Chapter-6 Estimation of telmisartan by RP – HPLCshodhganga.inflibnet.ac.in/bitstream/10603/20011/12... · Telmisartan, a non peptide molecule, is chemically described as “2 methyl-1H-1,3-benzodiazol-2-yl)

Chapter-6

Estimation of

telmisartan by

RP – HPLC

6.1 Introduction

Telmisartan, a non peptide molecule, is chemically described as “2

methyl-1H-1,3-benzodiazol-2-yl)

acid” [1]. Telmisartan is an angiotensin II receptor antagonist (

hypertension. 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. Telmisartan is used alone or in combination with other classes of antihyperte

the treatment of hypertension. Also used in the treatment of diabetic nephropathy in hypertensive

patients with type 2 diabetes mellitus, as well as the treatment of congestive heart failure (only in

patients who cannot tolerate ACE inhibitors).

of peroxisome proliferator-activated receptor gamma

of insulin and glucose metabolism. It is believed that telmisartan’s dual mode of action may

provide protective benefits against th

by diabetes and cardiovascular disease

its molecular weight is 514.63g and its chemical structure is as shown in

Figure : 6.1 Chemical

Telmisartan may mediate potent antiproliferative effects against renal cell carcinoma

(RCC) through peroxisome proliferator

potential target for prevention and treatment in RCC [3].

cancer cell growth through early apoptosis

patients with essential hypertension

Telmisartan, a non peptide molecule, is chemically described as “2-(4-

yl)-2-propyl-1H-1,3-benzodiazol-1-yl]methyl}phenyl)benzoic

Telmisartan is an angiotensin II receptor antagonist (ARB) used in the management of


1 (AT1) receptors with high affinity, causing inhibition of the action of


pressure. Telmisartan is used alone or in combination with other classes of antihyperte



patients who cannot tolerate ACE inhibitors). Telmisartan acts as a selective modulator

activated receptor gamma (PPAR-γ), a central regulator

metabolism. It is believed that telmisartan’s dual mode of action may

provide protective benefits against the vascular and renal damage caused by diabetes and

cardiovascular disease (CVD) [2]. Telmisartan empirical formula is C

its molecular weight is 514.63g and its chemical structure is as shown in Figure: 6.1.

Figure : 6.1 Chemical structure of telmisartan


peroxisome proliferator-activated receptor-γ (PPAR- γ) ligand.

potential target for prevention and treatment in RCC [3]. Telmisartan inhibits human urological

cancer cell growth through early apoptosis [4]. Telmisartan improves endothelial function in

patients with essential hypertension [5]. Telmisartan monotherapy improves cardiometabolic

-{[4-methyl-6-(1-

yl]methyl}phenyl)benzoic

) used in the management of


1 (AT1) receptors with high affinity, causing inhibition of the action of


pressure. Telmisartan is used alone or in combination with other classes of antihypertensives for



Telmisartan acts as a selective modulator

), a central regulator

metabolism. It is believed that telmisartan’s dual mode of action may

e vascular and renal damage caused by diabetes and

empirical formula is C33H30N4O2,

Figure: 6.1.


Telmisartan is a

Telmisartan inhibits human urological

Telmisartan improves endothelial function in

rtan monotherapy improves cardiometabolic

profile in obese hypertensive patients by improving insulin sensitivity and increasing plasma

adiponectin concentration, including its high-molecular-weight fraction, and by suppressing

microinflammation[6]. Telmisartan improves insulin sensitivity [7] and increases adiponectin

levels [8].Telmisartan has a number of pharmacological properties (longest plasma half-life,

highest lipophilicity and strongest receptor binding affinity) that distinguish it from other

angiotensin II receptor blockers (ARBs). Telmisartan is the only ARB with a broad indication for

cardiovascular (CV) risk reduction in patients with atherothrombotic disease or diabetes with

end-organ damage[9, 10]. Telmisartan has a more potent antioxidative effect through its ability

to enhance superoxide dismutase (SOD) activity in type 2 diabetic patients with

microalbuminuria [11]. It shows peculiar features that go beyond blood pressure control. It

presents promising and unique protective properties against target end-organ damage, potentially

able to open a scenario of new therapeutic approaches to cardiovascular disease [12].

Telmisartan prevented unfavorable cardiac remodeling through a reduction of cardiac

hypertrophy and fibrosis [13]. Telmisartan improves walking distance and endothelial function,

the ankle-brachial index (ABI) in non-diabetic patients and it may prevent further loss of quality

of life in patients with advanced peripheral artery disease (PAD) [14]. The telmisartan results in

a greater reduction of urinary markers than does enalapril and this effect occurs by a mechanism

independent of blood pressure reduction [15]. Telmisartan is a safe and effective alternative for

the treatment of hypertension. Moreover, due to its good tolerability, an increasing use of

telmisartan in cardiovascular high risk patients can be anticipated [16]. In hypertensive patients

with impaired glucose tolerance (IGT) telmisartan, compared to losartan, improved endothelial

function and insulin resistance independently, supporting the hypothesis that glucometabolic and

vascular insulin resistance are differentially regulated [17].

A good number of analytical methods have been developed for the determination of

telmisartan in pure form and in pharmaceutical dosage forms. An UV Spectrophotometric

method for the determination of telmisartan in tablet formulation was reported by Sagar Tatane

[18] in which the absorbance maximum was found at wavelength 230nm. Rajiv et al developed

and validated a UV spectrophotometric method by using methanol as a solvent for estimation of

telmisartan in API and in pharmaceutical dosage form [19]. Rekha Gangola et al reported a

spectrophotometric dual wavelength method [20] for simultaneous determination of

hydrochloride and telmisartan in combined dosage form. An UV spectrophotometric method was

reported for the estimation of telmisartan in bulk and tablet dosage form by Ajit Pandey et al [21]

in which 0.1N NaOH was used as a diluent and the analysis was carried out at 234nm. Several

chromatographic methods were developed and reported for the estimation of telmisartan. Komal

Patel et al reported a UV spectrophotometry method for estimation of telmisartan in bulk and

pharmaceutical dosage forms in which methanol was used as a solvent and the absorbance was

measured at 296 nm [22]. Palled et al reported a spectrophotometric method for the

determination of telmisartan in tablet dosage forms [23]. Kondawar et al reported a multi

wavelength analysis for the simultaneous determination of amlodipine besylate and telmisartan

in bulk drug and dosage form by using UV spectrophotometer [24]. Ilingo et al reported a UV

spectrophotometric method for the simultaneous determination of telmisartan and

hydrochlorothiazide in a pharmaceutical dosage form [25]. Thomas et al reported a simultaneous

spectrophotometric method for the estimation of amlodipine besylate and telmisartan in a tablet

dosage form [26]. Shah et al reported a method for simultaneous estimation of telmisartan and

hydrochlorothiazide using high performance thin layer chromatography (HPTLC) [27]. Prabhu et

al reported a method for the determination of telmisartan by HPTLC method [28]. A HPTLC

method was proposed by Chabukswar et al for the simultaneous determination of telmisartan and

amlodipinebesylate [29]. Patel et al [30] developed a HPTLC method for the simultaneous

estimation of telmisartan and ramipril in combined dosage form. A RP-HPLC method was

reported by Vekariya et al for the determination of telmisartan in solid dosage forms in the

concentration range 2-14µg mL-1. The observed retention time of telmisartan was about 7

minutes and the theoretical plate count at lower side was about 3345 [31]. Phani kishore et al

reported a stability indicating method for the estimation of telmisartan related substances in

tablet formulations which was well established to separate and determine about 7 related

substances of telmisartan with the run time of 50 minutes [32]. Shen et al [33] developed a

HPLC method for the assay of telmisartan in human plasma. Fluorescent detector was used for

the detection of telmisartan and naproxen was used as internal standard in this method. A reverse

phase liquid chromatographic method for quantitative estimation of telmisartan in human plasma

was reported by Kabra et al [34]. Vijay kumar et al developed a reverse phased high performance

liquid chromatographic method for the estimation of telmisartan in serum samples [35] which

involves of 10 minutes run time. The robustness of the method was not demonstrated. An

isocratic RP-HPLC method was reported for the assay of telmisartan in pharmaceutical

formulation by Narendra et al [36]. The run time of the method was 10 minutes. Sunil et al [37]

reported a HPLC method for simultaneous estimation of telmisartan and ramipril in

pharmaceutical formulations. The run time observed as 15 minutes with 1.5mL minute-1 flow

rate and the detection was done at 210 nm. Gupta et al [38] developed a RP-HPLC method for

determination of telmisartan in a tablet dosage form. Patel Amit et al [39] reported a RP-HPLC

method for the simultaneous determination of telmisartan and indapamide in a pharmaceutical

dosage form which requires 15 minutes of run time. Swamy et al reported a RP-HPLC method

for simultaneous determination of hydrochlorothiazide and telmisartan in pharmaceutical dosage

form in which the active drug components were separated within the 15 minutes [40]. Palled et al

reported a RP-HPLC method for the determination of telmisartan in tablet dosage forms [41].

Kurade et al reported a RP-HPLC method for simultaneous determination of ramipril and

telmisartan in tablet dosage forms [42]. Rao et al reported a HPLC method for the determination

of telmisartan in bulk and pharmaceutical formulations [43]. Sujana et al reported a RP-HPLC

method for the determination of telmisartan in pure and pharmaceutical formulations [44].

Kavitha et al developed a HPLC method for simultaneous determination of telmisartan and

hydrochlorothiazide in tablets with 15 minutes run time [45]. Bhaskararaju et al [46] developed a

HPLC method for the estimation of telmisartan in bulk and its pharmaceutical formulations in

which the theoretical plates were found at lower side with poor peak shape. Patil et al [47]

reported a liquid chromatographic method for simultaneous estimation of telmisartan and

ramipril in dosage forms. Bankey et al developed a spectrophotometric method for the

simultaneous determination of telmisartan and ramipril [48]. Wankhade et al reported a RP-

HPLC method for simultaneous estimation of telmisartan and hydrochlorothiazide in a combined

tablet dosage form [49]. Nalwade et al developed an ultra performance liquid chromatographic

(UPLC) method for simultaneous determination of telmisartan, amlodipine besylate and

hydrochlorothiazide in a combined poly pill dosage form [50]. Li et al developed a liquid

chromatography-tandem mass spectrometric method for the determination of telmisartan in

human plasma [51]. Yan et al reported a liquid chromatography-tandem mass spectrometry

method for simultaneous determination of telmisartan and hydrochlorothiazide in human plasma

[52]. Jat et al [53] reported a method for quantitative estimation of telmisartan in bulk drug by

using an UV spectrophotometer. Komal patel et al reported an absorbance correction method for

the simultaneous estimation of telmisartan and metoprolol succinate in combined dosage form

[54]. A spectrophotometric method for simultaneous estimation of amlodipine besylate and

telmisartan in tablet dosage form was reported by Pratap Y. Pawar et al [55]. An UPLC method

for estimation of related substances in telmisartan tablet dosage form was reported by Bhavani et

al [56]. Basaveswara Rao et al reported a RP-HPLC method for estimation of telmisartan in a

tablet dosage form [57]. A RP-HPLC method for simultaneous estimation of cilnidipine and

telmisartan in combined tablet dosage form was reported by Pawar et al [58]. A stability-

indicating simultaneous determenation of hydrochlorothiazide, ramipril and telmisartan in

combined dosage form by ultra performance liquid chromatography was reported by Vani et al

[59]. Pradhan et al [60] reported an UV specctrophotometric method for estimation of

telmisartan in bulk and pharmaceutical dosage form. Niranjan et al [61] and Chavan et al [62]

developed a UV spectrophotometric method for estimation and validation of telmisartan.

A large number of assay methods are available in literature for the determination of

telmisartan. Most of them were developed by using spectrophotometric and high performance

thin layer chromatographic techniques. The analytical techniques mentioned above are time

consuming processes, less sensitive, less precise and are not robust when compared with the

liquid chromatographic methods. High performance liquid chromatographic (HPLC) methods are

more preferred over spectrophotometric and thin layer chromatographic methods as they are

more sensitive, accurate, precise and specific. Multi component formulations are difficult to be

determined by spectrphotometric method as they might have similar ƛmax values which can be

separated and determined by high performance chromatography based on their retention times.

HPLC has been used for telmisartan determination. Although large numbers of HPLC methods

are reported for the determination of telmisartan in biological matrices and pharmaceutical

formulations, most of them are characterized by relatively long analysis times and not extended

the studies for validation for routine testing of telmisartan as per the regulatory requirements. As

such, no validated method is available, which is having short analysis time to estimate the assay

of telmisartan with good precision and accuracy as part of routine testing which is meets the

requirement of validation as required by regulatory and is more useful in commercial aspect. The

reported liquid chromatography-mass spectrometry (LC-MS) methods are quite suitable for the

determination of telmisartan in biological matrices and pharmaceutical formulations. But this

technique involves huge cost for analysis which may be a limitation for routine quality control

applications. Therefore, it is very imperative to develop a suitable analytical method for

telmisartan which can be easily adapted for routine and in-process quality control analysis or

similar studies.

The objective of the present study is to develop a rapid, simple, precise and accurate high

performance liquid chromatographic (HPLC) method for the determination of telmisartan in

pharmaceutical formulations. The proposed method is validated according to International

Conference on Harmonization (ICH) guidelines [63] in terms of specificity, precision, accuracy,

linearity, range, ruggedness and robustness including the stability of mobile phase, standard and

sample solutions.

6.2. Experimental

6.2.1. Reference substances, chemicals, reagents and samples

The complete analysis was carried out by using “Class A” volumetric glassware.

Analytical reagent grade potassium dihydrogen phosphate procured from Merck specialities

private limited, analytical reagent grade ortho phosphoric acid procured from Fisher scientific

private limited was used for buffer preparation. Water was purified by Milli Q water

purification system manufactured by Millipore Corporation located at Barnstead. HPLC grade

acetonitrile was procured from Rankem, New Delhi, India. Membrane filter with 0.45 µm size

(Millipore) was used for the mobile phase filtration and degassed under vacuum. Telmisartan

gift sample was provided by Dr. Reddy`s laboratories limited, Hyderabad, India. Telmisartan

formulation tablets (Targit) were purchased from Pfizer limited, India.

6.2.2. Instrumentation

Waters HPLC instrument with PDA detector and model number e2695 (Milford, MA,

USA), consisting of quaternary solvent manager, sample manager and column heating

compartment was used for chromatographic analysis and Empower software was used to operate

the Waters HPLC instrument. Semi micro analytical balance manufactured by Labindia was used

for weighing of standard and sample preparations, Labindia pH meter was used to adjust the

buffer pH as required. Ultrasonic bath manufactured by Lifecare equipments was used to

sonicate the sample preparation. A Waters Sunfire C18, 250mm length, 4.6mm internal diameter

column packed with porous silica of particle size 5µm was used as stationary phase.

6.2.3. Blank, standard and sample solution preparation

Telmisartan active pharmaceutical ingredients (API), formulated tablets and the

corresponding placebo (without API) were used throughout the development and validation. All

the samples were treated according to test solution preparation.

6.2.3.1. Standard solution preparation

8 mg of telmisartan were dissolved in a mixture of 15mL of phosphate buffer and 10mL

of acetonitrile in a 25mL volumetric flask and sonicated for 10 minutes followed by filtration

through 0.45µm membrane filter. The concentration of the prepared standard solution was

0.32mg mL-1.

6.2.3.2. Blank solution preparation

The mixture of phosphate buffer and acetonitrile (60:40 v/v) was used as mobile

phase as well as diluent. The same solution was used as blank solution also.

6.2.3.3. Sample solution preparation

The sample solution of telmisartan in pure form was prepared as described in standard

solution preparation (6.2.3.1). The sample preparation of telmisartan in pharmaceutical

formulations was a critical step as it involves the extraction of telmisartan from other formulated

compositions. An accurately weighed ten number of telmisartan pharmaceutical dosage units

were taken into a mortar and were crushed uniformly by using a clean pestle. The crushed tablet

powder equivalent to a single dosage unit was transferred in to a 25 mL volumetric flask,

dissolved and diluted to the mark with the mixture of phosphate buffer and acetonitrile (60:40

v/v). The above sample solution was subjected for sonication for 10 minutes using an ultrasonic

bath. The final solution was filtered through 0.45µm membrane filter.

6.2.4. Chromatographic conditions

A high performance liquid chromatograph (HPLC) was used to carry out the

chromatographic analysis. The telmisartan was separated from its formulated impurities by using

a Sunfire C18, 250mm x 4.6mm, 5µm, column manufactured by Waters. An isocratic pumping

program with a flow rate of 1.0 mL minute-1 at a column temperature of 45°C resulted the

retention time of telmisartan at 3.4 minutes and hence the run time of chromatographic analysis

was fixed at 5 minutes. The absorbance of telmisartan was measured at 242 nm. The injection

volume was set as 10µL. Buffer was prepared by dissolving 174.18 mg of potassium dihydrogen

phosphate in 1000mL of water and the pH of the resultant solution was adjusted to 4.4 with

dilute ortho phosphoric acid. The mobile phase was prepared by mixing buffer and acetonitrile in

the ratio 60:40 (v/v).

6.2.5. Evaluation of blank

10µL of mobile phase was used as blank solution and is injected into high performance

liquid chromatograph and the chromatogram was recorded.

6.2.6. Evaluation of system suitability

10µL of standard solution were injected into chromatographic system for six times and

monitored the tailing factor and theoretical plates for telmisartan peak. The relative standard

deviation of peak areas of telmisartan for six replicate injections was evaluated.

6.2.7. Procedure

Separately injected the standard preparation and the sample preparation into the high

performance liquid chromatograph and areas of the major peaks were recorded. The diluent

chromatogram was examined for any extraneous peaks and corresponding peaks observed in the

sample chromatogram were ignored. The retention time of telmisartan peak under the present

chromatographic conditions should be about 3.4 minutes.

6.2.8. Quantitation

Telmisartan peak areas were recorded for standard and sample injections. Respective

peak areas were taken into account to quantitate the amount of telmisartan present in the sample

as follows:

At Cs P

Amount of telmisartan (%) = ------ x ------- x ------ x 100

As Ct 100

Where, At = Telmisartan peak area obtained from the sample preparation;

As = Telmisartan peak area obtained from the standard preparation;

Cs = Concentration of telmisartan in standard solution;

Ct = Concentration of telmisartan in sample solution and

P = Telmisartan standard purity in percentage.

6.3 Results and discussion:

6.3.1. Method development and optimization

The important criteria for the development of successful high performance liquid

chromatographic (HPLC) method for telmisartan was that the method should be able to

determine the drug in a single run with good amount of resolution and that should be accurate,

reproducible, robust, stability indicating, free from interference (blank/ placebo/ other unknown

product) and should be straight forward enough for routine use in quality control laboratory. To

develop the stability indicating method, the retention behaviour of the compound with change in

percentage of organic solvent (acetonitrile) and change in pH of buffer was studied on Waters

Sunfire C18 column (250 mm × 4.6 mm, 5 µm). Telmisartan is usually present at trace levels in

complex biological matrix or pharmaceutical dosage forms. The solubility of telmisartan in

aqueous medium is very low (0.078 mg mL-1). The solubility of telmisartan in aqueous solutions

is strongly pH-dependent, with maximum solubility observed at high and low pH. The selection

of the stationary phase depends up on the nature of the sample, molecular weight and solubility.

The drug telmisartan is non-polar and hence preferably analyzed by reverse phase column, C18.

Non-polar compound is very attractive with reverse phase columns. So the elution of the

compound from the column was influenced by polar mobile phase. Mixture of phosphate buffer

and acetonitrile was selected as mobile phase and the effect of the composition of mobile phase

on the retention time of telmisartan was thoroughly investigated. The concentration of the

phosphate buffer and acetonitrile were optimized to give symmetric peak with short run time.

The optimized conditions were derived from the following trials.

6.3.1.1. Optimization of chromatographic parameters:

� Trial – I

The analysis carried out by using high performance liquid chromatograph (HPLC) with

the following chromatographic conditions was arrived at based on the telmisartan solubility and

its polarity.

Mobile phase : Mixture of phosphate buffer (pH 4.4) and acetonitrile (80:20 v/v).

Detection : PDA detector with 200 nm to 400 nm range

Column : 250mm length, 4.6mm internal diameter, 5µm particle size,

Sunfire C18

Flow rate : 0.6 mL minute-1

Injection volume : 20µL

Run time : 20 minutes

Column temperature : 25°C

The absorbance was found satisfactory at 242nm at which the peak response was found

on higher side due to injection overload. The back pressure of the column was found

significantly low and the retention time was observed as 13 minutes.

� Trial – II

Based on the observed ƛmax, the optimum wavelength for the detection was set as 242nm.

The injection volume was reduced to 10µL to optimize the injection load and flow rate was

increased to 0.8mL minute-1 to increase the desired back pressure to the column and to reduce

the retention time of telmisartan.

Mobile phase : Mixture of pH 4.4 phosphate buffer and acetonitrile (80:20 v/v).

Detection : 242nm


Sunfire C18





The peak symmetry of telmisartan and response were found satisfactory. The retention

time of telmisartan was found about 7 minutes which need to be reduced.

� Trial – III

To reduce the retention time of telmisartan, the mobile phase was optimized by

increasing the acetonitrile composition in the mobile phase and increased the column

temperature to 45°C.


Detection : 242nm


Sunfire C18





The chromatogram for the standard was recorded under the above mentioned

conditions. The peak symmetry was found good with the retention time of telmisartan at about

5minutes.

� Trial – IV

To decrease the retention time of telmisartan peak, the flow rate was increased to 1.0mL

minute-1.


Detection : 242nm


Sunfire C18





The telmisartan peak was eluted at about 3.4 minutes. The peak shape was good. Based

on the results of all the trial runs (I to IV), the results obtained under the optimal conditions of

trail IV were found to be satisfactory for the method validation of telmisartan.

6.3.2. Method validation

The developed test method was validated to include the requirements of ICH guidelines

[53]. The parameters like specificity, linearity, precision, accuracy, range, robustness,

ruggedness and system suitability were examined.

6.3.2.1. Specificity

The interference of placebo with telmisartan peak was verified as part of specificity study

by duplicate preparation of placebo as per the developed procedure. The placebo sample

solutions were prepared at various concentrations in the same way as described in the sample

preparation by taking placebo without telmisartan and were injected into chromatographic

system and recorded the chromatograms. The obtained chromatograms (Figure 6.2 to 6.4) have

shown that there were no interferences due to placebo and sample diluents at the retention time

of telmisartan.

6.3.2.2. Precision

6.3.2.2.1. System precision

Five replicate samples of telmisartan standard solution were injected into a high

performance liquid chromatographic system and chromatograms were recorded to ensure the

performance of the system under proposed chromatographic conditions and calculated the

relative standard deviation, tailing factor and theoretical plates of telmisartan peak. The observed

relative standard deviation, tailing factor and theoretical plates were found to be 0.1%, 1.079 and

8941 respectively which are satisfactory against the prescribed limits of not more than 2.0% for

relative standard deviation, not more than 2.0 for tailing factor and not less than 5000 theoretical

plates.

6.3.2.2.2. Methodprecision (repeatability)

The precision (repeatability) of the developed method tested by preparing six sample

solutions using drug product (telmisrtan tablets) and analyzing as per the test method. The assay

and relative standard deviation were calculated from six sample preparations. The results are

shown in table 6.1.

Table 6.1: Method precision results of telmisartan

S. No. % of telmisartan assay

1 98

2 98

3 98

4 97

5 98

6 97

Average 98

% RSD 0.4

The developed method was found to be precise as the relative standard deviation was

<0.5%. Assay of telmisartan was obtained between 97.0% and 98.0%. The obtained results

reveal that the developed method is precise.

6.3.2.3. Accuracy

To demonstrate the accuracy of the proposed method, sample solutions were prepared by

using telmisartan tablets, ranging from 50% to 150% (50%, 100% and 150%) of the target test

concentration as per test method. Six sample solutions at lower (50%) and higher (150%) levels

and three sample solutions at intermediate level (100%) were prepared and the chromatograms

were recorded. The % recovery at each level was calculated and tabulated in table 6.2.

Table 6.2: Accuracy results of telmisartan

Sample No.

Spike level

Added (µg mL-1)

Found (µg mL-1)

Individual recovery (%)

Statistical Analysis

1 50% 160.0 155.9 97.4 Mean* 97.6

2 50% 160.5 156.8 97.7

3 50% 160.3 156.2 97.4 SD* 0.2

4 50% 160.2 156.7 97.8

5 50% 160.0 156.5 97.8 % RSD* 0.2

6 50% 160.0 156.0 97.5

1 100% 320.6 311.6 97.2 Mean^ 97.1

2 100% 321.7 311.7 96.9 SD^ 0.2

3 100% 320.6 311.6 97.2 % RSD^ 0.2

1 150% 480.0 465.6 97.0 Mean* 97.2

2 150% 480.8 467.7 97.3

3 150% 479.8 466.0 97.1 SD* 0.1

4 150% 480.8 466.8 97.1

5 150% 480.8 467.7 97.3 % RSD* 0.1

6 150% 480.1 466.3 97.1

Overall statistical analysis

Mean$ 97.3

SD$ 0.3

% RSD$ 0.3

* : For six replicates

^ : For three replicates $ : For twenty one replicates

The results of accuracy as determined by both the calculation methods revealed that, the

average recovery at each level was between 97.0% and 98.0% with relative standard deviation at

each level as ≤1%. No significant difference was seen between both theoretical and practical

calculation methods. The results obtained from accuracy study confirm that the method is

accurate throughout its range.

6.3.2.4. Linearity

To demonstrate the linearity for telmisartan, five standard solutions of telmisartan were

prepared with concentration ranging from 50 % to 150 % of the target standard concentration

and injected into the chromatographic system and the chromatograms were recorded. The

coefficient of determination and bias were calculated at 100% concentration response. The

results are shown in table 6.3.

Table 6.3: Statistical data of telmisartan linearity study

S. No. Linearity

level Concentration

(µg mL-1)

Telmisartan peak area

(absorbing units)

Coefficient of determination (r2)

1 50% 160 4383539

0.9997

2 75% 240 6624439

3 100% 320 8840998

4 125% 400 10919787

5 150% 480 13295865

Figure 6.5: The linearity graph of telmisartan

R² = 0.9997

2000000

4000000

6000000

8000000

10000000

12000000

14000000

100 150 200 250 300 350 400 450 500

Concentration (µg mL-1)

Tel

mis

arta

n p

eak a

rea

(AU

)

The linearity curve (Figure 6.5) was prepared by plotting the values of concentration (µg

mL-1) on X-axisand the obtained peak areas of the chromatograms (Absorbing Units) on Y-axis.

The obtained coefficient of determination (R2) is 0.9997, which shows that the calibration curve

is very much linear in the concentration range mentioned.

6.3.2.5. Range

To demonstrate the range of the analytical method, data from six values of lower and

higher concentration solutions of accuracy preparation was considered. The obtained mean

recovery at lower level and higher level was found between 97.0% and 98.0% with the

coefficient of determination 0.9997 and relative standard deviation (for six preparations) at each

level ≤1% which shows that the analytical method is more accurate and precise throughout its

range of 160 µg mL-1 to 480 µg mL-1 of telmisartan.

6.3.2.6. Robustness of the method

6.3.2.6.1. Effect of variation in mobile phase composition (acetonitrile):

To demonstrate the robustness, the system suitability parameters were evaluated by

injecting standard solution, using two mobile phases, one containing 90% acetonitrile and other

contains 110% acetonitrile. The system suitability parameters were evaluated and tabulated the

results in table 6.4. The observed results of system suitability parameters from the variations of

mobile phase compositions were found satisfactory which shows that the mobile phase

preparation is robust for the intended purpose.

6.3.2.6.2. Effect of variation in flow rate

Robustness of flow rate was evaluated by varying the flow rate from 0.8mL minute-1 to

1.2mL minute-1. Standard solution was injected with different flow rates and evaluated the

system suitability parameters. The results are presented in Table 6.4. The obtained results

showed that the method is robust against the variation in flow rate of the mobile phase.

6.3.2.6.3. Effect of pH variation

To study the effect of the pH variation on the robustness of the proposed method, the

system suitability parameters were measured by injecting standard and system suitability

solutions into the chromatographic system with mobile phase having buffer of pH 4.3, 4.4 and

4.5. The results are tabulated in Table 6.4. The observed results of system suitability parameters

from the variations of mobile phase pH were found satisfactory as there is no significant change

in the system suitability results which show that the method is robust against slight variations in

pH.

6.3.2.6.4. Effect of variation in column oven temperature

The stability of the proposed method against the variations in column temperature was

studied by varying the temperature of the separation column between 40°C to 50°C. Standard

solution was prepared and injected into the chromatograph under prescribed conditions of

various column oven temperatures (40°C, 45°C and 50°C) and chromatograms were recorded.

The parameters like tailing factor, plate count and relative standard deviation were evaluated

from the chromatograms and presented in table 6.4. The observed results of system suitability

parameters at different temperatures were found satisfactory which shows the method is robust

against temperature fluctuations of the column.

Table 6.4: Summary results of robustness study

Robustness

Condition Variation USP Tailing USP plate count % RSD

As per test Method - 1.1 8941 0.1

Mobile phase

(buffer:acetonitrile)

60:40 v/v

- 10% (acetonitrile)

1.1 9054 0.1

+10% (acetonitrile) 1.2 8216 0.3

Flow Rate

(1.0 mL minute-1)

- 0.2 mL minute-1 1.1 8837 0.2

+0.2 mL minute-1 1.2 8459 0.2

pH (4.4)

- 0.1 1.1 8144 0.3

+ 0.1 1.0 7985 0.2

Column oven temperature (45°C)

- 5°C 1.2 8613 0.2

+ 5°C 1.1 9152 0.1

6.3.3. Comparison of results

A number of methods were reported for the determination of telmisartan present in both

plasma and in pharmaceutical formulations. Most of the reported methods are based on

spectrophotometric and HPTLC techniques. In general, HPLC methods are found to be better

with respect to precision and accuracy when compared to HPTLC and spectrophotometric

methods for quality control applications. Vekariya et al [31] have reported a precise single HPLC

method for the determination of telmisartan in pharmaceutical formulation. This method is found

to be superior over other reported methods. The results of proposed method are compared with

those reported by Vekariya et al and tabulated the observation in table 6.5.

Table 6.5: Comparison between reported method [31] and present method.

Parameter Reported method Present method Observations

Column Luna C18, 250mm

length, 4.6mm

internal diameter

packed with porous

silica with particle

size 5µm.

C18 sunfire column,

250mm length, 4.6mm

internal diameter

packed with porous

silica with particle size

5µm.

Sunfire columns have low

residual silanol

concentrations which

results in symmetric peak

shape.

Mobile

phase

5mM phosphate

buffer (pH adjusted

to 7.4) and

acetonitrile in the

ratio 60:40 v/v.

Phosphate buffer (pH

4.4) and acetonitrile in

the ratio 60:40 v/v.

Buffers with low pH are

more suitable for better life

of columns packed with

C18.

Diluents Methanol Mobile phase itself

was used as the

diluent.

In the reported method, the

mobile phase and diluent

are of different composition

and hence the complete

miscibility is doubtful. In

the present method the

mobile phase and diluent

are one and the same and

hence no problem of

miscibility.

Data

Acquisition

time

8 minutes per

injection

5 minutes per injection Less run time reduces

solvent consumption, and

saves analysis time

Method

validation

Linearity, accuracy,

limit of detection and

limit of quantification

were studied.

Specificity, precision,

linearity, accuracy,

range and robustness

of the method were

studied.

More validation parameters

(robustness, specificity)

were evaluated in the

present method

6.4 Conclusions:

A validated isocratic RP-HPLC method has been developed for the determination of

telmisartan in pharmaceutical finished dosage form. The analytical method was validated

according to the ICH guidelines and found that the method is selective, precise and accurate. The

proposed method is very simple, rapid, and specific. Its chromatographic run time of 5

minutes allows the analysis of a large number of samples in a short period of time. Therefore, it

is suitable for the routine analysis of pharmaceutical dosage forms. The simplicity of the method

allows its application in laboratories that lack sophisticated analytical instruments such as LC–

MS/MS or GC–MS/MS which are costly and time consuming. The method is exclusively useful

for quality control applications where multiple samples need to be analyzed within a short time.

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Documents

Chapter-6 Estimation of telmisartan by RP – HPLCshodhganga.inflibnet.ac.in/bitstream/10603/20011/12... · Telmisartan, a non peptide molecule, is chemically described as “2 methyl-1H-1,3-benzodiazol-2-yl)