Research Article
Available online at www.ijpras.com
Volume 3, issue 3 (2014):33-40
ISSN 2277-3657
International Journal of
Pharmaceutical Research &
Allied Sciences
Validated RP-HPLC Method for Simultaneous Determination of
Telmisartan and Hydrochlorothiazide in Pharmaceutical
Formulation
1
K. Megala1, A. Chenthilnathan1* and A. Sathish babu2
Department of Pharmaceutical Chemistry, Manonmaniam Sundaranar University, Tirunelveli,
Tamil Nadu, India
2
The Madras Pharmaceuticals, Chennai – 600 096, Tamil Nadu, India
Email: ala.chenthil@gmail.com
Subject: Analytical chemistry
Abstract
This paper deals with a simple, feasible and sensitive reverse-phase high-performance liquid
chromatographic method for the simultaneous determination of Telmisartan and Hydrochlorothiazide in
bulk and in pharmaceutical formulation. The chromatography was carried out by using HPLC system
(Shimadzu LC2010HT) with UV- Visible dual absorbance detector (PDA), using Inertsil 250 x 4.6 mm 5µm packing L11 column. The mobile phase consisting of buffer and mixture, acetonitrile and methanol in
the ratio of (50:50) [adjust pH to 3.0 with ortho phosphoric acid] and it was flowed at 1.2 ml/min. The
chromatographic detection was made at 298 nm for telmisartan and 270 nm for hydrochlorothiazide. The
proposed method was validated by parameters such as suitability, specificity, linearity, accuracy and
precision over a linearity range 50–150 µg/ml and stability according to the ICH guidelines (r > 0.9990).
The retention times of telmisartan and hydrochlorothiazide were found to be 18.43 min and 8.11 min
respectively. Hence, the method could be successfully applied for routine analysis of telmisartan and
hydrochlorothiazide in pharmaceutical formulation.
Keywords: Telmisartan, Hydrochlorothiazide, Pharmaceutical formulation, RP-HPLC, Validation.
Introduction
Telmisartan (Fig.1) chemically1, 4'-[(1, 4-dimethyl2'-propyl [2, 6’-1H-benzimidazol]-1'-yl) methyl][1, 1’-biphenyl]-2-carboxylic acid, is a non peptide
molecule under the class of angiotensin II receptor
antagonist. It is used for the treatment of essential
hypertension as alone or in combination with other
agents 2. Hydrochlorothiazide (Fig.2) chemically 3,
6-chloro-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine7-sulfonamide 1, 1-dioxide, is a widely used
thiazide diuretic. The combination of telmisartan
and hydrochlorothiazide is useful mainly in the
treatment of mild to moderate hypertension 4.
Figure 1: Structure of Telmisartan
Figure 2: Structure of Hydrochlorothiazide
Literature survey revealed that few analytical
methods such as HPTLC 5, LC-MS 6,
spectrofluorimetry 7, capillary electrophoresis 8,
spectrophotometry 9 and HPLC 10, have been
reported for telmisartan as alone and in
combination with hydrochlorothiazide. In this
present work, an attempt was made to develop a
simple, feasible and sensitive reverse-phase highperformance liquid chromatographic method for
the quantitative determination of Telmisartan and
Hydrochlorothiazide in bulk drug and in
pharmaceutical dosage forms.
The proposed
method was validated according to ICH guidelines.
33
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Materials and Methods
Experimental
Chemicals and reagents
Acetonitrile of HPLC grade and methanol were
purchased from E.Merck (India) Ltd., Mumbai.
Orthophosphoric acid of AR grade was obtained
from Qualigens Fine Chemicals Ltd., Mumbai.
Telmisartan and Hydrochlorothiazide were a gift
sample by The Madras Pharmaceuticals, Chennai –
600 096, Tamil Nadu, India. The commercially
available tablets containing Telmisartan and
Hydrochlorothiazide were procured from the local
market.
Instrumentation
and
chromatographic
conditions
The chromatography was carried out by using
HPLC system (Shimadzu LC2010HT) with UVVisible dual absorbance detector (PDA), using
Inertsil 250 x 4.6 mm 5-µm packing L11 column.
The mobile phase consisting of acetonitrile and
methanol in the ratio of (50:50) [adjust pH to 3.0
with ortho phosphoric acid] and filtered through
0.45µ membrane filter before use, degassed and
were pumped into the column at a flow rate of 1.2
ml/min. The chromatographic detection was made
at 298 nm for telmisartan and 270 nm for
hydrochlorothiazide. The volume of injection loop
was 20 µl prior to the injection of the drug
solution; the column was equilibrated for at least
15 min. with the mobile phase following through
the system.
Sample Preparation
Twenty tablets were randomly selected and
transferred into 500 ml volumetric flask. 25ml of
0.1N sodium hydroxide solution was added and
shake until the tablets have completely
disintegrated. Methanol (80% of the total volume
of the flask) was added, Sonicated for 10 minutes
and stirred vigorously for 30 minutes. Allowed to
cool to room temperature, diluted with methanol to
volume and mixed well. The concentration of the
sample stock solution is about 1.6mg/ml of
Telmisartan and 0.5 mg/ml of Hydrochlorothiazide.
A portion of the solution was centrifuged at 4000
RPM.
10 ml of above solution was taken in 50
ml volumetric flask and make up the volume with
1:1 solution of buffer and solution contains
acetonitrile: methanol (1:1). (320 µg/ml for
Telmisartan
and
100
µg/ml
for
Hydrochlorothiazide)
Calculation
The amount of telmisartan and hydrochlorothiazide
present in each tablet formulation was calculated
by using the formula:
Spl. area
---------- ×
Std. area
Std.dil Purity Avg.wt
-------- × -------- × ---------- × 100
Spl.dil
100
L.C
The proposed method was validated by parameters
such as suitability, specificity, linearity, accuracy
and precision (repeatability & reproducibility) and
stability according to the ICH guidelines.
Preparation of standard stock solution:
Results and Discussion
Accurately weighed about 320 mg of telmisartan
WS and 100 mg of hydrochlorothiazide WS in to a
clean 100 ml volumetric flask. 20 ml of standard
stock solution (12.50mg of Benzothiadiazine
related compound A USP in 500ml of diluents)
was added. The volume was finally made up with
diluents. 10 ml of above resulting solution was
taken in 100 ml volumetric flask and make up the
volume with a 1:1 solution of buffer and solution
contains acetonitrile
: methanol
(1:1).
(Telmisartan 0.32mg/ml, Hydrochlorothiazide
0.1mg/ml and Benzothiadiazine related compound
A 0.5 µg/ml)
All of the analytical validation parameters for the
proposed method were determined according to
International Conference on Harmonization (ICH)
guidelines 11 .
System Suitability
It is essential for the assurance of the
quality performance of chromatographic system.
Five injections of standard drug solutions,
Telmisartan and Hydrochlorothiazide were given
separately to the system. The mean area, standard
deviation and %RSD were calculated for the
standard drug solutions and mentioned in Table
1and 2. It was observed that all the values are with
in the limits.
34
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Table 1: System suitability for Telmisartan
S.No.
1.
2.
3.
4.
5.
Standard
Standard -1
Standard -2
Standard -3
Standard -4
Standard -5
Peak area
response
16068395
15994039
16129546
16034289
16069044
System suitability parameters
Number of
Retention time
theoretical plates
(min)
18.606
85986
80611
18.602
80639
18.603
18.617
78931
18.612
88691
Mean
18.608
Standard deviation
0.0063
RSD in %
0.03
Table 2: System suitability for Hydrochlorothiazide
S.No.
1.
2.
3.
4.
5.
Standard
Standard -1
Standard -2
Standard -3
Standard -4
Standard -5
Peak area
response
6289389
6293528
6309963
6319474
6278727
Specificity
The specificity of the HPLC method is illustrated
in Fig. 3, where a complete separation of
Telmisartan and Hydrochlorothiazide were
noticed in presence of other inactive excipients
used in tablets. In addition, there was no any
interference at the retention time of in the
chromatogram of placebo solution. In peak purity
analysis with PDA, purity angle was always less
System suitability parameters
Number of
Retention time
theoretical plates
(min)
8.666
6247
8.700
6464
8.653
6416
6454
8.654
6608
8.664
Mean
8.667
Standard deviation
0.0191
RSD in %
0.22
than purity threshold for the analyte. This shows
that the peaks of analyte were pure and excipients
in the formulation does not interfere the analyte.
The data were presented in the Table 3 and 4. It
was observed that all the values are within the
limits.
Table 3: Specificity for Telmisartan
S.No.
1.
2.
3.
4.
Name
Blank
Placebo
Standard
Sample
No. of Injections
1
1
1
1
Area
Nil
Nil
16129546
16129550
Table 4: Specificity for Hydrochlorothiazide
S.No.
1.
2.
3.
4.
Name
Blank
Placebo
Standard
Sample
No. of Injections
1
1
1
1
Area
Nil
Nil
6289389
6289392
35
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Figure 3 : Typical HPLC Chromatogram of Sample Tablets
(Telmisartan and Hydrochlorothiazide)
Linearity
The Linearity of this method was determined at
five levels from 50%– 150% of operating
concentrations
for
Telmisartan
and
Hydrochlorothiazide and it was shown in Table 5
and 6 . The plots of peak area of each sample
against respective concentrations of Telmisartan
and Hydrochlorothiazide were found to be linear
(Figure 4 and 5) in the range of 50%– 150% of
operating concentrations. Beer’s law was found to
be obeyed over this concentration range. The
.
linearity was evaluated by linear regression
analysis using least square method. The regression
equations were found to be Y= 46366x+1E+06 and
Y= 61940x+57848, for Telmisartan and
Hydrochlorothiazide respectively and correlation
coefficient of the standard curves were found to be
0.9995 and 1.000 for Telmisartan and
Hydrochlorothiazide respectively. It observed that
correlation coefficient and regression analysis are
within the limits.
Table 5: Linearity of response for Telmisartan
S.No
Target Level (%)
1.
Linearity
Level
Linearity -1
2.
50
Concentration
(µg/ml)
160
Area
8398067
Linearity -2
75
240
12167558
3.
Linearity -3
100*
320
16034230
4.
Linearity -4
125
400
19730215
5.
Linearity -5
150
480
23163318
Table 6: Linearity of response for Hydrochlorothiazide
S.No
Target Level (%)
1.
Linearity
Level
Linearity -1
2.
50
Concentration
(µg/ml)
25
Area
1614164
Linearity -2
75
37.5
2370008
3.
Linearity -3
100*
50
3156705
4.
Linearity -4
125
62.5
3925706
5.
Linearity -5
150
75
4707534
* Operating concentration
36
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y = 46366x + 1E+06
R2 = 0.9995
25000000
Area
20000000
15000000
10000000
5000000
0
0
100
200
300
400
500
600
Concentration (µg/ml)
Figure 4: Linearity of response for Telmisartan
Figure 5: Linearity of response for Hydrochlorothiazide
Accuracy
Accuracy of the method was found out by recovery
study by standard addition method. The known
amounts
of
standards,
Telmisartan
and
Hydrochlorothiazide were added to pre-analysed
samples at a level from 80% up to 120% and then
subjected to the proposed HPLC method
individually. The results of recovery studies were
shown in Table 7 and 8. It was observed that the
mean percentage recoveries were found to be for
Telmisartan and Hydrochlorothiazide which
demonstrated that the method was highly accurate.
Table 7: Accuracy for Telmisartan
S.No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Target level
80%
80%
80%
100%
100%
100%
120%
120%
120%
Mean
Standard deviation
RSD %
Area
13694177
13751455
13729102
16881622
17236505
16804965
19734447
19856807
21008516
Drug Recovery (%)
101.59
101.81
101.35
100.19
101.71
99.35
98.58
98.57
101.66
100.53
1.380
1.37
37
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Table 8: Accuracy for Hydrochlorothiazide
S.No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Target level
80%
80%
80%
100%
100%
100%
120%
120%
120%
Mean
Standard deviation
RSD %
Area
4898396
4884222
4884780
6349232
6472426
6301825
7370832
7464171
7768496
Precision
The precision of an analytical procedure expresses
the closeness of agreement between a series of
measurements obtained from multiple sampling of
the homogenous sample under the prescribed
conditions.
Drug Recovery (%)
99.34
98.75
99.51
100.87
100.50
100.51
99.32
100.68
101.28
100.08
0.8663
0.87
Reproducibility
Examines the precision between laboratories and is
often determined in collaborative studies.
Reproducibility data for Telmisartan and
Hydrochlorothiazide were shown in Table 9 and
10. This indicated that method was highly precise.
Table 9: Precision - Reproducibility for Telmisartan
S.No. Sample Name
Area
1.
Sample -1
15771133
2.
Sample -2
15883503
3.
Sample -3
16017056
4.
Sample -4
15588983
5.
Sample -5
15756517
6.
Sample -6
15910440
Mean
Standard deviation
RSD %
Drug Recovery (%)
99.26
99.96
100.80
100.00
99.16
100.13
99.88
0.6053
0.61
Table 10: Precision - Reproducibility for Hydrochlorothiazide
S.No. Sample Name
1.
Sample -1
2.
Sample -2
3.
Sample -3
4.
Sample -4
5.
Sample -5
6.
Sample -6
Mean
Standard deviation
RSD %
Area
5938671
5927322
5960768
5925739
5952645
5910516
Drug Recovery (%)
98.58
98.39
98.95
98.37
98.81
98.11
98.535
0.3094
0.31
38
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Stability
Standard and sample solutions to be used in the
analytical method were scrutinized for their
solution’s stability. This study was performed by
injecting standard and sample solution for the
period of 24 hours and results were presented in the
Table 11 and 12. It was found that there were no
remarked changes in the system suitability
parameters.
Table 11: Stability results obtained for Telmisartan standard solution
Condition
Average standar
response
Response
Average
Response
%Recovery % Difference
from initial
15739257
Initial
16059062
12th hr
16469413
24th hr
15850204
15803009
16300189
16315144
15834255
15815221
15771133
99.26
-
16307666
99.65
0.39
15824738
100.05
0.79
Table 12: Stability results obtained for Hydrochlorothiazide standard solution
Condition
Average standar
response
Response
Average
Response
%Recovery % Difference
from initial
5938578
Initial
6298216
12th hr
6533690
24th hr
6271969
5938765
6031753
6015389
5862660
5860338
Conclusion
The Proposed study describes a simple, feasible
and sensitive reverse-phase high-performance
liquid chromatographic method for the
quantitative determination of Telmisartan and
Hydrochlorothiazide
in
bulk
and
in
pharmaceutical formulations. The method was
validated as per ICH guidelines and found to be
simple, sensitive, accurate and precise. Therefore
the proposed method can be successfully used for
the routine analysis of Telmisartan and
Hydrochlorothiazide in solid dosage form without
interference.
98.58
-
6023571
98.96
0.38
5861499
98.08
0.50
“Cite this article”
K. Megala, A. Chenthilnathan, A. S. Babu
“Validated Rp-Hplc Method for Simultaneous
Determination
of
Telmisartan
And
Hydrochlorothiazide
In
Pharmaceutical
Formulation” Int. J. of Pharm. Res. & All. Sci.
2014;3(3):33-40
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Acknowledgements
The authors are thankful to the
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Chennai – 600 096, Tamil Nadu, India for
providing the necessary facilities to carry out for
the research work.
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