Simultaneous Determination and Validation of ... - Ijpwr.com

ISSN 0976-111X
INTERNATIONAL JOURNAL OF PHARMA WORLD RESEARCH
(An International Quarterly Published Online Research Journal)
www.ijpwr.com
E-mail:editorijpwr@gmail.com
Title:
SIMULTANEOUS DETERMINATION AND VALIDATION OF
TELMISARTAN AND RAMIPRIL IN PHARMACEUTICAL DOSAGE
FORM BY RP – HPLC AND HPTLC
B.Raj kumar*, M.Priyanka 1 , K.V.Subrahmanyam 2 , Syed Mujtaba Ahmed 3 ,
Ch.RakeshReddy 1 , R.Prem Sagar 1
Department of Pharmaceutical Analysis
Mits College of Pharmacy, kodad, Nalgonda
Email id: raj_u_001@yahoo.co.in
Mobile no: 9966544051, 9000004806
1. Department of Pharmaceutics
Mits College of Pharmacy, Kodad, Nalgonda
2. Department of Pharmaceutical Analysis
PIPS, Suryapet, Nalgonda
3. Department of Pharmaceutical chemistry
Netaji college of Pharmacy, Choutuppal, Nalgonda
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ABSTRACT:
The present work deals with the studies carried out on the development, optimization
and validation of RP-HPLC and HPTLC methods for the simultaneous estimation of Telmisartan
and Ramipril in combined dosage form. Market is folded with combination of drugs in various
dosage forms. The multi-components formulations have gained a lot of importance now days due
to greater patient acceptability, increased potency, multiple action, fewer side effects and quicker
reliefs. For simultaneous estimation of drugs present in multi-component dosage form, High
Pressure Liquid Chromatography (HPLC) and High Pressure Thin Layer Chromatography
(HPTLC) methods are considered to be most suitable since it is extremely precise, accurate,
sensitive, linear and rapid. The literature survey carried out and it revealed that several analytical
methods have been reported for estimation of these drugs as individual or in combination with
other drugs. So the objective of the work is to develop HPLC and HPTLC methods for
simultaneous estimation of drugs in multi-component dosage form for which no analytical
method has been previously reported. Hence, present study have been planned to develop a
specific, precise, accurate, linear, simple and rapid HPLC and HPTLC methods for simultaneous
estimation of Telmisartan and Ramipril in tablet dosage form.
Keywords:
RP-HPLC
HPTLC
TELM
RAMI
UV
I.P.
B.P.
Reverse Phase High Performance Liquid Chromatography
High Performance Thin Layer Chromatography
Telmisartan
Ramipril
Ultra-Violet
Indian Pharmacopoeia
British Pharmacopoeia
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INTRODUCTION:
The present work deals with the studies carried out on the development, optimization and
validation of RP-HPLC and HPTLC methods for the simultaneous estimation of Telmisartan and
Ramipril in combined dosage form.
Market is folded with combination of drugs in various dosage forms. The multicomponents
formulations have gained a lot of importance now a day due to greater patient
acceptability, increased potency, multiple action, fewer side effects and quicker relief.
Classification of Instrumental Methods of Analysis 9-11
Most of the instrumental techniques fit in to one of the three principal areas such as
Spectroscopy
Electrochemistry
Chromatography
Spectroscopy
Spectroscopy is the measurement and interpretation of electromagnetic radiation
absorbed, scattered, or emitted by atoms, molecules or other chemical species.
Examples: UV Spectrophotometry, Atomic Spectrometry, Infrared Spectrometry, Raman
Spectrometry, X-Ray Spectrometry, Nuclear Magnetic Resonance Spectrometry, Electron Spin
Resonance Spectrometry.
Electrochemistry
In this, each basic electrical measurement of current like resistance and voltage has
been measured alone or in combination for analytical purposes.Examples: Potentiometry,
Voltametric Techniques, Amperometric Techniques, Electrogravimetry and Conductance
Techniques
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CHROMATOGRAPHY
The term ‘Chromatography’ covers those processes aimed at the separation of the various
species of a mixture on the basis of their distribution characteristics between a stationary and a
mobile phase. Chromatographic methods can be classified most practically according to the
stationary and mobile phases, as shown in the following table
Classification of Chromatographic methods
Stationary phase Mobile phase Method
Solid
Liquid
Liquid
Liquid
Gas
Adsorption column, thin-layer, ion exchange,
High performance liquid chromatography.
Partition, column, thin-layer, HPLC, paper
chromatography.
Gas – Liquid Chromatography.
A. Method Development in Chromatography (HPLC and HPTLC) 1-12
Modes of Chromatography
Modes of chromatography are defined essentially according to the nature of the
interactions between the solute and the stationary phase, which may arise from hydrogen
bonding, Vander walls forces, electrostatic forces or hydrophobic forces or basing on the size of
the particles (e.g. Size exclusion chromatography).
Different modes of chromatography are as follows:
• Normal Phase Chromatography
• Reversed Phase Chromatography
• Reversed Phase – ion pair Chromatography
• Ion Chromatography
• Ion-Exchange Chromatography
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• Affinity Chromatography
• Size Exclusion Chromatography
Adsorption chromatography or normal phase chromatography
In normal phase chromatography, the stationary phase is a polar adsorbent and the mobile
phase is generally a mixture of non-aqueous solvents.
The silica structure is saturated with silanol groups at the end. These OH groups are
statistically disturbed over the whole of the surface. The silanol groups represent the active sites
(very polar) in the stationary phase. This forms a weak type of bond with any molecule in the
vicinity when any of the following interactions are present.
→ Dipole-induced dipole,
→ Dipole-dipole,
→ Hydrogen bonding,
→ π-Complex bonding,
These situations arise when the molecule has one or several atoms with lone pair electron
or a double bond. The absorption strengths and hence k’ values (elution series) increase in the
following order. Saturated hydrocarbons < olefins < aromatics < organic halogen compounds <
sulphides < ethers< esters < aldehydes and ketones < amines < sulphones < amides < carboxylic
acids. The strength of interactions depends not only on the functional groups in the sample
molecule but also on steric factors. If a molecule has several functional groups, then the most
polar one determines the reaction properties.
Chemically modified silica, such as the aminopropyl, cyanopropyl and diol phases is
useful alternatives to silica gel as stationary phase in normal phase chromatography.
The aminopropyl and cyanopropyl phases provide opportunities for specific interactions
between the analyte and the stationary phases and thus offer additional options for the
optimizations of separations. Other advantages of bonded phases lie in their increased
homogeneity of the phase surface.
Reversed Phase Chromatography
In 1960’s chromatographers started modifying the polar nature of silanol group by
chemically reacting silica with organic silanes. The objective was to make less polar or non polar,
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so that polar solvents can be used to separate water-soluble polar compounds. Since the ionic
nature of the chemically modified silica is now reversed i.e. it is non-polar or the nature of the
phase is reversed. The chromatographic separation carried out with such silica is referred to as
reversed- phase chromatography. The retention of the compounds decreases in the following
order: aliphatics > induced dipoles (i.e. CCl4) > permanent dipoles (e.g.CHCl3) > weak lewis
bases (ethers, aldehydes, ketones) > strong lewis bases (amines) > weak lewis acids (alcohols,
phenols) > strong lewis acids (carboxylic acids). Also the retention increases as the number of
carbon atoms increases.
As a general rule the retention increases with increasing contact area between sample
molecule and stationary phase i.e. with increasing number of water molecules, which are released
during the absorption of a compound. Branched chain compounds are eluted more rapidly than
their corresponding normal isomers.
In reversed phase systems the strong attractive forces between water molecules arising
from the 3-dimentional inter molecular hydrogen bonded network, from a structure of water that
must be distorted or disrupted when a solute is dissolved. Only higher polar or ionic solutes can
interact with the water structure.
Chemically bonded octadecyl silane (ODS) an alkaline with 18 carbon atoms, it is the
most popular stationary phase used in pharmaceutical industry. Since most pharmaceutical
compounds are polar and water soluble, the majority of HPLC methods used for quality
assurance, decomposition studies, quantitative analysis of both bulk drugs and their formulations
use ODS HPLC columns. The solvent strength in reversed phase chromatography is reversed
from that of adsorption chromatography (silica gel) as stated earlier. Water interacts strongly
highly with silanol groups, so that, adsorption of sample molecules become highly restricted and
they are rapidly eluted as a result. Exactly opposite applies in reversed phase system; water
cannot wet the non-polar (hydrophobic) alkyl groups such as C18 of ODS phase and therefore
does not interact with the bonded moiety. Hence water is the weakest solvent of all and gives
slowest elution rate. The elution time (retention time) in reversed phase chromatography
increases with increasing amount of water in the mobile phase.
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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 1
High Performance Liquid Chromatography (HPLC) is the fastest growing analytical
technique for the analysis of drugs. Its simplicity, high specificity and wide range of sensitivity
make it ideal for the analysis of many drugs in both dosage forms and biological fluids. In HPLC
the separation is about 100 times faster than the conventional liquid chromatography due to
packing of stationary phase particles in the range of 5-10µm.
Schematic Representation of HPLC
Solvent container Pump Damping unit Injection port
|
Column
|
Recorder Detector
|
Effluent
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY 2
High Performance Thin Layer Chromatography (HPTLC) is a powerful separation tool
for quantitative analysis. It can simultaneously handle several samples even of divergent nature
and composition. HPTLC is the most simple separation technique to day available to the analyst.
Features of HPTLC
quantitative analysis:
The following are some of the special features of HPTLC, which make it suitable for
Simultaneous processing of sample and standard.
Better analytical precision and accuracy, less need for internal standard.
Lower analysis time and less cost for analysis.
Simple sample preparation.
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Can handle samples of divergent nature.
No prior treatment for solvents like filtration and degassing.
Low mobile phase consumption for sample.
Visual detection is possible.
Since fresh stationary and mobile phase are used for each analysis there are no chances
for contamination.
Schematic procedure for HPTLC
The various steps involved in HPTLC are schematically represented as follows:
Sample and standard
Preparation
Selection of
chromatographic layer
Layer pre-washing
Layer pre-conditioning
Application of sample and standard
Chromatographic development
Detection
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Steps involved in HPTLC
Selection of chromatographic layer
Sample preparation
Pre-washing of plates
Activation of plates
Application of sample and standard
Pre-conditioning
Chromatographic development
Detection
Scanning
Selection of Chromatographic Layer
Pre-coated plate
With the availability of pre-coated plates commercially, the use of laboratory hand made
plates is on decline. The pre-coated plates with different support material (glass, aluminium and
plastic) and different sorbent layers are available in different format and thickness by various
manufacturers. Usually plates with sorbent thickness of 100 to 200µm are used for qualitative and
quantitative analysis, however for preparative work, plates with sorbent thickness of 1 to 2mm
are available in addition to chemically modified layers.
Sample Preparation: The sample preparation procedure is to dissolve the dosage form with
complete recovery of intact compound(s) of interest and minimum of matrix with a suitable
concentration of analyte(s) for direct application on HPTLC plate. The choice of suitable solvent
is very important.
For normal phase chromatography, solvent for dissolving sample should be nonpolar
and volatile as far as possible. For reverse phase chromatography, polar solvents are used to
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SIMULTANEOUS ESTIMATION OF TELMISARTAN AND RAMIPRIL
BY RP-HPLC
Materials and Methods
Instrumentation
software.
Shimadzu HPLC-LC 2010 CHT with class VP version 6.12 with chemstation
Reagents and Chemicals
• Acetonitrile HPLC grade
• Orthophospharic acid AR rgade
• Potassium di-hydrogen phosphate AR grade
• Methanol HPLC grade
• Water –Milli Q grade
Reference Standards
TELMISARTAN (TELM)
Purity - 99.42%
Loss on Drying - 0.5%
RAMIPRIL (RAMI)
Purity - 100.12%
Loss on Drying - 0.2%
OPTIMIZED CHROMATOGRAPHIC CONDITIONS
Column
: Hypersil ODS C 18 ; 4.6 x 150 mm, 5microns
Mobile Phase : 10 mM pot. Di hydrogen phosphate: acetonitrile(60:40).
PH : 3.0 ±0.01
Flow rate
: 1 ml/min
Detector
: UV
Injection volume : 20µl
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Column temperature
Wavelength
Run time
: Ambient
: 245 nm
: 15 minutes.
LABEL CLAIM
TELM - 40 mg
RAMI - 5 mg
PREPARATION OF BUFFER
10mM potassium di-hydrogen phosphate solution is prepared in water. i,e 1360.1 mg
dissolved in 1000 ml of distilled water PH is adjusted to 3.0 ±0.01with orthophosphoric acid and
filtered through 0.45 µm membrane filter.
Preparation of Mobile Phase
(60:40).
Mobile phase is prepared by mixing 600 ml of buffer and 400 ml of acetonitrile
Preparation of Standard Stock Solution
An accurately weighed quantity of 40 mg of Telmisartan and 5 mg of Ramipril is
transferred into a 100 ml volumetric flask. Dissolved with 25 ml of methanol and diluted to
required volume with mobile phase, having the concentration of 0.4 mg/ml of Telmisartan and
0.05 mg/ml of Ramipril.
Preparation of Standard Solution
From the standard stock solution 5 ml is pipetted out into 100 ml volumetric flask
and made up the volume with mobile phase, having the concentration of 0.02 mg/ml of
Telmisartan and 0.0025 mg/ml of Ramipril.
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Preparation of Sample Solution
Twenty tablets were weighed and ground to a fine powder. An amount of power
equivalent to 40 mg of Telmisartan and 5 mg of Ramipril were weighed accurately and
transferred into a 100 ml volumetric flask containing 25 ml of methanol and sonicated for 30 min.
and diluted to 100 ml with mobile phase, then the solution was filtered through 0.45 µm
membrane filter and 5 ml of filtrate taken into 100 ml volumetric flask and made up to the
volume with mobile phase.
Estimation Method
The standard stock solution is diluted to the working concentration equivalent to that
of sample. 20 µl of the standard and sample are injected separately and chromatograms are
generated, with peak area obtained for standard and sample the content of Telmisartan and
Ramipril in each tablet is calculated using the following
Amount of drug present
in each tablet =
Sample area x Std. Conc. x Std. Purity x
(1000-Std.Lod) x Avg. weight
Std. area x Sample conc.x100x100
Amount present
Percentage label claim = x 100
Label claim
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SIMULTANEOUS ESTIMATION OF TELMISARTAN AND RAMIPRIL
BY HPTLC
Materials and Methods
Instrumentation
Application mode : CAMAG Linomat IV Sample applicator
Scanner mode : CAMAG TLC Scanner III
Development mode : CAMAG Twin trough chamber
Reagents and Chemicals
Choloroform (AR Grade)
Methanol (AR Grade)
Ethly acetate (AR Grade)
Reference Standards
Telmisartan (TELM)
Purity - 99.42%
Loss on Drying (Lod) - 0.5%
Ramipril (RAMI)
Purity - 100.12%
Loss on Drying (Lod) - 0.2%
Optimized Chromatographic Conditions
Stationary phase : Pre-coated Silica get plat 60 F 254 prewashed
with methanol.
Mobile Phase : Ethyl Acetate: Chloroform: Methanol(10:3:1)
Distance between bands : 14 mm
Plate width : 20 X 20 cm
Spotted technique : Ascending development
Scanning mode : Absorbance
Lamp : Deuterium
Wavelength : 272 nm
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Lable Claim
TELM - 40 mg
RAMI - 5 M
Preparation of Mobile Phase Ten volumes of ethyl acetate, three volumes of chloroform and
one volume of methanol are mixed thoroughly and used as mobile phase.
Diluent
Methanol and chloroform in the ratio of 1:1 is used as diluent.
Preparation of Standard Stock Solution
An accurately weighed quantity of 40 mg of Telmisartan (working standard and 5 mg of
Ramipril (working standard) were dissolved in diluent [methanol and chloroform (1:1)] taken in
20ml volumetric flask. Then the volume is made up to 20 ml with diluent, having the
concentration of 2 and 0.25 mg/ml for Telmisartan and Ramipril, respectively.
Preparation of Standard Solution
1m of standard stock solution is transferred to 10 ml volumetric flask. Then it is made up
to volume with the diluent, having the concentration of 0.2 and 0.025 mg/ml for Telmisartan and
Ramipril, respectively.
Preparation of Sample Solution
Twenty tablets are weighed and powdered. The powder equivalent to 40 mg of
Telmisartan and 5 mg of Ramipril (average weight of tablet) was transferred to 20 ml volumetric
flask. The contents were dissolved in diluent and the volume is made up to the mark.1m of the
above solution is transferred to 10 ml volumetric flask. Then it is made up to volume with the
diluent.The contents were mixed well using ultra-sonicator and filtered through Whatman filter
paper number: 42.
Estimation Method
The sample was spotted on the chromplate with help of Linomate IV spotting system. The
chromatograms were recorded and the peak area for TELM and RAMI area values of sample
with that of standard using the formula:
Amount of drug present
in each tablet =
Sample area x Std. Conc.x Std. Purity x
(1000-Std.Lod) x Avg.weight
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Std. area x Sample conc.x100x100
Amount present
Percentage label claim = x 100
Label claim
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RESULTS AND DISCUSSIONS:
The assay values are tabulated in Table 1
Chromatogram of Telmisartan & Ramipril Formulation
TABLE 1
QUANTITATIVE ESTIMATION
Tablet
Sample
TELM
Lable
Claim (mg)
40
Amount present
(mg/tablet)
40.20
%Lable Claim
100.49
%Deviation
+ 0.49
RAMI
5
5.0099
100.19
+0.19
Each value is mean of three readings
The values obtained for the assay are statistically validated and tabulated in Table 2
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TABLE 2
STATISTICAL DATA FOR QUANTITATIVE ESTIMATION BY RP-HPLC
Tablet
%Label
Standard
%Relative Standard
Standard
sample
Claim (mg)
Deviation
Deviation
Error
TELM
100.49
±1.30
1.29
0.750
RAMI
100.19
±0.1069
0.106
0.016
Validation
For validating the developed method the parameters like linearity, range, suitability, system
precision and assay (recovery studies) are studied .The validation procedures are carried out as
follows.
Linearity and Range
The linearity of the analytical procedure is its ability (with in given range) to obtain the test
results which are directly proportional to the concentration of analyte in the sample. Linearity was
assessed by performing single measurement at several analyte concentrations. A minimum of five
concentrations were recommended for linearity studies.
To evaluate the linearity range of Telmisartan and Ramipril, varying concentrations of
standard stock solution is diluted with mobile phase to give minimum of five concentrations in
the range of 16 to 24µg/ml for Telmisartan and 2 to 3 µg for Ramipril. A calibration curve was
constructed for each sample by plotting the peak area obtained against the concentration.
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The linearity data for Telmisartan and Ramipril are presented as follows
TELMISARTAN
There exists a linear relationship in the concentration range of 16 to 14µg/ml for
Telmisartan. The data are tabulated
7000
6000
5000
Peak area
4000
3000
2000
1000
0
y = 237.91x + 1.2888
R 2 = 0.9998
0 5 10 15 20 25 30
Concentration(mg/ml)
Linearity of Telmisartan
LINEARITY DATA FOR TELMISARTAN
CONCENTRATION
(µg/ml)
16
PEAK AREA
3838.48
18
4286.70
20
4700.65
22
5237.62
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24 5735.72
From the data obtained correlation coefficient, y-intercept and slope were calculated to
provide mathematical estimates of linearity for Telmisartan and tabulated
PARAMETERS
Linear Dynamic range
Correlation coefficient
Slope(m)
Intercept(c)
TELMISARTAN
16-24µg/ml
0.9998
237.91
1.288
ANALYTICAL PERFORMANCE PARA METERS OF TELMISARTAN
RAMIPRIL
There exists a linear relationship in the concentration range of 2 to 3µg/ml for
Ramipril. The data are tabulated in Table 4a
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Peak area
3500
3000
2500
2000
1500
1000
500
0
y = 1088.6x + 4.8739
R 2 = 0.9999
0 0.5 1 1.5 2 2.5 3 3.5
Concentration(mcg/ml)
Linearity of Ramipril
CONCENTRATION (µg/ml)
2.00
2.25
2.50
2.75
3.00
PEAK AREA
2200.66
2450.88
2725.44
2995.46
3264.802
LINEARITY DATA FOR RAMIPRIL
from the data obtained correlation coefficient, y-intercept and slope were calculated to provide
mathematical estimates of linearity for Ramipril and tabulated
PARAMETERS
RAMIPRAMIL
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Linear Dynamic range
2 – 3 µg/ml
Correlation coefficient (r)
0.999
Slope (m)
1088.6
Intercept (c)
4.8739
SUITABILITY
System suitability parameters are tabulated in Table 5
Parameter TELM RAMI
Resolution 4.38
Asymmetry factor 1.48 1.56
No. of Theoretical plates 2945 4738
Tailing factor 1.2 1.32
SYSTEM PRECISION
TABLE -5 - SYSTEM SUITABILITY
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Standard with the concentration same as that used for assay is injected in
replicate and area of peak developed are noted down. The values of peak and percentage relative
standard deviation are calculated and results are tabulated in Table-6
TABLE -6
SYSTEM PRECISION
S. No Area of TELM Area of RAMI
1 4943.94 2875.08
2
4950.50
2880.50
3
4835.60
2875.52
4
4965.08
2872.62
5
4998.50
2908.63
Mean 4938.724 2882.464
Standard
Deviation 61.376 14.898
%
Standard
Deviation
1.242 0.516
METHOD PRECISION
Sample solution of the working concentration is injected in to replicate and
percentage label claim is calculated for both the drugs. The mean, standard deviation and
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percentage standard deviation are calculated for percentage label claim of the drug by using the
same formula that is used for assay calculation and results are tabulated in
S. No Label claim of TELM Label claim of RAMI
1
2
3
4
5
101.11
101.38
99.00
101.65
102.34
100.13
100.32
100.14
100.04
101.30
Mean
101.096 100.368
Standard
Deviation 1.125 0.0520
%
Standard
Deviation
1.11 0.518
METHOD PRECISION
RECOVERY STUDIES
To ensure that the reliability and accuracy of the method, recovery studies
are carried out by mixing a known concentration of standard drug with the pre analysed sample
and the content were reanalyzed by the proposed method.The study was conducted using 5%,
10% and 15% recovery. Three sets of 20 tablets were crushed and each set is mixed with 40 mg
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of Telmisartan and 5mg of Ramipril, 80 mg of Telmisartan and 10 mg of Ramipril and 120 mg of
Telmisartan and 15 mg of Ramipril for 5%, 10% and 15% recovery respectively. The method is
processed same as assay method. The results are tabulated in Table-8.
The percentage recovery is calculated using the formula:
Amount of drug found
Percentage Recovery = x 10
Amount of drug added
Chromatogram of Recovery Studies at 5% Level
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Chromatogram of Recovery Studies at 10% Level
Chromatogram of Recovery Studies at 15% Level
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Sample
Amt. of
Amt. of drug
% Recovery % Mean
% RSD
standard
recovered (mg)
Recovery
added (mg)
2
1.986
99.30
2
1.982
99.70
99.16
2
1.970
98.50
4
3.938
98.45
TELMI
4
3.968
99.20
98.90
0.209
4
3.963
99.07
6
5.98
99.6
6
5.94
99.00
99.31
6
5.96
99.33
0.25
0.246
98.40
0.25
0.249
99.60
99.06
0.25
0.248
99.20
0.5
0.498
99.60
RAMI
0.5
0.492
98.40
99.50
0.242
0.5
0.495
99.00
0.75
0.747
99.60
0.75
0.735
98.00
99.11
0.75
0.748
99.73
RECOVERY STUDIES
LIMITS OF MEASUREMENT
There are two important categories within the level of
measurement. They are Limit of detection (LOD) and Limit of quantification (LOQ).
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LOD can be defined as the smallest level analyte that gives a measurable response and can be
calculated using the formula:
LOD = 3 x Standard deviation
Slope
LOQ can be defined as the smallest concentration of analyte, which gives a response that can be
accurately quantified and can be calculated using the formula:
LOQ = 10 x Standard deviation
Slope
TABLE 8
LIMITS OF MEASUREMENTS
Sample
TELM
RAMI
Limit of
Detection (µg)
9.47
1.16
Limit of
Quantification (µg)
31.57
3.88
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RESULTS AND DISCUSSIONS: HPTLC
Densitogram of Telmisartan and Ramipril Formulation
1. Telmisartan
2. Ramipril
QUANTITATIVE ESTIMATION
Tablet
sample
TELM
RAMI
Label
Claim (mg)
40
5
Amount present
(mg/tablet)*
39.70
4.98
Each value is mean of three readings.
%Label
claim*
99.25
99.60
%Deviation*
-0.75
-0.40
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The value obtained for the assay are statistically validated and tabulated in table
STATISTICAL DATA
%
Tablet
sample
%Lable
Claim
Standard
deviation
Relative
standard
Standard
Error
deviation
TELM
RAMI
99.25
99.60
±0.605
±0.290
0.60
0.29
0.34
0.17
Validation
Linearity and Range
The linearity of an analytical procedure is its ability (within a given range) to obtain the
test results, which are directly proportional to the concentration of analyte in the sample.
Linearity was assessed by performing single measurement at several analyte concentrations. A
minimum of five concentration were recommended for linearity studies
To evaluate the linearity range of Telmisartan and Ramipril, varying concentration of
standard stock solution is diluted with mobile phase to give minimum of five concentrations in
the range of 120 to 280 µg/ml of Telmisartan and 15 to 35 µg/mlof Ramipril. A calibration curve
was constructed for each sample for each samSple by plotting the peak areas obtained against the
concentration.
Telmisartan
There exists a linear relationship in the concentration range of 120 to 280µg/ml for
Telmisartan. The data are tabulated shows the line of best fit for Telmisartan.
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Peak area
30000
25000
20000
15000
10000
5000
0
y = 94.728x + 116.59
R 2 = 0.9995
0 50 100 150 200 250 300
Concentration(mcg/ml)
Linearity of Telmisartan
LINEARITY DATA FOR TELMISARTAN
Concentration
(µg/ml)
Peak area
120
160
200
240
280
11518.6
15320.3
19205.7
23099.9
26282.9
From the data obtained Correlation coefficient, Y-intercept and Slope were calculated to
provided mathematical estimates of the degree of linearity for Telmisartan and it is tabulated
ANALYTICAL PERFORMANCE PARAMETERS OF TELMISARTAN
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Parameters
Linear Dynamic Range
Correlation coefficient (r)
Slope (m)
Intercept (c)
Telmisartan
120-280 µg/ml
0.9995
94.728
116.59
Ramipril
There exists a linear relationship in the concentration range of 15 to 35µg/ml for
Ramipril. The data are tabulated shows the line of best fit for Ramipril...
12000
10000
Peak area
8000
6000
4000
2000
y = 297.16x + 13.154
R 2 = 0.9996
0
0 5 10 15 20 25 30 35 40
Concentration(mcg/ml)
LINEARITY DATA FOR RAMIPRIL (RAMI)
Concentration
Peak area
(µg/ml)
15 4407.1
20 6012.3
25 7498.1
30 8994.4
35 1031.2
From the data obtained Correlation coefficient, Y-intercept and Slope were calculated
to provide mathematical estimates of the degree of linearity for Ramipril and it is tabulated
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Parameters
Linear Dynamic Range
Correlation coefficient (r)
Slope (m)
Intercept (c)
Ramipril
15 - 35µg/ml
0.9996
297.16
13.154
Analytical Performance Parameters Of Ramipril
Following fig shows the overlain densitogram for linearity of TELM and RAMI.
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Overlain densitogram for Linearity of Telmisartan and Ramipril
SYSTEM SUITABILITY
System suitability parameters like resolution and asymmetry factor or tailing factor are
studied and tabulated in following table.
SYSTEM SUITABILITY DATA
Parameters TELM RAMI
Resolution 2.85
Tailing factor 1.6 1.2
SYSTEM PRECISION
Standard solution with the concentration same as that used for assay is spotted in replicate
and the areas of peak in the developed chromatogram are noted down. The values of mean and
percentage relative standard deviation are calculated and tabulated in following table
SYSTEM PRECISION
S.NO Area of TELM Area of HCTZ
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1
2
3
4
5
19195.6
19189.7
19220.4
19230.5
19180.4
7480.4
7485.6
7392.8
7479.6
7486.4
Mean 19203.32 7464.96
%R.S.D 0.11 0.54
METHOD PRECISION
Sample solution at the working concentration is spotted in replicate and percentage label
claim is calculated for both the drugs. The mean and percentage relative standard deviation are
calculated for percentage label claim calculated for the drugs.
The percentage label claims for the drugs are calculated using the same formula, used for
assay calculation.
The average/mean, percentage relative standard deviation for the percentage label claims
are calculated for both the drugs and the data are tabulated in Table
METHOD PRECISION DATA
S.NO
%Label claim
TELM
HCTZ
1
2
3
4
5
99.01
98.99
99.15
98.20
98.94
99.06
99.12
97.90
99.04
99.14
Mean 99.05 98.85
%R.S.D 0.37 0.53
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RECOVERY STUDIES
To ensure the reliability of the method, recovery were carried out by mixing a
known quantity of standard drug with the pre-analyzed sample and the content were reanalyzed
by the proposed method.
The study was conducted using 10% recovery. 10 tablets were crushed along with 40mg
of Telmisartan and 5mg of Ramipril (for 10%). The method of analysis is same as that of assay.
The percentage recovery is calculated using the formula:
Amount of drug received
Percentage Recovery = _______________________ x 100
Amount of drug added
The results are tabulated in table and for densitogram
Recovery Densitogram of Telmisartan and Ramipril
1. Telmisartan
2. Ramipril
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Sample
TELM
RAMI
RECOVERY STUDIES
Amt. of std. Amt. of drug %
added (mg) recovered (mg) Recovery
4
3.989
99.72
4
3.982
99.55
4
4.012
100.3
0.5
0.496
99.2
0.5
0.501 100.2
0.5
0.499
99.8
Mean%
Recovery
99.85
99.73
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SUMMARY AND CONCLUSION
The results of proposed RP-HPLC method are summarized in following table
SUMMARY FOR RP-HPLC
Observation
Parameter
TELMI RAMI
Label claim (mg/tab) 40 5
% Label claim
100.49 100.19
% RSD (NMT 2%) 1.29 0.106
Linearity range (µg/ml) 16 to 24 2 to 3
Correlation coefficient (NLT 0.999) 0.9997 0.9998
Resolution 4.38
Asymmetry factor (NMT 2%) 1.48 1.56
Number of Theoretical Plates
2945 4738
(NLT 2000)
System precision % RSD (NMT 2%) 1.242 0.516
Method precision % RSD (NMT 2%) 1.11 0.518
% Recovery (98 to 102%) 99.12 99.22
Limit of Detection (µg) 9.47 1.16
Limit of Quantification (µg) 31.57 3.88
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The results of proposed RP-HPTLC method are summarized in following table
SUMMARY FOR RP-HPTLC
Observation
Parameter
TELMI RAMI
Label claim (mg/tab) 40 5
% Label claim
99.25 99.60
% RSD (NMT 2%) 0.60 0.29
Linearity range (µg/ml) 120 to280 15 to35
Correlation coefficient (NLT 0.999) 0.9995 0.9996
Resolution 2.85
System precision % RSD (NMT 2%) 0.11 0.54
Method precision % RSD (NMT 2%) 0.37 0.53
% Recovery (98 to 102%) 99.85 99.73
Limit of Detection (µg) 187 30
Limit of Quantification (µg) 623 103
CONCLUSION
The proposed HPLC and HPTLC methods were found to be simple, specific, precise,
accurate and rapid for determination of Telmisartan and Ramipril in combined tablet dosage
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form. The mobile phase is simple to prepare and economical. The sample recoveries in all
formulations were in good agreement with their respective label claims and they suggested non –
interference of formulation excipients in the estimation.
Hence, this method can be easily and conveniently adopted for routine analysis of
Telmisartan and Ramipril in combined tablet dosage form.
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