ISSN   0974-3618  (Print)                    www.rjptonline.org

            0974-360X (Online)

 

 

RESEARCH ARTICLE

 

Method Development and Validation for the Estimation of Telmisartan and Chlorthalidone in Bulk and Pharmaceutical Dosage form by HPTLC Method

 

Mrs. Aishwarya P. Joglekar

Shree Ambabai Talim Sanstha’s Diploma in Pharmacy College. Miraj, Maharashtra 416414

 *Corresponding Author E-mail: aishwarya3001@gmail.com

 

ABSTRACT:

In this study, the author has developed a HPTLC method for the estimation of Telmisartan and Chlorthalidone in commercial brand of ERITEL-CH40 mg containing 40 mg of Telmisartan and 12.5 mg of Chlorthalidone. The values obtained suggested that the proposed HPTLC method was simple, precise, rapid and robust for determination of Telmisartan and Chlorthalidone. The mobile phase was simple to prepare and economical. The authors then validated the method as per ICH guidelines and correlated the obtained values with standard values. Satisfactory results were obtained. The sample recoveries in all formulations were in good agreement with their respective label claims and they suggested noninterference of formulation excipients in the estimation. Hence, the method can be easily and conveniently adopted for routine estimation of Telmisartan and Chlorthalidone in tablet dosage form.

 

KEYWORDS: Telmisartan, Chlorthalidone, method development, validation and HPTLC.

 

 


INTRODUCTION:

Telmisartan is an Angiotension Receptor Bloker (ARB) chemically it is 4'[(1,4'-Dimethyl-2'-propyl[2,6'-bi-1H- benzimidazol]-1'-yl)methyl] [1,1'-biphenyl]-2- carboxylic acid , it shows high affinity for the angiotension II type 1 (AT1) receptors, has a long duration of action, and has the longest half-life of any ARB. In addition to blocking the Renin-Angiotension System (RAS), telmisartam acts as a selective modulator of Peroxisome proliferator-activated receptor gamma (PPAR- γ), a central regulator of insulin and glucose metabolism.1 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). Telmisartan has binding affinity 3000 times with AT-2 receptor than AT-1 receptor.

 

 

 

 

 

Received on 07.01.2015       Modified on 20.01.2015

Accepted on 13.02.2015      © RJPT All right reserved

Research J. Pharm. and Tech. 8(4): April, 2015; Page 376-381

DOI: 10.5958/0974-360X.2015.00063.3

 

 

Telmisartan is also having maximum half life in sartans – 24 Hrs. Telmisartan is indicated in the treatment of essential hypertension. Chlorthalidone is an oral diuretic with prolonged action (48–72 hours) and low toxicity. Chemically it is 2-chloro-5(1-hydroxy-3-oxo-1-isoindolinyl) benzenesulfonamide. The major portion of the drug is excreted unchanged by the kidneys. The diuretic effect of the drug occurs in approximately 2.6 hours and continues for up to 72 hours. The mean half-life following a 50 to 200 mg dose is 40 hours. In the first order of absorption, the elimination half-life is 53 hours following a 50 mg dose, and 60 hours following a 100 mg dose. Approximately 75 percent of the drug is bound to plasma proteins, 58 percent of the drug being bound to albumin. This is caused by an increased affinity of the drug to erythrocyte carbonic anhydrase.1 Nonrenal routes of elimination have yet to be clarified. Data are not available regarding percentage of dose as unchanged drug and metabolites, concentration of the drug in body fluids, degree of uptake by a particular organ or in the fetus, or passage across the blood-brain barrier. The drug produces copious diuresis with greatly increased excretion of sodium and chloride. At maximal therapeutic dosage, Chlorthalidone is approximately equal in its diuretic effect to comparable maximal therapeutic doses of benzothiadiazine diuretics. The site of action appears to be the cortical diluting segment of the ascending limb of Henle's loop of the nephron. 1

 

MATERIAL AND METHODS:

Materials:

Telmisartan and chlorthalidone were generoumacy gifted by Eris Life Sciences Pvt. Ltd., Ahmedabad and the formulation (Eritel-CH40 mg) containing 40mg telmisartan and 12.5 mg chlorthalidone of from local pharmacy. All the chemicals used were of analytical grade. The chemicals used for the study were; Methanol (Ranbaxy), 2-Propanol (Merck), Toluene (Merck), 25% Ammonia (Merck), Double distilled water.

 

Instrumentation:

A calibrated High Performance Thin Layer Liquid Chromatography instrument consisting of a Camag Linomat IV sample applicator with Camag TLC scanner 3 controlled by Wincats Software Version 1.2.3, and Twin trough chambers, make Switzerland, was used for all the experiments7. A calibrated analytical balance, manufactured by Mettler (Model AE-204), Switzerland. A hair dryer, Silencio 1000, manufactured by Braun. All the glassware used for the experiment were, manufactured by Borosil, India.

 

Methods Employed1:

HPTLC Technique for the estimation of Telmisartan and Chlorthalidone.

 

Selection of Solvent:

Ideal properties of solvent employed for HPTLC are,

·        Drug should be soluble in solvent used.

·        Drug should show stability in solvent used.

·        Solvent should be volatile.

 

Accordingly, methanol was selected as the solvent for further studies.

 

Selection of Wavelength:6, 7

The sensitivity of HPTLC method that uses UV detector depends upon the proper selection of wavelength. An ideal wavelength is the one that gives maximum absorbance and good responses for the drug detected at lower concentration also. UV spectra of Telmisartan and Chlorthalidone were recorded from that 257 nm was selected for further studies. The overlay spectrum is shown in Fig-1.

 

Preparation of Standard Solution:8

100 mg of Telmisartan and 50 mg of Chlorthalidone were accurately weighed and dissolved in 50 ml of methanol to produce Telmisartan (2000 μg/ml or 2000 ng/μL) and Chlorthalidone (1000 μg / ml or 1000 ng / μL) respectively.

Preparation of Sample Solution:

Twenty tablets each containing quantity equivalent to 40 mg of Telmisartan and 12.5mg of Chlorthalidone were weighed, powered and average weight was calculated. Quantity equivalent to 40mg of Telmisartan and 12.5mg of Chlorthalidone was weighed and transferred to 100 volumetric flask. The drug was extracted by addition of methanol with shaking and finally volume was made up to the mark. The solution was filtered through Whatman filter paper (No.14), and the resulting solution was used for the analysis.

 

Optimized Chromatographic Conditions:

A solvent system that would give dense compact spots, good separation from each other and separation from solvent front and application position was to be selected. Initially different solvent systems were tried (Table: 1), where bands closer to the solvent front and diffused bands were observed. Finally, Toluene: 2-Propanol: 25%Ammonia (6.5:3.5:0.2v/v/v) gave good sharp and symmetrical peak.

 

Table: 1 Different Solvent System Tried4.

Solvent system tried

Observations

Methanol: Chloroform :Glacial acetic acid

Poor separation

Toluene: Methanol: Ethyl acetate

Tailing

Toluene : Methanol: Chloroform

Poor separation

Toluene : Methanol: Glacial acetic acid

Lack of repeatability

Toluene: 2-Propanol: 25%Ammonia

Good separation with symmetric peaks

 

Among these systems, Toluene: 2-Propanol: 25%Ammonia was selected because in this system good, compact, dense spots were obtained with good resolution between analytes, good separation from solvent front and sample application positions.

 

Fixed Experimental Conditions:6,7

Stationary phase: TLC aluminium plate pre coated with silica gel 60F254 (Make: Merck) 

 

Mobile phase: Toluene: 2-Propanol: 25%Ammonia (6.5:3.5:0.2v/v/v) 

Diluent: Methanol 

Development Chamber: Twin through chamber  saturation: 30 minutes with filter paper  Separation Technique: Ascending chromatography development to a distance of 70% from line of application at room temperature

Migration time: 15 minutes

Band width: 6mm under stream of nitrogen using Linomat IV

Application Rate: 0.1μL/S using Linomate IV

Application Volume: 0.1μL using Linomate IV

Densitometric Mode: Absorbance

Scanning Speed: 20mm / S

Slit dimensions: 6× 0.45 mm

Detection Wavelength: UV 257 nm using Densitometer

Lamp Source: Deuterium lamp using Densitometer

Analysis of Marketed Formulation:8

Twenty tablets each containing quantity equivalent to 40 mg of Telmisartan and 12.5mg of Chlorthalidone were weighed, powered and average weight was calculated. Quantity equivalent to 40mg of Telmisartan and 12.5mg of Chlorthalidone was weighed and transferred to 100 volumetric flask. The drug was extracted by addition of methanol with shaking and finally volume was made up to the mark. The solution was filtered through Whatman filter paper (No.14). The formulation was assayed by spotting 1μL / S of the solution on to the plate followed by development and scanning (Fig.5). The amount of the drugs were calculated from the peak area obtained. (Table. 4)

 

Method Validation:

The validation of the developed method was carried out in terms of linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), inter and intraday precision, repeatability of sample application and stability studies as per ICH guidelines.3

 

Linearity:

Aliquots 1μL of standard solutions were applied on the plate. The linear regression data showed good linear relationship over a concentration range of 200 to 500 ng/spot for Telmisartan and 50-250 ng/spot for Chlorthalidone. The slope, intercept and correlation co-efficient values for Telmisartan and Chlorthalidone were shown (Table.3).

 

Recovery Studies:

Recovery studies of the drugs were carried out for the accuracy parameters. It was done by mixing a known quantity of standard drug with the pre analyzed sample formulation and the contents were reanalyzed by the proposed method. This was carried out at 80%, 100% and 120% levels. The percentage recovery was calculated and the values were shown in (Table-5).

 

Limit of Detection (LOD) and Limit of Quantification (LOQ):

LOD and LOQ were determined by applying decreasing amount of the drug in triplicate on the plate. The lowest concentration at which the peak is detected is called ‘Limit of Detection’ which was found to be 15 ng/spot for Telmisartan and 20 ng/spot for Chlorthalidone. The lowest concentration at which the peak is quantified is called ‘Limit of Quantification’ which was found to be 45 ng/spot and 60 ng/spot for Telmisartan and Chlorthalidone respectively.

 

RESULTS AND DISCUSSION:

Simple precise and accurate HPTLC method was developed for the estimation of Telmisartan and Chlorthalidone.

Fig.1. Overlain absorption spectra of Telmisartan and Chlorthalidone in methanol

 

  

UV 366nm                                     UV 254nm

Fig: 2 Developed Chromatographic Plate.

 

The solvent system that would gives dense and compact spots with significant Rf values was desired for quantification of Telmisartan and Chlorthalidone in pharmaceutical formulation. The mobile phase consisting of Toluene: 2-propanol: 25% Ammonia (6.5:3.5:0.2 %v/v/v) gave Rf values of 0.09 and 0.38 for Telmistartan and Chlorthalidone respectively. Fig.3 The linearity regression data showed a good linear relationship over a concentration range of 100 ng/spot – 500 ng / spot and 50 ng / spot – 250 ng / spot for Telmisartan and Chlorthalidone respectively. Table 3, the calibration curves were shown in Fig-4.1 and 4.2. The accuracy of the method was determined by recovery experiments. The recovery study was carried out and the percentage recovery range found to be within the limit. 98.61 % for Telmisartan and 99.42 % for Chlorthalidone (Table 5). The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The Detection Limit (LOD) was found to be 15 ng / spot for Telmisartan and 20 ng / spot for Chlorthalidone respectively (Table-2). The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (signal to noise ratio of 10). The quantitation limit (LOQ) was found to be 45 ng / spot for Telmisartan and 60 ng / spot for Chlorthalidone respectively (Table-2).

 

Table-2: Summery of linear regression and validation data.

Sr.No

Parameters

Telmisartan

Chlorthalidone

1.

Linearity range (ng/spot)

100-500 ng/spot

50-250 ng/spot

2.

Correlation Coefficient

0.9996

0.9995

3.

Slop

2550

3340

4.

Intercept

1894

2920

5.

LOD

15 ng/spot

20 ng/spot

6.

LOQ

45 ng/spot

60 ng/spot

 


Fig.3: Standard drugs chromatogram

 

Fig: 4.1: Linearity graph telmisartan

 

Fig: 4.2: Linearity graph chlorthalidone.

 

Fig: 5 Chromatogram of Formulation.

 

Sr.No

Telmisartan Concentration in ng/spot

Peak area

Chlorthalidone Concentration in ng/spot

Peak area

1.

100

2914

50

745

2.

200

3899

100

1702

3.

300

4995

150

2780

4.

400

5927

200

3724

5.

500

6985

250

4804

 

Slope

2550

Slope

3340

 

Intercept

1894

Intercept

2920

 

Co-relation coefficient

0.9996

Co-relation coefficient

0.9995

 

 

Table-4: Estimation of formulation.

Drug

Amount mg/tablet

% Drug content

SD

%RSD

Label claim

Estimated

Telmisartan

40

39.98

99.95

0.54

0.52

Chlorthalidone

12.5

12.35

98.80

0.47

0.45

 

Table -5: Recovery Study data.

Drug

Amount Added

Amount recovered

% Recovery

SD

%RSD*

Telmisartan

Lable claim

40 mg/tablet

32mg (80%)

31.5

98.43

 

 

40mg (100%)

39.3

98.25

0.68

0.67

48mg (120%)

47.6

99.16

 

 

Chlorthalidone

10mg (80%)

9.6

96.00

 

 

Lable claim

12.5 (100%)

12.7

101.60

0.87

0.86

12.5mg/tablet

15mg (120%)

15.1

100.66

 

 

*% RSD should not be more than 2

 


CONCLUSION:

A combination Telmisartan and Chlorthalidone is currently available for the treatment of hypertension. Author’s concluded that as there were no reported methods for their simultaneous estimation, a High performance thin layer chromatography (HPTLC) method was developed and validated for the determination of Telmisartan and Chlorthalidoneon pre-coated silica gel TLC plates using toluene: 2-propanol:25% ammonia (6.5: 3.5: 0.2 v/v/v) as the mobile phase with densitometry detection at 257nm. The developed method was found to be simple, rapid, selective, sensitive and suitable for simultaneous determination of Telmisartan and Chlorthalidone. The HPTLC method offered several advantages over liquid chromatographic methods such as the possibility of simultaneous analysis of sample and standard on the same plate, short system equilibrium time, multiple/repeated scanning of chromatograms, higher mobile phase pH, large sample capacity, short run time, minimum solution consumption and no prior treatment for solvents like filtration and degassing1.

 

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