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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
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
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
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
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
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
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
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
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
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
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
11
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
12
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
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
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
15
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
16
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
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
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.)
19
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
20
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
35
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
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
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
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
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
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)
41
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
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
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
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
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.
46
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
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
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
Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 49
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
5. UV-Light Degradation:
Fig.17. Chromatogram of UV radiation treated AMLO and OLME
Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 51
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.
Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 52
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Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 55
Development of spectroscopic and chromatographic methods for simultaneous estimation of amlodipine besilate and olmesartan medoxomil in tablet dosage form” 56