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1. DRUG PROFILE

1.1. Telmisartan

Telmisartan [Figure 4.1], is an angiotensin-II receptor blocker (ARB) used in the

management of hypertension [1]. Generally, angiotensin II receptor blockers (ARBs)

such as Telmisartan bind to the angiotensin II type 1 (AT1) receptors with high affinity,

causing inhibition of the action of angiotensin II on vascular smooth muscle, ultimately

leading to a reduction in arterial blood pressure [2]. Recent studies suggest that

Telmisartan may also have PPAR-gamma agonistic properties that could potentially

confer beneficial metabolic effects [3-4].

Figure 4.1: Molecular structure of Telmisartan

Molecular formula : C33H30N4O2

Molecular weight : 514.63

Chemical name : 4'-[(1,4'-dimethyl-2'-propyl[2,6'-bi-1H-benzimidazol]-1'-

yl)methyl]-[1,1'-biphenyl]-2-carboxylic acid.

Solubility : Telmisartan is insoluble in water and in the pH range of 3

to 9, sparingly soluble in strong acid except in hydrochloric

acid and soluble in strong base.

120

Table 4.1: List of brand names of formulations of Telmisartan [5-6]

S. No Brand names Formulation Available strength

Address of Manufacturer

1 MACSART Tablet 20 mg

40 mg

80 mg

Macleods

Pharmaceuticals

2 TELISTA Tablet 20 mg

40 mg

Lupin

Pharmaceuticals

3 TELMA Tablet 20 mg

40 mg

80 mg

Glenmark

Pharmaceuticals

4 TELMICHEK Tablet 40 mg Indoco

Pharmaceuticals

5 TELMISAT Tablet 20 mg

40 mg

80 mg

Biocon

Pharmaceuticals

6 TELPRES Tablet 20 mg

40 mg

80 mg

Abbott Healthcare

Private Limited

7 TELSAR Tablet 20 mg

40 mg

80 mg

Unichem

Pharmaceuticals

8 TELSAT Tablet 40 mg Sain

Pharmaceuticals

9 TELSITE Tablet 20 mg

40 mg

80 mg

Sanofi

Pharmaceuticals

10 TEMAX Tablet 20 mg

40 mg

80 mg

Wockhardt

Pharmaceuticals

121

2. LITERATURE SURVEY

Several analytical methods have been reported for the determination of

Telmisartan in pure drug, pharmaceutical dosage forms and in biological samples using

spectrophotometry [7-9], HPLC [10-18] and LC-MS [19-21] have been reported for the

determination of Telmisartan in dosage forms.

Sunil et al [7] developed UV first derivative spectrophotometric methods for the

determination of Telmisartan in pharmaceutical formulation. The solutions of standard

and sample were prepared in 0.1 M sodium hydroxide. In the UV spectrophotometric

method, the quantitative determination of the drug was carried at 295 nm and the linearity

range was found to be 4-20 µg/mL. For the first order derivative spectrophotometric

method, the drug was determined at 311 nm with the linearity ranges 4-20 µg/mL. The

calibration graphs constructed at their wavelength of determination were found to be

linear for UV and derivative spectrophotometric methods. The proposed methods have

been extensively validated. There was no significant difference between the performance

of the proposed method regarding the mean values and standard deviations. The

described method can be readily utilized for analysis of pharmaceutical formulation.

Kalyankar et al [8] developed a simple, accurate, cost efficient and reproducible

spectrophotometric method for the estimation of ammonia in Telmisartan in bulk and

pharmaceutical dosage form. UV spectrophotometric method, which is based on

measurement at maximum wavelength λmax 425 nm shows linearity in a concentration

range of 12.5-37.5 µg/mL and in aqueous solutions presents a square correlation

coefficient (r2) of 0.9999. The mean % recovery was found to be 100.03 to 99.92 and the

precision expressed as relative standard deviation (%R.S.D.) 0.1044%. In addition, the

122

proposed method was simple, easy to apply, low-cost, does not use polluting reagents and

requires relatively inexpensive instruments. Results of the analysis were validated

statistically and by recovery study.

Palled [9] developed difference spectrophotometric method for the estimation of

Telmisartan in bulk drug and in pharmaceutical formulations. Telmisartan exists in two

different forms in acidic and basic mediums that differ in their UV spectra. Difference

spectrum, obtained by keeping Telmisartan in 0.01 N NaOH in reference cell and

Telmisartan in 0.01 N HNO3 in sample cell, showed two characteristic peaks at 295 nm

and 327 nm with positive and negative absorbance respectively. Difference of absorbance

between these two maxima was calculated to find out the amplitude, which was plotted

against concentration. The method was found to be linear in the range of 2-12 µg/mL.

Gupta et al [10] developed a simple, rapid, precise, sensitive and reproducible

reverse phase high performance liquid chromatographic (RP-HPLC) method for

determination of Telmisartan in tablet dosage form and validated. Chromatographic

separation was achieved on a 250 × 4.6 mm, 5µ, Waters symmetry column in gradient

mode, with mobile phase consisting of a mixture of solution (10 mM potassium

dihydrogen phosphate, pH 3.5 ± 0.01): acetonitrile (64:40 v/v) was used. The quantitation

performed at flow rate of 1.0 mL/min at 230 nm and run time was 12 min. The analytical

method was validated as per ICH guideline for linearity, accuracy, precision, specificity,

limit of detection, limit of quantification, robustness and stability and method can be

extended to the analysis of Telmisartan in tablet formulations. The relative standard

deviation values for precision was less than 2% and % recovery was greater than 98% for

Telmisartan.

123

Varma et al [11] developed a simple, fast and precise reverse phase, isocratic

HPLC method for the separation and quantification of Telmisartan and

Hydrochlorothiazide in pharmaceutical dosage form. The quantification was carried out

using ProntoSIL C18 EPS 150 X 4.6 mm, 3µm enhanced polar selectivity column and

mobile phase comprised of potassium dihydrogen phosphate buffer pH adjusted to 3.2 ±

0.5 with orthophosphoric acid and acetonitrile in proportion of ratio 55:45 v/v and

degassed under ultrasonication. The flow rate was 0.8 mL/min and the effluent was

monitored at 271 nm. The retention time of Telmisartan and Hydrochlorothiazide were

5.01±0.5 min and 2.94±0.5 min respectively. The method was validated in terms of

linearity, precision, accuracy, specificity, limit of detection and limit of quantitation.

Linearity of Telmisartan and Hydrochlorothiazide were in the range of 15.01 to 75.05

µg/mL and 5.02 to 25.10 µg/mL respectively. The percentage recoveries of both the

drugs were 100.8% and 99.5% for Telmisartan and Hydrochlorothiazide respectively

from the tablet formulation. The proposed method is suitable for simultaneous

determination of Telmisartan and Hydrochlorothiazide in pharmaceutical dosage form.

Alankar et al [12] developed a RP-HPLC method for the simultaneous estimation

of Ramipril and Telmisartan in tablet dosage forms, using UV-detector. The developed

method was validated as per ICH guidelines and specificity, linearity & range, accuracy,

precision and robustness was performed. Specificity was determined by comparing the

results obtained by running the placebo solution with that of standard and method was

found to be specific due to no interference between placebo peaks and drugs peaks.

Linearity range was found to be 4 to16 µg/mL and 32 to128 µg/mL of Ramipril and

Telmisartan respectively. In the linearity study, regression equation and coefficient of

124

correlation for Ramipril and Telmisartan were found to be (Y = 924480X - 151831, r

=0.9997) and (Y = 2901878.3558X + 3803877, r = 0.9996) respectively. This developed

method was successfully utilized for the simultaneous estimation of Ramipril and

Telmisartan in pharmaceutical tablet dosage forms.

Anand kumar et al [13] developed a simple, sensitive and specific liquid

chromatographic method with UV detection for the simultaneous estimation of

Hydrochlorothiazide, Ramipril and Telmisartan in tablet dosage form and Bisoprolol as

internal standard. Separation was achieved with an Phenomenex luna 5µ C18, 250 X 4.6

mm 5µm size column, ambient temperature with a low pressure gradient mode with

mobile phase containing acetonitrile, 0.5% of potassium dihydrogen phosphate buffer pH

3.5 adjusted with orthophosphoric acid (50:50 v/v). The flow rate was 1 mL/min and

eluent was monitored at 220 nm. The selected chromatographic conditions were found to

effectively separate Hydrochlorothiazide, Ramipril and Telmisartan with retention time

of 3.1, 5.0 and 13.8 min respectively. The linearity range of Hydrochlorothiazide,

Ramipril and Telmisartan found in the range of 2-10 µg/mL, 5-25 µg/mL and 10-50

µg/mL respectively. The proposed method was found to be accurate, precise,

reproducible and specific and can also be used for routine quality control analysis of

these drugs in combination tablets.

Kurade et al [14] developed a rapid high performance liquid chromatographic

method and validated for the estimation of Ramipril and Telmisartan simultaneously in

combined dosage form. A Genesis C18 column having dimensions of 250 × 4.6 mm and

particle size of 5 µm in isocratic mode, with mobile phase containing a mixture of 0.01 M

potassium dihydrogen phosphate buffer (adjusted to pH 3.4 using orthophosphoric acid):

125

methanol:acetonitrile (15:15:70 v/v/v) was used. The mobile phase was pumped at a flow

rate of 1.0 mL/min and the eluents were monitored at 210 nm. The selected

chromatographic conditions were found to effectively separate Ramipril (Rt: 3.68 min)

and Telmisartan (Rt: 4.98 min) having a resolution of 3.84. The method was validated in

terms of linearity, accuracy, precision, specificity, limit of detection and limit of

quantitation. Linearity for Ramipril and Telmisartan were found in the range of 3.5-6.5

µg/mL and 28.0-52.0 µg/mL, respectively. The percentage recoveries for Ramipril and

Telmisartan ranged from 99.09-101.64% and 99.45-100.99%, respectively. The limit of

detection and the limit of quantitation for Ramipril was found to be 0.5 µg/mL and 1.5

µg/mL respectively and for Telmisartan was found to be 1.5 µg/mL and 3.0 µg/mL

respectively. The method was found to be robust and can be successfully used to

determine the drug content of marketed formulations.

Ramesh et al [15] developed an accurate, precise, simple and economical High

Performance Liquid Chromatographic method for the estimation of Telmisartan and

Hydrochlorothiazide and validated. The method so developed is Reverse Phase High

Performance Liquid Chromatographic method using Inertsil ODS column (Length: 250

mm, Diameter: 4.6 mm, Particle size: 5 µm) with a simple Trifluoro acetic acid buffer.

Buffer and acetonitrile were mixed in a proportion of 70:30 v/v as mobile phase. The

method so developed was validated in compliance with the National, Regional and

International Regulatory Guidelines by using very well developed analytical method

validation tool which comprises with the analytical method validation parameters like

specificity , forced degradation, system suitability, method precision, linearity, accuracy,

range, robustness, ruggedness, solution stability, filter validation. The results obtained

126

were well with in the acceptance Criteria.

Shen [16] developed a sensitive, simple, and accurate HPLC method was

developed for the assay of Telmisartan in human plasma using Naproxen as internal

standard, the assay involved liquid-liquid extraction of the compound from acidified

plasma into organic solvent and reversed-phase chromatography with fluorescence

detection. The assay was shown to be linear from 0.5 to 1000 ng/mL. In 24 healthy

volunteers, the plasma concentrations of the drug were determined after a single oral dose

of 160 mg.

Palled [17] developed a simple fast and precise reverse phase high performance

liquid chromatographic method was developed for the determination of Telmisartan from

tablet dosage forms. The column used was Hypersil C18 BDS (250 x 4.6 mm) from

Thermo. In isocratic mode, mobile phase used was acetonitrile: methanol (60:40 v/v) was

used. The flow rate was 1.2 mL/min, and eluent monitored at 245 nm.

Torrealday et al [18] developed a high performance liquid chromatographic

method with fluorimetric detection has been developed for the quantitation of the

angiotensin II receptor antagonist (ARA II) 4-((2-n-propyl-4-methyl-6-(1-

methylbenzimidazol-2-yl)-benzimidazol-1-yl)methyl)biphenyl-2-carboxylic acid

(Telmisartan) in urine, using a Novapak C18 column 150 x 3.9 mm, 4 µm. The mobile

phase consisted of a mixture acetonitrile-phosphate buffer (pH 6.0, 5 mM) (45:55, v/v)

pumped at a flow rate of 0.5 mL/min. Effluent was monitored at excitation and emission

wavelengths of 305 and 365 nm, respectively. Separation was carried out at room

temperature. Chromatographic variables were optimised by means of experimental

design. A clean-up step was used for urine samples consisting of a solid-phase extraction

127

procedure with C8 cartridges and methanol as eluent. This method proved to be accurate

(RE from -12 to 6%) and precise (intra- and inter-day coefficients of variation (CV) were

lower than 8%) to be applied to the determination of the active drug in urine samples

obtained from hypertensive patients. Concentration levels of Telmisartan at different time

intervals (from 0 up to 36 h after oral intake) were monitored.

Chen et al [19] developed a sensitive liquid chromatographic–electrospray

ionization mass spectrometric method and validated for fast determination of Telmisartan

in human plasma. Plasma of 0.1 mL was deprotienated with methanol, centrifugation,

evaporation to dryness and dissolving in mobile phase, samples were separated using a

Hypersil-Keystone C18 reversed-phase column (150 × 2.1 mm i.d., 5 µm), together with

a mobile phase containing of acetonitrile:10mM ammonium acetate (42:58, v/v), 0.2%

acetic acid and was isocratically eluted at a flow rate of 0.2 mL/min. Telmisartan and its

internal standard, Valsartan, were measured by electrospray ion source in positive

selective ion monitoring mode. The method demonstrated linearity from 1 to 2000 ng/mL

(r = 0.9988). The limit of quantification for Telmisartan in plasma was 1 ng/mL with

good accuracy and precision. The mean sample extract recovery of the method was

higher than 82% and 78% for Telmisartan and internal standard (IS), respectively. The

within-run and between-run precision ranged from 3.4 to 8.9% and 5.9 to 11.2% (relative

standard deviation, R.S.D.), respectively.

Li et al [20] developed a rapid, selective and sensitive method for the

determination of the angiotensin II receptor antagonist, Telmisartan, in human plasma has

been developed. Telmisartan and the internal standard, Diphenhydramine, were extracted

from plasma using diethyl ether–dichloromethane (60:40, v/v), and separated on a Zorbax

128

extend C18 column using methanol: 10 mM ammonium acetate (85:15, v/v) adjusted to

pH 4.5 after mixing with formic acid as mobile phase. Detection was carried out by

multiple reaction monitoring on a Q-trap™ LC–MS/MS system with an ESI interface.

The assay was linear over the range 0.5–600.0 ng/ml with a limit of quantitation of

0.5 ng/mL and a limit of detection of 0.05 ng/mL. Intra- and inter-day precision were

<6.7% and <8.1%, respectively, and the accuracy was in the range 88.9–111.0%. The

assay was applied to a pharmacokinetic study of Telmisartan given as a single oral dose

(80 mg) to healthy volunteers.

3. EXPERIMENTAL

3.1. Instrumentation

The author had attempted to develop and validate a liquid chromatographic

method for determination of Telmisartan using an isocratic Waters HPLC system on an

Xterra C8 column (150 mm x 4.6 mm, 3 µm). The instrument is equipped with a 2695

binary pump with inbuilt degasser, 2487 Dual absorbance detector and Rheodyne injector

with 20 µL sample loop. A 20 µL Hamilton syringe was used for injecting the samples.

Data was analysed using Waters Empower 2 software. A double-beam Elico SL-159 UV-

Visible spectrophotometer was used for spectral studies. Degassing of the mobile phase

was done by using an ultrasonic bath sonicator. A Shimadzu balance was used for

weighing the materials.

3.2. Chemicals and Solvents

The reference sample of Telmisartan (API) was obtained from Sun

Pharmaceutical Industries Ltd., Borada, India. The branded formulations (tablets)

(TELMA and TELISTA tablets containing 40 mg of Telmisartan) were procured from the

129

local market. HPLC grade acetonitrile and analytical grade potassium dihydrogen

Phosphate was obtained from Qualigens Fine Chemicals Ltd, Mumbai, India.

Hydrochloric acid, sodium hydroxide, hydrogen peroxide and triethyl amine of analytical

grade were obtained from Merck Chemicals Ltd, Mumbai, India. Milli-Q water was used

throughout the experiment dispensed through 0.22 µ filter of the Milli-Q water

purification system from Millipore, Merck KGaA, Darmstadt, Germany.

3.3. The Phosphate buffer solution

Weigh about 7.0 grams of Potassium dihydrogen Phosphate and transfer to 1000

mL standard flask, add 400 mL of Milli-Q water mix and dilute to volume with Milli-Q

water, sonicate for five minutes and cool to room temperature, measure the pH of above

buffer solution and finally adjusted the pH to 3.0±0.05 with Triethyl amine solution and

filtered through 0.45 µ nylon filter.

3.4. The mobile phase

A mixture of potassium dihydrogen phosphate buffer pH 3.0 and acetonitrile in

the ratio of 40:60 v/v was prepared and used as mobile phase.

3.5. The diluent

The potassium dihydrogen phosphate buffer pH 3.0 and acetonitrile mixture in the

ratio of 40:60 v/v was used as diluent.

3.6. Preparation of standard solution of the drug

About 40 mg of Telmisartan was accurately weighed and transferred into a 100

mL clean dry volumetric flask containing 50 mL of diluent. The solution was sonicated

130

for 5 min and then volume was made up to the mark with a further quantity of the diluent

to get a concentration of 400 µg/mL for Telmisartan (Stock solution). Further pipette 1

mL of the above stock solution into a 10 mL volumetric flask and the volume was made

up to the mark with the diluent.

3.7. Preparation of sample (tablet) solution

Twenty tablets were weighed and finely powdered. An accurately weighed

portion of powder sample equivalent to 40 mg of Telmisartan was transferred to a 100

mL volumetric flask containing 50 mL of the diluent. The contents of the flask were

sonicated for about 10 min for complete solubility of the drug and volume made up with

further quantity of diluent. Then this mixture was filtered through 0.45 µ membrane

filter. Pipette 1 mL of the above stock solution into a 10 mL volumetric flask and the

volume was made up to the mark with the diluent.

4. METHOD DEVELOPMENT

For developing the method, a systematic study of the effect of various factors was

undertaken by varying one parameter at a time and keeping all other conditions constant.

Method development consists of selecting the appropriate wave length and choice of

stationary and mobile phases. The following studies were conducted for this purpose.

4.1. Detection wavelength

The spectrum of diluted solution of the Telmisartan in diluent was recorded on

UV spectrophotometer. The peak of maximum absorbance was observed. The spectra of

Telmisartan showed that a balanced wavelength was found to be 229 nm.

131

4.2. Choice of stationary phase

Preliminary development trials have performed with octadecyl columns and octyl

columns with different types, configurations and from different manufacturers. Finally

the expected separation and shapes of peak was succeeded in Xterra C8 column.

4.3. Selection of the mobile phase

In order to get sharp peak and base line separation of the components, the author

has carried out a number of experiments by varying the composition of various solvents

and its flow rate. To effect ideal separation of the drug under isocratic conditions,

mixtures of solvents like water, methanol and acetonitrile with or without different

buffers in different combinations were tested as mobile phases on a C8 stationary phase.

A mixture of Potassium di hydrogen phosphate buffer pH 3.0 and acetonitrile in the ratio

of 40:60 v/v was proved to be the most suitable of all the combinations since the

chromatographic peaks obtained were better defined and resolved and almost free from

tailing.

4.4. Flow rate

Flow rates of the mobile phase were changed from 0.5-2.0 mL/min for optimum

separation. A minimum flow rate as well as minimum run time gives the maximum

saving on the usage of solvents. It was found from the experiments that 0.9 mL/min flow

rate was ideal for the successful elution of the analyte.

132

4.5. Run time

No interference in blank and placebo solutions for the drug peak in the trail

injections with a runtime of 6.0 min.

4.6. Optimized chromatographic conditions

Chromatographic conditions as optimized above were shown in Table 4.2. These

optimized conditions were followed for the determination of Telmisartan in bulk samples

and its combined tablet formulations. The chromatogram of standard and sample

solutions of Telmisartan was shown in Figure 4.2 and Figure 4.3. The chromatograms of

stability studies of Telmisartan were shown from in Figure 4.7 to Figure 4.10.

133

Table 4.2: Optimized chromatographic conditions for the estimation of Telmisartan

in tablet dosage form

Mobile phase : Potassium di hydrogen phosphate buffer pH 3.0:acetonitrile,

40:60 v/v

Pump mode : Isocratic

pH of Buffer : 3.0±0.05

Diluent : Potassium di hydrogen phosphate buffer:acetonitrile, 40:60 v/v

Column : Xterra C8 column, 150 mm x 4.6 mm, 3 µm

Column Temp : Ambient

Wavelength : 229 nm

Injection Volume : 20 µl

Flow rate : 0.9 mL/min

Run time : 6 min

Typical tR :-

Telmisartan : 2.736±0.5 min

134

Figure 4.2: Chromatogram of standard solution of Telmisartan

Figure 4.3: Chromatogram of sample solution of Telmisartan

5. VALIDATION OF THE PROPOSED METHOD

The proposed method was validated as per ICH [21-22] guidelines. The

parameters studied for validation were specificity, linearity, precision, accuracy,

robustness, system suitability, limit of detection, limit of quantification, and solution

stability.

5.1. Specificity

A study conducted to establish specificity of the proposed method involved

injecting blank and placebo using the chromatographic conditions defined for the

135

proposed method. It was found that there is no interference due to excipients in the tablet

formulation and also found good correlation between the retention times of standard and

sample. The specificity results are shown in Table 4.3. The chromatograms of blank and

placebo for Telmisartan was shown in Figure 4.4 and Figure 4.5.

Table 4.3: Specificity study

Name of solution Retention time (min)

Blank No peaks

Telmisartan 2.74

Figure 4.4: Chromatogram showing no interference of blank for Telmisartan

136

Figure 4.5: Chromatogram showing no interference of placebo for Telmisartan

5.2. Linearity

Linearity was performed by preparing standard solutions of Telmisartan at

different concentration levels including working concentration mentioned in experimental

condition from 20.0 to 60.0 µg/mL Twenty micro litres of each concentration was

injected in duplicate into the HPLC system. The response was read at 229 nm and the

corresponding chromatograms were recorded. From these chromatograms, the mean peak

areas were calculated and linearity plots of concentration over the mean peak areas were

constructed individually. The regressions of the plots were computed by least square

regression method. Linearity results were presented in Table 4.4 and linearity plots are

shown in Figure 4.6.

137

Table 4.4: Linearity study of Telmisartan

Figure 4.6: Linearity plot of Telmisartan

Level Concentration of Telmisartan (µg/mL) Mean peak area

Level-1 20 2395392

Level-2 30 3474808

Level-3 40 4677202

Level-4 50 5699253

Level-5 60 6937725

Slope 113091

Intercept 11323

Correlation Coefficient 0.9996

138

5.3. Precision

Precision is the degree of repeatability of an analytical method under normal

operational conditions. Precision of the method was performed as system precision,

method precision and intermediate precision.

5.3.1. System precision

To study the system precision, five replicate standard solutions of Telmisartan

was injected. The percent relative standard deviation (% RSD) was calculated and it was

found to be 0.04 for Telmisartan, which is well within the acceptable criteria of not more

than 2.0. Results of system precision studies are shown in Table 4.5.

Table 4.5: System precision

Injection number

Area of Telmisartan

Acceptance criteria

1 4700148

The %RSD of peak area

of Telmisartan should

not be more than 2.0

2 4696685

3 4700823

4 4699330

5 4700718

Mean 4699541

SD 1703

%RSD 0.04

139

5.3.2. Method precision

The method precision study was carried out on five preparations from the same

tablet samples of Telmisartan and percent amount of both were calculated. The %RSD of

the assay result of five preparations for Telmisartan in method precision study was found

to be 0.74 which is well within the acceptance criteria of not more than 2.0. The results

obtained for assay of Telmisartan is presented in Table 4.6.

Table 4.6: Method precision

Sample number %assay

Telmisartan

1 99.37

2 100.7

3 99.85

4 101.3

5 100.4

Mean 100.3

SD 0.7474

%RSD 0.74

5.3.3. Intermediate precision

The intermediate precision study was carried out by different analysts, different

columns, different reagents using different HPLC systems from the same tablet of

Telmisartan and the peak area of Telmisartan was calculated. The %RSD of the peak

areas of five preparations in intermediate precision study of Telmisartan was 0.39 which

is well within the acceptance criteria of not more than 2.0. The results of intermediate

precision study are reported in Table 4.7.

140

Table 4.7: Intermediate precision study of Telmisartan

Injection number

Area of Telmisartan

Acceptance criteria

1 4723338

The %RSD of peak area

of Telmisartan should not

be more than 2.0

2 4722324

3 4725118

4 4758167

5 4755466

Mean 4736883

SD 18250

%RSD 0.39

5.4. Accuracy

The accuracy of the method was determined by standard addition method. A

known amount of standard drug was added to the fixed amount of pre-analyzed tablet

solution. Percent recovery was calculated by comparing the area before and after the

addition of the standard drug. The standard addition method was performed at three

concentration levels of 50%, 100% and 150%. The solutions were analyzed in triplicate

at each level as per the proposed method. The percent recovery and %RSD at each level

was calculated and results are presented in Table 4.8. The percent recovery of

Telmisartan was 99.59 to 100.04 and the mean recovery was found to be 99.79 by the

proposed method. This indicates that the proposed method was accurate.

141

Table 4.8: Recovery study for Telmisartan

%Concentration(at specification

Level)

Mean peak area

Amount of Telmisartan

spiked (mg)

Amount of Telmisartan recovered

(mg)

%Recovery Mean

Recovery

50% 2379254 20.1 20.05 99.75%

99.79% 100% 4719780 40.0 40.10 100.04%

150% 6858762 59.80 59.55 99.59%

5.5. Robustness

The robustness study was performed by slight modification in flow rate of the

mobile phase and composition of the mobile phase. Sample of Telmisartan at 40 µg/mL

concentration were analyzed under these changed experimental conditions. It was

observed that there were no marked changes in chromatograms, which demonstrated that

the developed method was robust in nature. The results of robustness study are shown in

Table 4.9.

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Table 4.9: Robustness study for Telmisartan

Condition Mean Peak

area %assay %difference

Unaltered 4676205 99.8 -

Flow rate at 0.7 mL/min

Flow rate at 1.1 mL/min

4679623

4593681

100.1

98.4

0.3

1.4

Mobile phase:

• Buffer(45):Acetonitrile(55)

• Buffer(35):Acetonitrile(65)

4598964

4763582

98.3

101.8

1.5

2.0

5.6. System suitability

System suitability was studied under each validation parameters by injecting six

replicates of the standard solution. The system suitability parameters are given in Table

4.10.

Table 4.10: System suitability for Telmisartan

Parameter Tailing factor Theoretical plates

Specificity study 1.20 2347

Linearity study 1.16 2465

Precision study 1.18 2581

Robustness study

Flow rate at 0.7 mL/min

Flow rate at 1.1 mL/min

Mobile phase:

• Buffer(45):Acetonitrile(55)

• Buffer(35):Acetonitrile(65)

1.12

1.32

1.06

1.16

2201

2095

2156

2316

143

5.7. Limit of detection and Limit of quantification

Limit of detection (LOD) is defined as the lowest concentration of analyte that

gives a detectable response. Limit of quantification (LOQ) is defined as the lowest

concentration that can be quantified reliably with a specified level of accuracy and

precision. For this study six replicates of the analyte at lowest concentration were

measured and quantified. The LOD and LOQ of Telmisartan are given in Table 4.11.

Table 4.11: LOD and LOQ of Telmisartan

Parameter Measured value (µg/mL)

Limit of detection 0.010

Limit of quantification 0.033

5.8. Solution stability

To determine the stability of Telmisartan in solution, the standard and sample

solution were observed under room temperature. Any change in the retention time, peak

shape and variation in response was compared to the pattern of chromatogram of freshly

prepared solution. The solution stability results are shown in the Table 4.12.

Table 4.12: Solution stability of Telmisartan

Standard solution Sample solution

Time

(hours) Response % variation

Time

(hours) Response % variation

Initial 4700148 - Initial 4699527 -

12 4657824 0.9 12 4652389 1.1

24 4625315 1.6 24 4618546 1.8

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5.9. Stability studies

In order to demonstrate the stability of both standard and sample solutions during

analysis, both solutions were analyzed over a period of 24 hours at room temperature.

The results show that for both solutions, the retention time and peak area of Telmisartan

(%RSD less than 2.0) has no significant degradation within the indicated period, thus

indicated that both solutions were stable for at least 24 hours, which was sufficient to

complete the whole analytical process. Further forced degradation studies were

conducted indicating the stability of proposed method. The results of the degradation

studies are shown in the Table 4.13.

Table 4.13: Forced degradation study results for Telmisartan

Stress

Conditions

Degradation

Time (Hrs)

Telmisartan

%Assay %Degradation

Control -- 99.8 --

Acid 1 96.9 -2.9

Base 1 91.0 -8.8

Peroxide 1 82.9 -16.9

Thermal 48 86.9 -12.9

Control sample

Twenty tablets were weighed and finely powdered. An accurately weighed

portion of powder sample equivalent to 40 mg of Telmisartan was transferred to a 100

mL volumetric flask containing 50 mL of the diluent. The contents of the flask were

sonicated for about 10 min for complete solubility of the drug and volume made up with

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further quantity of diluent. Then this mixture was filtered through 0.45 µ membrane

filter. 5.0 mL of this filtrate was further diluted to 50 mL with mobile phase.

Acid degradation sample

Twenty tablets were weighed and finely powdered. An accurately weighed

portion of powder sample equivalent to 40 mg of Telmisartan was transferred to a 100

mL volumetric flask containing 50 mL of the diluent. The contents of the flask were

sonicated for about 10 min for complete solubility of the drug. Then 10 mL of 5N acid

(Hydrochloric acid) was added, refluxed for 60 minutes at 60°C, then cooled to room

temperature, neutralized with 5N base (Sodium hydroxide) and diluted to volume with

diluent. Filtered about 25 mL of the above sample solution through 0.45 µ membrane

filter. Pipetted 5 mL of the above filtered sample solution into a 50 mL volumetric flask

and diluted to volume with diluent. Typical chromatogram of acid degradation for

Telmisartan is shown in Fig. 4.7.

Figure 4.7: Chromatogram of acid degradation showing Telmisartan

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Base degradation sample

Twenty tablets were weighed and finely powdered. An accurately weighed

portion of powder sample equivalent to 40 mg of Telmisartan was transferred to a 100

mL volumetric flask containing 50 mL of the diluent. The contents of the flask were

sonicated for about 10 min for complete solubility of the drug. Then 10 mL of 5N base

(Sodium hydroxide) was added, refluxed for 60 minutes at 60°C, then cooled to room

temperature, neutralized with 5N acid (Hydrochloric acid) and diluted to volume with

diluent. Filtered about 25 mL of the above sample solution through 0.45 µ membrane

filter. Pipetted 5 mL of the above filtered sample solution into a 50 mL volumetric flask

and diluted to volume with diluent. Typical chromatogram of base degradation for

Telmisartan is shown in Fig. 4.8.

Figure 4.8: Chromatogram of base degradation showing Telmisartan

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Peroxide degradation sample

Twenty tablets were weighed and finely powdered. An accurately weighed

portion of powder sample equivalent to 40 mg of Telmisartan was transferred to a 50 mL

volumetric flask containing 50 mL of the diluent. The contents of the flask were

sonicated for about 10 min for complete solubility of the drug. Then 4 mL of 30%

hydrogen peroxide was added, refluxed for 60 minutes at 60°C, then cooled to room

temperature and diluted to volume with diluent. Filtered about 25 mL of the above

sample solution through 0.45 µ membrane filter. Pipetted 5 mL of the above filtered

sample solution into a 50 mL volumetric flask and diluted to volume with diluent.

Typical chromatogram of peroxide degradation for Telmisartan is shown in Fig. 4.9.

Figure 4.9: Chromatogram of oxidative degradation showing Telmisartan

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Thermal degradation sample

Twenty tablets were weighed and finely powdered. The powder was exposed to

heat at 105°C for about 2 days. An accurately weighed portion of powder sample

equivalent to 40 mg of Telmisartan was transferred to a 100 mL volumetric flask

containing 50 mL of the diluent. The contents of the flask were sonicated for about 10

min for complete solubility of the drug. Filtered about 25 mL of the above sample

solution through 0.45 µ membrane filter. Pipetted 5 mL of the above filtered sample

solution into a 50 mL volumetric flask and diluted to volume with diluent. Typical

chromatogram of thermal degradation for Telmisartan is shown in Fig. 4.10.

Figure 4.10: Chromatogram of thermal degradation showing Telmisartan

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6. RESULTS AND DISCUSSION

The present study was aimed at developing a simple, sensitive, precise and

accurate HPLC method for the estimation of Telmisartan from bulk samples and tablet

dosage forms. A non-polar C8 analytical chromatographic column was chosen as the

stationary phase for the separation and determination of Telmisartan. Mixtures of

commonly used solvents like water, methanol and acetonitrile with or without buffers in

different combinations were tested as mobile phases. The choice of the optimum

composition is based on the chromatographic response factor, a good peak shape with

minimum tailing. A mixture of buffer and acetonitrile in the ratio of 60:40 v/v was

proved to be the most suitable of all the combinations since the chromatographic peak

obtained was well defined, better resolved and almost free from tailing. The retention

time of Telmisartan was found to be 2.74 min.

The linearity was found satisfactory for the drug in the range 20.0-60.0 µg/mL

(Table 4.4). The regression equation of the linearity curve of Telmisartan between

concentrations over its peak areas was found to be Y=113091X+113232 (where Y is the

peak area and X is the concentration of Telmisartan in µg/mL). Precision of the method

was studied by repeated injection of tablet solution and results showed lower %RSD

values (Table 4.5-4.7). This reveals that the method is quite precise. The percent

recoveries of the drug solutions were studied at three different concentration levels. The

percent individual recovery and the %RSD at each level were within the acceptable limits

(Table 4.8). This indicates that the method is accurate. The absence of additional peaks in

the chromatogram indicates non-interference of the commonly used excipients in the

tablets and hence the method is specific.

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The deliberate changes in the method have not much affected the peak tailing,

theoretical plates and the percent assay. This indicates that the present method is robust

(Table 4.9). The system suitability studies were carried out to check various parameters

such as theoretical plates and tailing factor (Table 4.10). The lowest values of LOD and

LOQ as obtained by the proposed method indicate that the method is sensitive (Table

4.11). The solution stability studies indicate that the drug was stable up to 24 hours

(Table 4.12). The forced degradation studies indicate that the drug was stable in stability

studies (Table 4.13).

7. CONCLUSION

The proposed stability-indicating RP-HPLC method was simple, specific,

sensitive, accurate and precise and can be used for analysis of Telmisartan in bulk

samples and its tablet dosage forms.

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