Post on 28-Jul-2018
CHAPTER – 4
SIMULTANEOUS DETERMINATION OF RELATED SUBSTANCES OF
TELMISARTAN AND HYDROCHLOROTHIAZIDE IN TABLET
DOSAGE FORM BY USING REVERSE PHASE HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
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SPP SPTM, SVKM’s NMIMS, Mumbai 17
CHAPTER – 4
Simultaneous determination of related substances of Telmisartan and Hydrochlorothiazide in tablet dosage form by using reverse phase high
performance liquid chromatography 4.0 INTRODUCTION The objective was to develop a single method for simultaneous determination of related
substances of Telmisartan and Hydrochlorothiazide in a tablet dosage form of two
strengths (80mg-12.5mg and 40mg-12,5mg). The method was validated as per ICH
guidelines Q2 (R1).
4.1 Drug Profile 4.1.1 Telmisartan Telmisartan, 4-[(1,4-dimethyl-2-propyl[2,6- bi-1H-benzimidazol]-1-yl) methyl-[1,1-
biphenyl]-2-carboxylic acid, is a potent, long-lasting, nonpeptide antagonist of the
angiotensin II type-1 (AT1) receptor that is indicated for the treatment of essential
hypertension. It selectively and insurmountably inhibits stimulation of the AT1 receptor
by angiotensin II without affecting other receptor systems involved in cardiovascular
regulation. In clinical studies, Telmisartan shows comparable antihypertensive activity
to members of other major antihypertensive classes, such as angiotensin-converting
enzyme (ACE) inhibitors, beta-blockers and calcium channel blockers. Telmisartan
(TE) is widely used in the treatment of hypertension.
Telmisartan is official in USP and EP General Name : Telmisartan Chemical Structure :
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SPP SPTM, SVKM’s NMIMS, Mumbai 18
Chemical Name : 4′-[[4-Methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-
propyl- 1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxylic acid
Molecular Formula : C33H30N4O2 Molecular Weight : 514.6 CAS Number : 144701-48-4 Description : White or slightly yellowish, crystalline powder Solubility : Practically insoluble in water, slightly soluble in methanol, sparingly soluble in methylene chloride. It dissolves in 1 M sodium hydroxide Drug Category : Angiotensin II (AT1) receptor antagonist (Antihypertensive) Listed Impurities in Monograph (EP/BP): Impurities A, B, C and D are specified 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-1H- benzimidazol-1-yl]methyl]biphenyl-2-carboxylic acid
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SPP SPTM, SVKM’s NMIMS, Mumbai 19
Impurity C. 1,1-dimethylethyl 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2- propyl-1Hbenzimidazol-1-yl]methyl]biphenyl-2-carboxylate
Impurity D. Unidentified Impurity
Impurity E. 1-[(2′-carboxybiphenyl-4-yl)methyl]-4-methyl-2-propyl-1H-enzimidazol- 6-carboxylic acid
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SPP SPTM, SVKM’s NMIMS, Mumbai 20
Impurity F. 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H- benzimidazol-1-yl]methyl]biphenyl-2-carboxamide
Impurity G. 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carbonitrile
Impurity F. 1,1-dimethylethyl 4′-(bromomethyl)biphenyl-2-carboxylate
The EP impurity identification mixture when injected in the EP API monograph method
was expected to generate the following chromatogram. The chromatogram given below
is obtained from EP.
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4.1.2 Hydrochlorothiazide Hydrochlorothiazide (HCTZ) (6-chloro-3, 4-dihydro-2H-1, 2, 4-benzo-thiadiazine-7-
sulfonamide 1,1-dioxide) is a widely prescribed diuretic. It is indicated for the treatment
of edema, control of essential hypertension and management of diabetes insipidus. [4]
Hydrochlorothiazide, a thiazide diuretic, is also used to treat mild to moderate
hypertension, usually in combination with other antihypertensive agents with different
mechanisms of action. [5] Blood pressure control is often inadequate using monotherapy.
Combination therapy can simplify dosing regimens, improve compliance, and decrease
side effects.
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SPP SPTM, SVKM’s NMIMS, Mumbai 22
Hydrochlorothiazide is official in USP and EP/ BP General Name : Hydrochlorothiazide (HCTZ) Chemical Structure :
Chemical Name : 6-Chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7- sulphonamide 1,1-dioxide
Molecular Formula : C7H8ClN3O4S2 Molecular Weight : 297.7 CAS Number : 58-93-5 Description : White or almost white, crystalline powder Solubility : Very slightly soluble in water, soluble in acetone, sparingly soluble in ethanol (96 per cent). It dissolves in dilute solutions of alkali hydroxides Drug Category : Thiazide diuretic Listed Impurities in Monograph (EP/BP):
Impurities A, B and C are specified impurities.
Impurity A. chlorothiazide
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SPP SPTM, SVKM’s NMIMS, Mumbai 23
Impurity B. 4-amino-6-chlorobenzene-1,3-disulphonamide (salamide)
Impurity C. 6-chloro-N-[(6-chloro-7-sulphamoyl-2,3-dihydro-4H-1,2,4-benzothiadiazin- 4-yl 1,1- dioxide)methyl]-3,4-dihydro-2H-1,2,4-benzothiadiazine-7- sulphonamide 1,1-dioxide
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SPP SPTM, SVKM’s NMIMS, Mumbai 24
4.2 LITERATURE SURVEY
The literature survey reveals that, Telmisartan and Hydrochlorothiazide are reported in
British Pharmacopoeia. [6, 7] There have been several publications describing analytical
methods for the determination of HCTZ and TE individually or with other drugs as
combination.
Although there are a few papers published on simultaneous determination of TE and
HCTZ in formulation most of them deal with the assay of each constituent. Several
methods are reported for the determination of TE like Spectrophotometric [7] and HPLC. [9-11] The other methods available in the literature are based on Linear Sweep
polarography, [12] LC–MS. [13] Articles on the determination of HCTZ in combination
with other drugs by HPLC are also reported in literature. [14, 15]
However the exhaustive literature survey revealed that none of the most recognized
pharmacopoeias or any journals includes these drugs in combination for the
simultaneous determination of related substances of TE and HCTZ and the information
regarding the stability of the drugs is not available@. The Regulatory agencies
recommend the use of stability indicating analytical methods (SIAMs) [16] for the
analysis of stability samples [17]. This requires stress studies (in order to generate the
potential related impurities under stressed conditions), method development and
validation .With the advent of the International Conference on Harmonization (ICH)
guidelines [18], requirements for the establishment of SIMs have become more clearly
mandated. The production of the potential impurities in a drug product generally takes
place under various environmental conditions like exposure to light, heat, hydrolysis or
oxidation. Hence Stress testing can help identify degradation products and provide
important information about intrinsic stability of the drug product.
Several methods have been studied earlier for simultaneous determination of
Telmisartan and Hydrochlorothiazide, but there is no report on method for related
substances of these drugs in combination. So the aim of our study is to develop simple,
fast, accurate and specific reversed phase High Performance Liquid Chromatographic
method for simultaneous determination of related substances of Telmisartan and
Hydrochlorothiazide in tablet dosage form.
---------------------------------------------------------------------------------------------------------- @ This search was performed at the start and during the course of the study. A draft monograph has been published in USP Forum Vol
36(3). The Monograph is now official as per USP 34, from December 2011. A comparison of methods is given in the next section.
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Table 4.2.1: Related Substances – Pharmacopeia methods for Drug Substance
Telmisartan HCTZ
BP 2012 USP 35 BP 2012 USP 35
Method HPLC HPLC HPLC HPLC
Column C18
125mm x 4.0mm; 5µ; 10nm
poresize
C18
125mm x 4.0mm; 5µ; 10nm
poresize
C18
100mm x 4.6mm; 5µ;
C18
50mm x 4.6mm; 3.5µ
Column Temp 40°C 40°C Not Mentioned 35°C
Mobile Phase Mobile phase A: 2.0 g of
potassium dihydrogen
phosphate and 3.8 g of
sodium pentanesulphonate
monohydrate in 1000ml
water, pH 3.0 adjusted with
dilute
Phosphoric acid.
—mobile phase B:
methanol : acetonitrile
(20:80 V/V)
Solution A: 2.0 g of
monobasic potassium
phosphate and 3.8 g of
sodium 1-pentanesulfonate
in 1 L of water. pH adjusted
with 1 M phosphoric acid to
3.0.
Solution B: Acetonitrile and
methanol (4:1)
—mobile phase A: to 940
ml of phosphate buffer
solution pH 3.2 add 60.0 ml
of methanol and 10.0 ml of
tetrahydrofuran and mixed;
—mobile phase B: to a
mixture of 500 ml of
methanol and 500 ml of
phosphate buffer solution
pH 3.2 50.0 ml of
tetrahydrofuran was added
and mixed
Solution A— A mixture of
acetonitrile and methanol
(3:1) was prepared and
degassed.
Solution B— A solution of
anhydrous formic acid in
water (5 in 1000) was
prepared and degassed.
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SPP SPTM, SVKM’s NMIMS, Mumbai 26
Gradient Time
(min)
%A %B
0 70 30
3 70 30
28 20 80
Re-equilibrate to initial
conditions
Time
(min)
%A %B
0 70 30
2 70 30
27 20 80
32 20 80
32.1 70 30
37 70 30
Time
(min)
%A %B
0 100 0
17 55 45
30 55 45
35 100 0
50 100 0
Time
(min)
%A %B
0 3 97
5 3 97
14 36 64
18 3 97
20 3 97
Flow Rate 1ml/min 1ml/min 0.8ml/min 1ml/min
Wavelength 230nm 230nm 224nm 275nm
Inj Vol (µL) 10 10 10 10
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SPP SPTM, SVKM’s NMIMS, Mumbai 27
Table 4.2.2: Related Substances – Pharmacopeia methods for Drug Product
Telmisartan HCTZ Telmisartan-HCTZ
BP 2012 USP 35 BP 2012 USP 35 USP 35
Method Not Available HPLC HPLC HPLC HPLC
Column Not Applicable C18
40mm x 4.0mm; 5µ
C18
100mm x 4.6mm; 3µ
C18
250mm x 4.6mm;
C8
125mm x 4.0mm; 5µ
Column Temp Not Applicable 40°C Ambient Not Mentioned 40°C
Mobile Phase Not Applicable Buffer - 2.0 g/L
ammonium
dihydrogen
phosphate was
prepared. Adjusted
the pH with 1 M
phosphoric acid to
a 3.0
Mobile Phase:
Methanol and Buffer
(7:3)
--mobile phase A: A
mixture of 10
volumes of
tetrahydrofuran, 60
volumes of methanol
and 940 volumes of
phosphate buffer
solution pH 3.2 was
prepared.
--mobile phase B: A
mixture of 50
volumes of
A mixture of 0.1 M
monobasic sodium
phosphate and
acetonitrile (9:1) was
prepared and
degassed. The pH
adjusted with
phosphoric acid to
3.0 ± 0.1
Buffer: 2.0 g/L of
ammonium
dihydrogen
phosphate was
prepared. The pH
was adjusted with
phosphoric acid to
3.0.
Solution A: Methanol
and acetonitrile (1:1)
was mixed.
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SPP SPTM, SVKM’s NMIMS, Mumbai 28
tetrahydrofuran, 500
volumes of methanol
and 500 volumes of
phosphate buffer
solution pH 3.2 was
prepared.
Gradient Not Applicable Isocratic (Run time
not mentioned)
Isocratic (Run time
not mentioned)
Time
(min)
%A %B
0 85 15
3.50 85 15
3.51 45 55
7.70 45 55
7.71 20 80
12.0 20 80
12.1 85 15
15.5 85 15
Flow Rate Not Applicable 0.7ml/min 0.8ml/min 2ml/min 1.2ml/min
Wavelength Not Applicable 298nm 224nm 254nm 270-Telmisartan
298- HCTZ
Inj Vol (µL) Not Applicable 5 20 20 10
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SPP SPTM, SVKM’s NMIMS, Mumbai 29
4.3 PRESENT WORK AND DISCUSSION 4.3.1. Selection of Chromatographic Method Official methods and methods published in literature for Telmisartan and
Hydrochlorothiazide were based on reverse phase chromatographic (RPC) separation.
So, all development was conducted using reverse phase chromatography. Reverse Phase
chromatography is a choice because of its ease of handling and robust nature.
4.3.2 Selection of Stationary Phase
USP, EP monograph and reported HPLC methods of Telmisartan or
hydrochlorothiazide recommended use of C18 column for the purpose of determination
of related impurities and assay. So C18 columns were preferred as stationary phase. The
EP impurity mixture was used to understand the separation of Telmisartan impurities.
Since EP API method was based on Kromasil C18 column, this column was given
higher preference over other C18 brands to ensure similarity of elution to EP
monograph.
4.3.3 Selection of Wavelength for Analysis
The optimum wavelength selected was 270 nm which represents the wavelength where
all impurities have suitable responses in order to permit simultaneous determination of
related impurities of Telmisartan and HCTZ. The overlain UV spectrum for HCTZ, TE
and their related substances were recorded. All overlaid components showed desirable
absorbance at 270nm. Hence 270nm was selected for method development. 270nm is
also the wavelength which has an absorption maximum for hydrochlorothiazide and an
absorption minimum for Telmisartan. Thus at this wavelength, minor changes will not
affect peak areas and thus the final result. This will in effect produce a robust method
with respect to wavelength.
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Figure: 4.3.3.1 Overlaid UV spectrum of Telmisartan and Hydrochlorothiazide
4.3.4 Selection and Optimization of Mobile Phase From literature survey, it was observed that Hydrochlorothiazide is poorly retained on
C18 stationary phase whereas Telmisartan is strongly retained. Thus a gradient elution
program was selected over isocratic elution. A high aqueous percentage was used in the
initial period to retain the hydrochlorothiazide and its impurities while a higher organic
was used to elute the Telmisartan and its impurities in the later period.
Various gradient trials were taken. It was observed that there was inadequate separation
between Hydrochlorothiazide and its related impurities. Additionally they were eluting
very close to the dead volume. To overcome this problem, initial trials were conducted
with high percentage of aqueous phase. It led to longer retention time for Telmisartan
and its impurities. Further trials were taken by using cationic ion pairing agent (Sodium
salt of hexane sulphonic acid). This not only served to retain the Hydrochlorothiazide
better, it also showed a better separation of the Telmisartan impurity peaks. The run
time was found considerably less as compared to previous trials. The use of ion pairing
reagent permitted usage of higher percentage of organic phase during the start of the
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SPP SPTM, SVKM’s NMIMS, Mumbai 31
gradient. This facilitated earlier elution of Telmisartan and its impurities with desirable
separation.
The above developed method was tried for separation of known impurities and
unknown impurities generated by forced degradation.
The major challenge faced during the development of a single method for simultaneous
determination of related substances of more than one API is not only the separation of
impurities from each other and the main peaks but also to identify the source of
unknown impurities. It is of critical importance to allot an unknown impurity to the
correct API component since the estimation of impurities is based on the label claim of
the API in the finished product. The limits of the unknown impurities are in turn based
on daily doses of the individual API. A wrong identification can pass an out of
specifications (OOS) batch or erroneously reject a correct batch.
This critical determination was handled in the following manner by analysing three
placebo preparations apart from the sample.
1. Placebo which has all the excipient except for the APIs
2. Placebo which has all excipient and Telmisartan API spiked in the same
quantitative composition as in the finished product. This is the placebo for
Hydrochlorothiazide part.
3. Placebo which has all excipient and Hydrochlorothiazide API spiked in the same
quantitative composition as in the finished product. This is the placebo for
Telmisartan part.
The forced degradation studies were performed on all the above preparations in addition
to the finished product to determine the source of the unknown impurities.
The next challenge was in selection of common diluent for both the drugs in the tablet.
Initially 0.1M Ethanolic sodium hydroxide was used as a common diluent for the tablet,
since Telmisartan is soluble is basic alcoholic diluents. Though this diluent was suitable
for dissolving both the drug, it was observed that in this diluent Hydrochlorothiazide
degraded rapidly. This problem was overcome by using a weaker (0.01M) Ethanolic
sodium hydroxide solution.
Initial gradient trials were conducted using buffer (pH = 3) containing ion pair reagent
as majority of the methods reported in the literature used the same strategy. Sodium
Hexane sulphonate, monohydrate was chosen due to its easy availability in pure form
and stability. Purity of ion pair reagent used is very critical for method development and
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SPP SPTM, SVKM’s NMIMS, Mumbai 32
further routine analysis. The ion paring capacity of the reagent directly depends on its
purity.
Initial trials showed a slight fronting on the main peaks, probably due to action of the
ion paring reagent. This was overcome by increasing the column temperature to 40°C.
Various gradient elution programs were tried to obtain desirable results. The final
optimized chromatographic conditions are given below.
Optimized Chromatographic Conditions:
Chromatographic condition for related substances:
Instruments/Equipment : HPLC, Make – Waters, Alliance, 2695 Separation
Module, (UV/PDA), or equivalent.
Analytical Balance, Make –Mettler Toledo, Model-
XS205DU, or equivalent.
Column : Kromasil C18, 250 x 4.6 mm, 5µm or equivalent
Flow rate : 1.0 ml/minute
Column temperature : 40°C
Wavelength : 270 nm
Sample temperature : 20°C
Injection volume : 10 µl
Run time for sample : 45 minutes
Gradient Program:
Time in minutes Mobile Phase A % Mobile Phase B %
0.01 94 6
15 94 6
35 20 80
40 20 80
42 94 6
45 94 6
Mobile Phase A : 2.0 gm of Potassium dihydrogen phosphate anhydrous and 1.04 gm of Sodium 1- Hexane sulphonic acid monohydrate, in 1000ml of water; pH 3.0 with ortho phosphoric acid Mobile Phase B : A mixture of Acetonitrile: Methanol in the ratio 80:20 v/v
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SPP SPTM, SVKM’s NMIMS, Mumbai 33
4.4 FORCED DEGRADATION STUDIES
The forced degradation studies were carried out to achieve adequate degradation of the
individual drugs. They were carried out on the higher strength tablets (80mg-12.5mg)
and chromatographed along with a non-stressed sample (control). The role of placebo
degradation is a significant part of method development for multi-drug formulation.
4.4.1 Telmisartan Degradation
4.4.1.1 Hydrolytic conditions: acid-, base-induced degradation.
Acid Degradation
10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml volumetric
flask. 10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with intermittent
shaking till the tablets disintegrated. The solution was cooled to room temperature. About
10 ml of diluent C was added. 5 ml of 5N Hydrochloric acid was added and the solution
was heated in the oven at 70°C for 24 hours. The solution was then cooled and neutralized
with same volume and same strength alkali and made up the volume with diluent-D. Further
3 ml was diluted to 25 ml with diluent-D and filtered through 0.45 Nylon filter.
Placebos were treated similarly.
Base Degradation
10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml volumetric flask.
10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with intermittent shaking till
the tablets disintegrated. The solution was cooled to room temperature. About 10 ml of diluent
C was added. 5 ml of 5N Sodium Hydroxide was added and the solution was heated in the oven
at 70°C for 24 hours. The solution was then cooled and neutralized with same volume and same
strength acid and made up the volume with diluent-D. Further 3 ml was diluted to 25 ml with
diluent-D and filtered through 0.45 Nylon filter.
Placebos were treated similarly.
4.4.1.2 Oxidative condition: hydrogen peroxide-induced degradation.
10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml volumetric flask.
10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with intermittent shaking till
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SPP SPTM, SVKM’s NMIMS, Mumbai 34
the tablets disintegrated. The solution was cooled to room temperature. About 10 ml of diluent
C was added. 5 ml of 50 % Hydrogen peroxide was added. The solution was heated in the oven
at 70°C for 24 hours. The solution was then cooled and made up the volume with diluent-D
Placebos were treated similarly.
4.4.1.3 Thermal degradation.
10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml
volumetric flask. 10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with
intermittent shaking till the tablets disintegrate. The solution was cooled at room
temperature and 10 ml of diluent C was added. The solution was heated in the oven at
70°C for 24 hours, cooled and volume was made up the with diluent-D. Further 3 ml of
this solution was diluted to 25 ml with diluent-D
Placebos were treated similarly.
4.4.1.4 Photolytic degradation.
As per guidelines for photostability testing of new drug substances and products,
samples
should be exposed to light providing an overall illumination of not less than 1.2 million
lx hours and an integrated near ultraviolet energy of not less than 200Wh/m2 to allow
direct comparisons to be made between the drug substance and drug product. [19]
For photo stability testing 10 tablets of Telmisartan + Hydrochlorothiazide were
transferred into each of 100 ml clear glass, 100 ml flask covered with aluminium foil
and 100 ml amber colour volumetric flask. 10 ml of diluent-B was added and
sonnicated for 5 to 10 minutes with intermittent shaking till the tablets disintegrated.
The solution was cooled at room temperature and 10 ml of diluent C was added. These
flasks were kept under UV and white light for 1.2 million lux hours in photo stability
chamber/ 200Wh m-2. After study the sample was cooled and diluted upto the mark
with diluent-D. Further 3 ml of this solution was diluted to 25 ml with diluent-D and
filtered through 0.45μ Nylon filter.
Placebos were treated similarly.
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4.4.2 Hydrochlorothiazide Degradation
4.4.2.1 Hydrolytic conditions: acid-, base-induced degradation.
Acid Degradation
10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml volumetric
flask. 10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with intermittent
shaking till the tablets disintegrated. The solution was cooled to room temperature. About
10 ml of diluent C was added. 5 ml of 5N Hydrochloric acid was added and the solution
was heated in the oven at 70°C for 1 hour. The solution was then cooled and neutralized
with same volume and same strength alkali and made up the volume with diluent-D. Further
3 ml was diluted to 25 ml with diluent-D and filtered through 0.45 Nylon filter.
Placebos were treated similarly.
Base Degradation
10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml volumetric flask.
10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with intermittent shaking till
the tablets disintegrated. The solution was cooled to room temperature. About 10 ml of diluent
C was added. 5 ml of 5N sodium hydroxide was added and the solution was heated in the oven
at 70°C for 6 hours. The solution was then cooled and neutralized with same volume and same
strength acid and made up the volume with diluent-D. Further 3 ml was diluted to 25 ml with
diluent-D and filtered through 0.45 Nylon filter.
Placebos were treated similarly.
4.4.2.2 Oxidative condition: hydrogen peroxide-induced degradation.
10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml volumetric flask.
10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with intermittent shaking till
the tablets disintegrated. The solution was cooled to room temperature. About 10 ml of diluent
C was added. 5 ml of 50 % Hydrogen peroxide was added. The solution was heated in the oven
at 70°C for 6 hours. The solution was then cooled and made up the volume with diluent-D
4.4.2.3 Thermal degradation.
10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml
volumetric flask. 10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with
intermittent shaking till the tablets disintegrate. The solution was cooled at room
temperature and 10 ml of diluent C was added. The solution was heated in the oven at
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SPP SPTM, SVKM’s NMIMS, Mumbai 36
70°C for 6 hours, cooled and volume was made up with diluent-D. Further 3 ml of this
solution was diluted to 25 ml with diluent-D
4.4.2.4 Photolytic degradation.
As per guidelines for photostability testing of new drug substances and products,
samples should be exposed to light providing an overall illumination of not less than 1.2
million lx hours and an integrated near ultraviolet energy of not less than 200Wh/m2 to
allow direct comparisons to be made between the drug substance and drug product. [19]
For photo stability testing 10 tablets of Telmisartan + Hydrochlorothiazide were
transferred into each of 100 ml clear glass, 100 ml flask covered with aluminium foil
and 100 ml amber colour volumetric flask. 10 ml of diluent-B was added and
sonnicated for 5 to 10 minutes with intermittent shaking till the tablets disintegrated.
The solution was cooled at room temperature and 10 ml of diluent C was added. These
flasks were kept under UV and white light for 1.2 million lux hours in photo stability
chamber/ 200Wh m-2. After study the sample was cooled and diluted upto the mark
with diluent-D. Further 3 ml of this solution was diluted to 25 ml with diluent-D and
filtered through 0.45μ Nylon filter.
Placebos were treated similarly.
4.4.3 Observations in forced degradation studies.
It was observed that overall Telmisartan is a much stable molecule than
Hydrochlorothiazide under the harsh conditions. Hydrochlorothiazide degraded in most
conditions. The chromatograms are given in the below figures.
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SPP SPTM, SVKM’s NMIMS, Mumbai 37
Figure-4.4.1: Chromatogram of Blank.
Figure-4.4.2: Chromatogram of Placebo without Telmisartan and Hydrochlorothiazide.
Figure-4.4.3: Chromatogram of Placebo with Telmisartan.
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SPP SPTM, SVKM’s NMIMS, Mumbai 38
Figure-4.4.4: Chromatogram of Placebo with Hydrochlorothiazide.
Figure-4.4.5: Chromatogram of Standard solution.
Figure-4.4.6: Chromatogram of Control Sample solution.
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SPP SPTM, SVKM’s NMIMS, Mumbai 39
Figure-4.4.7: Chromatogram of Impurities mixture with Telmisartan and Hydrochlorothiazide.
Name Retention Time USP Resolution
HCTZ-IMP-B 10.674 HCTZ-IMP-A 14.909 6.23
HYDROCHLOROTHIAZIDE 17.918 3.72 TELMISARTAN IMP-A 27.582 20.43
HCTZ-IMP-C 27.812 2.05 TELMISARTAN IMP-E 31.130 26.65 TELMISARTAN IMP-F 31.497 2.73 TELMISARTAN IMP-B 32.911 10.35
TELMISARTAN 34.704 11.92 TELMISARTAN IMP-C 36.157 9.53
Figure-4.4.8: Chromatogram of sample in Acid for Telmisartan.
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SPP SPTM, SVKM’s NMIMS, Mumbai 40
Figure-4.4.9: Chromatogram of sample in Acid for Hydrochlorothiazide.
Figure-4.4.10: Chromatogram of sample in Base for Telmisartan.
Figure-4.4.11: Chromatogram of sample in Base for Hydrochlorothiazide.
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Figure-4.4.12.1: Chromatogram of sample in Peroxide for Telmisartan.
Figure-4.4.12.2: Chromatogram of sample in Peroxide for Telmisartan (Zoom Scale from 20 to 40 mintues)
Figure-4.4.13.1: Chromatogram of sample in Peroxide for Hydrochlorothiazide.
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SPP SPTM, SVKM’s NMIMS, Mumbai 42
Figure-4.4.13.2: Chromatogram of sample in Peroxide for Hydrochlorothiazide (Zoom Scale from 20 to 40 mintues)
Figure-4.4.14: Chromatogram of sample Heat for Telmisartan.
Figure-4.4.15: Chromatogram of sample Heat for Hydrochlorothiazide.
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SPP SPTM, SVKM’s NMIMS, Mumbai 43
Figure-4.4.16: Chromatogram of sample in Photolytic study.
Telmisartan
The major degradation was observed under acidic and oxidative stress conditions. There
was no significant degradation observed under other stress conditions.
Under acidic stress conditions one degradant was generated and eluted at RRT of about
1.05. Since Telmisartan is a relatively stable molecule further investigation was carried
out to find out the nature of this degradant. The sample was treated under same stress
conditions by replacing the methanol in the diluents with Acetonitrile. This particular
degradant was not generated under this stress condition. This study confirmed that the
observed peak was actually an ester formed in situ due to interaction between methanol
and Telmisartan. Please refer to Figures 4.4.17 and 4.4.18 respectively.
Based on the above facts, the following impurity formation pathway can be predicted:
N
N
CH3
N
N
CH3
CH3
O
OH
Methanol
Hydrochloric
acid
N
N
CH3
N
N
CH3
CH3
O
O CH3
Telmisartan Methyl ester of Telmisartan
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 44
Figure-4.4.17: Degradation of Telmisartan under Acidic condition with diluents containing
Acetonitrile in place of methanol.
Under oxidative conditions none of the individual degradant generated was significant
to perform degradant qualification.
Hydrochlorothiazide
Hydrochlorothiazide, unlike Telmisartan, degraded under most of the stress conditions.
A common degradant formed under all the stress conditions was Impurity B, i.e. 4-
Amino-6-chloro-1,3-benzenedisulfonamide.
Peak Observed when methanol is present in Diluents
Peak is not observed when Acetonitrile is present in Diluents instead of Methanol
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 45
This is a known and qualified impurity as per USP/ EP
A short summary of the observation is given in the table 4.4.1.
Table: 4.4.1 Samples injected under different stress conditions
Condition % Degradation % Degradation
Telmisartan Hydrochlorothiazide
Acidic 6.65% 20.90%
Basic Not Significant 29.86%
Oxidation 6.18% 20.11%
Thermal Not Significant 22.25%
Photolytic Not Significant 13.18% (with
respect to control)
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 46
4.5 EXPERIMENTAL WORK 4.5.1 Instrumentation
Equipment Make Model
HPLC Waters 2695Alliance Separation
Module, (PDA/UV
Detector) 2996/2487
Column AKZO
NOBEL
Kromasil C-18,
250 x 4.6 mm, 5µm
pH meter Thermo
Electron
Corp.
Orion-4star 1117000
Analytical
Balance
Mettler
Toledo
XS205DU
Micro Balance Mettler
Toledo
UMX-2
Ultrasonnicator Spectralab -
Photostability
Chamber
Thermolab 400litr
Water Bath Spectralab
4.5.2 Chemicals and Reagents
Name Grade Manufacturer
Potassium dihydrogen phosphate GR Merck Sodium 1- Hexane sulphonic acid monohydrate HPLC Alfa Aesar Methanol HPLC, Gradient grade Merck Acetonitrile HPLC, Gradient grade Rankem Monobasic sodium phosphate GR Merck
Ortho-Phosphoric acid GR Merck Sodium Hydroxide GR Merck Hydrochloric acid GR Merck 50% Hydrogen peroxide GR Merck Water HPLC milli-Q In-house
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 47
4.5.3 Working Standard
Standard Lot .No. Potency (as is) %
Telmisartan TE0010108 98.7
Hydrochlorothiazide HCT/60914 99.5
HCTZ-Impurity-B CRD/HCTZ/01/30/01 99.5
4.5.4 Solution Preparation Preparation of Diluent
Diluent – A: 0.1 N alcoholic NaOH
Weigh accurately and transfer 4 gm of Sodium hydroxide in 1000 ml volumetric flask
add 40 ml water sonnicate to dissolve and make up to mark with methanol.
Diluent – B: 0.005 N alcoholic NaOH
Dilute 50 ml of Diluent A to 1000 ml volumetric flask and make up to mark with
Methanol.
Diluent – C
Prepare a mixture of Buffer: Acetonitrile in the ratio 70:30 v/v.
Diluent – D
Prepare a mixture of Methanol: Water in the ratio 50:50 v/v.
Diluent – E
Prepare a mixture of Acetonitrile: Water in the ratio 50:50 v/v.
Preparation of Mobile Phase
Mobile Phase A: Buffer (pH – 3.0)
Weigh accurately 2.0 gm of Potassium dihydrogen phosphate anhydrous and 1.04 gm
of Sodium 1- Hexane sulphonic acid monohydrate, transfer into 1000ml of water.
Adjust the pH 3.0 with ortho phosphoric acid.
Mobile Phase B
Prepare a mixture of Acetonitrile: Methanol in the ratio 80:20 v/v. Mix and degas.
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 48
Preparation of Reference Solution – Hydrochlorothiazide
Weigh accurately and transfer about 1.0 mg of Hydrochlorothiazide Impurity B in 10
ml volumetric flask and dilute to volume with Diluent E.
Preparation of Impurity Mixture Solution
Take 150 µl of Impurity B and 1850 µl of Hydrochlorothiazide API.
Preparation of Standard solution
Preparation of Standard Stock Solution - Telmisartan
Weigh accurately about 40.0 mg of Telmisartan working standard and transfer into a
200ml volumetric flask. Add about 70 ml of Diluent - A, sonnicate to dissolve and
make up the volume up to the mark with Diluent - A. Further dilute this solution 5 ml
to 50 ml with Diluent – D.
Preparation of Standard Stock Solution - Hydrochlorothiazide
Weigh accurately about 60.0 mg of Hydrochlorothiazide working standard and
transfer into a 200 ml volumetric flask. Add about 70 ml of in Diluent -E, sonnicate to
dissolve and make up the volume up to the mark with Diluent E. Further dilute this
solution 5 ml to 50 ml with Diluent E.
Preparation of Standard Solution
Take 15 ml Standard stock solution of Telmisartan and 5 ml Standard stock solution
of Hydrochlorothiazide in 100 ml volumetric flask, make the volume with Diluent -D.
Preparation of Sample solution
For 80 – 12.5 mg
Weigh accurately 10 tablets and transfer in 100 ml volumetric flask. Add 10 ml of
Diluent-B, sonnicate for 5 to 10 minutes with intermittent shaking till the tablets
disintegrate. Cool the solution at room temperature, add 10 ml of Diluent C and make
up the volume with Diluent-D. Further dilute 3 ml of this solution to 25 ml with
Diluent -D. Filter through 0.45 µ Nylon filter and inject.
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 49
For 40 – 12.5 mg
Weigh accurately 10 tablets and transfer in 50 ml volumetric flask. Add 5 ml of
Diluent - B, sonnicate for 5 to 10 minutes with intermittent shaking till the tablets
disintegrate. Cool the solution at room temperature, add 5 ml of Diluent C and make
up the volume with Diluent-D. Further dilute 3 ml of this solution to 25 ml with
Diluent -D. Filter through 0.45 µ Nylon filter and inject.
Preparation of Placebo solution
For Telmisartan
Weigh Placebo sample equivalent to 10 tablets and transfer in 100 ml volumetric
flask. Add 10 ml of Diluent - B, sonnicate for 5 to 10 minutes with intermittent
shaking till the placebo disperses. Cool the solution at room temperature, add 10
ml of Diluent C and make up the volume with Diluent-D. Further dilute 3 ml of this
solution to 25 ml with Diluent -D. Filter through 0.45 µ Nylon filter and inject.
For Hydrochlorothiazide
Weigh Placebo sample equivalent to 10 tablets and transfer in 100ml volumetric flask.
Add 10 ml of Diluent - B, sonnicate for 5 to 10 minutes with intermittent shaking
till the placebo disperses. Cool the solution at room temperature, add 10 ml of Diluent
C and make up the volume with Diluent-D. Further dilute 3 ml of this solution to 25
ml with Diluent -D. Filter through 0.45 µ Nylon filter and inject.
For Placebo without Telmisartan + Hydrochlorothiazide
Weigh Placebo sample equivalent to 10 tablets and transfer in 100ml volumetric flask.
Add 10 ml of Diluent - B, sonnicate for 5 to 10 minutes with intermittent shaking till
the placebo disperses. Cool the solution at room temperature, add 10 ml of Diluent C
and make up the volume with Diluent-D. Further dilute 3 ml of this solution to 25 ml
with Diluent -D. Filter through 0.45 µ Nylon filter and inject.
Procedure
Separately inject equal volumes of Blank (Diluent), Placebo solution and sample
solution and measure the area counts for the impurity peaks. The retention time of
Telmisartan Peak is about 34.85 minutes. The retention time of Hydrochlorothiazide
Peak is about 17.93 minutes.
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 50
When the chromatograms are recorded under the prescribed conditions, the
approximate relative retention times, in the chromatogram of Impurity mixture
preparation are:
Name Relative retention
time
Relative response
factor
Impurity B About 0.6 0.74
Evaluation of System Suitability
Inject the Standard solution six times into the chromatograph and record the
chromatogram. The relative standard deviation for six replicate injections of standard
solution should not be more than 5.0 %.The system suitability has to be ensured at the
beginning of the exercise as well as at the end of the analysis. For a lengthy sample
sequence system suitability need to be ensured during the analysis as well.
Calculation
Calculate the % Impurities as follows:
For Telmisartan (80-12.5 mg)
AT Std. Wt. 5 15 100 25 P 100 1
% Impurity = ------ x ------------ x ------ x ------ x --------- x ------ x ------- x ------- x ------
AS 200 50 100 10 3 100 LC RRF
For Telmisartan (40-12.5 mg)
AT Std. Wt. 5 15 50 25 P 100 1
% Impurity = ------ x ------------ x ------- x ------ x --------- x ------ x ----- x ------- x ------
AS 200 50 100 10 3 100 LC RRF
Where,
AT : Peak area of Impurity in the chromatogram of sample solution.
AS : Average area count of Telmisartan peak in the chromatogram of
standard solution.
Std.wt : Weight of Telmisartan taken in mg
P : Percent potency of Telmisartan working standard on as such basis.
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 51
LC : Label claim of Telmisartan in mg
RRF : Relative response factor
Note: For impurities where RRF value is not mentioned, take it as 1.
For Hydrochlorothiazide (80-12.5 mg)
AT Std.Wt. 5 5 100 25 P 100 1
% Impurity = ------ x --------- x ------- x ------ x ------- x ------ x -----x ------- x -----
AS 200 50 100 10 3 100 LC RRF
For Hydrochlorothiazide (40-12.5 mg)
AT Std.Wt. 5 5 50 25 P 100 1
% Impurity = ------ x --------- x ------- x ------ x ------- x ------ x -----x ------- x -------
AS 200 50 100 10 3 100 LC RRF
Where ,
AT : Peak area of Impurity in the chromatogram of sample solution.
AS : Average area count of Hydrochlorothiazide peak in the
chromatogram of standard solution.
Std.wt : Weight of Hydrochlorothiazide taken in mg
P : Percent potency of Hydrochlorothiazide working standard on as
such basis.
LC : Label claim of Hydrochlorothiazide in mg
RRF : Relative response factor
Note: For impurities where RRF value is not mentioned, take it as 1.
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 52
4.6 VALIDATION OF THE DEVELOPED METHOD 4.6.1 Validation Parameters and Acceptance Criteria The forced degradation studies showed only one major degradant originating due to hydrochlorothiazide. This was 4-Amino-6-chloro-1,3-benzenedisulfonamide or Impurity B. None of the known impurities due to Telmisartan were generated during forced degradation. Thus during validation, only this impurity was identified as “known”, the rest were included under “unknown” or “unspecified” impurities whose limits are governed by ICH guidelines. In the specificity study the Telmisartan impurity mixture (obtained from EP) was injected to confirm separation of individual impurities.
Table 4.6.1: The validation summary
Sr.No. Parameters Acceptance
criteria
Result obtained
1.0 System suitability
% RSD for Standard
solution.
USP Tailing Factor
USP Plates
NMT 5.0 %
NMT 2.0
NLT 500000 for
Telmisartan and
10000 for HCTZ.
Telmisartan HCTZ
0.60
1.03
740969
2.63
0.95
17221
2.0
2.1
Specificity
Identification
Results should be
comparable with
respect to the
retention time and
relative retention
time.
Component RT RRT
Telmisartan 34.716 -
HCTZ 17.871 -
Impurities mixture
HCTZ-IMP-B 10.674 0.60*
HCTZ-IMP-A 14.909 0.83*
HCTZ 17.918 -
HCTZ-IMP-C 27.812 1.55*
TELMI-IMP-A 27.582 0.79°
TELMI-IMP-E 31.130 0.90°
TELMI-IMP-F 31.497 0.91°
TELMI-IMP-B 32.911 0.95°
TELMISARTAN 34.704 -
TELMI-IMP-C 36.157 1.04°
2.2
Interference
No interference
from blank and
placebo to main
component and
impurities
Complies
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 53
2.3
Peak purity
Purity angle
should be less
than purity
threshold.
Standard peak
should be pure.
Telmisartan HCTZ
Purity angle 0.472 2.449
Purity
Threshold 1.288 3.538
XX*= RRT with respect to Hydrochlorothiazide. XX°= RRT with respect to Telmisartan.
Table 4.6.1: The validation summary (continued)
Sr.No. Parameters Acceptance
criteria
Result obtained
2.4
Forced degradation
The peak due to
known impurities
should be pure as
shown on the
PDA.
Telmisartan
Condition % Total impurities
Control 0.04
Acid 6.65
Base 0.03
Peroxide 6.18
Heat 0.05
Photolytic 0.12
Hydrochlorothiazide
Control 0.43
Acid 20.90
Base 29.86
Peroxide 20.11
Heat 22.25
Photolytic 12.81
3.0 Limit of Detection
% RSD for LOD:
between 10% and
33%
Component % RSD Conc. (%)
HCTZ-IMP-B 25.37 0.007
HCTZ 15.68 0.020
Telmisartan 17.26 0.010
4.0 Limit of Quantitation % RSD for LOQ:
NMT 10%
HCTZ-IMP-B 7.36 0.027
HCTZ 5.43 0.060
Telmisartan 5.42 0.031
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 54
Table 4.6.1: The validation summary (continued)
Sr.No. Parameters Acceptance criteria Result obtained
5.0 Linearity
Response should be
Linear Response is linear
Correlation coefficient
should not be less than
0.99.
HCTZ-IMP-B 0.9998
HCTZ 1.0000
Telmisartan 0.9940
% Limit of Y-
Intercept should be
within ± 10.0% of the
corresponding Y-co-
ordinate of the
working level.
HCTZ-IMP-B -1.11
HCTZ -1.53
Telmisartan 0.68
6.0 Accuracy
(Recovery)
At LOQ Level mean
recovery should be in
the range 75.0 % to
125.0 %.
% Mean Recovery
mg HCTZ
IMP-B HCTZ Telmisartan
80 97.8 100.6 100.4
40 87.7 106.4 106.4
Mean recovery should
be in the range of
80.0%- 120.0%.
80 102.8 98.1 100.3
40 96.1 97.8 102.0
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 55
Table 4.6.1: The validation summary (continued)
Sr.
No
Parameters Acceptance criteria Result obtained
7.0
7.1
7.2
System Precision
System suitability
% RSD for Standard solution.
USP Tailing Factor
USP Plates
NMT 5.0 %
NMT 2.0
NLT 500000 for
Telmisartan and 10000
for HCTZ.
Telmisartan HCTZ
0.60
1.03
740969
2.63
0.95
17221
Method Precision
RSD for % Impurity content.
NMT 10.0%.
Component % RSD
Telmisartan
Strength 80/
12.5 40/ 12.5
Single max
imp 0.00 0.00
HCTZ
HCTZ-
IMP-B 4.43 2.00
Single max
imp 7.91 6.20
7.3 Intermediate Precision
(Ruggedness)
System suitability
% RSD for Standard solution.
USP Tailing Factor
USP Plates
RSD for % Impurity content.
RSD for pooled result
( Analyst-I and II )
NMT 5.0 %
NMT 2.0
NLT 500000 for
Telmisartan and 10000
for HCTZ.
NMT 10.0%.
NMT 10.0%.
Telmisartan HCTZ
0.97
0.94
765481
0.78
0.95
14693
Component % RSD
Telmisartan
Strength 80/
12.5 40/ 12.5
Single max 0.00 0.00
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 56
imp
Pooled result
Single max
imp 0.00 0.00
HCTZ
HCTZ-
IMP-B 2.03 0.52
Single max
imp 6.20 5.96
Pooled result
HCTZ-
IMP-B 4.49 2.93
Single max
imp 7.07 6.14
Table 4.6.1: The validation summary (continued)
Sr.
No
Parameters Acceptance criteria Result obtained
8.0 Stability in analytical
solution
The % Difference of Initial and
after 24 hours result should be
0.05.
Telmisartan HCTZ
Sample and
Standard
solutions are
stable for at
least upto
24hours at
20°C
Standard solutions
are stable for at
least upto 24hours
at 20°C and
Sample Solutions
are stable upto
6hours at 20°C
9.0 Filter compatibility
% Difference for impurity content
of Centrifugate and filtered should
be within 0.05.
Complies Complies
10.
0
Robustness
Change in Flow rate
(± 0.1 ml/min)
Change in wavelength
There should be no significant
change in system suitability
parameters.
There should be no significant
No significant change
No significant change
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 57
(265 and 272 nm)
Change in Buffer pH
(± 0.2)
Column oven
temperature (± 5°C)
change in system suitability
parameters.
There should be no significant
change in system suitability
parameters.
There should be no significant
change in system suitability
parameters.
No significant change
No significant change
4.6.2 System Suitability Single injection of Blank (Diluent) and six replicate injections of standard solution were
made on the system to demonstrate that the instrument is suitable for carrying out analytical
work. The data obtained is summarized in Table. 4.6.2.1
Table 4.6.2.1: System suitability
Standard Solution
Telmisartan Hydrochlorothiazide
USP Tailing 1.03 0.95
USP Plates 740969 17221
Area
69682 55157
68722 53993
69827 53093
69291 51298
69544 52004
69027 52601
Mean 69349 53024
SD 418.95 1392.25
%RSD 0.60 2.63
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 58
4.6.3 Specificity Peak Purity:
Blank (diluent), Placebo without Telmisartan and Hydrochlorothiazide, Placebo with
Telmisartan, Placebo with Hydrochlorothiazide, Standard solution, individual impurities,
mixture of all impurities with sample and sample solution were injected.
The data demonstrate that there is no interference in blank, placebo, Telmisartan,
Hydrochlorothiazide, Known and unknown impurities peaks. All peaks are well resolved.
The data obtained is summarized in Table 4.6.3.1, 4.6.3.2 and 4.6.3.3.
Table 4.6.3.1: Specificity (Identification and Interference)
Component Retention
time (min)
RRT Peak Purity
Telmisartan 34.716 - Pass
Hydrochlorothiazide 17.871 - Pass
Impurities mixture
HCTZ-IMP-B 10.674 0.60* Pass
HCTZ-IMP-A 14.909 0.83* Pass
Hydrochlorothiazide 17.918 -
HCTZ-IMP-C 27.812 1.55* Pass
TELMI-IMP-A 27.582 0.79° Pass
TELMI-IMP-E 31.130 0.90° Pass
TELMI-IMP-F 31.497 0.91° Pass
TELMI-IMP-B 32.911 0.95° Pass
Telmisartan 34.704 -
TELMI-IMP-C 36.157 1.04° Pass
XX*= RRT with respect to Hydrochlorothiazide. XX°= RRT with respect to Telmisartan.
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 59
Forced degradation data
Table 4.6.3.2: For Telmisartan
Condition % Impurity
Unk-1 Unk-2 Unk-3 Unk-4 Unk-5 Unk-6 Unk-7
RRT 0.79 0.82 0.83 0.85 0.86 0.87 0.88
Control
% Impurity - - - - - - -
Acid degradation
% Impurity 0.01 - - - - - -
Base degradation
% Impurity - - - - - - -
Peroxide degradation
% Impurity 0.82 0.45 1.96 0.56 0.38 0.33 0.20
Heat degradation
% Impurity 0.04 - - - - - -
Photolytic degradation
% Imp
Control
(Aluminium)
- - - - - - -
% Imp Amber
colour flask - - - - - - -
% Impurity in
Clear glass - - - - - - -
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 60
Table 4.6.3.2: For Telmisartan (continued)
Condition % Impurity
Unk-8 Unk-9 Unk-10 Unk-11 Unk-12 Unk-13 Unk-14
RRT 0.90 0.91 0.92 0.93 0.94 0.95 0.97
Control
% Impurity - - 0.02 - - - -
Acid degradation
% Impurity - - - - - - -
Base degradation
% Impurity - - - - - - -
Peroxide degradation
% Impurity 0.14 0.07 0.19 0.38 0.50 0.01 0.47
Heat degradation
% Impurity - - - - - - -
Photolytic degradation
% Imp
Control
(Aluminium)
- - 0.01 - 0.02 - -
% Imp Amber
colour flask - - 0.01 - 0.02 - -
% Impurity in
Clear glass - - 0.06 - 0.03 - -
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 61
Table 4.6.3.2: For Telmisartan (continued)
Condition % Impurity
Unk-15 Unk-16 Unk-17 Unk-18 Peak Purity
Standard
Solution
% Total
Impurity RRT 0.98 0.99 1.01 1.05
Control
% Impurity - - - 0.02 Peak Pure 0.04
Acid degradation
% Impurity - - - 6.64 Peak Pure 6.65
Base degradation
% Impurity - - - 0.03 Peak Pure 0.03
Peroxide degradation
% Impurity 0.06 0.04 0.54 0.05 Peak Pure 6.18
Heat degradation
% Impurity - - - 0.01 Peak Pure 0.05
Photolytic degradation
% Imp
Control
(Aluminium)
- - - 0.02 Peak Pure 0.05
% Imp Amber
colour flask - - - 0.03 Peak Pure 0.06
% Impurity in
Clear glass - - - 0.03 Peak Pure 0.12
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 62
Table 4.6.3.3: For Hydrochlorothiazide
Condition % Impurity
Imp-B Unk
1
Unk
2
Unk
3
Unk
4
Unk
5
Unk
6
Peak
Purity
%
Total
Impuri
ty
RRT 0.59 0.65 0.69 0.75 0.80 0.90 1.53
Control
%
Impurity 0.35 - - - - - 0.08
Peak
Pure 0.43
Acid degradation
%
Impurity 20.44 - - - 0.10 0.24 0.12
Peak
Pure 20.90
Base degradation
%
Impurity 24.42 0.49 0.39 0.42 3.75 0.39 -
Peak
Pure 29.86
Peroxide degradation
%
Impurity 17.63 0.17 1.83 - 0.21 0.13 0.14
Peak
Pure 20.11
Heat degradation
%
Impurity 20.71 - - - 0.24 1.30 -
Peak
Pure 22.25
Photolytic degradation
% Imp
Control
(Alumini
um foil
wrapped)
11.77 - - - 0.21 1.03 0.03 Peak
Pure 13.04
% Imp
Clear
glass
flask
14.53 - - - 0.22 11.4 0.04 Peak
Pure 26.22
%
Impurity
in Amber
flask
11.71 - - - 0.09 0.98 0.03 Peak
Pure 12.81
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 63
4.6.4 Determination of Limit of Detection (LOD) and Limit of Quantitation (LOQ) For determining LOD and LOQ, A series of dilutions with decreasing concentrations were
injected into the system and the areas were determined. Graph of concentration vs. area
were plotted and SLOPE of the line was calculated. Also the STEYX of this line
(Correction for the residual error of the peak areas and concentration) was determined.
The data obtained are summarized in Table 4.6.4.1.
Table 4.6.4.1: Prediction Linearity
Sr.No. HCTZ-IMP-B HCTZ Telmisartan
Conc.
(ppm)
Area Conc.
(ppm)
Area Conc.
(ppm)
Area
1 0.52 12473 0.50 17219 0.51 13108
2 0.41 9774 0.40 13126 0.41 10566
3 0.31 7490 0.30 10156 0.30 8940
4 0.21 4936 0.20 6080 0.20 7401
5 0.10 2504 0.10 2801 0.10 6315
CORREL 0.9998
0.9989
0.9886
STEYX 91.129 302.212 466.990
SLOPE 23819.686 35882.000 16269.506
PREDICTED LOD
(ppm) 0.01 0.03 0.09
PREDICTED LOQ
(ppm) 0.04 0.08 0.29
PREDICTED LOD
(%) 0.008 0.019 0.010
PREDICTED LOQ
(%) 0.026 0.056 0.030
Six replicates of above predicted LOD and LOQ solution were done on system and
calculated % RSD are summarized in Table 4.6.4.2 and 4.6.4.3. A percentage RSD of
below 10 is required for LOQ whereas a percentage RSD value from 10 to 30 is required
for LOD. Both the criteria had been fulfilled.
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 64
Table 4.6.4.2: Precision for LOD.
Sr.No. HCTZ-IMP-B HCTZ Telmisartan
Concentration (ppm) 0.010 0.030 0.100
Conc. w.r.t sample (%) 0.007 0.020 0.010
227 962 4246
535 1165 4904
457 969 4852
511 816 3949
420 1064 6218
433 763 5715
Mean 431 957 4981
SD 109.21 149.93 859.85
% RSD 25.37 15.68 17.26
Table 4.6.4.3: Precision for LOQ.
Sr.No. HCTZ-IMP-B HCTZ Telmisartan
Concentration (ppm) 0.041 0.090 0.300
Conc. w.r.t sample (%) 0.027 0.060 0.031
1338 2993 8483
1438 3063 9002
1231 2889 9611
1303 2954 9372
1185 2943 9076
1213 3346 9904
Mean 1285 3031 9241
SD 94.49 164.64 500.51
% RSD 7.36 5.43 5.42
Impurities LOD LOQ
Hydrochlorothiazide and its Related Impurities
Impurity B 0.007% (0.010ppm) 0.027% (0.041ppm)
HCTZ (Unknown) 0.020% (0.030ppm) 0.060% (0.090ppm)
Telmisartan and its Related Impurities
Telmisartan (Unknown) 0.010% (0.100ppm) 0.031% (0.300ppm)
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 65
4.6.5 Linearity and Range The Linearity of response was determined by preparing different concentrations of standard
stock solution ranging from LOQ to 200% of the limit concentration. The data summarized
in Table 4.6.5.1, 4.6.5.2 and 4.6.5.3. The data shows that the response is found to be linear;
Correlation coefficient is more than 0.99.
Table 4.6.5.1: Linearity of HCTZ-IMPURITY-B
Level (%) Concentration w.r.t
sample (%) Area
LOQ 0.03 1285
50.7 0.51 19731
68.0 0.68 26080
102.0 1.02 39030
135.3 1.35 51620
169.3 1.69 63000
203.3 2.03 77273
406.7 4.07 157727
CORRELATION COEFFICIENT (r) 0.9998
Y-INTERCEPT -502.40
SLOPE 38643.69
MEDIAN (AREA) 45325
% LIMIT OF Y-INTERCEPT ± 10% OF MEDIAN -1.11
Figure 4.6.5.1 Linearity of HCTZ Impurity B
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 66
Table 4.6.5.2: Linearity of HCTZ
Level (%) Concentration w.r.t
sample (%) Area
LOQ 0.06 3031
50.7 0.51 25697
67.3 0.67 35110
100.7 1.01 52425
134.7 1.35 69854
201.3 2.01 102554
336.0 3.36 175453
537.3 5.37 286142
1008 10.08 530695
CORRELATION COEFFICIENT (r) 1.0000
Y-INTERCEPT -1066.00
SLOPE 52848.28
MEDIAN (AREA) 69854
% LIMIT OF Y-INTERCEPT ± 10% OF MEDIAN -1.53
Figure 4.6.5.2 Linearity of HCTZ
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 67
Table 4.6.5.3: Linearity of Telmisartan
Level (%) Concentration w.r.t
sample (%) Area
LOQ 0.031 9241
51.0 0.05 13443
76.0 0.08 18009
102.0 0.11 22762
152.0 0.16 31932
203.0 0.21 41345
305.0 0.32 60045
508.0 0.53 116242
CORRELATION COEFFICIENT (r) 0.9940
Y-INTERCEPT 184.82
SLOPE 208947.31
MEDIAN (AREA) 27347
% LIMIT OF Y-INTERCEPT ± 10% OF MEDIAN 0.68
Figure 4.6.5.3 Linearity of Telmisartan
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 68
4.6.6 Accuracy
Thirteen samples of dosage in form of spiked placebo with HCTZ-IMP-B,
Hydrochlorothiazide and Telmisartan at four different levels for two strengths, each level
in triplicate were prepared. Single placebo preparation (unspiked), 3x LOQ, 3x 50%, 3x
100% and 3x 200% spiked placebo of the limit concentration were prepared. From the
amount added and the amount found, the percentage recovery was calculated along with
mean recovery. The results obtained were summarized in Table 4.6.6.1 (for system
suitability of 80-12.5 strength), 4.6.6.2, 4.6.6.3, 4.6.6.4, 4.6.6.5, 4.6.6.6, 4.6.6.7 and
4.6.6.8.
For 80/12.5 mg:
Table 4.6.6.1: System suitability
Standard Solution
Telmisartan Hydrochlorothiazide
USP Tailing 0.98 0.95
USP Plates 753230 14182
Area
54128 55008
53361 54571
54033 54809
53510 54702
54061 55456
53198 54297
Mean 53715 54807
SD 406.46 397.10
%RSD 0.76 0.72
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 69
Table 4.6.6.2: Accuracy for HCTZ-IMP-B
Level
%
Amount
added
%
Response
Amount
recovered
%
%
Recovery
Mean recovery
%
LOQ
0.031 1263 0.031 99.4
97.8 0.031 1206 0.029 94.9
0.031 1259 0.031 99.0
50 %
0.582 23787 0.580 99.7
102.8
0.582 23975 0.585 100.5
0.582 24280 0.592 101.7
100 %
1.163 48683 1.187 102.1
1.163 49539 1.208 103.9
1.163 49358 1.204 103.5
200 %
2.326 99818 2.434 104.7
2.326 99757 2.433 104.6
2.326 99575 2.428 104.4
Table 4.6.6.3: Accuracy for Hydrochlorothiazide
Level
%
Amount
added
%
Response Amount
recovered
%
%
Recovery
Mean recovery
%
LOQ
0.050 2825 0.051 102.0
100.6 0.050 2787 0.050 100.6
0.050 2747 0.050 99.1
50 %
0.495 26238 0.473 95.7
98.1
0.495 26490 0.478 96.6
0.495 26591 0.480 96.9
100 %
0.991 53521 0.966 97.5
0.991 54009 0.975 98.3
0.991 54030 0.975 98.4
200 %
1.981 108561 1.959 98.9
1.981 110176 1.988 100.4
1.981 110142 1.988 100.3
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 70
Table 4.6.6.4: Accuracy for Telmisartan
Level
%
Amount
added
%
Response
Amount
recovered
%
%
Recovery
Mean recovery
%
LOQ
0.031 5488 0.032 103.0
100.4 0.031 5329 0.031 100.0
0.031 5227 0.030 98.1
50 %
0.051 9348 0.054 105.3
100.3
0.051 9328 0.054 105.1
0.051 9375 0.054 105.6
100 %
0.103 17400 0.101 98.0
0.103 17647 0.102 99.4
0.103 17569 0.101 98.9
200 %
0.205 34162 0.197 96.2
0.205 34607 0.200 97.4
0.205 34469 0.199 97.1
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 71
For 40/12.5 mg:
Table 4.6.6.5: System suitability
Standard Solution
Telmisartan Hydrochlorothiazide
USP Tailing 0.98 1.00
USP Plates 778385 12225
Area
52731 55504
52786 54547
53642 55366
53324 55582
53703 56439
54607 55032
Mean 53466 55412
SD 694.52 630.37
%RSD 1.30 1.14
Table 4.6.6.6: Accuracy for HCTZ-IMP-B
Level
%
Amount
added
%
Response
Amount
recovered
%
%
Recovery
Mean recovery
%
LOQ
0.015 2179 0.013 87.6
87.7 0.015 2188 0.013 88.0
0.015 2179 0.013 87.6
50 %
0.582 45178 0.545 93.6
96.1
0.582 42373 0.511 87.8
0.582 50621 0.610 104.9
100 %
1.163 92948 1.121 96.4
1.163 91496 1.103 94.9
1.163 90611 1.093 94.0
200 %
2.326 188991 2.279 98.0
2.326 187841 2.265 97.4
2.326 189530 2.286 98.3
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 72
Table 4.6.6.7: Accuracy for Hydrochlorothiazide
Level
%
Amount
added
%
Response
Amount
recovered
%
%
Recovery
Mean recovery
%
LOQ
0.025 5996 0.027 107.0
106.4 0.025 5859 0.026 104.6
0.025 6036 0.027 107.7
50 %
0.495 52358 0.467 94.4
97.8
0.495 50013 0.446 90.2
0.495 52975 0.473 95.5
100 %
0.991 109678 0.979 98.8
0.991 107620 0.960 96.9
0.991 106648 0.952 96.0
200 %
1.981 229187 2.045 103.2
1.981 228791 2.042 103.1
1.981 226977 2.026 102.3
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 73
Table 4.6.6.8: Accuracy for Telmisartan
Level
%
Amount
added
%
Response
Amount
recovered
%
%
Recovery
Mean recovery
%
LOQ
0.026 4734 0.027 107.1
106.4 0.026 4770 0.028 108.0
0.026 4603 0.027 104.2
50 %
0.051 9523 0.055 107.8
102.0
0.051 9110 0.053 103.1
0.051 9599 0.056 108.6
100 %
0.103 17768 0.103 100.5
0.103 17764 0.103 100.5
0.103 17521 0.102 99.1
200 %
0.205 35262 0.205 99.8
0.205 35181 0.204 99.5
0.205 34942 0.203 98.8
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 74
4.6.7 Precision 4.6.7.1 System Precision:
Single injection of Blank (Diluent) and six replicate injections of standard solution were
made on the system. The data obtained is summarized in Table 4.6.7.1.1. The data
demonstrated that the system was suitable.
Table 4.6.7.1.1: System precision
Standard Solution
Telmisartan Hydrochlorothiazide
USP Tailing 1.03 0.95
USP Plates 740969 17221
Area
69682 55157
68722 53993
69827 53093
69291 51298
69544 52004
69027 52601
Mean 69349 53024
SD 418.95 1392.25
%RSD 0.60 2.63
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 75
4.6.7.2 Method Precision:
Six sample solution, were prepared and injected on the HPLC. The data obtained is
summarized in Table 4.6.7.2.1, 4.6.7.2.2, 4.6.7.2.3 and 4.6.7.2.4. All data are well within
acceptance criteria.
For 80/12.5 mg Tablets:
Table 4.6.7.2.1: Method precision (Telmisartan)
Spl. No. Unknown-1 Unknown-2 Unknown-3 % Total
impurities RRT-0.92 RRT-1.05 RRT-1.07
1 0.01 0.02 0.01 0.04
2 0.01 0.02 0.01 0.04
3 0.01 0.02 0.01 0.04
4 0.01 0.02 0.01 0.04
5 0.01 0.02 0.01 0.04
6 0.01 0.02 0.01 0.04
Mean 0.01 0.02 0.01 0.04
SD 0.000 0.000 0.000 0.000
% RSD 0.00 0.00 0.00 0.00
Table 4.6.7.2.2: Method precision (Hydrochlorothiazide)
Spl. No.
HCTZ-IMP-B Unknown-1 % Total
impurities
% Total Impurities
(Telmisartan +
HCTZ) RRT-0.60 RRT-1.51
1 0.34 0.08 0.42 0.46
2 0.35 0.08 0.43 0.47
3 0.34 0.08 0.42 0.46
4 0.34 0.09 0.43 0.47
5 0.38 0.08 0.46 0.50
6 0.35 0.07 0.42 0.46
Mean 0.35 0.08 0.43 0.47
SD 0.015 0.006 0.015 0.015
% RSD 4.43 7.91 3.60 3.30
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 76
For 40/12.5 mg Tablets:
Table 4.6.7.2.3: Method precision (Telmisartan)
Spl. No. Unknown-1 Unknown-2 Unknown-3 % Total
impurities RRT-0.92 RRT-1.05 RRT-1.07
1 0.01 0.02 0.01 0.04
2 0.01 0.02 0.01 0.04
3 0.01 0.02 0.01 0.04
4 0.01 0.02 0.01 0.04
5 0.01 0.02 0.01 0.04
6 0.01 0.02 0.01 0.04
Mean 0.01 0.02 0.01 0.04
SD 0.000 0.000 0.000 0.000
% RSD 0.00 0.00 0.00 0.00
Table 4.9.6.7.4: Method precision (Hydrochlorothiazide)
Spl. No. HCTZ-IMP-B Single max imp % Total
impurities
% Total Impurities
(Telmisartan +
HCTZ) RRT-0.60 RRT-0.85
1 0.75 0.08 0.95 0.99
2 0.74 0.09 0.95 0.99
3 0.74 0.09 0.95 0.99
4 0.78 0.08 0.98 1.02
5 0.76 0.08 0.96 1.00
6 0.75 0.08 0.95 0.99
Mean 0.75 0.08 0.96 1.00
SD 0.015 0.005 0.012 0.012
% RSD 2.00 6.20 1.27 1.22
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 77
4.6.7.3 Intermediate Precision (Ruggedness):
Same procedure of system precision and method precision was followed by another Analyst
on different instrument and on different day. The data obtained from Analyst-II are
summarized in Table 4.6.7.3.1, 4.6.7.3.2, 4.6.7.3.3, 4.6.7.3.4 and 4.6.7.3.5. Table 4.6.7.3.1
demonstrates the system was suitable for the experiment.
Table 4.6.7.3.1: System suitability
Standard Solution
Telmisartan Hydrochlorothiazide
USP Tailing 0.94 0.95
USP Plates 765481 14693
Area
59038 53876
59296 53502
60409 54612
59058 53512
59999 54133
59111 54051
Mean 59485 53948
SD 578.91 419.51
%RSD 0.97 0.78
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 78
For 80/12.5 mg Tablets:
Table 4.6.7.3.2: Intermediate precision (Telmisartan)
Spl. No. Unknown-1 Unknown-2 Unknown-3 % Total
impurities RRT-0.92 RRT-1.05 RRT-1.07
1 0.01 0.02 0.01 0.04
2 0.01 0.02 0.01 0.04
3 0.01 0.02 0.01 0.04
4 0.01 0.02 0.01 0.04
5 0.01 0.02 0.01 0.04
6 0.01 0.02 0.01 0.04
Mean 0.01 0.02 0.01 0.04
SD 0.000 0.000 0.000 0.000
% RSD 0.00 0.00 0.00 0.00
Table 4.6.7.3.3: Intermediate precision (Hydrochlorothiazide)
Spl. No. HCTZ-IMP-B Single max % Total
impurities
% Total
Impurities
(Telmisartan +
HCTZ) RRT-0.62 RRT-1.51
1 0.37 0.08 0.45 0.49
2 0.36 0.08 0.44 0.48
3 0.38 0.09 0.47 0.51
4 0.37 0.08 0.45 0.49
5 0.37 0.09 0.46 0.50
6 0.38 0.08 0.46 0.50
Mean 0.37 0.08 0.46 0.50
SD 0.008 0.005 0.010 0.010
% RSD 2.03 6.20 2.31 2.12
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 79
For 40/12.5 mg Tablets:
Table 4.6.7.3.4: Intermediate precision (Telmisartan)
Spl. No. Unknown-1 Unknown-2 Unknown-3 % Total
impurities RRT-0.92 RRT-1.05 RRT-1.07
1 0.01 0.02 0.01 0.04
2 0.01 0.02 0.01 0.04
3 0.01 0.02 0.01 0.04
4 0.01 0.02 0.01 0.04
5 0.01 0.02 0.01 0.04
6 0.01 0.02 0.01 0.04
Mean 0.01 0.02 0.01 0.04
SD 0.000 0.000 0.000 0.000
% RSD 0.00 0.00 0.00 0.00
Table 4.6.7.3.5: Intermediate precision (Hydrochlorothiazide)
Spl. No. HCTZ-IMP-B Single max % Total
impurities
% Total Impurities
(Telmisartan +
HCTZ) RRT-0.60 RRT-0.85
1 0.79 0.09 1.01 1.05
2 0.79 0.09 1.01 1.05
3 0.79 0.08 1.00 1.04
4 0.79 0.08 1.00 1.04
5 0.79 0.09 1.02 1.06
6 0.80 0.09 1.02 1.06
Mean 0.79 0.09 1.01 1.05
SD 0.004 0.005 0.009 0.009
% RSD 0.52 5.96 0.89 0.85
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 80
The pooled data obtained from Analyst-I and Analyst-II is summarized in Table 4.6.7.3.6 and
4.6.7.3.7. All data are within acceptance limits.
Table 4.6.7.3.6: Pooled data (80/12.5 mg Tablets)
Analyst
% Impurity of Telmisartan% Impurity of
HCTZ % Total
impuritiesUNK-1 UNK-
2 UNK-
3 HCTZ-IMP-B
Single max
RRT-0.92
RRT-1.05
RRT-1.07
RRT-0.60
RRT-1.51
I
0.01 0.02 0.01 0.34 0.08 0.46
0.01 0.02 0.01 0.35 0.08 0.47
0.01 0.02 0.01 0.34 0.08 0.46
0.01 0.02 0.01 0.34 0.09 0.47
0.01 0.02 0.01 0.38 0.08 0.50
0.01 0.02 0.01 0.35 0.07 0.46
II
0.01 0.02 0.01 0.37 0.08 0.49
0.01 0.02 0.01 0.36 0.08 0.48
0.01 0.02 0.01 0.38 0.09 0.51
0.01 0.02 0.01 0.37 0.08 0.49
0.01 0.02 0.01 0.37 0.09 0.50
0.01 0.02 0.01 0.38 0.08 0.50
Mean 0.01 0.02 0.01 0.36 0.08 0.48
SD 0.000 0.000 0.000 0.016 0.006 0.018
% RSD 0.00 0.00 0.00 4.49 7.07 3.76
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 81
Table 4.6.7.3.7: Pooled data (40/12.5 mg Tablets)
Analyst
% Impurity of Telmisartan% Impurity of
HCTZ % Total
impurities UNK-
1 UNK-
2 UNK-
3 HCTZ-IMP-B
Single max
RRT-0.92
RRT-1.05
RRT-1.07
RRT-0.60
RRT-0.85
I
0.01 0.02 0.01 0.75 0.08 0.99
0.01 0.02 0.01 0.74 0.09 0.99
0.01 0.02 0.01 0.74 0.09 0.99
0.01 0.02 0.01 0.78 0.08 1.02
0.01 0.02 0.01 0.76 0.08 1.00
0.01 0.02 0.01 0.75 0.08 0.99
II
0.01 0.02 0.01 0.79 0.09 1.05
0.01 0.02 0.01 0.79 0.09 1.05
0.01 0.02 0.01 0.79 0.08 1.04
0.01 0.02 0.01 0.79 0.08 1.04
0.01 0.02 0.01 0.79 0.09 1.06
0.01 0.02 0.01 0.80 0.09 1.06
Mean 0.01 0.02 0.01 0.77 0.09 1.02
SD 0.000 0.000 0.000 0.023 0.005 0.030
% RSD 0.00 0.00 0.00 2.93 6.14 2.90
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 82
4.6.8 Stability in Analytical solution:
The sample solution was kept at sample temperature for 24 hours were injected on to the
HPLC. The data obtained are summarized in Table 4.10.1 and 4.10.2. Sample solution was
stable for 24 hours with respect to Telmisartan while it was stable for 6 hours with respect
to Hydrochlorothiazide.
Table 4.6.8.1 Stability in analytical solution (Telmisartan)
Condition Standard
Area
Cumulative
RSD of
Standard
% Impurity
Single max imp
RRT-1.05
%
Cumulative
Difference
INITIAL 69682 - 0.02 -
6Hrs 69027 0.67 0.02 0.00
10Hrs 71511 1.83 0.02 0.00
15Hrs 72166 2.48 0.02 0.00
20Hrs 72891 3.18 0.02 0.00
24Hrs 70526 0.85 0.01 0.01
Table 4.6.8.2 Stability in analytical solution (Hydrochlorothiazide)
Condition Standard
Area
Cumulative
RSD of
Standard
% Impurity
% HCTZ-IMP-B
%
Cumulative
Difference
Single max imp
RRT-1.51
%
Cumulative
Difference
INITIAL 55157 - 0.34 - 0.08 -
6Hrs 52601 3.35 0.35 0.01 0.07 0.01
10Hrs 53964 1.55 0.41 0.07 0.07 0.01
15Hrs 53814 1.74 0.54 0.20 0.07 0.01
20Hrs 52749 3.16 0.63 0.29 0.05 0.03
24Hrs 55102 0.07 0.80 0.46 0.05 0.03
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 83
4.6.9 Filter Compatibility
Injection of spiked sample solution filtered through different types of filters
(Centrifuged, Glass, Nylon, PVDF and Teflon) were done on HPLC. Calculate % difference
against centrifuged sample solution. All the tested filters were found to be compatible with
the sample.
4.6.10 Robustness Small, deliberate changes in the chromatographic conditions were made and the effect of those
changes on the system suitability parameters was monitored by injecting system suitability
solutions. The data obtained are summarized in Tables 4.12.1 and 4.12.2. All results are within
acceptable limits for Telmisartan. For Hydrochlorothiazide the wavelength variation was
limited to ±2nm since at ±5nm the response of impurity B was changing.
Table 4.6.10.1 Robustness (Telmisartan)
System suitability solution
Changes in
parameters Values
Retention
Time of
Telmisartan
Tailing
Factor
USP
Plates
%
RSD
STD
area
Single max Impurity
RRT-1.05
% Cumulative
Difference
Control As per
method 34.716 1.03 740969 0.60 0.02 -
Flow
(ml/min)
0.9 35.510 0.99 707554 0.73 0.01 0.01
1.1 34.157 1.03 739536 1.26 0.02 0.00
Wavelength
(nm)
265 34.716 1.02 744020 0.42 0.02 0.00
272 34.716 1.03 723823 0.91 0.02 0.00
Temperature
°C
35 34.768 1.05 704933 0.50 0.01 0.02
45 34.763 1.01 790608 0.34 0.02 0.00
Buffer pH 2.8 34.043 0.99 792305 4.98 0.02 0.00
3.2 35.321 1.07 762886 2.39 0.01 0.01
CHAPTER - 4
SPP SPTM, SVKM’s NMIMS, Mumbai 84
Table 4.6.10.2 Robustness (Hydrochlorothiazide)
System suitability solution
Changes in
parameters Values
RT of
HCTZ
Tailing
Factor
USP
Plates
%
RSD
STD
area
HCTZ-IMP-B
RRT-0.60
Single max
Impurity
RRT-1.51
%
Cu
mu
lati
ve
Dif
fere
nce
%
Cu
mu
lati
ve
Dif
fere
nce
Control As per
method 17.871 0.95 17221 2.63 0.35 - 0.08 -
Flow
(ml/min)
0.9 19.484 0.97 23726 1.61 0.35 0.00 0.04 0.04
1.1 16.449 1.03 17994 2.04 0.40 0.05 0.05 0.03
Wavelength
(nm)
265 17.874 1.05 23045 2.75 0.39 0.04 0.06 0.02
272 17.878 1.04 22727 2.41 0.31 0.04 0.09 0.01
Temp.
°C
35 20.484 0.95 82012 0.88 0.34 0.01 0.06 0.02
45 15.126 1.02 16436 1.91 0.39 0.04 0.08 0.00
Buffer pH 2.8 17.747 0.96 16297 2.06 0.35 0.00 0.08 0.00
3.2 18.265 1.06 18625 2.80 0.38 0.03 0.07 0.01
4.6.11 CONCLUSIONS:
The method has been shown to be specific for Telmisartan + Hydrochlorothiazide
Tablets.
The method has been shown to be Linear, precise and accurate across the suitable analytical range.
Solution has been shown to be stable for at least upto 24 hours at 20°c for Telmisartan
and stable for at least upto 6 hours at 20°C for Hydrochlorothiazide. Thus sample has to be injected within 6 hours of preparation.
The method has been shown to be robust towards deliberate minor changes in the
method parameters.
Limit of detection and Limit of quantification concentration have been set.
The method can be used in quality control laboratory for release of production batches.