Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
INTERNATIONAL JOURNAL OF
CHCHROMATOGRAPHIC TECHNOLOGIES
WWW.IJCT.CO.IN
ISSN: 1234-1234
Research Article
Development and validation of stability indicating HPLC method for
simultaneous estimation of Telmisartan and Hydrochlorothiazide
related substances in Telmisartan/Hydrochlorothiazide tablets
Palakurthi Ashok Kumar*1, Sivaranjani Thiagarajan1, Y Ravindra Kumar1,
Anireddy Jaya shree2.
1 Branded
formulations, Analytical Research & Development, Dr.Reddy’s Laboratories, IPDO, Bachupally,
Hyderabad-500072.India
2 Center
for Chemical Sciences and Technology Institute of Science & Technology Jawaharlal Nehru Technological
University, Kukatpally, Hyderabad -500 085.India
*Corresponding Author: Email:ashokchem2000@gmail.com, Contact: +91-8008166887
ABSTRACT
A simple and gradient RP-LC method has been developed and validated for the simultaneous estimation of
Telmisartan and Hydrochlorothiazide related substances in combination tablets dosage form of Telmisartan and
Hydrochlorothiazide. The method can able be separate thirteen impurities of Telmisartan and three impurities of
Hydrochlorothiazide with not less than 2.5 resolution. The separation was achieved by using Inertsil ODS-3V
250x4.6mm, 5µm column at a flow rate of 1.0 mL-1 with the mobile phase-A consisting of 0.02M potassium
dihydrogen phosphate buffer solution with pH of 3.0 and mobile phase-B consisting a mixture of water and
Acetonitrile in the ratio 10: 90 v/v respectively. Detection was carried out at 230 nm. The column temperature
was maintained at 40oC.The molecules are forced to all stress conditions such as acid, base, oxidation, heat and
photolysis as per the recommendations of ICH guidelines. All degradants are well separated from the main
analytes and also from all impurities. The method is proved to be robust with respect to change in flow rate, pH,
organic phase composition and column temperature. The proposed method is found to be sensitive, precise,
rapid, reproducible, and offers good column life.
INTRODUCTION
The multi component dosage for have gained a lot
of importance due to greater patients acceptability,
multiple action and quicker relief. Telmisartan is
an angiotension receptor blocker that shows high
affinity for the angiotension II type 1 receptors, has
a long duration of action, and has the longest halflife of an ARB. In addition to blocking the ReninAngiotensin System (RAS), Telmisartan acts as a
selective modulator of Peroxisome proliferatoractivated receptor gamma (PPAR-γ), a central
regulator of insulin and glucose metabolism.
Hydrochlorothiazide is a diuretic (water pill) that
helps control blood pressure by getting rid of
excess salt and water. In the present study
Telmisartan + Hydrochlorothiazide in a bilayered
tablet formulation was used to evaluate the
chromatographic separation of Telmisartan,
Hydrochlorothiazide and its related impurities.
Telmisartan, a non-peptide molecule, is chemically
described as 4'-[(1, 4’-dimethyl-2'-propyl [2, 6’-bi1Hbenzimidazol]-1'-yl) methyl]-[1, 1’-biphenyl]-2carboxylic acid. Its empirical formula is
C33H30N4O2, its molecular weight is 514.63, and
Telmisartan is a white to slightly yellowish solid. It
is practically insoluble in water and in the pH
range of 3 to 9, sparingly soluble in strong acid
(except insoluble in hydrochloric acid), and soluble
in strong base.
Page 1
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
Telmisartan
List of Telmisartan impurities
Impurity A 4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H-benzimidazole
Impurity B 4′-[[7-methyl-5-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1Hbenzimidazol-1-yl]
methyl]biphenyl-2-carboxylic acid
Impurity-C 1,1-dimethylethyl 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H
benzimidazol-1-yl]methyl]biphenyl-2-carboxylate
Impurity E 1-[(2′-carboxybiphenyl-4-yl) methyl]-4-methyl-2-propyl-1H-enzimidazol6-carboxylic acid
Impurity F 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1Hbenzimidazol-1-yl]
methyl]biphenyl-2-carboxamide
Page 2
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
Impurity G 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1Hbenzimidazol-1yl]methyl]biphenyl-2-carbonitrile
Impurity H 1,1-dimethylethyl 4′-(bromomethyl)biphenyl-2-carboxylate
TEL 2 Impurity : Methyl-4’-2[2n-propyl-4-methyl-6-(1-methylbenzimidazole-2-yl)-benzimidazol1-yl-methyl-biphenyl-2-carboxylate
CH3
N
CH3
N
N
N
N
N
Cl
COOH
N
N
COOH
COOH
CH3
Diacid impurity
Chloro analog impurity
Hydrochlorothiazide
Hydrochlorothiazide is a white, or practically white, practically odorless, crystalline powder with a
molecular weight of 297.74. It is slightly soluble in water, and freely soluble in sodium hydroxide
solution. Hydrochlorothiazide is chemically described as 6-chloro-3, 4-dihydro-2H-1, 2, 4benzothiadiazine-7sulfonamide 1,1-dioxide. Its empirical formula is C7H8ClN3O4S2, and its
structural formula is
Hydrochlorothiazide
Page 3
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
List of Hydrochlorothiazide impurities:
Impurity A Chlorothiazide.
Literature reveals few RP-HPLC methods
and is not capable of producing proper
resolution between impurity F and impurity
E. The main objective is to develop and
validate a simple, effective and reproducible
HPLC method for the determination of
Telmisartan, Hydrochlorothiazide and all
related impurities in tablet dosage
formulation.
The highly polar molecule requires aqueous
mobile phase. The mobile phase-A consisting
of 0.02M potassium dihydrogen phosphate
buffer solution with pH of 3.0 and mobile
phase-B consisting a mixture of water and
Acetonitrile in the ratio 10: 90 v/v
respectively .The quantification was carried
out on Inertsil ODS-3V 250x4.6mm 5µm
column was selected to enhance retention
capacity, sensitivity and specificity of the
analytes and their related substances.
Gradient flow was used to separate the all
impurities with proper separation.
The developed method is found to be
specific, reproducible and stability indicating.
MATERIALS AND
METHODS
Materials reagents
Impurity- B. 4-amino-6-chlorobenzene1,3-disulphonamide (DSA)
impurity and impurity D is unspecified
impurity. Chloro analogue, diacid impurity and
TEL2
are
API
process
impurities.
Hydrochlorothiazide
(purity-99.5%)
and
impurities
are
Chlorothiazide
and
Disulphonamide. Telmisartan Impurity E and
impurity F are obtained from Synpure
laboratories. Telmisartan, impurity A, impurity
B, Chloro analogue, Diacid impurity and TEL2
are obtained from Dr. Reddy’s laboratories Ltd.
Potassium dihydrogen phosphate (AR gradeMerck (India) limited, All other chemicals and
solvents used were of analytical grade or HPLC
grade.
Apparatus
The analysis was carried out on waters
Alliance HPLC systems 2695 separation
module connected to 2996 Photo diode array
detector. Data acquisition was carried out
using Empower software.
Different chromatographic
during trials were
column
used
i) Kromosil C18, 125 x 4.0 mm, 5 µm.
(make-Waters),
ii) Waters Symmetry Shield RP-18,
250x4.6mm, 5µ (make-Waters),
iii) Inertsil ODS 3V, 250x4.6mm, 5µ
(make-GL
Sciences)
Telmisartan (purity-99.2%) and impurities A, B,
C, D, E, F, G and H are official in European
Pharmacopoeia. impurity C is a process related
Page 4
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
but 20mM concentration
sensitivity of the method.
Optimization of Chromatographic
conditions:
increased the
Column Selection
Buffer pH Selection
Different HPLC columns were used for
optimizing the chromatographic condition.
To elute highly non polar impurities gradient
programme was selected. Different gradient
programmes were used to obtain good
resolution between the impurities. In Inertsil
ODS 3V column all impurities are separated
with not less than 2.5 resolutions. The
sensitivity of the method also increased with
this column in comparison with other
columns. The final method conditions
Chromatogram shown in Figure1.
The pH had an effect on the retention times
of the Telmisartan, Hydrochlorothiazide and
their related compounds. Resolutions and
peak symmetry are found good at pH 3.0.
Buffer Selection
Different buffers such as ammonium
dihydrogen phosphate, potassium dihydrogen
phosphate, sodium per chlorate were
evaluated for separation of Impurities and
system suitability parameters and overall
chromatographic performance. In the
sequential trials potassium dihydrogen
phosphate was found to be suitable for
effective separation of parent peak and
impurities. It was observed that change in the
buffer concentration did not offer significant
changes in the elution pattern and resolution,
Time (minutes)
0
2
12
30
40
50
51
60
61
65
Mobile phase A
85
85
65
50
40
20
0
0
85
85
Chromatograms of Blank, Placebo, Diluted
standard, Telmisartan impurities and
Telmisartan
&
Hydrochlorothiazide
Impurities spiked Chromatograms are given
Optimized Chromatographic conditions
The
separation
of
Telmisartan,
Hydrochlorothiazide
and
all
related
substances were achieved using 20mM
Potassium dihydrogen phosphate buffer, pH
adjusted to 3.5 using 1% ortho phosphoric
acid solution as mobile phase-A. Water and
acetonitirile in the ratio of 10:90 as mobile
phase–B at a flow rate of 1.0mL /minute
(gradient (table 1)).Detection and purity
establishment of the main drug and
impurities were achieved using photo diode
array (PDA) detector at 230nm.The drug
samples and formulation samples were
prepared in 0.01M HCl: Methanol (80:20)
which is used as a diluent to achieve a
concentration of 500µg/mL of Telmisartan
and 200 µg/mL and 10µL of the sample were
injected. The run time optimized was found
to be 65 minutes
Gradient programme:
Mobile phase B
15
15
35
50
60
80
100
100
15
15
in (figure 1 to figure 5). Relative Retention
times (RRTs), Resolutions (R) and Tailing
factors (F) of individual peaks are given in
(table 2).
Page 5
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
Figure 1: Blank
Figure 2: Placebo
Figure 3: Telmisartan Impurity D Chromatogram
Figure 4: Telmisartan Impurities Chromatogram
Page 6
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
Figure 5: Telmisartan & Hydrochlorothiazide Impurities spiked Chromatogram
Table2:
Component Name
DSA
Chlorothiazide
Telmisartan Impurity A
Telmisartan Impurity E
Telmisartan Impurity F
Telmisartan Impurity B
Telmisartan Impurity G
TEL 2
Dimer acid
Telmisartan Impurity C
Chloroanalog
TEL 1
Telmisartan Impurity H
RRT
0.82
0.90
0.37
0.67
0.76
0.85
1.10
1.17
1.40
1.50
1.54
1.62
1.77
Resolution
2.6
10.8
36.4
8.5
7.6
7.2
5.0
16.4
5.5
3.1
6.2
18.1
Tailing
1.2
1.2
1.1
1.1
1.1
1.1
1.1
1.1
1.2
1.0
1.1
1.1
1.0
VALIDATION OF METHOD
Specificity
The Forced degradation of placebo and formulation was carried out as per ICH guidelines (ICH
Q2B). The acid, base, water and oxidation stress conditions were studied out by refluxing both
the APIs. The thermal degradation was carried out by heating the APIs at 105◦C.All the stress
conditions with purity angle and purity threshold are reported in (table3, 4 & 5).
Table 3: Peak purity of Telmisartan in stressed condition.
Stress condition
As such Sample
Acid Degradation
(1.7 N HCl, refluxed at 60ºC for 10hours)
Base Degradation
(1.7 N HCl, refluxed at 60ºC for 10hours)
Peroxide degradation
(1.7% H2O2 , refluxed at 60ºC for 10hours)
Heat degradation (105 ºC for 24hours)
Water degradation
(10mL H2O , refluxed at 60ºC for 10hours)
NA
Purity
angle
0.847
Purity
threshold
1.001
1.5
0.613
1.006
No
2.6
0.508
1.008
No
5.2
0.089
1.018
No
1.4
0.178
1.010
No
0.1
0.131
1.009
No
%Degradation
Purity flag
No
Page 7
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
Table 4: Peak purity of Hydrochlorothiazide in stressed condition
NA
Purity
angle
0.376
Purity
threshold
0.985
Purity
flag
No
20.7
0.107
1.054
No
13.2
0.122
1.061
No
23.7
0.159
1.83
No
12.2
0.277
1.170
No
11.6
0.096
1.075
No
Stress condition
%Degradation
As such Sample
Acid Degradation
(1.7 N HCl, refluxed at 60ºC for 1hour)
Base Degradation
(1.7 N HCl, refluxed at 60ºC for 1hour)
Peroxide degradation
(1.7% H2O2 , refluxed at 60ºC for 1hour)
Heat degradation (105 ºC for 6hours)
Water degradation (10mL H2O , refluxed at 60ºC for
10hours)
Table 5: % of Impurities formed in stressed condition
Name of the Impurity
DAS
Chlorothiazide
Impurity-A
Impurity-E
Impurity-F
Impurity-B
TEL2
Dimer Acid
Chloro analogue
Impurity-D
Acid
20.7
0.1
1.1
-
Base
9.9
0.1
2.5
-
Robustness
The robustness was investigated by varying
the conditions of change in the flow rate, pH,
column temperature and organic phase
composition. The study was conducted at
different flow rates of 0.8mL/min, and
1.2mL/min.The mobile phase pH was
modified to 2.8 and 3.2 and column
temperature was adjusted to 35°C and 45°C
to study the effect of pH and column
temperature respectively. Organic phase
composition was varied to 90% and 110% in
mobile phase-B to study the effect of organic
phase composition variation. The method
% of Impurities
Thermal
6.5
0.7
0.1
Peroxide
13.6
0.4
0.9
0.2
0.1
0.1
0.1
Water
42.0
0.1
-
was found to be robust with respect to flow
rate, pH, column temperature and organic
phase composition without any changes in
system suitability parameters such as tailing
factor and resolution (table 6 & 7).
Note:
1) Relative retention times of Telmisartan
impurities are calculated with respect to
Telmisartan retention time.
2) Relative
retention
times
hydrochlorothiazide
impurities
calculated
with
respect
hydrochlorothiazide retention time.
of
are
to
Table-6: Robustness
Parameters
Changes in parameters
The tailing factor for
Hydrochlorothiazide
The tailing factor for Telmisartan
RRT of DSA
RRT of Chlorthalidone
Flow rate(mL/min)
0.8
1.2
Between 0.8
Between 0.8
to 1.5
to 1.5
Between 0.8
Between 0.8
to 1.5
to 1.5
0.84
0.81
0.91
0.88
pH
2.8
Between 0.8
to 1.5
Between 0.8
to 1.5
0.82
0.89
3.2
Between
0.8 to 1.5
Between
0.8 to 1.5
0.83
0.90
Page 8
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
RRT of Imp-A
RRT of Imp-E
RRT of Imp-F
RRT of Imp-B
RRT of TEL 2
RRT of Dimer acid
RRT of Chloro analogue
0.39
0.68
0.78
0.85
1.17
1.39
1.52
0.35
0.67
0.76
0.85
1.18
1.44
1.58
0.37
0.70
0.81
0.90
1.22
1.47
1.62
0.37
0.66
0.73
0.81
1.15
1.34
1.45
Table-7: Robustness
Parameters
Changes in parameters
The tailing factor for
Hydrochlorothiazide
The tailing factor for Telmisartan
RRT of DSA
RRT of Chlorthalidone
RRT of Imp-A
RRT of Imp-E
RRT of Imp-F
RRT of Imp-B
RRT of TEL 2
RRT of Dimer acid
RRT of Chloro analogue
Column Temperature
35ºC
Between 0.8
to 1.5
Between 0.8
to 1.5
0.85
0.91
0.39
0.68
0.79
0.85
45ºC
Between 0.8
to 1.5
Between 0.8
to 1.5
0.83
0.90
0.38
0.68
0.77
0.83
1.15
1.37
1.53
1.17
1.37
1.49
Organic phase ratio in mobile
phase-B
90%
110%
Between 0.8
Between 0.8
to 1.50
to 1.5
Between 0.8
Between 0.8
to 1.5
to 1.5
0.82
0.82
0.90
0.89
0.35
0.38
0.68
0.68
0.77
0.77
0.86
0.84
1.16
1.35
1.46
1.18
1.44
1.61
Robustness Chromatograms:
Flow 0.8 mL/minute
Page 9
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
Flow 1.2 mL/minute
Buffer pH-2.8
Buffer pH-3.2
Column Temperature 35°C
Page 10
Palakurthi Ashok Kumar et all/IJCT 2014 1(1)
Column Temperature 45°C
CONCLUSION
The method provides selective quantification
of Telmisartan and Hydrochlorothiazide
impurities without interference of blank,
placebo,
thereby
affirming
stabilityindicating nature of the method. The
proposed method is highly selective,
ACKNOWLEDGEMENTS
The author wish to thank the management of
Dr.Reddy’s Laboratories Ltd. for supporting
this work.
REFERENCES
i)
ii)
iii)
ICH Q1B Photo stability testing of new
active substances and medicinal
products.
ICH Q2B Validation of Analytical
Procedures: Methodology International
Conference on Harmonization of
Technical requirements for registration
of Pharmaceuticals for Human use,
Geneva, Switzerland, 1996.
Kiran R. Patil, Vipul P. Rane,
Jaiprakash N. Sangshetti and
reproducible, specific and rapid. The
developed method was robust in the
separation and quantification of Telmisartan
and Hydrochlorothiazide related impurities.
This method can be used in the routine
analysis of production samples.
Devanand B. Shinde ,journal of
Chromatographia,2008,67:455-459
iv) Leena R. Bhat ,Rahul K. Godge ,Asfak
T. Vora ,Mrinalini C. Damle ,Journal of
liquid chromatography and related
technologies,2007,30:3059-3067
v) SB Wankhede, MR Tajne, KR Gupta,
SG Wadodkar,Indian journal of
pharmaceutical sciences,2007,69:298300
vi) European pharmocopiea, 6.3:2154
vii) NJ Shah, BN Suhagia, RR Shah, PB
Shah, ,Indian journal of pharmaceutical
sciences,2007,69:202-205
viii) Ben-mei Chen, Yi-zeng Liang , Ya-li
Wang, Fu-Liang Deng, Ping Zhou, Fangqiu Guo and Lan-fang Huang, Analytica
Chimica Acta,2005,540:367-373
Page 11