rp hplc metod of amlodipine

1. Development and Validation of RP-HPLC Method for
the Simultaneous Estimation of Atenolol and
Amlodipine in Tablet Dosage Form
DEPARTMENT OF PHARMACEUTICEUTICS
MR.TANMAY PANIGRAHY

2. CONTENT
1. INTRODUCTION
2. DRUG PROFILE
3. LITERATURE REVIEW
4. AIM & OBJECTIVE
5. PLAN OF WORK
6. MATERRIAL & METHOD
7. RESULT & DISCUSSION
8. CONCLUSION
9. REFERENCE

3. 1. INTRODUCTION
Pharmaceutical analysis
Pharmaceutical Analysis may be defined as the application of analytical procedures used
to determine the purity, safety and quality of drugs and chemicals .
There are main two types of chemical analysis.
1. Qualitative (identification)
2. Quantitative (estimation)
This course has access to the full range of Titration method , Spectroscopic technique ,
Chromatographic method , Spectrophotometry etc.
Chromatographic
Chromatography is usually based on principle of partition of solute between two phases.
It usually consists of a Mobile Phase and a Stationary Phase.
The Mobile Phase usually refers to the mixture of the substances to be separated
dissolved in a liquid or a gas .
The Stationary Phase is a porous solid matrix through which the sample contained in the
mobile phase percolates.

4. HPLC
High Performance Liquid Chromatography [HPLC] is principle is based
on adsorption as well as partition chromatography is depending on the
nature of stationary phase, if stationary phase is solid principle is based
on adsorption chromatography and if stationary phase is liquid principle
is based on partition chromatography.
PRINCIPLE OF RP-HPLC METHOD
 The principle of separation in normal phase mode and reverse phase
mode is adsorption.
 When a mixture of components are introduced in to a HPLC column,
they travel according to the stationary phase.

5. INSTRUMENTATION
1. PUMP-SOLVENT DELIVERY SYSTEM
2. SAMPLE INJECTION SYSTEM
3. GUARD COLUMN
4. ANALYTICAL COLUMNS
5. DETECTORS
6. RECORDER AND INTEGRATORS

6. Parameters for the method validation
1. System suitability.
2. Selectivity.
3. Linearity.
4. Range
5. Accuracy.
6. Precision.
7. Repeatability
8. Reprodutibility
9. Robustness.

7. 2. DRUG PROFILE
ATENOLOL
Structure :-
2-(4-{2-hydroxy-3-[(propan-2 yl) amino] propoxy}phenyl) acetamide
Molecular Formula :- C14H22N2O3
Molecular Weight :-266.34
Melting Point:- 158-160OC
Storage:- at room temperature between 68°F and 77°F (20°C and 25°C).
Bioavailability:- 40–50%
Protein binding:- 6–16%

8. Mechanism of action:- Atenolol belongs to a class of drugs
known as beta blockers. It works by blocking the action of certain
natural chemicals in your body, such as epinephrine, on the heart and
blood vessels. This effect lowers the heart rate, blood pressure, and
strain on the heart.
Uses:- Atenolol belongs to a group of medicines called beta
blockers. It's used to treat high blood pressure and irregular heartbeats
. It can also be used to prevent chest pain caused by angina. If you
have high blood pressure, taking atenolol helps prevent future heart
disease, heart attacks and strokes.

9. AMLODIPINE
Struture :-
3-Ethyl 5-methyl-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-
dihydropyridine-3,5-dicarboxylate
Molecular Formula :- C20H25CIN2O5
Molecular Weight :- 408.88 g.mol-1
Storage:- at room temperature between 59°F and 86°F (15°C and 30°C).
Protein binding:- 93%

10. Mechanism of action:-Amlodipine is an angioselective calcium
channel blocker and inhibits the movement of calcium ions into vascular
smooth muscle cells. This causes vasodilation and a reduction in
peripheral vascular resistance, thus lowering blood pressure. Its effects on
cardiac muscle also prevent excessive constriction in the coronary
arteries.
Uses :- Amlodipine is used in the management of hypertension and
coronary artery disease in people with either stable angina or vasospastic
angina and without heart failure. It can be used as either monotherapy or
combination therapy for the management of hypertension or coronary
artery disease. Amlodipine can be administered to adults and children 6–
17 years of age.

11. Sl. No. AUTHOR TITLE CONCLUSION
1 Md. Ahsanul Haque ,
AsmaNaznin et al.
Development and validation of
RP-HPLC method for
simultaneous estimation of
Atenolol and Amlodipine in tablet
dosage form.
used as a more
convenient and
efficient option for
the analysis of
atenolol and
amlodipine in tablet
dosage form.
2 Mohammed Imran,
Dinesh M Sakarkar et
al.
RP-HPLC method for
simultaneous estimation of
amlodipine and metoprolol in
tablet Formulation
percentage
recoveries obtained
for amlodipine and
metoprolol ranges
from 100.04 to
100.57 %.
3 M.M. Eswaruduet al., Validated RP-HPLC Method for
Simultaneous Estimation of
Atenolol, Hydrochlorothiazide and
Losartan Potassium in Bulk and
Pharmaceutical Dosage Form
used for routine
quality control
analysis of these
drugs in biological
samples either alone
or in combined
pharmaceutical
dosage forms.
LITERATURE REVIEW

12. Sl. No. AUTHOR TITLE CONCLUSION
4 PRAJWALA NAIKINI,
ANUSHAAKULA et al.,
RP-HPLC METHOD
DEVELOPMENT AND VALIDATION
FOR THE SIMULTANEOUS
ESTIMATION OFAMLODIPINE
AND ATENOLOL IN BULK AND
TABLET DOSAGE FORMS
The proposed method
was validated in
accordance with ICH
guidelines and hence
can be successfully
applied to the
simultaneous estimation
of Amlodipine and
Atenolol in tablet
formulations.
5 GUNASEKAR
MANOHARAN et al.,
DEVELOPMENT AND VALIDATION
OF RP-HPLC METHOD FOR THE
SIMULTANEOUS ESTIMATION OF
AMLODIPINE AND NEBIVOLOL IN
RAW AND TABLET FORMULATION
Linearity range for
Amlodipine and
Nebivolol were 10-
50 g/ml, 10–50 g/ml.
6 Mustafa Çelebier et al., HPLC method development for the
simultaneous analysis of amlodipine
and valsartan in combined dosage
forms and in vitro dissolution studies
found to be linear
within the range 0.1 –
50 μg mL-1 for
amlodipine, and 0.05 –
50 μg mL-1 for
valsartan.

13. Sl. No. AUTHOR TITLE CONCLUSION
7 RAVISANKAR P ,
ASWINI M,
SRINIVASA BABU P,
et al.,
DEVELOPMENT AND
VALIDATION OF RP-HPLC
METHOD FOR
SIMULTANEOUS
DETERMINATION OF
METOPROLOL AND
AMLODIPINE IN TABLET
DOSAGE FORM
The method was
very sensitive with
regard to limit of
detection 0.125
μg/mL, 0.102 μg/mL
and limit of
quantitation 0.381
μg/mL, 0.311μg/mL
respectively.
8 K. Krishna Chaitanya
et al.,
RP-HPLC METHOD
DEVELOPMENT AND
VALIDATION OF AMLODIPINE
AND
LOSARTAN IN BINARY
MIXTURE
The calibration
curve was linear over
the concentration
range 1.25-7.5 μg/mL
and 12.5-75 μg/mL
for Amlodipine and
Losartan
respectively.

14. 4. AIM & OBJECTIVE
Aim
 To develop simple ,rapid , specific and sensitive RP-HPLC method for the
determination of Atenolol and Amlodipine in pharmaceutical formulations.
 According to the literature survey it was found that there was very few methods
for the determination of Atenolol and Amlodipine by RP-HPLC .
 To validate the method according to ICH guidelines.
Objective
 The scope of developing and validating an analytical method is to ensure a
suitable method for a particular analyte more specific, accurate and precise. The
main objective for that is to improve the inferences, which should be followed in
the development and validation.
 The survey of literature reveals that simultaneous analytical methods are
available for the drug atenolol and amlodipine , but the methods for
determination of single drug is still emerging. The existing methods are
inadequate to meet the requirements ; hence it is proposed to improve the
existing methods and to develop new methods for the determination of atenolol
and amlodipine.

15. 5. PLAN OF WORK:
1. Plan of work consists of the following steps:
 Method development
a. Method development by reverse phase HPLC
b. Selection of suitable wavelength
c. Selection of stationary phase
d. Selection of mobile phase
 Optimization of chromatographic condition
a. Development of simple. Cost eflective and accurate U spectroscopic method.
b. Selection of suitable wavelength
c. Selection of suitable Solvent
d. Optimization of proposed method

16. 2. Method validation
 Validation of the HPLC method using following parameters
a. System suitability
b. Accuracy
c. Linearity
d. Precision
• System precision
• Method precision
• Intermediate precision
e. Robustness
• Variation in flow rate
f. Solution stability.
3. Summarize the validation data and report.

17. 6. MATERIALAND METHOD
1. HPLC METHOD DEVELOPMENT:
 Mobile Phase Optimization
The mobile phase, a mixture of buffer, Methanol and Acetonitrile
(40:30:30v/v) pumped at a flow rate of 1.0ml/min through the column (BDS
C18;5μ,4.6x250mm)at ambient temperature.
 Wave Length Selection
The overlain spectrum of the two candidate drugs were obtained. The overlain
spectrum shows iso absorptive point at 232.2 nm. The two wavelengths were
selected one as 232.2 nm (Isoisorptive point) and other as 224 nm.
 Optimized HPLC conditions:
Inertsil C18 (250 mm x 4.6 mm, 5μm) column maintained at ambient
temperature was used as stationary phase. An isocratic mobile phase
constituting Triethylamine Buffer (pH adjusted to 3.0 with Ortho-Phosphoric
Acid), Acetonitrile and Methanol in ratio 4:3.5:2.5 (v/v/v), at a flow rate of 1.0
ml/min was used.

18.  Preparation of standard solution.
For 100% standard solution of target concentration 50 mg atenolol and 5 mg
amlodipine (as amlodipine besylate) were weighed, dissolved and sonicated in
mobile phase to produce 100 ml. 80%, 90%, 110%, and 120% standard solutions
of target concentration were also prepared in the same way.
 Preparation of sample solution.
20 tablets were accurately weighed and the average weight was calculated. The
tablets were grinded to a fine powder with the help of mortar and pestle.
 Preparation of standard drug solution
Standard stock solutions containing atenolol (ATN) and amlodipine besylate
(AMN) were prepared individually by dissolving 10 mg of ATN and quantity of
AMN separately in 0.1 N HCL Solution. It was then sonicated for 10 min and
final volume of both the solutions were made up to 100 ml with 0.1 N HCL
Solution to get stock solutions containing 100μg/ml each of ATN and AMN in
two different 100 ml volumetric flask.

19. Preparation of mixed stock solution:
An accurately weighed 10 mg each of AT and AB working standards were transferred
into a 100 ml volumetric flask. 20 ml of methanol was added into it and sonicated for
awhile for dissolving the drugs. The flask was made up to 100 ml with methanol so as
to get a concentration of 100 μg/ml.
Preparation of mixed standard solution:
From the stock solution, 1ml was transferred into a 10 ml volumetric flask and volume
was made up to mark with methanol to get a final concentration of 10 μg/ml. The
resulting solution containing 10 μg/ml of AT and 10 μg/ml of AB were scanned in UV-
Visible spectrophotometer from 400-200 nm to determine the wavelength of maximum
absorption of both the drugs in combination. Both drugs in combination showed
maximum absorption at 225 nm. The values are shown in table 1 and the spectra of
combination of AT and AB was appended in Fig. 2.

20. TABLE 1: OBSERVATION FROM UV-VISIBLE SPECTRO
PHOTOMETER
Sl. No. Wavelength (nm) Absorbance of
Combination
1 200 0.601
2 210 0.744
3 220 0.702
4 225 0.705
5 230 0.687
6 250 0.229
7 350 0.135
8 400 0.023

21. Fig. 1: Overlain spectra of ATN and AMN in 0.1 N HCL
FIG. 2: COMBINED SPECTRA OF ATENOLOLAND AMLODIPINE
BESYLATE

22. Standard preparations:
Standard Stock Solution:
Standard stock solution was prepared by transferring 14 mg of AB working standard into 20 ml
volumetric flask, added methanol and sonicated for 5 min for dissolving the drug and volume was
made up with mobile phase.
Working Standard Solution:
Working standard solution was prepared by transferring 10 mg of AT into 100 ml volumetric flask,
added methanol and sonicated for 5 min for dissolving the drug. 2 ml of AB stock solution was
added and volume was made up with mobile phase.
Sample preparation for Tablet Analysis:
Tablet powder equivalent to weight of one tablet was accurately weighed and transferred into 100 ml
volumetric flask, 60 ml of mobile phase was added and sonicated for 15 min; volume was made up
with mobile phase. Solution was filtered through 0.45μ nylon filter. 5 ml of the filtrate was diluted to
25 ml with mobile phase. 10 μl of the blank, placebo, standard and sample solution were injected
separately into the chromatographic system and chromatograms were recorded and the peak areas
were measured. A typical chromatogram of AT and AB was appended in Fig. 3.

23. FIG. 3: TYPICAL CHROMATOGRAM OF ATENOLOLAND AMLODIPINE
BESYLATE
METHOD VALIDATION:
System suitability.
TABLE 2: SYSTEM SUITABILITY STUDIES
Sl. No. Parameters ATa ABb
1 USP Plate Count 3527 4441
2 Peak Asymmetry
Resolution
1.46 1.36
3 Resolution 0.0 9.9

24. Specificity (Sensitivity or Selectivity):
Specificity was performed to detect the presence of interference peak (blank and placebo peaks) at
the retention time of the analyte peak.
Precision:
Repeatability (System precision & Method precision):
The precision of the method was determined by repeatability (intra-day) and intermediate precision
(inter-day) study. Repeatability was determined by performing four repeated analysis of the three
standard solutions (90%, 100% and 110% of target concentration) of atenolol and amlodipine on the
same day, under the same experimental conditions.
Ruggedness (Intermediate precision):
The ruggedness of the test method was determined by carrying out precision study in six replicates
of assay on a single batch sample in different days by two different analysts, on two different
columns and on two different instruments. The difference in the average assay of method precision
and intermediate precision for AT and AB is 0.19 and 0.18 respectively which is not more than the
limit 2. The values were given in table 4.

25. TABLE 3: PRECISION STUDIES (REPEATABILITY)
Sl. No.
System Precision Method Precision System Precision Method Precision
Standard Area %Assay Standard Area %Assay
AT AB AT AB
1 100.3 99.8 100.0 99.6
2 99.9 99.4 99.9 99.6
3 99.5 99.4 99.2 100.2
4 100.3 99.6 99.9 99.6
5 100.0 99.4 99.7 99.5
6 100.0 99.5 100.1 99.6
Mean 100.0 99.5 99.8 99.7
SD 0.30 0.16 0.32 0.26
%RSD 0.30 0.16 0.32 0.26

26. TABLE 4: PRECISION STUDIES (RUGGEDNESS)
Sl. No. Analyst 1, Day 1 Analyst 2, Day 2
% Assay % Assay
AT AB AT AB
1 100.3 99.8 100.0 99.6
2 99.9 99.4 99.9 99.6
3 99.5 99.4 99.2 100.2
4 100.3 99.6 99.9 99.6
5 100.0 99.4 99.7 99.5
6 100.0 99.5 100.1 99.6
Mean 100.0 99.5 99.8 99.7
SD 0.30 0.16 0.32 0.26
%RSD 0.30 0.16 0.32 0.26

27. Accuracy (Recovery or Trueness):
TABLE 5: RECOVERY STUDIES (ACCURACY)
Spike Level (%)
Added
Amt.(ppm)
Recovered c
Amt. (ppm)
Mean %
Recovery
%RSD
AT 50 49.75 50.15 100.80 0.52
100 99.50 99.07 99.57 0.25
150 149.25 149.09 99.90 0.44
AB 50 5.02 5.00 99.67 0.06
100 10.11 10.10 99.87 0.57
150 14.95 14.99 100.23 0.15
Linearity
The linearity of detector response for AT and AB was determined by preparing a series
of solution of AT and AB working standards over the range of 50% to 150% of targeted
concentration. These solutions were injected into the chromatographic system and
response area was recorded. The regression equation for AT and AB were found to be y
= 20158x+33346 and y = 20124x + 453.44 with correlation coefficient 0.9992 and
0.9998, respectively. A linearity plot for AT and AB was shown in Fig. 4 and 5, and the
correlation co-efficient was evaluated. The values were given in table 6.

28. TABLE 6: LINEARITY STUDIES
Linearity
Level (%)
AT AB
Conc. Aread Conc. Areae
50 50 1078115 5 99431
75 75 1571158 7.5 153203
100 100 2026472 10 203238
125 125 2573288 12.5 252746
150 150 3030131 15 300303
R2 y = 20158x + 33346 y = 20124x + 453.44
m 20158 20124
c 33346 453.44
r 0.9992 0.9998
d, e = mean of 3 readings Conc. = Concentration in ppm; R2 = Regression
Equation; m = Slope; c = Intercept; r = Correlation Coefficient

29. FIG. 4: LINEARITY PLOT FOR ATENOLOL
FIG. 5: LINEARITY PLOT FOR AMLODIPINE BESYLATE

30. Robustness:
Effect of variation in flow rate:
Effect of variation in temperature:
TABLE 7: ROBUSTNESS (FLOW RATE) STUDIES
Flow Rate
(ml/min)
AT AB
Std Areaf Tailingg Std Areah Tailingi
0.8 2526823 1.50 255095.2 1.40
%RSD 1.21 0.47 1.29 0.32
1.0 2000287 1.45 197704.4 1.35
%RSD 0.60 1.15 1.35 0.84
1.2 1650522 1.40 161985.6 1.19
%RSD 0.52 0.39 0.92 0.59
f, g, h, i = mean of 5 readings

31. TABLE 8: ROBUSTNESS (TEMPERATURE) STUDIES
Flow
Rate
(ml/min)
AT AB
Std Areaj Tailingk Std Areal Tailingm
20 2072960 1.89 194664 1.53
%RSD 0.20 0.24 0.93 0.36
Ambient 2005386 1.47 194000 1.36
%RSD 0.20 0.57 1.25 0.33
30 2204814 1.44 216892 1.10
%RSD 0.14 0.69 0.70 0.76
j, k, l, m = mean of 5 readings; Temp. = Temperature

32. Limit of detection and limit of quantitation
Limit of detection and limit of quantitation represent the concentration of
analyte that would yield signal to noise ratio of 3 for LOD and 10 for LOQ
respectively. To determine LOQ and LOD serial dilutions of mixed standard
solution of Amlodipine and Atenolol was made from standard solution. The
samples were injected in LC system and measured signal from the samples
was compared with those of blank samples.

33. 7. RESULTS AND DISCUSSION
Method Development
System suitability
Table 10. Result of system suitability tests of atenolol and amlodipine
Name of drug-atenolol
Injection No.
Retention
time
Area
Theoretical
plates
Tailing factor
1 1.677 3686546.00 3078.025 1.406
2 1.675 3687323.00 3077.843 1.405
3 1.676 3685214.00 3078.468 1.406
4 1.678 3685944.00 3079.15 1.406
5 1.674 3686674.00 3077.843 1.405
6 1.676 3687404.00 3078.468 1.406
Average 1.676 3686517.50 3078.300 1.406
SD 0.001 835.228 0.504 0.001
%RSD 0.084 0.023 0.016 0.037

34. Name of drug-amlodipine
Injection No.
Retention
time
Area
Theoretical
plates
Tailing factor
1 5.035 1353496.00 2982.438 1.403
2 5.016 1354432.00 2978.862 1.405
3 5.005 1354197.00 2976.722 1.407
4 4.997 1353220.00 2983.002 1.406
5 4.989 1352243.00 2989.282 1.408
6 4.981 1351266.00 2995.562 1.4091
Average 5.004 1353142.33 2984.311 1.406
SD 0.020 1203.005 6.983 0.002
%RSD 0.390 0.089 0.234 0.155
Selectivity.
Selectivity is the ability to assess the analyte in the presence of components that may
be expected to be present. Typically these might include impurities, degraded products,
matrix, etc. Standard solutions (100%) containing both the drugs was injected first.
Then drugs solution containing three placebo formulations were injected one after
another.

35. Linearity.
Fig 6: It shows linearity plot of Amlodipine
Fig 7: It shows linearity plot of Atenolol

36. Accuracy.
Figure 8. Accuracy curve of atenolol
Figure 9. Accuracy curve of amlodipine

37. Precision.
The precision of an analytical method is the degree of agreement among individual test
results when the method is applied repeatedly to multiple samplings of a homogeneous
mixture. The precision of an analytical method is usually expressed as the standard
deviation or relative standard deviation (coefficient of variation) of a series of
measurements. Precision was measured by repeatability and intermediate precision.
Repeatability.
Repeatability expresses the precision under the same operating conditions over a short
interval of time. It is also termed intra-assay precision. Repeatability is usually
demonstrated by repeated measurements of a single sample. Minimum four
determinations at each of three concentrations across the intended range, or a
minimum of six determinations at the test concentration are recommended

38. Intermediate precision.
Table 11. Inter-day variability (three different concentrations of standard
solution of atenolol, injected on different days).
Days Std conc.
Mean peak area of
atenolol (n=4)
% RSD
1st
90% 3311515.25 0.0797
100% 3686255.75 0.0243
110% 4031586.50 0.1105
2nd
90% 3312999.75 0.0529
100% 3686025.25 0.1518
110% 4033851.00 0.0854
3rd
90% 3332855.75 0.0767
100% 3706295.75 0.0556
110% 4069579.75 0.069

39. Table 12. Inter-day variability (three different concentrations of standard solution
of amlodipine injected on different days)
Days Std conc.
Mean peak area of
atenolol (n=4)
% RSD
1st
90% 1299744.00 0.1667
100% 1353835.75 0.0422
110% 1613112.00 0.1691
2nd
90% 1300537.00 0.076
100% 1377701.25 1.5183
110% 1616511.00 0.2024
3rd
90% 1315026.75 0.1273
100% 1368271.75 0.0341
110% 1633389.75 0.1308

40. Robustness
As part of the robustness, deliberate changes in the flow rate and detection wavelength
were made to evaluate the impact on the method and retention times were significantly
changed.
Table 13 : Robustness test of Amlodipine and Atenolol
Parameters Changes
%Recovery of
amlodipine
% recovery of
atenolol
%target
Flow
rate(ml/min)
0.9 99.8 99.3 100%
1.1 99.7 99.4 100%
Column
temperature(0c)
28 99.7 99.5 100%
30 99.6 99.5 100%
Limit of detection and Limit of quantitation
Table 14: It shows LOD and LOQ for Amlodipine & Atenolol
mcg Area
Amlodipine
LOD 0.40 10.45
LOQ 1.22 31.65
Atenolol
LOD 5.26 104.46
LOQ 15.94 316.54

41. Analysis of market products
Table 15. Determination of atenolol and amlodipine in commercial
formulations by high performance liquid chromatography
Brand Atenolol Amlodipine
Labeled
amount(m
g)
Observed
amount
(mg)
Purity
Labeled
amount
(mg)
Observed
amount
(mg)
Purity
T1 50.00 50.01 100.02 5.00 4.98 99.60
T2 50.00 49.51 99.02 5.00 5.01 100.20
T3 50.00 49.54 99.08 5.00 4.98 99.60
T4 50.00 49.57 99.14 5.00 4.97 99.40
T5 50.00 49.37 98.74 5.00 5.02 100.40
T6 50.00 49.44 98.88 5.00 4.87 97.40

42. 8. CONCLUSION
The proposed high-performance liquid chromatographic method has been evaluated
over the accuracy, precision and linearity and proved to be more convenient and
effective for the quality control and identity of atenolol and amlodipine in
pharmaceutical dosage forms. The measured signals were shown to be precise,
accurate and linear over the concentration range tested (80–120% of target
concentration) with a correlation coefficient better than 0.999. Moreover, the lower
solvent consumption along with the short analytical run time of 10 minutes leads to
an environmentally friendly chromatographic procedure that allows the analysis of a
large number of samples in a short period of time. Therefore, this HPLC method
can be used as a routine sample analysis. Additionally in this method, there was no
interference from matrix sources.

43. 9. REFERENCES
1. Brunton, L.L. and Parker, K.L. 1996. Editors. Goodman & Gilman’s Manual of
Pharmacology and Therapeutics. New York: McGraw Hill.
2. Tripathi K.D. 2003. Essential of Medical Pharmacology, 5th Ed., Jaypee Brothers
Medical Publishers (P) Ltd., New Delhi
3. Williams A. 2004. Foye’s Principles of Medicinal Chemistry, 5th Ed., published by B.I.
publications Pvt. Ltd.
4. Prisant, L.M. 2002. Fixed low-dose combination in firstline treatment of hypertension.
J. Hypertens. 20, S11-S19.
5. Li-Ping, X., Fu-Ming, S.H., Chao-Yu, M., Yúan-Ying, J. and Ding-Feng, S. (2005).
Synergism of atenolol and amlodipine on lowering and stabilizing blood pressure in
spontaneously hypertensive rat. Acta. Pharmacologica. Sinica, 26, 1303-1308.
6. Md. AhsanulHaque, AsmaNaznin, A.N.M HamidulKabir , Md. Khalid Hossain and
S.M. Ashraful Islam Department of Pharmacy, The University of Asia Pacific,
Dhanmondi, Dhaka-1209, Bangladesh

44. 7. Blessen Philip, Juddy Joseph and M. Sundarapandian RP-HPLC method Development
and validation for simultaneous estimation of Atenolol and Amlodipine Besylate in
Pharmaceutical dosage forms. International journal of Pharmaceutical sciences and
Research 2011; Vol. 2(8): 2156-2161.
8. Md. AhsanulHaque, AsmaNaznin, A.N.M HamidulKabir, Md. Khalid Hossain and
S.M. Ashraful Islam Development and validation of RP-HPLC method for
simultaneous estimation of Atenolol and Amlodipine in tablet dosage form. Dhaka
Univ. J. Pharm. Sci 2010; 9(2): 131-138.
9. Ranjan Kumar Barman, M. Anwar ul Islam, Maruf Ahmed, Mir Imam Ibne Wahid and
Robiul Islam Simultaneous high performance liquid chromatographic determination
of Atenolol and Amlodipine in pharmaceutical dosage form. J. Pak. J. Pharm. Sci
2007; Vol.20 (4), 274-279.
10. Prianshutangri, Mohit Arya and Lakshmayya Simultaneous estimation of Atenolol an
Amlodipine in combined dosage form. J. International journal of pharmaceutical
Research and Development 2011; Vol 4(03): may-2012(296-301).