WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Konidala et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 3, 1962-1972.

Research Article

ISSN 2278 4357

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR

THE SIMULTANEOUS DETERMINATION OF AMLODIPINE
BESYLATE AND TELMISARTAN IN BULK AND
PHARMACEUTICAL DOSAGE FORMS
Sathish Kumar Konidala*, Santosh Kumar Vobbilireddi, Risy Namratha
Jammulamudi, Sujana K1 and Dr. A. Prameela Rani2
1

Asst. Professor, Department of Pharmaceutical Analysis, University College of

Pharmaceutical Sciences.

Principal, University College of Pharmaceutical Sciences, Acharya Nagarjuna

University, Nagarjuna Nagar, Guntur, AP, India-523 510.

Article Received on
01 February 2014,
Revised on 26 February 2014
Accepted on 15 March 2014

ABSTRACT
A simple, validated RP-HPLC method for the simultaneous estimation
of Amlodipine Besylate and Telmisartan in pharmaceutical dosage
form and bulk was developed for routine analysis. This method was
developed by selecting Chromosil C18 (2504.6mm column as

*Correspondence for Author

Mr. Sathish kumar konidala

stationary phase and ACN: MeOH: water (65: 30: 5 v/v/v) as mobile

Department of Pharmaceutical

phase. Flow rate of mobile phase was maintained at 1 ml/min at

Analysis, University College of

ambient temperature throughout the experiment. Quantification was

Pharmaceutical Sciences.

achieved with ultraviolet (DAD) detection at 240 nm. The retention

times of Amlodipine Besylate and Telmisartan were found as 4.56 min

and 6.59 min respectively. The detector response was linear in the concentration range of 220 g/ml and 16-160 g/ml, has regression coefficients 0.998 and 0.999 for Amlodipine
Besylate and Telmisartan respectively. From recovery studies we concluded that the recovery
of amlodipine besylate and telmisartan has no interference with any excepients in the
formulation. This method has been validated according to ICH guidelines and shown to be
Specific, Sensitive, Precise, Accurate, Rugged and Robust. Hence, this method can be applied
for routine quality control of Amlodipine Besylate and Telmisartan in dosage forms as well
as in bulk drug.
KEY WORDS: Amlodipine Besylate, Telmisartan, RP-HPLC, Twynsta tablets.

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1962

Konidala et al.

World Journal of Pharmacy and Pharmaceutical Sciences

INTRODUCTION
Amlodipine Besylate1 is a drug belongs to anti-hypertensive class used to treat hypertension
by blocking the calcium channels. It has the structural formula and shown in Fig. 1.

Figure 1: Chemical strutre of amlodipine besylate

The chemical name of Amlodipine Besylate is 2-[(2-aminoethoxy)-methyl]-4-(2chlorophenyl)-1, 4-dihydro-6-methyl-3, pyridine dicarboxylic acid 3-ethyl 5-methyl ester
benzene sulphonate. The molecular formula of Amlodipine Besylate is C20H25ClN2O5.
C6H6O3S and it has the molecular weight of 567.1 g/mol. It is slightly soluble in water and in
isopropyl alcohol, sparingly soluble in dehydrated alcohol, freely soluble in methyl alcohol.

Figure 2: Chemical structure of Telmisartan

Telmisartan[2] is a drug belongs to anti-hypertensive class used to treat hypertension by
blocking the angiotensin II receptors. It has the structural formula and shown in Fig. 2.
The chemical name Telmisartan 2-(4-{[4-methyl-6-(1-methyl-1H-1,3-benzodiazol-2-yl)-2propyl-1H-1,3-benzodiazol-1-yl]methyl}phenyl)benzoic acid. The molecular formula of
Telmisartan is C33H30N4O2 and it has the molecular mass of 514.617 g/mol. It is soluble in
methanol and in chloroform, insoluble in water.

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World Journal of Pharmacy and Pharmaceutical Sciences

The two drugs which are mentioned above are official in Indian Pharmacopia[3] and United
States Pharmacopoeia[4]. From the literature survey, we found that Amlodipine Besylate and
Telmisartan were estimated by different analytical methods like RP-HPLC[5]-[13],
spectrophotometry[14]-[24] and HPTLC[25]. The availability of an HPLC method for
simultaneous estimation of above mention cardiovascular drugs will be very much useful for
the determination in bulk and pharmaceutical formulations. This study aimed to develop a
simple, precise, accurate and validated Reversed-Phase HPLC method for the simultaneous
estimation of Amlodipine Besylate and Telmisartan in bulk and pharmaceutical dosage form
as per ICH guidelines[26]-[27]. The statistical analysis proved that method is reproducible and
selective for the simultaneous analysis of Amlodipine Besylate and Telmisartan in bulk and
formulations.
MATERIALS AND METHODS
Gift samples of Pharmaceutical grade Amlodipine Besylate and Telmisartan were supplied by
Hetero Drugs Ltd., Hyderabad, India. The Methanol (HPLC grade), Acetonirile (HPLC
grade) were purchased from MERCK and distilled water was collected from in- house
production. The commercially available TWYNSTA tablets (one equivalent to 5mg
Amlodipine Besylate and of 40mg of Telmisartan) which are manufactured by Boehringer
ingelheim was purchased from market for analysis.
Instruments
The instruments that are used in proposed method are HPLC system PEAK LC 7000 isocratic
system connected to DAD detector and LC-GC AGN204PO balance was used for all
weighing.
Method Development
Chromatographic Conditions
Chromatographic separation was achieved by using Chromosil C18 (2504.6mm, 5) column
as stationary phase and composition of ACN: MeOH: H2O (65: 30: 5 v/v/v) as mobile phase.
Flow rate was maintained at 1 ml/min at ambient temperature and the injection volume used
was 20l. The detection was carried out at 240 nm. Diluent was prepared by mixing 300 ml
of methanol, 650 ml of Acetonitrile and 50 ml of triple distilled water, filtered through
Whattman filter paper (0.45m) and degassed before use. Typical chromatogram of standard
drug is as shown in Fig. 3.

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1964

Konidala et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Preparation of stock solution

Accurately weighed quantity of each drug (10 mg) was transferred to 10.0 ml volumetric
flask. Then small amount of methanol was added and ultra-sonicated for 5 min and diluted
upto the mark with methanol (Concentration: 1000g/ml).
Preparation of standard working solution
From the stock solution 1ml was pipette out into 10 ml volumetric flask and diluted to final
volume using methanol as solvent (100 g/ml).
Preparation of mobile phase
The mobile phase was prepared by mixing 300 ml of methanol, 650 ml of Acetonitrile and 50
ml of triple distilled water, filtered through Whattman filter paper (0.45m) and degassed
before use.

Figure 3: Typical chromatogram of standard drugs

Preparation of working sample solution
Select 20 tablets of TWYNSTA (containing 5mg of Amlodipine and 40mg of Telmisartan).
Weigh accurately each tablet and calculate the average content of 20 tablets. Weigh
accurately powder equivalent to 5mg of Amlodipine and 40mg of Telmisartan and transferred
to 10ml standard volumetric flask. Then add small amount methanol into the volumetric
flask, sonicate for about 15min, and the final volume was made with same to obtain solution
having the concentration of 500g/ml of Amlodipine and 4000 g/ml of Telmisartan. The
mixture was then filtered through Whattman filter paper (0.45m). The above solution was
suitably diluted with mobile phase to obtain the solution having the final concentration of

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World Journal of Pharmacy and Pharmaceutical Sciences

5g/ml of Amlodipine and 40g/ml of Telmisartan. A typical chromatogram of TWYNSTA

formulation (sample) drug was shown in Fig. 4. The assay results are shown in table 1.

Figure 4: typical chromatogram of drugs formulation

Table 1: Analysis of Formulation
S.NO

Drug

1
Amlodipine
2
Telmisartan
* mean of three readings

Label
claim(mg)
5
40

Amount
% of Assay
found(mg)*
4.94
98.80
39.69
99.24

METHOD VALIDATION
The method was validated for its linearity range, accuracy, precision, sensitivity and
specificity. Method validation is carried out as per ICH guidelines26.
Linearity
Calibration curve was constructed by plotting peak area Vs concentration of drug solutions,
and the regression equation was calculated. The calibration curve was plotted over the
concentration range 2-20g/ml for Amlodipine Besylate and 16-160g/ml for Telmisartan.
The diluted final concentration solutions of each drug were prepared carefully from their
working standard solutions. Each 20l aliquots of solution were injected into the HPLC
system which is operated according to chromatographic condition as described above. The
constructed calibration curves of Amlodipine Besylate and Telmisartan were shown in Fig. 5
and 6.

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Konidala et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Figure 5: Calibration curve of Amlodipine Besylate

Figure 6: Calibration curve of Telmisartan

Accuracy
The accuracy of the methods was determined by calculating recovery values of Amlodipine
Besylate and Telmisartan by the standard addition method. The accuracy of the method was
determined by preparing solutions of different concentrations in which the amount of
marketed formulation(TWYNSTA) was kept constant (5mg of Amlodipine and 40mg of
Telmisartan) and the amount of pure drug was varied that is 80%, 100% and 120%
respectively. The solutions were prepared in triplicates and the accuracy was indicated by %
recovery was shown in table 4.
Precision
The precision of the instruments was checked by repeatedly injecting (n=3) solutions of
Amlodipine Besylate and Telmisartan (10 and 80 g/ml).

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1967

Konidala et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Intermediate Precision (Reproducibility)

The intra-day and inter-day precision of the proposed methods were determined by the
corresponding responses three times on the same day and on three different days over a
period of one week for three different concentration of Amlodipine Besylate and Telmisartan
(10 and 80 g/ml).
Robustness
Robustness of the method was determined by carrying out the analysis at three different
wavelengths (i.e. 2402 nm) and three different mobile phase pH (i.e. 4.20.2).
Ruggedness
Ruggedness of the method was determined by carrying out the analysis by two different
analysts and the respective peak areas were noted. The result was indicated by % RSD.
Limit of detection and limit of quantification
The limit of detection (LOD) and limit of quantification (LOQ) of the drug were calculated
using the following equations as per International Conference of Harmonization (ICH)
guidelines.
LOD = 3.3 X /S
LOQ = 10 X /S
RESULTS AND DISCUSSION
To optimize the RP-HPLC parameters, several mobile phases of different compositions were
tried. A satisfactory separation and good peak symmetry for Amlodipine Besylate and
Telmisartan were obtained with a mobile phase consisting of ACN: MeOH: H2O (65: 30: 5
v/v/v). Quantification was achieved with UV detection at 240nm based on peak area.
Complete resolution of the peaks with clear baseline was obtained. System suitability
parameters was calculated and compared with the standard limit as per ICH. The following
tables shows the results obtained which are related different analytical method validation
parameters

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Konidala et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Table 2: Linearity of Amlodipine

S.No
Concentration of Amlodipine (g/mL)
1
2
2
4
3
6
4
8
5
10
6
12
7
14
8
16
9
18
10
20
* mean of two readings

Peak Area SD
485740.611
835730.432
1161610.463
1467470.297
1775300.609
2116560.113
2470260.980
2786070.675
3181290.547
3499270.603

Table 3: Linearity of Telmisartan

S.No Concentration of Telmisartan (g/mL)
1
16
2
32
3
48
4
64
5
80
6
96
7
112
8
128
9
144
10
160
* mean of two readings

Peak AreaSD
1405150.598
2241270.207
3294760.398
4212890.189
5265200.596
6293380.132
6992160.876
8150660.549
9057920.342
10136080.139

Table 4: Accuracy results of AML by RP-HPLC method

Amount of
Amount of
sample taken
standard added
(g/mL)
(g/mL)
4
2
4
4
4
6
* mean of three readings

% of std
added
50
100
150

Amount
recovered
(g/mL)*
1.97
4.03
6.01

% amount
recovered*

% RSD

98.5
100.45
100.1

1.16

Table 5: Accuracy results of TELM by RP-HPLC method

Amount
Amount
of
standard
sample taken
added
(g/mL)
(g/mL)
32
16
32
32
32
48
* mean of three readings

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of

Amount
% of std
recovered
added
(g/mL)*

%
amount
% RSD
recovered*

50
100
150

98.9
101.6
101.43

15.83
32.52
48.69

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World Journal of Pharmacy and Pharmaceutical Sciences

Table 6: precision studies of AML and TELM by RP-HPLC method

Amount of std
taken
(g/ml)
4
8
AML
14
32
64
TELM
92
* mean of three readings
Drug

Intra-day precision
Mean*% RSD

Inter-day precision
Mean*% SD

99.910.580
101.510.498
100.230.970
99.830.457
100.450.467
98.930.655

100.030.618
99.650.260
99.340.686
101.030.543
99.030.986
99.730.342

Table 6: Ruggedness studies

S.No
1
2
3
4
5
6

Condition
MP1-(MeOH:ACN:H2O
35:60:5 v/v/v)
MP2-(MeOH:ACN:H2O
25:70:5 v/v/v)
Wavelength 238nm
Wavelength 242nm
pH 4.0
pH 4.4

Amlodipine
Area

%
Assay

Telmisartan
Area

%
Assay

100667

100.35

279846

101.1

97869

98.16

279223

100.89

100266
98069
100865
101265

100.49
98.29
101.15
101.5

278462
279085
277216
277631

100.61
100.83
100.15
100.32

Table 7: LOD and LOQ of Amlodipine Besylate and Telmisartan

STD
Amlodipine Besylate
Telmisartan

LOD (g/mL)
0.17
0.52

LOQ (g/mL)
0.74
2.25

CONCLUSION
A simple, accurate, precise, selective and sensitive RP- HPLC assay method with DAD
detection for simultaneous estimation of Amlodipine Besylate and Telmisartan in
pharmaceutical dosage form has been developed and validated. The method will be
extensively used for the simultaneous estimation of Amlodipine Besylate and Telmisartan in
bulk and pharmaceutical formulation.
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World Journal of Pharmacy and Pharmaceutical Sciences

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World Journal of Pharmacy and Pharmaceutical Sciences

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1972