International Standard Serial Number (ISSN): 2249-6793

International Journal of Universal Pharmacy and Life Sciences 3(6): November-December 2013

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND LIFE SCIENCES
Pharmaceutical Sciences Research Article……!!!

Received: 19-10-2013; Revised; Accepted: 23-11-2013

GRADIENT RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF
AMLODIPINE AND CANDESARTAN IN TABLETS
R. Kalaichelvi1*, G. Srilakshmi1 and E. Jayachandran2
1. K.C.Reddy Institute of Pharmaceutical Sciences, Jangamguntla Palem, Medikonduru Mandal, Guntur-
522 438, India.
2. S.C.S. College of Pharmacy, Harapanahalli-583131. Devanagari Dist, Karnataka, India.
Keywords: ABSTRACT
Amlodipine, candesartan, A simple, rapid and precise gradient RP-HPLC method is
RP-HPLC developed and validated for the simultaneous estimation of
amlodipine and candesartan in pure drug and combined solid
For Correspondence: dosage form. The method is based on High Performance Liquid
R. Kalaichelvi Chromatography on a reversed-phase column Phenomenex Luna,
C18, 150 × 4.6 mm, 5 μm, using a mobile phase of 0.1% ortho
K.C.Reddy Institute of
phosphoric acid (pH 3.0) and acetonitrile in gradient programme.
Pharmaceutical Sciences,
The flow rate was 1.0 mL/min, column temperature was 30˚C and
Jangamguntla Palem,
detector wavelength was 230 nm. Both the drugs were well
Medikonduru Mandal, Guntur-
resolved on the stationary phase and the retention time was around
522 438, India
2.67 minute for amlodipine and 7.73 minute for candesartan. The
E-mail:
method validation carried out by performing precision, linearity,
rkselvi1983@gmail.com specificity and robustness parameters. The linear calibration range
was found to be 12.5 to 75 μg/mL for amlodipine and 20 to 120
μg/mL for candesartan. The proposed method has been
successfully applied to the pharmaceutical dosage form containing
the above-mentioned drugs in combination without any
interference of the excipients.

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 International Standard Serial Number (ISSN): 2249-6793

INTRODUCTION
Amlodipine1 is chemically 3-ethyl 5-methyl 2- [(2-aminoethoxy) methyl]-4-(2-
chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate,is a calcium channel blocking
agent. It inhibits the influx of extracellular calcium across the myocardial and vascular
smooth muscle cell membranes. The decrease in extracellular calcium inhibits the contractile
processes of the myocardial smooth muscle cells, causing dilation of the coronary and
systemic arteries, increased oxygen delivery to the myocardial tissue, decreased total
peripheral resistance, decreased systemic blood pressure, and decreased after load.
Amlodipine occupies the plasma membrane dihydropyridine receptor and causes competitive
blockade of the voltage- operated slow calcium channel. Candesartan2 (CDS), 1-
[[(cyclohexyloxy) carbonyl]oxy]ethyl 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-
yl]methyl]-1H-benzimidazole-7-carboxylate is an angiotensin II receptor antagonist, selective
for AT1 receptors, with tight binding to and slow dissociation from the receptor. The
literature survey reveals that, there have been several publications describing analytical
methods for the determination of AML3-10 and CDS11-18 individually or with other drugs as
combination. Based on literature review the present work aims to develop and validate
simple, fast, accurate and specific reversed phase high performance liquid chromatographic
method for simultaneous determination of AML and CDS in pure drugs and tablet
formulation.
MATERIALS AND METHODS
Instrumentation
Waters 2695 HPLC system equipped with Phenomenex luna, C18, 150 × 4.6 mm, 5µm,
column, Rheodyne injector with 10 µL loop, 2996 PDA detector and Empower-2 software
was used. The pH meter and sonicator and filtration unit also used for the filtrating the
solutions.
Materials
Acetonitrile, methanol and water were of HPLC grade and were purchased from s.d fine-
chem Limited, Mumbai, India. Orthophosphoric acid was of analytical-reagent grade and
purchased from Thermo fisher Scientific india pvt.Ltd, Mumbai, India. Tablets for the study
were purchased from pharmacy. Each tablet was labeled to contain 8 mg CDS and 5mg of
AML.

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 Intternationall Standard Serial Num
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93

EXPERIIMENTAL METHOD
Chroma
atographic Conditions
C
hod is based on High Performance L
The meth Liquid Chrom
matography on a reverseed-phase OD
DS
(C18), 150
1 mm × 4.6 mm, 5 μm coluumn, using a mobile phase
p soluttion of 0.1%
%
orthophoosphoric acidd solution pH
H 3.0, and aacetonitrile in
n gradient pprogramme. The flow raate
was 1.0 ml/min,
m colu
umn temperaature at 30˚C
C and detectoor wavelengtth of 230 nm
m.
Mobile Phase
P
The mob
bile phase was prepared by dissolvinng 1ml orthoo phosphoricc acid in litttle quantity oof
HPLC grrade water and
a volume made upto 1000 ml byy the same ppH 3.0. Acetonitrile waas
filtered and
a degassedd. The two soolvents weree supplied in
n gradient proogramme.
Preparattion of Stan
ndard solutiions:
Accurateely weighed and transferrred 40 mg oof CDS and 25 mg of A
AML standarrds into a 1000
ml clean dry volumettric flask, addd 70 ml of m
methanol, soonicated for 5 minutes an
nd make up to
t
the final volume withh 100 ml off methanol. F
From this soolution 0.5, 11.0, 1.5, 2.0,, 2.5 and 3 ml
m
solution was
w taken annd further diiluted to 10 m
ml with the same methanol. This givves 12.5 to 75
7
μg/mL for
f AML annd 20 to 1220 μg/mL foor CDS. Th
he typical chhromatogram
ms for blankk,
standard and sample are given inn Fig: 1, 2 annd Fig: 3.

Fig.. 1: Typical chromatogra

c am showing no interference of blankk for AML annd CDS.

Fig. 2: Typical
T chro
omatogram oof standard showing
s AM
ML and CDS..

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 Intternationall Standard Serial Num
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Fig. 3: Typical chromatogram of sample shhowing AML

L and CDS.
System Suitability
S T
Tests
System suitability
s waas verified by
b injecting standard sollution. The ppercent RSD
D was verifieed
from the replicate injjections of sttandard soluution. The ressults are given in Table 1.
Table 1: System suiitability parrameters forr AML and CDS by prooposed method
Parameterss A
AML CDS
Theoretical Plates 7994 114341
Reten
ntion Time (min) 2.67 7.733
Asym
mmetry 1.32 1.222
Estimatiion of AML and CDS from
fr Tablett Formulatioon
wenty tablets were weighhed accuratelly and finelyy powered. Tablet
Randomlly picked tw T powdeer
equivalen
nt to 4 mg of
o CDS and 2.5
2 mg of A
AML were weighed
w and placed into clean and drry
10 mL vo
olumetric flaask. The pow
wder was firrst dissolvedd in sufficiennt volume off methanol bby
sonicatioon and the vo
olume was made
m to10 m
mL with sam
me. Further, filtered
f throuugh Whatmaan
0.45μ filtter paper an
nd 1mL of reesultant soluution was diiluted methaanol. The sam
mple solutioon
(10 μL) was injectedd and the chhromatogram
m was recordded. The peeak area of the
t drugs waas
calculateed and the am
mount of eacch drug preseent per tablett was estimaated.
METHO
OD VALIDA
ATION
The deveeloped metho
od was validdated by evaaluating speccificity, lineaarity, precisiion, accuracyy,
limit of detection (L
LOD), limit of quantificcation (LOQ
Q), robustness and systeem suitabilitty
parameteers in accord
dance with thhe ICH guideeline.
Linearityy and Rang
ge:
Linearityy was determ
mined by plootting the staandard curvee in the concentration rannge of 12.5 to
t
75 μg/m
mL for AML
L and 20 to mL for CDS. The linearrity of the methods waas
o 120 μg/m
evaluatedd by linear reegression an
nalysis, usingg least squarre method.

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 International Standard Serial Number (ISSN): 2249-6793

Accuracy
This parameter is performed to determine the closeness of test results with that of the true
value which is expressed as % recovery. These studies were performed at three different
levels (50%, 100% and 150%) and the % recovery of AML and CDS was calculated.
Precision
The injection repeatability was established by six replicate injections of the standard solution
containing both the analytes of study. The sample repeatability was established by
performing the analysis six times.
Specificity
The chromatogram produced by standard drug solution and sample solution of test
concentration were compared at the retention time of the drugs.
Limit of Detection and Limit of Quantitation
The limit of detection (LOD) and the limit of quantitation (LOQ) for AML and CDS were
determined from standard deviation of the response and the slope.
RESULTS
Method development
A variety of mobile phases were investigated in the development of HPLC method for the
analysis of AML and CDS in tablet dosage form. The suitability of mobile phase was decided
on the basis of selectivity and sensitivity of the assay. The maximum absorption wavelength
of the reference drug solution was found to be 230 nm. This was observed from the UV
absorption spectra and was selected as detection wavelength for LC analysis. During the
optimization of method, the chromatographic conditions were fixed by trial and error method.
Method validation
The calibration plot for the method was linear over the concentration range of 12.5 to 75
μg/mL for AML and 20 to 120 μg/mL for CDS. The linearity data for the developed method
is given in Table 2. The determination of coefficient (r2) was 0.999 for both drugs. Assay
results for tablets are in Table 3. Accuracy was calculated by recovery studies for AML and
CDS at three levels and presented in Table 3.
Table 2. Linearity study for AML and CDS by proposed method
S. no. AML CDS
Concentration Peak Area Concentration Peak Area
(µg/ml) (µg/ml)
1 12.5 689130 20 487532
2 25 1256712 40 955476
3 37.5 1937389 60 1501282
4 50 2511924 80 2023565
5 62.5 3132313 100 2507087
6 75 3744708 120 3010334

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Table 3: Recovery studies, assay and precision for AML and CDS by proposed method
Drug Labeled amount (%) label claim % Recovery Precision
(mg/ tablet) ± S.D (% RSD) (n=6)

AML 5 99.96 ± 0.2351 99.14-100.86 0.7069

CDS 8 99.98 ± 0.3574 99.07-101.81 0.9028
For precision and intermediate precision, % RSD of AML and CDS were within 2.0% thus
confirming good precision of the analytical method development. In Specificity there was no
any interference at the retention time of AML and CDS in the chromatogram of sample and
standard solution. Robustness of the method was performed by making deliberate changes in
flow rate and composition of mobile phase and it was by calculating established % RSD
values and was within acceptance criteria range of 2.0%.
CONCLUSION
The developed HPLC technique is precise, specific and accurate. Statistical analysis proves
that the method is suitable for the analysis of both AML and CDS in pure and pharmaceutical
formulation without any interference from the excipients. The method has been found to be
better than previously reported methods, because of use of a less economical and readily
available mobile phase, no internal standard. All these factors make this method suitable for
quantification of AML and CDS in pure drugs and in pharmaceutical dosage form.
ACKNOWLEDGEMENT
The authors express their sincere thanks to the management, K.C.Reddy Institute of
Pharmaceutical Sciences, Guntur, for providing the necessary facilities to carry out the
research work.
REFERENCES
1. http://www.drugbank.ca/drugs/DB00381.
2. The Merck Index, 14th ed., Whitehouse Station, NJ: Merck Research Laboratories
3. Alnowaiser M.A., Simultaneous determination of Amlodipine and atorvastatin in caduet
tablets using HPLC, American Journal of Applied Sciences, 2013; Vol. 10(8): 849-852.
4. Wankhede S.B., Chitlange S.S., Spectrophotometric and HPLC Methods for Simultaneous
Estimation of Amlodipine Besilate, Losartan Potassium and Hydrochlorothiazide in Tablets,
Indian Journal of Pharmaceutical Sciences, 2010; Vol. 72(1): 136-140.
5. Chaudhari B.G., Patel N.M., and Shah P.B., Stability Indicating RP-HPLC Method for
Simultaneous Determination of Atorvastatin and Amlodipine from Their Combination Drug
Products, Chemical and Pharmaceutical Bulletin, 2007; Vol. 55(2): 241-246.

20  Full Text Available On www.ijupls.com

 International Standard Serial Number (ISSN): 2249-6793

6. Kumar G.V.S., Development and Validation of RP-HPLC Method for Simultaneous

Estimation of Amlodipine and Indapamide in Bulk and Tablet Dosage Form, Asian Journal of
Biomedical and Pharmaceutical Sciences, 2012; Vol 2(12): 55-59.
7. Hussain S., Munjewar R.R., Farooqui M., Development and Validation of a Simultaneous
HPLC Method for Quantification of Amlodipine Besylate and Metoprolol Tartrate in Tablets,
Journal of PharmaSciTech, 2012; Vol. 1(2):1-5.
8. Patil A.E., Patil S.V., Bari M.M., Barhate S.D., Validated RP-HPLC method for
simultaneous estimation of amlodipine besylate and clopidogrel bisulphate in bulk and tablet
dosage form, International Journal of Bioassays, 2013; Vol. 02 (02): 412-415.
9. Inglot T., Mączka P., Rutkowska E., New HPLC Method with Experimental Design and
Fluorescence Detection for Analytical Study of Antihypertensive Mixture, Amlodipine and
Valsartan, American Journal of Analytical Chemistry, 2013; Vol. 4, 17-23.
10. Patel G., Patel S., Prajapati D., and Mehta R., RP-HPLC Method for Simultaneous
Estimation of Amlodipine Besylate and Hydrochlorothiazide in Combined Dosage Forms,
Stamford Journal of Pharmaceutical Sciences, 2010; Vol. 3(1): 49-53.
11. Annapurna M.M., Ravi Kumar K, liquid chromatographic method for the simultaneous
quantitative determination of candesartan cilexetil and hydrochlorthiazide in pharmaceutical
dosage forms, Journal of Drug Delivery and Therapeutics, 2012; Vol. 2(2): 48-54.
12. Qutab S.S., Razzaq S.N., Ashfaq M., Shuja Z.A., and Khan I.U., Simple and sensitive
LC–UV method for simultaneous analysis of hydrochlorothiazide and candesartan cilexetil in
pharmaceutical formulations, Acta Chromatographica, 2007; Vol. 19: 119-129.
13. Balamuralikrishna K., Development and validation of high performance liquid
chromatographic method for Simultaneous Estimation of Candesartan-Hydrochlorothiazide
in combined tablet dosage form, Der Pharma Chemica, 2010; Vol. 2(6): 231-237.
14. Erk N., Simultaneous Analysis of Candesartan Cilexetil and Hydrochlorothiazide in
Human Plasma and Dosage Forms Using HPLC with a Photodiode Array Detector, Journal of
Liquid Chromatography and Related Technologies, 2003; Vol. 26(15): 2581-2591.
15. Devanaboyina N., Ganga Rao B., Simultaneous Determination of Candesartan and
Hydrochlorothiazide in combined Pharmaceutical Dosage form by New RP-HPLC Method,
Research Journal of Pharmaceutical, Biological Sciences, 2012; Vol. 3(1): 270-278.
16. Rao B.A., and Surya Kumar T.A.D., Development and validation of a RP-HPLC method
for estimation of hydrochlorothiazide and candesartan cilexetil in pharmaceutical dosage
form, The International Journal of Pharmaceutics, 2013; Vol. 3(1): 166-169.

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 International Standard Serial Number (ISSN): 2249-6793

17. Sunandamma Y., Single RP-HPLC method for the quantification of candesartan and
hydrochlorothiazide in formulations, The Experiment, 2013; Vol. 7(4): 428-437.
18. Mary Sudha T., Subba Rao G., Vineetha P., Spandana K., Tasleem S.K., Ashapaul B.,
Simulataneous estimation and validation of candesartan and hydrochlorthiazide in tablet
dosage form by RP-HPLC method, An International Journal of Advances in Pharmaceutical
Sciences, 2012; Vol. 3(5): 325-332.

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