UV-absorbance difference method for simultaneous estimation of

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Der Pharmacia Lettre, 2015, 7 (1):280-284
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ISSN 0975-5071
USA CODEN: DPLEB4
UV-absorbance difference method for simultaneous estimation of atenolol and
amlodipine besylate in combined dosage forms
P. Suresh1, B. Hari Babu2* and I. E. Chakravarthy3
1
Dept. of Chemistry, Govt. Degree College, Proddatur, YSR Kadapa, AP, India
2
Dept .of Chemistry, Acharya Nagarjuna University, Guntur, AP, India
3
Dept .of Chemistry, Rayalaseema University, Kurnool, AP, India
_____________________________________________________________________________________________
ABSTRACT
A new UV-spectrophotometric method for the simultaneous and separate estimation of Atenolol and amlodipine
besylate in binary tablet formulations has been described. The method is based on the estimation of one drug in
presence of another drug by absorbance method. In this method, two wavelengths 209 and 244.5 nm were selected
for Atenolol and at these wavelengths the absorbance difference was found to be almost zero. Similarly, the two
wavelengths 265 and 278 nm were selected for Amlodipine besylate and the absorbance difference was almost zero.
In the mixture of Atenolol and amlodipine besylate solution the absorbance values of four wave lengths 209, 244.5,
265 and 278 nm were measured. The amount of Atenolol is directly proportional to the absorbance difference
between 265 and 278 nm. Similarly, the amount of Amlodipine besylate is directly proportional to the absorbance
difference between 209 and 244.5 nm.
Keywords: Spectrophotometry, Atenolol, Amlodipine besylate, absorbance difference method.
_____________________________________________________________________________________________
INTRODUCTION
Atenolol [1] is an antihypertensive, anti IC anginal, and antiarrhythmdrug; chemically, it is 4-(2-hydroxy-3isopropyl amino propoxy)-phenyl acetamide. Amlodipine besylate [2] is calcium antagonist and chemically, it is 3ethyl-5-methyl-(4 RS)-2-[(2-amino ethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1, 4-dihydropyridine-3,5dicarboxylate benzene sulphonate. The Indian Pharmacopoeia described a non-aqueous titration method for the
assay of atenolol. The British Pharmacopoeia examines amlodipine besylate by liquid chromatography. HPLC [3],
reversed phase HPLC [4], colorimetric method [5, 6, 7], HPTLC [8, 9], gas liquid chromatography [10], difference
spectrophotometric estimation [11] are few of the methods reported in literature for the analysis of atenolol and
amlodipine besylate from their respective formulations. HPTLC [12], reversed phase HPLC [13], HPTLC [14] and
vierodt’s [15,16,17] methods are reported for simultaneous estimation of atenolol and amlodipine besylate in
combined dosage form, but no method is reported for simultaneous estimation of these drugs in combination by
absorbance difference method by spectrophotometry. The aim of the present work is to develop a simple, rapid,
precise reproducible and economically viable method for the simultaneous analysis of the selected binary drug
formulation by using absorbance difference method without any interference from each other.
MATERIALS AND METHODS
Spectronics-2080 double beam UV-VIS spectrophotometer with spectral band width of 0.1nm with a pair of 10 mm
quartz cells used for all spectral and absorbance measurements. All chemicals used were of analytical grade.
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Preparation of standard Atenolol solution. Pure Atenolol (50mg) was dissolved in 50 mL ethanol. Further the stock
solution is diluted with ethanol to get a working concentration of 20 µg/mL.
Preparation of standard Amlodipine besylate solution. Pure Amlodipine besylate (50mg) was dissolved in 50 mL
ethanol. Further the stock solution was diluted with ethanol to get a working concentration of 0.35 µg/mL.
Preparation of mixed solution. Two solutions, the first containing 50 µg/mL of Atenolol and the second containing
0.35 µg/mL of Amlodipine besylate were used as mixed solution. Four mixed standard solutions were made by
taking 4, 3, 2 and 1mL of Atenolol solution in series of test tubes and the Amlodipine besylate solution was added to
a series of test tubes to keep the total volume at 5mL.
Preparation of Atenolol curve. Various aliquots of (5, 6, 7 and 8mL) of Atenolol solution were transferred into a
series of 10mL standard flasks and the volume in each flask was adjusted to 10mL with ethanol. The absorbance of
these solutions was scanned over the range 200-320 nm. The absorbance spectrum of Atenolol was shown in Fig. 1.
Sample-1
1.2
1.1
Absorbance(Abs)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
200
205
210
215
220
225
230
235
240
245
250
255
260
265
270
275
280
285
290
295
300
305
310
315
320
Wavelength(nm)
Fig. 1: Absorbance spectrum of Atenolol
Preparation of Amlodipine bseylate curve. Various aliquots of (5, 6, 7 and 8mL) of Amlodipine besylate solution
were transferred into a series of 10mL standard flasks and the volume in each flask was adjusted to 10mL with
ethanol. The absorbance of these solutions was scanned over the range 200-300 nm. The absorbance spectrum of
Amlodipine besylate was shown in Fig.2
Sample-1
0.9
0.8
Absorbance(Abs)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
200
205
210
215
220
225
230
235
240
245
250
255
260
265
270
275
280
285
290
295
300
305
310
315
320
Wavelength(nm)
Fig.2: Absorbance spectrum of Amlodipine besylate
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Two wavelengths 209 and 244.5 nm was selected for Atenolol; at these two wavelengths the absorbance difference
was almost zero and in case of Amlodipine besylate there is maximum absorbance difference. A calibration curve
was drawn between the absorbance difference values of Amlodipine besylate and the amount of Amlodipine
besylate in µg/mL. The amount of Amlodipine besylate present in the sample was estimated from the calibration
curve. Similarly, the two wavelengths 265 and 278 nm were selected for Amlodipine besylate; at these two
wavelengths the absorbance difference was almost zero and in case of Atenolol there is a maximum absorbance
difference. A calibration curve was drawn between the absorbance difference values of Atenolol and the amount of
Atenolol in µg/mL. The amount of Atenolol present in the sample was estimated from the calibration curve.
Various aliquots of mixtures of Atenolol and Amlodipine besylate solutions in different proportions were transfered
into a series of test tubes and the volume in each case was made 5mL (Table 1). The absorbance values were
measured at two wavelengths 209 nm and 244.5 nm for Atenolol and two wavelengths 265 nm and 278 nm for
estimation Amlodipine besylate. A calibration curve was drawn between the absorbance difference values of
Atenolol and the amount of Atenolol in µg/mL. A calibration curve was drawn between the absorbance difference
values of Amlodipine besylate and the amount of Amlodipine besylate in µg/mL. A linear curve in each case was
obtained. The linearity of the curves obtained indicated that it obeys Beer’s law and the suitability of this method for
the simultaneous determination of two drugs in a mixture
Sample-1
Sample-1
1.1
1.0
0.8
Absorbance(Abs)
Absorbance(Abs)
0.9
0.7
0.6
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.1
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
200
205
210
215
220
225
230
235
240
245
250
255
260
265
270
275
280
285
290
295
300
305
310
315
320
0.0
200
205
210
215
220
225
230
235
240
245
250
255
260
265
270
275
280
285
290
295
300
305
310
315
320
0.5
0.4
0.3
0.2
Wavelength(nm)
Wavelength(nm)
Fig 3: Over lain spectrum of Atenolol and amlodipine besylate
Table 1.
Standard
No.
1
2
3
4
Concentration of the two components in the four mixed standards
Volume of AT* Amount of AT* Volume of AM+
(mL)
(µg/mL)
(mL)
4
80
1
3
60
2
2
40
3
1
20
4
*=Atenolol, += Amlodipine besylate
Amount of AM+
(µg/mL)
3.5
7.0
10.5
14
Table 2. Estimation of atenolol and amlodipine besylate in pharmaceutical preparations
Sample
Tableta
Amount found by proposed method(mg)**
AT1
AM2
49.96
4.98
% Recovery by proposed method*
AT1
AM2
99.82
99.92
Tabletb
50
5
49.90
5.02
99.96
99.85
c
50
5
50.02
4.94
99.90
99.88
Tablet
a
Label Claim(mg/tab)
AT 1
AM2
50
5
49.9
4.96
99.70
99.90
Tabletd
50
5
amlokind AT marketed by mankind pharma, bstamloBeta marketed by Dr.Reddy’s Labs Ltd, cAngicam-Beta marketed by Blue Cross Labs LTD,
d
amlovas-AT marketed by macleods pharmaceuticalsLTD ; 1Atenolol, 2Amlodipine ; *Average of five determinations; **determination of
Atenolol and Amlodipine besylate in combined dosage form pharmaceutical preparations by proposed method.
Estimation in the marketed formulation. 20 Tablets were weighed and crushed into fine powder. An average weight
of the tablets containing the two drugs atenolol and amlodipine besylate in the ratio 1:3 were taken and an amount
of 50mg dissolved in 50mL ethanol by vigorously shaking and the volume was made up to the mark. The solution
was then filtered through the Whatmann filter paper N0.41 and the solution was diluted to get a final concentration
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of 20 µg/mL of Atenolol and 0.35 µg/mL of Amlodipine besylate. The sample solutions were measured at 209 and
244.5 nm for Atenolol and 265 and 278 nm for amlodipine besylate. The results are presented in table 2.
Validation of the method. The method was validated in terms of linearity, accuracy, precision, specificity and
reproducibility of the sample applications. The linearity of the method was investigated by serially diluting the stock
solutions of Atenolol (20 µg/mL) and Amlodipine besylate (0.35 µg/mL) and measured the absorbance values at 209
and 244.5 nm for Atenolol and 265 and 278 nm for amlodipine besylate. Calibration curves were constructed by
plotting the absorbance difference values against the amount of drug in µg/mL.
ATENOLOL
AMLODIPINEBESYLATE
120
260
90
Absorbance Difference
Absorbance Difference
220
180
60
140
100
30
60
0
20
0 10 20 30 40 50 60 70 80 90 100
0
Concentration (µg/ml)
3
6
9
Concentration (µg/ml)
12
15
.
Fig. 4. Calibration curves of atenolol and amlodipine besylate
Statistical analysis. A statistical analysis was performed on the statistically significant variables using the statistical
software. The parameters determined were coefficient of variation, standard deviation and student t-test.
Recovery experiment. To ensure the accuracy and reproducibility of the results obtained, recovery experiments were
performed by adding a known amount of standard drug to previously analysed pharmaceutical preparation. The
results are recorded in table 2.
RESULTS AND DISCUSSION
The present study was carried out to develop a simple, rapid, sensitive, precise, reproducible and accurate
spectrophotometric method for the estimation of Atenolol and Amlodipne besylate in combined dosage forms. The
proposed absorbance difference method was simple, less time consuming, low cost, found to be one of the best
analytical techniques employed for routine analytical purposes like assay and pharmaceutical formulations. At the
initial stage in the development of the method the Atenolol solution was first scanned over the range 200-320 nm
and two wavelengths 209 and 244.5 nm were selected for Atenolol having the absorbance almost zero at these
wavelengths. In case of Amlodipine besylate there should be a considerable absorbance difference; Amlodipine
besylate solution was scanned over the range 200-300 nm and similarly, two wavelengths 265 and 278 nm were
selected for estimation and the absorbance difference was almost zero at these wavelengths. The results obtained by
the proposed method are in good agreement with the label claim of the tablets. The additive and excipients usually
present in tablets do not found interfered. As a check on accuracy of the method, recovery experiment was
performed and percent recovery values also tabulated in table 2.
Table 3. Statistical analysis of estimation of atenolol and amlodipine besylate
Tablet 1
S.D*
ATb
AMc
0.3049 0.4560
C.V**
ATb
AMc
0.1524
0.1001
ATb
0.2934
AMc
1.1770
Tablet 2
0.4764
0.2701
0.2382
0.0450
0.3755
0.4971
Tablet 3
0.2449
0.3193
0.1224
0.0532
0.9132
0.1401
Sample
tcala
0.0894
0.0345 0.2503
0.4314
Tablet 4 0.1788 0.2073
*Standard deviation, ** Coeffcient of variation; aCalculated `t’ value by proposed method;
Theoritical values at 95% confidence limit,`t’ 2.78; bAtenolol, cAmlodipine
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The statistical analysis was studied by the proposed method (table 3). The values of standard deviation and
coefficient of variation were satisfactorily low, indicating the accuracy and the reproducibility of the method.
Student t-test showed that the calculated `t’ values are less than the theoretical value 2.78 with 4 degrees of freedom
at 5% level of significance, indicating that there is no significant difference between the proposed and official
values.
CONCLUSION
In Conclusion, the results indicate that the proposed absorbance difference method was found to be simple, rapid,
precise and accurate, and it can be used for the routine analysis of simultaneous estimation of Atenolol and
Amlodipine besylate.
Acknowledgements
The authors are highly thankful to Acharya Nagarjuna University for constant encouragement.
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