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Pad. Dr. D.Y. Patil Institute of Pharmaceutical Science & Research, Pimpri, Pune-18. A SEMINAR ON “DEVELOPMENT OF SPECTROSCOPIC AND CHROMATOGRAPHIC METHODS FOR SIMULTANEOUS ESTIMATION OF AMLODIPINE BESILATE AND OLMESARTAN MEDOXOMIL IN TABLET DOSAGE FORM” Dr. SAGAR B. WANKHEDE Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 1

SAGAR PPT

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Page 1: SAGAR PPT

Pad. Dr. D.Y. Patil Institute of Pharmaceutical Science &

Research, Pimpri, Pune-18.

A SEMINAR ON

“DEVELOPMENT OF SPECTROSCOPIC AND CHROMATOGRAPHIC METHODS FOR

SIMULTANEOUS ESTIMATION OF AMLODIPINE BESILATE AND OLMESARTAN MEDOXOMIL IN

TABLET DOSAGE FORM”

Dr. SAGAR B. WANKHEDE

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 1

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CONTENTS

1. INTRODUCTION

2. DRUG PROFILE AND LITERATURE SURVEY

3. OBJECTIVE AND PLAN OF WORK

4. EXPERIMENTAL AND RESULTS

UV- SPECTROPHOTOMETRY

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

5. SUMMARY AND CONCLUSION

6. REFERENCES

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 2

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INTRODUCTION

Analytical methods development & Validation plays important roles in the discovery, development and manufacturing of pharmaceuticals.

The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency and performance of drug products

Method validation is the process of proving (through scientific studies) that an analytical method is acceptable for its intended use.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 3

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Structure-

IUPAC Name- (R, S) 2-[(2-Aminoethoxy) methyl]-4 -(2-chlorophenyl)-1,4-dihydro-6- methyl-3,5- pyridinedicarboxylic acid 3-ethyl 5-methyl ester benzene sulphonate.44Molecular formula- C20H25CLN2O5.C6H5O3S. Molecular weight- 567.1

Description- a white crystalline powder. Melting Point- 195 - 204 0C

Solubility- Slightly soluble in water and in isopropyl alcohol,sparingly soluble in dehydrated alcohol,freely soluble in methyl alcohol.Sparingly soluble in ethanol.

Therapeutic category- a long-acting calcium channel blocker.

DRUG PROFILE

AMLODIPINE BESILATE (AMLO)

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 4

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Reported method of analysis of Amlodipine Besilate

Simple and rapid high performance liquid chromatography method for the determination of Amlodipine concentration in plasma and urine

Reverse phase high performance liquid chromatographic determination of Amlodipine and Benazepril HCl in tablets.

Stability indicating HPTLC method of analysis of Amlodipine and Benazepril HCl.

Reverse phase high performance liquid chromatographic determination of Amlodipine and Atenlol in tablets.

Stability indicating HPTLC method of analysis of Amlodipine and Atenolol HCl.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 5

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Effect of charged and uncharged chiral additives on the resolution of Amlodipine enantiomers in liquid chromatography and Capillary Electrophoresis.

Reverse phase high performance liquid chromatographic determination of Amlodipine and Hydrochlorthiazide in tablets.

Reverse phase high performance liquid chromatographic determination of Amlodipine and Ramiril HCl in tablets

Simultaneous spectrophotometric determination of amlodipine besilate and atorvastatin calcium in binary mixture

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 6

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Structure-

IUPAC Name- (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl 5-(2-hydroxypropan-2-yl)-2-propyl-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]ph=enyl]methyl]imidazole-4-carboxylate

Molecular formula- C29H30N6O6 . Molecular weight- 558.6

Description- white to light yellowish-white or crystalline powder.

Melting Point- 175-180oC

Solubility- It is practically insoluble in water and sparingly soluble in methanol.

Therapeutic category- Angiotensin II Receptor Blockers .

DRUG PROFILE

OLMESARATAN MEDOXOMIL(OLME)

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 7

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Reported method of analysis of Olmesartan Medoxomil

Development and validation of indicating HPLC method for simultaneous determination of Hydrochlorthiazide and Olmesartan.

Reverse phase high performance liquid chromatographic determination of Olmesartan and Hydrochlorthiazide in tablet dosage form

Difference spectrophotometric estimation of Olmesartan.

Stability indicating HPTLC method of analysis of Olmesartan and Hydrochlorthiazide.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 8

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AIMS AND OBJECTIVE

No analytical method have been reported for the simultaneous estimation of Amlodipine Besilate(AMLO) and Olmesaratan Medoxomil(OLME) in combined dosage formulation.

The aims and objective of the present study are : 1. To develop some -UV- Visible spectrophotometric methods. - RP-HPLC method. - HPTLC method.2. To employ the developed methods for the analysis of standard laboratory mixture and marketed formulation.3. Validation of the developed methods.4. To compare the developed methods using suitable statistical tools.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 9

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PLAN OF WORK

1. Selection of multicomponent formulation By literature and market survey.

2. Selection of analytical techniques: UV-Spectrophotometric method.

RP-HPLC method.

HPTLC method.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 10

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3. Method development by UV-Spectrophotometric methods: Selection of the solvent.

Selection of analytical wavelengths.

Study of Beer-Lambert’s Law.

To develop UV Spectrophotometric method for the analysis of

AMLO & OLME in tablet dosage formulations.

To perform analysis of standard laboratory mixture and tablet formulations

by proposed method.

To validate the developed methods by using different statistical parameters.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

PLAN OF WORK

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4. Method development by RP-HPLC method Selection of suitable detection wavelength.

Optimization of mobile phase composition.

Optimization of chromatographic conditions.

Study of system suitability parameters.

To determine linearity range of Amlodipine Besilate and Olmesartan

Medoxomil.

To perform analysis of standard laboratory mixture and tablet formulations by

proposed method.

To validate the developed methods by using different statistical parameters.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

PLAN OF WORK

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5. Method development by HPTLC method Selection of suitable detection wavelength.

Optimization of mobile phase composition.

Optimization of chromatographic conditions

To determine linearity range of Amlodipine Besilate and Olmesartan Medoxomil.

To perform analysis of standard laboratory mixture and tablet formulations by

proposed method

To validate the developed methods by using different statistical parameters.

Forced degradation studies.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

PLAN OF WORK

13

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Experimental and Results

I. UV-Visible Spectrophotometry

II. High Performance Liquid Chromatography

III. High Performance Thin Layer Chromatography

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 14

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INSTRUMENT USED

Make : Shimadzu

Model Name : UV-1700

Wavelength Range : 190-1100 nm

I. UV-Visible Spectrophotometry

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

UV-Spectrophotometric methods for the simultaneous estimation of OLME and AMLO: Simultaneous Equation methodArea Under Curve Method

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METHODS : A. SIMULTANEOUS EQUATION METHOD B. AREA UNDER CURVE METHOD

Solvent used: MethanolPreparation of Standard Stock Solution: 50 mg drug → 50.0 ml, 5.0 ml → 50.0 ml (100 g/ml of AMLO)50 mg drug → 50.0 ml, 5.0 ml → 50.0 ml (100 g/ml of OLME)

Selection of analytical wavelength: 20 g/ml of (AMLO and OLME)

AMLO : 237.5 nmOLME : 255.5 nm

Fig.1: Overlain Spectra of AMLO and OLME

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

Fig.2: Overlain Spectra of AMLO and OLME

AMLO : 242.5-232.5 nmOLME : 260.5-250.5 nm

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Study of Beer-Lambert’s Law: Simultaneous Equation Method

Fig. 3: Standard Calibration Curve for AMLO

10-50 g/ml solution → AMLO 10-50 g/ml solution → OLME

y = 0.031x + 0.020R² = 0.998

y = 0.019x + 0.016R² = 0.997

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 20 40 60

Abso

rban

ce

Concentration(µg/ml)

Calibration Curve of AMLO

AMLO(237.5)

AMLO(255.5)

y = 0.037x + 0.022R² = 0.997

y = 0.040x + 0.023R² = 0.997

0

0.5

1

1.5

2

2.5

0 20 40 60

Abso

rban

ceConcentration(µg/ml)

Calibration Curve of OLME

OLME(237.5)

OLME(255.5)

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

Fig. 4: Standard Calibration Curve for OLME

17

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Study of Beer-Lambert’s Law: Area Under Curve Method

Fig. 5: Standard Calibration Curve for AMLO

Fig. 6: Standard Calibration Curve for OLME

10-50 g/ml solution → AMLO 10-50 g/ml solution → OLME

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 18

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Determination of Absorptivity of Drug at Selected Wavelengths

Drug Absorptivity value* X values *

237.5 nm 255.5 nm 242.5-232.5 nm

260.5-250.5 nm

AMLO 41.86 ± 0.3890 28.25 ± 0.4207 414.13 ± 0.6127 283.08 ± 0.8170

OLME 39.00 ± 0.1998 41.39 ± 0.3406 389.32 ± 0.7570 408.97 ± 0.7357 * denotes average of six determination

Simultaneous Equation Method

AbsorbanceAbsorptivity : ---------------------

Conc. (g/lit.)

4 g/ml solution of AMLO and 16 g/ml OLME was prepared in methanol

Standard absorptivity values of AMLO and OLME

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

Area Under Curve Method AUC at selected wavelength range X : ------------------------------------------------ Concentration (g/ lit.)

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Analysis of Standard Laboratory Mixture:

10.0 mg AMLO + 40.0 mg OLME → 50 ml, 5.0 ml → 50.0 ml, 5.0 ml → 25.0 ml(Concentration: 4 µg/ml of AMLO and 16 µg/ml of OLME)

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

MethodsSimultaneous Equation

methodArea Under Curve

AMLO OLME AMLO OLME

Amount of Drug Estimated*(mg)

9.988 40.007 9.979 40.169

% Estimation* 99.88 100.02 99.79 100.42

S.D. ± 0.2182 ± 0.1576 ± 0.3048 ± 0.1097

C.V. 0.2185 0.1575 0.3054 0.1092

* denotes average of six determinations

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Analysis of Tablet Formulation

Twenty tablets of AMLO and OLME combination were weighed and average weight was

determined.

The tablet powder equivalent to 20.0 mg of OLME was transferred to 25 ml volumetric flask

and dissolved in methanol.

The solution was ultrasonicated for 20 min. and filtered through Whatman filter paper No. 42.

The filtrate was appropriately diluted with methanol to obtain final concentration within Beer

Lambert’s range, for each drug.

Absorbance of the diluted solution was recorded at the selected wavelengths and concentration of

each drug was then calculated using simultaneous equation and area under curve methods.

Results of tablet analysis are depicted in following Table.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 21

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Results of Analysis of Tablet Formulation

MethodsSimultaneous Equation

methodArea Under Curve

AMLO OLME AMLO OLME

Label Claim(mg)

5.0 20.0 5.0 20.0

Amount of Drug Estimated*(mg)

5.002 20.012 5.004 20.074

% label Claim* 100.04 100.06 100.08 100.37

S.D. ± 0.4810 ± 0.4235 ± 0.1472 ± 0.2039

C.V. 0.4808 0.4232 0.1471 0.2031 * denotes average of six determinationsDevelopment of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan

medoxomil in tablet dosage form” 22

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Recovery Studies: To ascertain accuracy of proposed method

Level of Recovery

Amount of Pure Drug Added (mg)

Simultaneous Equation Method

Area Under Curve Method

% Recovery % Recovery

AMLO OLME AMLO OLME AMLO OLME

80% 4.0 16.0 100.18 100.07 100.03 100.524.0 16.0 100.30 100.24 100.09 100.63

100% 5.0 20.0 100.65 100.29 100.03 100.425.0 20.0 100.44 100.51 100.30 100.23

120% 6.0 24.0 99.50 100.11 100.43 100.506.0 24.0 99.18 100.09 99.69 100.58

Mean % Recovery 100.04 100.22 100.09 100.48S.D. ± 0.5745 ± 0.1679 ± 0.2556 ± 0.1418C.V. 0.5743 0.1675 0.2554 0.1411Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan

medoxomil in tablet dosage form” 23

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Precision Studies

Intra Day Precision

Simultaneous Equation Method Area Under Curve Method

AMLO OLME AMLO OLME

% Label Claim 100.08 100.18 100.13 100.22

S.D. ± 0.0135 ± 0.2075 ± 0.0750 ± 0.0765

C.V. 0.0135 0.2071 0.0749 0.0763

Inter Day Precision

Simultaneous Equation Method Area Under Curve Method

AMLO OLME AMLO OLME

% Label Claim* 99.92 100.08 100.02 100.26

S.D. ± 10.096 ± 0.0820 ± 0.1287 ± 0.1165

C.V. 0.0917 0.0819 0.1287 0.1162

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

* indicates average of three determination.24

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

ParametersSimultaneous

Equation MethodArea Under Curve

MethodAMLO OLME AMLO OLME

Limit of Detection (µg/mL) 0.247 0.172 0.117 0.104

Limit of Quantification (μg/mL) 0.749 0.521 0.356 0.315

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 25

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Instrument Used

Make : Merck Hitachi.Pump : L – 7100 double reciprocating pump.UV detector : L – 7400 (190- 666 nm).Column : C18 Column.

LACHROME HPLC SYSTEM

High Performance Liquid Chromatography

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 26

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Preparation of Standard Stock Solution:

50.0 mg drug → 50.0 ml, 5.0 ml → 25.0 ml (200 g/ml of AMLO)50.0 mg drug → 50.0 ml, 5.0 ml → 50.0 ml (100 g/ml of OLME)

Optimized Chromatographic Conditions:20 g/ml of (AMLO and OLME)Mobile phase - 0.05 M Phosphate buffer: Acetonitrile

(50:50 v/v)Column - Kromasil C18 (4.6 mm. i.d. x 25 cm.)Flow rate - 1.0 ml/minDetection wavelength - 238 nmInjection volume - 20 µLRun –time - 8 min

METHOD:

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 27

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Fig.7: Typical Chromatogram of AMLO and OLME

System Suitability Parameters:

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 28

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Sr. No.

Parameters AMLO OLME

1. Retention time (Rt) 3.79 5.38

2. Resolution (R) 2.22

3. Tailing factor (T) 1.30 1.17

4. No. of theoretical plates (N)

3313 6979

10.0 mg AMLO + 40.0 mg OLME → 50 ml, 5.0 ml → 25.0 ml, 2.0 ml → 10.0 ml

Solution of 8 µg/ml AMLO and 32 µg/ml OLME was prepared

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 29

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Fig. 8: Standard Calibration Curve for AMLO

Fig. 9: Standard Calibration Curve for OLME

Study of Linearity Range:

8-40 g/ml solution → AMLO 10-50 g/ml solution → OLME

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 30

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Analysis of Standard Laboratory Mixture

Solution of 8 µg/ml AMLO and 32 µg/ml OLME were prepared

* denotes average of six determination

10.0 mg AMLO + 40.0 mg OLME → 50.0 ml, 5.0 ml → 25.0 ml, 2.0 ml → 10.0 ml

Drug Taken(mg)

Drug Estimated*(mg)

% Estimation* S.D. C.V.

AMLO OLME AMLO OLME AMLO OLME AMLO OLME AMLO OLME

10 40 10.004 40.117 100.04 100.30 ± 0.9140 ± 0.7382 0.9136 0.7360

Results of Standard Laboratory Mixture Analysis :

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 31

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Twenty tablets of AMLO and OLME combination were weighed and average weight was determined. The tablet powder equivalent to 20.0 mg of OLME was transferred to 25 ml volumetric flask and dissolved in Mobile phase.

The solution was ultrasonicated for 20 min. and filtered through Whatman filter paper No. 42.

Then tablet solution was further diluted with mobile phase to obtain final concentration within the linearity range. The diluted solution was filtered through 0.2 µ membrane filter. 20 µl of the solution was injected and chromatographed under optimized chromtographic conditions.

Chromatograms were recorded and amount of drug in mg/tablet and % label claim was calculated by comparing mean peak area of standard and sample solution..

Analysis of Tablet Formulation

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 32

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Results of Analysis of Tablet Formulation

Sr.No. Content Label

Claim (mg)

Amt. of drug estimated(mg/tab)

% Label

Claim*S.D. C.V.

1. AMLO 5.0 5.007 100.14 ± 0.5841 0.5833

2. OLME 20.0 20.056 100.28 ± 0.2702 0.2694

* denotes average of six determinations

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 33

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Results of Recovery Studies

Level of Recovery

Amount of Pure Drug Added (mg)

% Recovery

AMLO OLME AMLO OLME

80% 4.0 16.0 100.2 100.344.0 16.0 100.18 100.23

100% 5.0 20.0 100.02 100.15.0 20.0 100.04 100.15

120% 6.0 24.0 99.97 99.986.0 24.0 99.38 99.61

Mean % Recovery 99.97 100.07S.D. ± 0.3008 ± 0.2551C.V. 0.3009 0.2549

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 34

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Precision Studies

Intra Day PrecisionAMLO OLME

% Label Claim 100.06 100.07S.D. ± 0.3305 ± 0.0723C.V. 0.3303 0.0722

Inter Day PrecisionAMLO OLME

% Label Claim 99.88 99.96S.D. ± 0.1242 ± 0.1552C.V. 0.1243 0.1553

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

* indicates average of three determination.

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

Parameters AMLO OLME

Limit of Detection (µg/mL) 0.085 0.152

Limit of Quantification (μg/mL) 0.127 0.423

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 36

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Results of Robustness Studies

Chromatographic Changes

Factor Level Retention time Tailing factor

Flow Rate(ml/min) AMLO OLME AMLO OLME

0.9 – 0.1 4.18 6.03 1.45 1.34

1.0 0 3.79 5.38 1.30 1.17

1.1 + 0.1 3.44 4.98 1.36 1.23

Mean 3.80 5.46 1.37 1.25

S.D. ± 0.3702 ± 0.5299 ± 0.0755 ± 0.0862

Mobile Phase (v/v) AMLO OLME AMLO OLME

49: 51 – 1.0 3.59 5.24 1.43 1.22

50: 50 0 3.79 5.38 1.30 1.17

51: 49 +1.0 3.89 5.76 1.34 1.18

Mean 3.76 5.46 1.36 1.19

S.D. ± 0.1528 ± 0.2691 ± 0.0666 ± 0.0265

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 37

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Instrument Used

TLC Plate Used : Aluminium plates precoated with silica gel 60 F254 plates

Development Chamber : Camag twin trough glass development chamber with stainless steel lid

Sample Applicator : Camag Linomat V sample applicator

UV Cabinet : Camag hightec UV cabinet fitted with dual wavelength 254/366 nm,

8 volt UV lamp

TLC Scanner : Camag TLC scanner III (Densitometer) with WinCAT’s software

version 1.4.3.6336

CAMAG HPTLC SYSTEM

High Performance Thin Layer Chromatography

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 38

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Optimum Chromatographic Conditions

a) Stationary phase :Aluminium plate precoated with silica gel 60F254.

b) Plate size : 10 X 10 cm Thickness : 200 µm

c) Mobile phase : Chloroform: Acetonitrile: Methanol: Glacial acetic acid (4:4:1.5:0.5 v/v/v/v)

d) Mode of application : Band

e) Developing chamber : Twin trough glass development chamber with stainless steel lid (10 X 10 cm)

f) Saturation time : 10 min.

g) Separation technique : Ascending.

i) Migration distance : ≈ 80 mm

j) Scanning mode : Absorbance reflectance.

k) Slit dimensions : 4 X 0.45 mm

l) Scanning wavelength : 254 nm

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 39

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Fig. 10: Typical densitogram of AMLO and OLME.

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 40

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Study of Beer-Lambert’s Law:

Fig. 11: Standard Calibration Curve for AMLO

Fig. 12: Standard Calibration Curve for OLME

Linearity Range of AMLO → 200-1200 ng Linearity Range of OLME → 1600-3200 ng

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

20.0 mg drug → 50.0 ml, 5.0 ml → 10.0 ml (200 g/ml of AMLO)20.0 mg drug → 25.0 ml, 5.0 ml → 10.0 ml (400 g/ml of OLME)

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Analysis of Standard Laboratory Mixture

Solution of 60 g/ml AMLO and 240 g/ml OLME were prepared

* denotes average of six determination

6.0 mg AMLO + 24.0 mg OLME → 50.0 ml, 5.0 ml → 10.0 ml

Drug Taken(mg)

Drug Estimated*(mg)

% Estimation* S.D. C.V.

AMLO OLME AMLO OLME AMLO OLME AMLO OLME AMLO OLME

6.0 24.0 6.014 24.121 100.24 100.50 ± 0.7685 ± 0.2807 0.7646 0.2800

Results of Standard Laboratory Mixture Analysis :

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 42

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Twenty tablets of AMLO and OLME combination were weighed and average weight was determined. The tablet powder equivalent to 24.0 mg of OLME was transferred to 50 ml volumetric flask and dissolved in Mobile phase.

The solution was ultrasonicated for 20 min. and filtered through Whatman filter paper No. 42.

Then tablet solution was further diluted with mobile phase to obtain final concentration within the linearity range. On TLC plate two bands of standard and eight bands of sample solution, 10 µL each, were applied and the plate was developed and scanned under the optimized chromatographic conditions

Chromatogram were recorded and amount of drug in mg/tablet and % label claim was calculated by comparing mean peak area of standard and sample solution..

Analysis of Tablet Formulation

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 43

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Results of Analysis of Tablet Formulation

Sr.No. Content Label

Claim (mg)

Amt. of drug estimated(mg/tab)

% Label

Claim*S.D. C.V.

1. AMLO 5.0 5.005 100.10 ± 0.5717 0.5712

2. OLME 20.0 20.073 100.36 ± 1.1440 1.1399

* denotes average of six determinations

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 44

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Results of Recovery Studies

Level of Recovery

Amount of Pure Drug Added (mg)

% Recovery

AMLO OLME AMLO OLME

80% 4 16.0 100.01 100.084 16.0 100.01 100.14

100% 5 20.0 100.01 100.215 20.0 100.05 100.08

120% 6 24.0 99.72 99.986 24.0 99.75 99.93

Mean % Recovery 99.93 100.07S.D. ± 0.1483 ± 0.1024C.V. 0.1484 0.1023

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 45

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Precision Studies

Intra Day PrecisionAMLO OLME

% Label Claim 99.82 100.3S.D. ± 0.3262 ± 0.3439C.V. 0.3268 0.3429

Inter Day PrecisionAMLO OLME

% Label Claim 99.34 100.63S.D. ± 0.7186 ± 0.7257C.V. 0.7134 0.7231

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form”

* indicates average of three determination.

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

Parameters AMLO OLME

Limit of Detection (ng) 6.422 40.261

Limit of Quantification (ng) 19.461 121.99

Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 47

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Results of Robustness Studies

ParametersLevel

Rf valueMobile phase composition

(± 0.1 mL)AMLO OLME

3.9:3.9:1.4:0.2 – 0.1 0.20 0.75

4.0:4.0:1.5:0.5 0 0.21 0.78

4.1:4.1:1.6:0.2 + 0.1 0.22 0.80 Amount of mobile Phase (v/v)

(± 1 mL)AMLO OLME

9 – 1.0 0.20 0.78

10 0 0.21 0.78

11 +1.0 0.21 0.79 Duration for chamber saturation

(± 2 min)AMLO OLME

8 min – 2 0.20 0.77

10 min 0 0.21 0.78

12 min +2 0.22 0.80 Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan

medoxomil in tablet dosage form” 48

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Forced Degradation Studies

1. Acidic Degradation 2. Alkali Degradation:

Fig.13. Chromatogram of 0.1M HCL Fig.14. Chromatogram of 0.1M NaOH

treated AMLO and OLME treated AMLO and OLME

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3. Oxidative Degradation : 4. heat Degradation:

Fig.15. Chromatogram of 3 % H2O2 treated Fig.16. Chromatogram of dry heat treated AMLO and OLME AMLO and OLME Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan

medoxomil in tablet dosage form” 50

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5. UV-Light Degradation:

Fig.17. Chromatogram of UV radiation treated AMLO and OLME

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Summary and Conclusion

The proposed method UV-Spectrophotometric, HPLC and HPTLC methods yields accurate and precise results for quantitative estimation of both Amlodipine and Olmesartan in combined dose tablet formulation.

Proposed UV-Spectrophotometric, HPLC and HPTLC methods is very simple, specific, economical and less time consuming and can be used for routine quality control of Amlodipine and Olmesartan in tablet dosage form.

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• Beckett, A.H. and Stenlake, J.B., In; Practical Pharmaceutical Chemistry, 4th Edn., Part 2, CBS Publishers and Distributors, New Delhi, 2002, 275-278, 281-300.

• Sethi, P.D., In; HPLC ‘High Performance Liquid Chromatography’, Quantitative Analysis of Pharmaceutical Formulations, 1st Edn., CBS Publishers and Distributors, New Delhi, 2001, 3-72, 116-120.

• Skoog, D.A., West, D.M., Holler, F.J. and Crouch, S.R., In; Fundamentals of Analytical Chemistry, 5th Edn., Thomson Brooks/Cole, 971-995.

• British Pharmacopoeia, 2008, Vol. І, London: Her Majesty's Stationary Office,

Pg 137.• Budavari S, O’Neil MJ, Smith A, Heckelman PE, Kinneary JF. The Merck Index: An encyclopedia of chemicals, drugs and biologicals, Whitehouse Station,

NJ: Merck and Co. Inc; 2001. 13th ed., Pg. 86-87.• Brian B. Hoffman. Pharmacological basis of Therapeutics. Brunton LL, Lazo

JS, Parker KL. Mc Graw- Hill Medical Publishing Division, New York, 2001, 10th ed.

Pg. 832-839.

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Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 53

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• Gohil K., Trivedi P., Molvi K.I.: Spectrophotometric analysis of amlodipine besylate in bulk and in tablet dosage forms, Indian J. Pharma. Sci. 2005, 67(3), 376-378. 

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