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.
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
|
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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