About Author:
B. Raj kumar*, M. Priyanka¹, K. V. Subrahmanyam², Syed Mujtaba Ahmed³, Ch. RakeshReddy¹, R. Prem Sagar¹
*Department of Pharmaceutical Analysis
Mits College of Pharmacy, kodad, Nalgonda
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

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.

REFERENCE ID: PHARMATUTOR-ART-1138

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 multi-components 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

Ø Spectroscopy

Ø Electrochemistry

Ø 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

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

Adsorption column, thin-layer, ion exchange, High performance liquid chromatography.

Liquid

Gas

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

· 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,

® p-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, 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.

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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.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY¹
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.

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY²
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
The following are some of the special features of HPTLC, which make it suitable for quantitative analysis:
Ø Simultaneous processing of sample and standard.
Ø Better analytical precision and accuracy, less need for internal standard.
Ø Lower analysis time and less cost for analysis.
Ø Simple sample preparation.
Ø Can handle samples of divergent nature.
Ø No prior treatment for solvents like filtration and degassing.
Ø Low mobile phase consumption for sample.
Ø Visual detection is possible.
Ø 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:

Steps involved in HPTLC

Ø Selection of chromatographic layer

Ø Sample preparation

Ø Pre-washing of plates

Ø Activation of plates

Ø Application of sample and standard

Ø Pre-conditioning

Ø Chromatographic development

Ø Detection

Ø 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 non-polar and volatile as far as possible. For reverse phase chromatography, polar solvents are used to

SIMULTANEOUS ESTIMATION OF TELMISARTAN AND RAMIPRIL BY RP-HPLC

Materials and Methods

Instrumentation
Shimadzu HPLC-LC 2010 CHT with class VP version 6.12 with chemstation software.

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 C₁₈; 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

Column temperature : Ambient

Wavelength : 245 nm

Run time : 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
Mobile phase is prepared by mixing 600 ml of buffer and 400 ml of acetonitrile (60:40).

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.

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

Sample area x Std. Conc. x Std. Purity x
(1000-Std.Lod) x Avg. weight

Amount of drug present = ------------------------------------------------------
in each tablet Std. area x Sample conc.x100x100

Amount present

Percentage label claim = ----------------------- x 100

Label claim

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 F254 pre-washed 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:

Sample area x Std. Conc.x Std. Purity x
(1000-Std.Lod) x Avg.weight

Amount of drug present = -----------------------------------------------------------------
in each tablet Std. area x Sample conc.x100x100

Amount present

Percentage label claim = ------------------------- x 100

Label claim

RESULTS AND DISCUSSIONS:

The assay values are tabulated in Table 1

Chromatogram of Telmisartan & Ramipril Formulation

TABLE 1

QUANTITATIVE ESTIMATION

Tablet Sample

Lable Claim (mg)

Amount present

(mg/tablet)

%Lable Claim

%Deviation

TELM

RAMI

40.20

5.0099

100.49

100.19

+ 0.49

+0.19

Each value is mean of three readings

The values obtained for the assay are statistically validated and tabulated in Table 2

TABLE 2

STATISTICAL DATA FOR QUANTITATIVE ESTIMATION BY RP-HPLC

Tablet sample

%Label Claim (mg)

Standard Deviation

%Relative Standard Deviation

Standard Error

TELM

RAMI

100.49

100.19

±1.30

±0.1069

1.29

0.106

0.750

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.

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

Linearity of Telmisartan

LINEARITY DATA FOR TELMISARTAN

CONCENTRATION (µg/ml)

PEAK AREA

3838.48

4286.70

4700.65

5237.62

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

TELMISARTAN

Linear Dynamic range

Correlation coefficient

Slope(m)

Intercept(c)

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

Linearity of Ramipril

CONCENTRATION(µg/ml)

PEAK AREA

2.00

2.25

2.50

2.75

3.00

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

Linear Dynamic range

Correlation coefficient (r)

Slope (m)

Intercept (c

2 – 3 µg/ml

0.999

1088.6

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.

TABLE -5 - SYSTEM SUITABILITY

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SYSTEM PRECISION
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

4943.94

4950.50

4835.60

4965.08

4998.50

2875.08

2880.50

2875.52

2872.62

2908.6

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 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

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 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
PercentageRecovery = ----------------------------- X 10
Amount of drug added

Chromatogram of Recovery Studies at 5% Level

Chromatogram of Recovery Studies at 10% Level

Chromatogram of Recovery Studies at 15% Level

Sample

Amt. of standard added (mg)

Amt. of drug recovered (mg)

% Recovery

% Mean Recovery

% RSD

TELMI

1.986

1.982

1.970

99.30

99.70

98.50

99.16

0.209

3.938

3.968

3.963

98.45

99.20

99.07

98.90

5.98

5.94

5.96

99.6

99.00

99.33

99.31

RAMI

0.25

0.246

0.249

0.248

98.40

99.60

99.20

99.06

0.242

0.5

0.498

0.492

0.495

99.60

98.40

99.00

99.50

0.75

0.747

0.735

0.748

99.60

98.00

99.73

99.11

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).

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

Limit of

Detection (µg)

Limit of Quantification (µg)

TELM

RAMI

9.47

1.16

31.57

3.88

RESULTS AND DISCUSSIONS: HPTLC

Densitogram of Telmisartan and Ramipril Formulation
1. Telmisartan
2. Ramipril

QUANTITATIVE ESTIMATION

Tablet sample

Label

Claim (mg)

Amount present (mg/tablet)*

%Label claim*

%Deviation*

TELM

RAMI

39.70

4.98

99.25

99.60

-0.75

-0.40

Each value is mean of three readings.

The value obtained for the assay are statistically validated and tabulated in table

STATISTICAL DATA

Tablet sample

%Lable

Claim

Standard deviation

% Relative standard deviation

Standard Error

TELM

RAMI

99.25

99.60

±0.605

±0.290

0.60

0.29

0.34

0.17

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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/ml of 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.

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

Parameters

Telmisartan

Linear Dynamic Range

Correlation coefficient (r)

Slope (m)

Intercept (c)

120-280 µg/ml

0.9995

94.728

116.5

Ramipril

There exists a linear relationship in the concentration range of 15 to 35µg/ml for Ramipril. The data are tabulatedshows the line of best fit for Ramipril...

LINEARITY DATA FOR RAMIPRIL (RAMI)

Concentration (µg/ml)

Peak area

4407.1

6012.3

7498.1

8994.4

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

Parameters

Ramipril

Linear Dynamic Range

Correlation coefficient (r)

Slope (m)

Intercept (c)

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.

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
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
S.NO	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

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 tableand for densitogram

Recovery Densitogram of Telmisartan and Ramipril
1. Telmisartan
2. Ramipril

RECOVERY STUDIES

Sample

Amt. of std. added (mg)

Amt. of drug recovered (mg)

% Recovery

Mean% Recovery

TELM

3.989

3.982

4.012

99.72

99.55

100.3

99.85

RAMI

0.5

0.496

0.501

0.499

99.2

100.2

99.8

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

Parameter	Observation
Parameter	TELMI	RAMI
Label claim (mg/tab)	40	5
% Label claim % RSD (NMT 2%)	100.49 1.29	100.19 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 (NLT 2000)	2945	4738
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

The results of proposed RP-HPTLC method are summarized in following table

SUMMARY FOR RP-HPTLC

Parameter	Observation
Parameter	TELMI	RAMI
Label claim (mg/tab)	40	5
% Label claim % RSD (NMT 2%)	99.25 0.60	99.60 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 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 Ramiprilin combined tablet dosage form.

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