying the conditionsand was in accordance with the true value System suitabilitydata were also found to be satisfactory during variation ofthe analytical conditions (Table 2) The analytical methodtherefore remains unaffected by slight but deliberate changesin the analytical conditions

During the study of the stability of stored solutions ofstandards and test preparations for assay determination thesolutions were found to be stable for up to 48 hr Assay valuesobtained after 48 hr were statistically identical with the initialvalue without measurable loss (Table 3)

Before each measurement of validation data a systemsuitability test was performed by measurement of generalcharacteristics such as peak asymmetry number of the-oretical plates and RSD () of peak area observed for

ISRN Chromatography 5

Table 1 Evaluation data of accuracy study

Level () Theoretical concentrationa

(mgmL)Observed concentrationa

(mgmL) Recovery RSD

Telmisartan50 004998 004917 9837 168100 009995 009804 9809 058150 014993 014963 9979 120

Cilnidipine50 001253 001246 9969 049100 002505 002477 9889 013150 003752 003734 9952 015

aEach value corresponds to the mean of six determinations

Table 2 Evaluation data of robustness study

Robust conditions Assay System suitability parametersTheoretical plates Asymmetry

Flow 09mLmin 10031 3448 103Flow 11mLmin 9976 3372 098Buffer-ACN(34 66 vv) 9977 3397 102

Buffer-ACN(30 70 vv) 9976 3362 107

Column change 10037 3290 103

Table 3 Evaluation data of stability study

Intervals Assay for test solution

stored at 2ndash8∘C

Assay for test solutionstored at ambient

temperatureTelmisartan Cilnidipine Telmisartan Cilnidipine

Initial 9956 9971 9999 996612 h 9961 9985 10011 1001724 h 9954 9984 10014 997736 h 9998 9923 10018 994548 h 9974 10015 9989 10020

a standard solutionThe values obtainedwere satisfactory andin accordance with in-house limits (Tables 4 and 5)

4 Conclusion

This LC method for simultaneous assay validation of Telmis-artan and Cilnidipine in a tablet formulation was successfullydeveloped and validated for its intended purpose In thisstudy stability of Telmisartan and Cilnidipine in presentdosage form was established through employment of ICHrecommended stress condition The developed procedurehas been evaluated over the specificity linearity accuracyprecision and robustness in order to ascertain the stability ofthe analytical method It has been proved that it was specificlinear precise accurate robust and stability indicatingHence the method is recommended for routine qualitycontrol analysis and also stability sample analysis

Table 4 Evaluation data of system suitability study for Telmisartan

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 102 3628 124Linearity 053 3290 146Precision 020 3799 134Intermediateprecision 036 3730 138

Accuracy 083 3528 129Solution stability 016 3026 120Robustness 071 3413 145bMeans not more than and cmeans not less than

Table 5 Evaluation data of system suitability study for cilnidipine

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 026 10214 135Linearity 069 9823 123Precision 014 10195 129Intermediateprecision 022 10166 140

Accuracy 055 10081 138Solution stability 029 10372 115Robustness 062 10224 126bMeans not more than and cmeans not less than

Acknowledgments

The authors are thankful to Unique Chemicals Panoli Indiaand CPL Chemicals Ankleshvar India for providing Telmis-artan and Cilnidipine standard and facilities and Grantsgiven under UGC-Special Assistance Programme Depart-ment Research Support (DRS) and Department of Scienceamp Technology (DST) New Delhi Fund For Improvementof Science amp Technology (FIST) National Facility for DrugDiscovery (NFDD) and Department of Chemistry Saurash-tra University Rajkot-360 005 india for providing analyticalfacilities

6 ISRN Chromatography

References

[1] M RamaMohana Reddy A Praveen Kumar V Krishna Reddyand S Wasimul Haque ldquoStability-indicating hplc method forsimultaneous estimation of low level impurities of telmisartanand hydrochlorothiazide in tablet dosage formsrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 1pp 497ndash504 2012

[2] A Gupta R M Charde and M S Charde ldquoDeterminationof telmisartan and forced degradation behavior by RP-HPLCin tablet dosage formrdquo International Journal of PharmaceuticalChemistry vol 2 no 3 pp 93ndash99 2012

[3] M S Mohammed ldquoSpctrophotometric method for the estima-tion of cilnidipine in bulk and pharmaceutical dosage formsrdquoOriental Journal of Chemistry vol 29 no 1 pp 131ndash134 2013

[4] A S Rathore L Sathiyanarayanan and K R MahadikldquoStability-indicating high-performance thin-layer chromato-graphic method for quantitative estimation of emtricitabine inbulk drug and pharmaceutical dosage formrdquo ISRN Chromatog-raphy vol 2012 Article ID 278583 7 pages 2012

[5] P P Vekariya and H S Joshi ldquoDevelopment and validation ofRP-HPLCmethod for azilsartan medoxomil potassium quanti-tation in human plasma by solid phase extraction procedurerdquoISRN Spectroscopy vol 2013 Article ID 572170 6 pages 2013

[6] S Singh B Singh R Bahuguna L Wadhwa and R SaxenaldquoStress degradation studies on ezetimibe and development of avalidated stability-indicating HPLC assayrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 41 no 3 pp 1037ndash10402006

[7] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[8] C D Niranjan S M Patil J K Sabaji and A N ChivateldquoDevelopment of UV spectrophotometric method for estima-tion and validation of telmisartan as a pure APIrdquo Journal ofPharmacy Research vol 5 no 6 pp 3331ndash3333 2012

[9] K K Pradhan U S Mishra A Sahoo K C Sahu DMishra and R Dash ldquoMethod development and validation ofTelmisartan in bulk and pharmaceutical dosage forms by UVSpectrophotometric methodrdquo International Journal of Researchin Pharmaceutical Sciences vol 2 no 4 pp 526ndash530 2011

[10] G Mahathi Y Ravindra Reddy and P Uttam Prasad ldquoDevel-opment and validation of telmisartan and atorvastatin calciumin combined dosage form by RP-HPLCrdquo International Journalof Pharmacy and Technology vol 3 no 3 pp 3370ndash3389 2011

[11] A M Raja N Spoorthy D Banji K N V Rao C Vanita andS D Kumar ldquoSimultaneous estimaton of metroprolol succinateand telmisartan in tablet dosage form by RP-HPLCrdquo Journal ofPharmacy Research vol 5 no 8 pp 4585ndash4587 2012

[12] P Vani and S K Kalyana ldquoA rapid stability-indicating simul-taneous determenation of hydrochlorothiazide ramipril andtelmisartan in combined dosage form by ultra performanceliquid chromatographyrdquo Pharmacia Letter vol 5 no 3 pp 81ndash89 2013

[13] K E Parmar R S Mehta N D Patel and K E ParmarldquoDevelopment and validation of HPTLC method for simul-taneous determination of telmisartan and chlorthalidone inbulk and pharmaceutical dosage formrdquo International Journal ofPharmacy and Pharmaceutical Sciences vol 5 no 2 pp 420ndash425 2013

[14] H S Karmalkar V V Vaidya N A Gomes M P Choukekarand M B Kekare ldquoDetermination of cilnidipine from pharma-ceutical formulation by high performance thin layer chromato-graphicmethodrdquoAnalytical Chemistry vol 7 no 8 pp 573ndash5762008

[15] K-R Lee Y-J Chae J-H Lee et al ldquoQuantification ofcilnidipine in human plasma by liquid chromatography-massspectrometryrdquo Journal of Liquid Chromatography and RelatedTechnologies vol 35 no 2 pp 308ndash320 2012

[16] P P Chaudhari and A V Bhalerao ldquoMethod validation forspectrophotometric estimation of cilnidipinerdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 5pp 96ndash98 2012

[17] P Pawar S V Gandhi P B Deshpande B Padmanabh SVanjari and S U Shelar ldquoSimultaneous RP-HPLC estimationof cilnidipine and telmisartan in combined table dosage formrdquoChemica Sinica vol 4 no 2 pp 6ndash10 2013

[18] P Pawar P B Deshpande S Gandhi and V Bhavnani ldquoHighperformance hin layer chromatographic determenation of cilni-dipine and telmisartan in combined dosage formrdquo InternationalResearch Journal of Pharmacy vol 3 no 6 pp 219ndash222 2012

[19] G J Kher V R Ram K L Dubal A H Bapodara and HS Joshi ldquoValidation of a stability-indicating LC method forassay of leflunomide in tablets and for determination of contentuniformityrdquo International Journal of ChemTech Research vol 3no 2 pp 523ndash530 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

ISRN Chromatography 5

Table 1 Evaluation data of accuracy study

Level () Theoretical concentrationa

(mgmL)Observed concentrationa

(mgmL) Recovery RSD

Telmisartan50 004998 004917 9837 168100 009995 009804 9809 058150 014993 014963 9979 120

Cilnidipine50 001253 001246 9969 049100 002505 002477 9889 013150 003752 003734 9952 015

aEach value corresponds to the mean of six determinations

Table 2 Evaluation data of robustness study

Robust conditions Assay System suitability parametersTheoretical plates Asymmetry

Flow 09mLmin 10031 3448 103Flow 11mLmin 9976 3372 098Buffer-ACN(34 66 vv) 9977 3397 102

Buffer-ACN(30 70 vv) 9976 3362 107

Column change 10037 3290 103

Table 3 Evaluation data of stability study

Intervals Assay for test solution

stored at 2ndash8∘C

Assay for test solutionstored at ambient

temperatureTelmisartan Cilnidipine Telmisartan Cilnidipine

Initial 9956 9971 9999 996612 h 9961 9985 10011 1001724 h 9954 9984 10014 997736 h 9998 9923 10018 994548 h 9974 10015 9989 10020

a standard solutionThe values obtainedwere satisfactory andin accordance with in-house limits (Tables 4 and 5)

4 Conclusion

This LC method for simultaneous assay validation of Telmis-artan and Cilnidipine in a tablet formulation was successfullydeveloped and validated for its intended purpose In thisstudy stability of Telmisartan and Cilnidipine in presentdosage form was established through employment of ICHrecommended stress condition The developed procedurehas been evaluated over the specificity linearity accuracyprecision and robustness in order to ascertain the stability ofthe analytical method It has been proved that it was specificlinear precise accurate robust and stability indicatingHence the method is recommended for routine qualitycontrol analysis and also stability sample analysis

Table 4 Evaluation data of system suitability study for Telmisartan

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 102 3628 124Linearity 053 3290 146Precision 020 3799 134Intermediateprecision 036 3730 138

Accuracy 083 3528 129Solution stability 016 3026 120Robustness 071 3413 145bMeans not more than and cmeans not less than

Table 5 Evaluation data of system suitability study for cilnidipine

System suitability data RSD () Theoreticalplates Asymmetry

In-house limit NMTb 20 NLTc 2000 NMTb 20Specificity 026 10214 135Linearity 069 9823 123Precision 014 10195 129Intermediateprecision 022 10166 140

Accuracy 055 10081 138Solution stability 029 10372 115Robustness 062 10224 126bMeans not more than and cmeans not less than

Acknowledgments

The authors are thankful to Unique Chemicals Panoli Indiaand CPL Chemicals Ankleshvar India for providing Telmis-artan and Cilnidipine standard and facilities and Grantsgiven under UGC-Special Assistance Programme Depart-ment Research Support (DRS) and Department of Scienceamp Technology (DST) New Delhi Fund For Improvementof Science amp Technology (FIST) National Facility for DrugDiscovery (NFDD) and Department of Chemistry Saurash-tra University Rajkot-360 005 india for providing analyticalfacilities

6 ISRN Chromatography

References

[1] M RamaMohana Reddy A Praveen Kumar V Krishna Reddyand S Wasimul Haque ldquoStability-indicating hplc method forsimultaneous estimation of low level impurities of telmisartanand hydrochlorothiazide in tablet dosage formsrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 1pp 497ndash504 2012

[2] A Gupta R M Charde and M S Charde ldquoDeterminationof telmisartan and forced degradation behavior by RP-HPLCin tablet dosage formrdquo International Journal of PharmaceuticalChemistry vol 2 no 3 pp 93ndash99 2012

[3] M S Mohammed ldquoSpctrophotometric method for the estima-tion of cilnidipine in bulk and pharmaceutical dosage formsrdquoOriental Journal of Chemistry vol 29 no 1 pp 131ndash134 2013

[4] A S Rathore L Sathiyanarayanan and K R MahadikldquoStability-indicating high-performance thin-layer chromato-graphic method for quantitative estimation of emtricitabine inbulk drug and pharmaceutical dosage formrdquo ISRN Chromatog-raphy vol 2012 Article ID 278583 7 pages 2012

[5] P P Vekariya and H S Joshi ldquoDevelopment and validation ofRP-HPLCmethod for azilsartan medoxomil potassium quanti-tation in human plasma by solid phase extraction procedurerdquoISRN Spectroscopy vol 2013 Article ID 572170 6 pages 2013

[6] S Singh B Singh R Bahuguna L Wadhwa and R SaxenaldquoStress degradation studies on ezetimibe and development of avalidated stability-indicating HPLC assayrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 41 no 3 pp 1037ndash10402006

[7] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[8] C D Niranjan S M Patil J K Sabaji and A N ChivateldquoDevelopment of UV spectrophotometric method for estima-tion and validation of telmisartan as a pure APIrdquo Journal ofPharmacy Research vol 5 no 6 pp 3331ndash3333 2012

[9] K K Pradhan U S Mishra A Sahoo K C Sahu DMishra and R Dash ldquoMethod development and validation ofTelmisartan in bulk and pharmaceutical dosage forms by UVSpectrophotometric methodrdquo International Journal of Researchin Pharmaceutical Sciences vol 2 no 4 pp 526ndash530 2011

[10] G Mahathi Y Ravindra Reddy and P Uttam Prasad ldquoDevel-opment and validation of telmisartan and atorvastatin calciumin combined dosage form by RP-HPLCrdquo International Journalof Pharmacy and Technology vol 3 no 3 pp 3370ndash3389 2011

[11] A M Raja N Spoorthy D Banji K N V Rao C Vanita andS D Kumar ldquoSimultaneous estimaton of metroprolol succinateand telmisartan in tablet dosage form by RP-HPLCrdquo Journal ofPharmacy Research vol 5 no 8 pp 4585ndash4587 2012

[12] P Vani and S K Kalyana ldquoA rapid stability-indicating simul-taneous determenation of hydrochlorothiazide ramipril andtelmisartan in combined dosage form by ultra performanceliquid chromatographyrdquo Pharmacia Letter vol 5 no 3 pp 81ndash89 2013

[13] K E Parmar R S Mehta N D Patel and K E ParmarldquoDevelopment and validation of HPTLC method for simul-taneous determination of telmisartan and chlorthalidone inbulk and pharmaceutical dosage formrdquo International Journal ofPharmacy and Pharmaceutical Sciences vol 5 no 2 pp 420ndash425 2013

[14] H S Karmalkar V V Vaidya N A Gomes M P Choukekarand M B Kekare ldquoDetermination of cilnidipine from pharma-ceutical formulation by high performance thin layer chromato-graphicmethodrdquoAnalytical Chemistry vol 7 no 8 pp 573ndash5762008

[15] K-R Lee Y-J Chae J-H Lee et al ldquoQuantification ofcilnidipine in human plasma by liquid chromatography-massspectrometryrdquo Journal of Liquid Chromatography and RelatedTechnologies vol 35 no 2 pp 308ndash320 2012

[16] P P Chaudhari and A V Bhalerao ldquoMethod validation forspectrophotometric estimation of cilnidipinerdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 5pp 96ndash98 2012

[17] P Pawar S V Gandhi P B Deshpande B Padmanabh SVanjari and S U Shelar ldquoSimultaneous RP-HPLC estimationof cilnidipine and telmisartan in combined table dosage formrdquoChemica Sinica vol 4 no 2 pp 6ndash10 2013

[18] P Pawar P B Deshpande S Gandhi and V Bhavnani ldquoHighperformance hin layer chromatographic determenation of cilni-dipine and telmisartan in combined dosage formrdquo InternationalResearch Journal of Pharmacy vol 3 no 6 pp 219ndash222 2012

[19] G J Kher V R Ram K L Dubal A H Bapodara and HS Joshi ldquoValidation of a stability-indicating LC method forassay of leflunomide in tablets and for determination of contentuniformityrdquo International Journal of ChemTech Research vol 3no 2 pp 523ndash530 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 ISRN Chromatography

References

[1] M RamaMohana Reddy A Praveen Kumar V Krishna Reddyand S Wasimul Haque ldquoStability-indicating hplc method forsimultaneous estimation of low level impurities of telmisartanand hydrochlorothiazide in tablet dosage formsrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 1pp 497ndash504 2012

[2] A Gupta R M Charde and M S Charde ldquoDeterminationof telmisartan and forced degradation behavior by RP-HPLCin tablet dosage formrdquo International Journal of PharmaceuticalChemistry vol 2 no 3 pp 93ndash99 2012

[3] M S Mohammed ldquoSpctrophotometric method for the estima-tion of cilnidipine in bulk and pharmaceutical dosage formsrdquoOriental Journal of Chemistry vol 29 no 1 pp 131ndash134 2013

[4] A S Rathore L Sathiyanarayanan and K R MahadikldquoStability-indicating high-performance thin-layer chromato-graphic method for quantitative estimation of emtricitabine inbulk drug and pharmaceutical dosage formrdquo ISRN Chromatog-raphy vol 2012 Article ID 278583 7 pages 2012

[5] P P Vekariya and H S Joshi ldquoDevelopment and validation ofRP-HPLCmethod for azilsartan medoxomil potassium quanti-tation in human plasma by solid phase extraction procedurerdquoISRN Spectroscopy vol 2013 Article ID 572170 6 pages 2013

[6] S Singh B Singh R Bahuguna L Wadhwa and R SaxenaldquoStress degradation studies on ezetimibe and development of avalidated stability-indicating HPLC assayrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 41 no 3 pp 1037ndash10402006

[7] M Bakshi and S Singh ldquoDevelopment of validated stability-indicating assay methodsmdashcritical reviewrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 28 no 6 pp 1011ndash10402002

[8] C D Niranjan S M Patil J K Sabaji and A N ChivateldquoDevelopment of UV spectrophotometric method for estima-tion and validation of telmisartan as a pure APIrdquo Journal ofPharmacy Research vol 5 no 6 pp 3331ndash3333 2012

[9] K K Pradhan U S Mishra A Sahoo K C Sahu DMishra and R Dash ldquoMethod development and validation ofTelmisartan in bulk and pharmaceutical dosage forms by UVSpectrophotometric methodrdquo International Journal of Researchin Pharmaceutical Sciences vol 2 no 4 pp 526ndash530 2011

[10] G Mahathi Y Ravindra Reddy and P Uttam Prasad ldquoDevel-opment and validation of telmisartan and atorvastatin calciumin combined dosage form by RP-HPLCrdquo International Journalof Pharmacy and Technology vol 3 no 3 pp 3370ndash3389 2011

[11] A M Raja N Spoorthy D Banji K N V Rao C Vanita andS D Kumar ldquoSimultaneous estimaton of metroprolol succinateand telmisartan in tablet dosage form by RP-HPLCrdquo Journal ofPharmacy Research vol 5 no 8 pp 4585ndash4587 2012

[12] P Vani and S K Kalyana ldquoA rapid stability-indicating simul-taneous determenation of hydrochlorothiazide ramipril andtelmisartan in combined dosage form by ultra performanceliquid chromatographyrdquo Pharmacia Letter vol 5 no 3 pp 81ndash89 2013

[13] K E Parmar R S Mehta N D Patel and K E ParmarldquoDevelopment and validation of HPTLC method for simul-taneous determination of telmisartan and chlorthalidone inbulk and pharmaceutical dosage formrdquo International Journal ofPharmacy and Pharmaceutical Sciences vol 5 no 2 pp 420ndash425 2013

[14] H S Karmalkar V V Vaidya N A Gomes M P Choukekarand M B Kekare ldquoDetermination of cilnidipine from pharma-ceutical formulation by high performance thin layer chromato-graphicmethodrdquoAnalytical Chemistry vol 7 no 8 pp 573ndash5762008

[15] K-R Lee Y-J Chae J-H Lee et al ldquoQuantification ofcilnidipine in human plasma by liquid chromatography-massspectrometryrdquo Journal of Liquid Chromatography and RelatedTechnologies vol 35 no 2 pp 308ndash320 2012

[16] P P Chaudhari and A V Bhalerao ldquoMethod validation forspectrophotometric estimation of cilnidipinerdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 4 no 5pp 96ndash98 2012

[17] P Pawar S V Gandhi P B Deshpande B Padmanabh SVanjari and S U Shelar ldquoSimultaneous RP-HPLC estimationof cilnidipine and telmisartan in combined table dosage formrdquoChemica Sinica vol 4 no 2 pp 6ndash10 2013

[18] P Pawar P B Deshpande S Gandhi and V Bhavnani ldquoHighperformance hin layer chromatographic determenation of cilni-dipine and telmisartan in combined dosage formrdquo InternationalResearch Journal of Pharmacy vol 3 no 6 pp 219ndash222 2012

[19] G J Kher V R Ram K L Dubal A H Bapodara and HS Joshi ldquoValidation of a stability-indicating LC method forassay of leflunomide in tablets and for determination of contentuniformityrdquo International Journal of ChemTech Research vol 3no 2 pp 523ndash530 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of