Simultaneous UV Spectrophotometric Method for Estimation of ...

Asian J. Research Chem. 2(1): Jan.-March, 2009
,
ISSN 0974-4169
www.ajrconline.org
RESEARCH ARTICLE
Simultaneous UV Spectrophotometric Method for Estimation of Losartan
Potssium and Amlodipine Besylate in Tablet Dosage Form.
Priyanka R Patil*, Sachin U Rakesh, PN Dhabale, and KB Burade
Govt. College of Pharmacy, Karad- 415 124, (Satara), Maharashtra, India
*Corresponding Author E-mail: priyapatil5586@rediffmail.com
ABSTRACT
Two simple, accurate, precise, reproducible, requiring no prior separation and economical procedures for simultaneous
estimation of Losartan Potassium and Amlodipine Besylate in tablet dosage form have been developed. First method
employs formation and solving of simultaneous equation using 208 nm and 237.5 nm as two analytical wavelengths
for both drugs in methanol. The second method is Q value analysis based on measurement of absorptivity at 242.5 nm
(as an iso-absorptive point) and 237.5 nm. Losartan Potassium and Amlodipine Besylate at their respective max 208
nm and 237.5 nm and at isoabsorptive point 242.5 nm shows linearity in a concentration range of 2-20 g/mL.
Recovery studies range from 99.95% for Losartan Potassium and 99.33% for Amlodipine Besylate in case of
simultaneous equation method and 102.93% for Losartan Potassium and 101.02% for Amlodipine Besylate in case of
Q - analysis method confirming the accuracy of the proposed method. The proposed method is recommended for
routine analysis since it is rapid, simple, accurate and also sensitive and specific by no heating and no organic solvent
extraction.
KEY WORDS: Losartn Potassium, Amlodipine Besylate, max , Simultaneous equation method, Q analysis
INTRODUCTION:
Losartan (I, 2-n-butyl-4-chloro-1-[p-(o-1H-tetrazol- 5-
ylphenyl) benzyl]-imidazole-5methanol monopotassium salt)
is a highly selective, orally active, non-peptide angiotensin II
receptor antagonist indicated for the treatment of
hypertension. It has a more potent active metabolite
EXP3174 (II, 2-n-butyl-4-chloro-1-[2-(1H-tetrazol-5 yl)
biphenyl- 4-yl) methyl] imidazole-5-carboxyl acid) 1 . The
determination of Losartan has been carried out in tablets by
HPLC, capillary electrophoresis and super-critical fluid
chromatography 2,3 , in urine by gas chromatography- mass
spectrometry 4 and, simultaneously with its active metabolite
in biological fluids, by HPLC 5- 10 .
Amlodipine, chemically, 2-[(2- aminoethoxy) methyl]- 4-
(2-chlorophenyl) -1, 4-dihydro- 6-methyl-3, 5-
pyridinedicarboxylic acid 3-ethyl, 5-methyl ester, is an antihypertensive
and an antianginal agent in the form of the
besylate salt, Amlodipine besylate. It is not official in any
Pharmacopoeia. Various analytical methods have been
reported for the assay of Amlodipine besylate 11 in pure
form as well as in pharmaceutical formulations.
Received on 20.02.2009 Modified on 13.04.2009
Accepted on 24.05.2009 © AJRC All right reserved
Asian J. Research Chem. 2(2): April.-June, 2009 page 183-187
They include high performance liquid chromatography, 12-17
reversed phase high performance liquid chromatography, 18-
21 high performance thin layer chromatography, 22-25 gas
chromatography, 26 gas chromatography–mass
spectrometry, 27
liquid chromatography with tandem mass spectrometry 28
and fluorimetry, 29 derivative spectroscopy, 30,31
simultaneous multicomponent mode of analysis and
difference spectrophotometry 32-34 .
By these two methods no UV spectrophotometric study
on Losartan and Amlodipine in tablet dosage form in
pharmaceutical preparations has been found in literature
survey. There was only one method has been reported 35
for estimation of Losartan and Amlodipin in tablet by
absorption correction method, which prompted to pursue
the present work. The objective of the present work is to
develop and validate new analytical methods for
simultaneous determination of Losartan Potassium and
Amlodipine Besylate in tablet dosage form. This
communication forms the first report of two simple,
sensitive and reproducible methods for the simultaneous
estimation of Losartan Potassium and Amlodipine
Besylate from combined dosage form.
MATERIALS AND METHODS:
Materials:
Spectral runs were made on a Shimadzu UV-Visible
spectrophotometer, model- 1700 (Japan) was employed
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with spectral bandwidth of 0.5 nm and wavelength
accuracy of ± 0.3 nm with automatic wavelength
corrections with a pair of 10 mm quartz cells. Glasswares
used in each procedure were soaked overnight in a
mixture of chromic acid and sulphuric acid rinsed
thoroughly with double distilled water and dried in hot air
oven. Amlodipine besylate reference standard was kindly
provided by Shreya Life Sciences Pvt. Ltd. Aurangabad
(M. S.) while Losartan Potassium was provided by Lupin
Research Park, Pune. The pharmaceutical preparations of
combination of Losartan and Amlodipine that is Alsartan
AM tablet (ARISTO, Mumbai). Methanol of analytical
reagent grade was purchased by Loba Chemie Pvt. Ltd.
(India). All the solutions were protected for light and were
analyzed on the day of preparations.
Selection of common solvent:
Methanol of analytical reagent grade was selected as
common solvent for developing spectral characteristics
of drug. The selection was made after assessing the
solubility of both the drugs in different solvents.
Preparation of Standard Drug Solution:
Standard stock solutions containing Losartan Potassium
(LP) and Amlodipine besylate (AB) were prepared
individually by dissolving 2.5 mg of LP and quantity of
AB equivalent to Amlodipine base 2.5 mg separately in
20 ml of methanol. It was then sonicated for 10 minutes
and the final volume of both the solutions were made up
to 50 ml with methanol to get stock solutions containing
50 g/ mL each of LP and AB in two different 50 ml
volumetric flasks.
Asian J. Research Chem. 2(1): Jan.-March, 2009
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A 1 a x 2 – A 2 a x 1
Cy = a y 1 ax 2 - ay 2 a x 1
…………Eq. (ii)
Where, A 1 and A 2 are absorbances of mixture at 208 nm
and 237.5 nm respectively, ax 1 and ax 2 are absorptivities
of LP at 1 and 2 respectively and ay 1 and ay 2 are
absorptivities of AB at 1 and 2 respectively. Cx and Cy
are concentrations of LP and AB respectively.
Method II (Absorbance ratio or Q-analysis method):
From the overlain spectrum of LP and AB, two
wavelengths were selected one at 242.5 nm which is the
isoabsorptive point for both the drugs and the other at
237.5 nm which is max of AB . The absorbances of the
sample solutions prepared in a similar manner as in the
previous method, were measured and the absorptivity
values for both drugs at the selected wavelengths were
also calculated. The method employs Q values and the
concentrations of drugs in sample solution were
determined by using the following formula,
For LP
Q 0 – Q 2 a 1
C 1 = ×
Q 1 –Q 2 A
For AB
Q 0 – Q 1 a 2
C 2 = ×
Q 2 –Q 1 A
Where,
Absorbance of sample at 237.5 nm
Q 0 =
Absorbance of sample at 242.5 nm
Determination of Absorption Maxima:
By appropriate dilution of two standard drug solutions
with methanol, solutions containing 10 g ml -1 of LP
and 10 g ml -1 of AB were scanned separately in the
range of 200- 400 nm to determine the wavelength of
maximum absorption for both the drugs. LP and AB
showed absorbance maxima at 208 nm ( 1 ) and 237.5
nm ( 2) respectively. The overlain spectra showed max
of both drugs and also isoabsorptive points at 242.5 nm
(Fig. 1).
Method I (Simultaneous equation method):
Two wavelengths selected for the method are 208 nm
and 237.5 nm that are absorption maximas of LP and AB
respectively in methanol. The stock solutions of both
the drugs were further diluted separately with methanol
to get a series of standard solutions of 2-20 g /mL
concentrations. The absorbances were measured at the
selected wavelengths and absorptivities (A 1%, 1 cm)
for both the drugs at both wavelengths were determined
as mean of three independent determinations.
Concentrations in the sample were obtained by using
following equations-
A 1 ay 2 – A 2 a y 1
Cx =
…………Eq. (i)
ax 1 ay 2 -ax 2 ay 1
Q 1 =
Q 2 =
Absorptivity of LP at 237.5 nm
Absorptivity of LP at 242.5nm
Absorptivity of AB at 237.5 nm
Absorptivity of AB at 242.5nm
A = Absorbance of sample at isoabsorptive point,
a 1 and a 2 = Absorptivities of LP and AB respectively at
isoabsorptive point.
Application of the proposed method for the
determination of LP and AB in tablets:
Twenty tablets of marketed formulation Alsartan AM
(ARISTO, Mumbai) containing LP 50 mg and AB
equivalent to Amlodipine base 5 mg were weighted, and
finely powdered. For analysis of drug, a standard addition
method was used. An accurately weighted 2.5 mg of pure
AB was added to finely powdered samples to bring the
concentration of AB in linearity range. With this addition, the
ratio of LP and AB in the samples was brought to 1:1.
Quantity of powder equivalent to 5 mg of LP and 5 mg of
Amlodipine base was weighed and dissolved in 40 mL of
methanol and sonicated for 10 minutes. Then the solution
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Asian J. Research Chem. 2(1): Jan.-March, 2009
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Table 1: Linear regression analysis of calibration curves with their respective absorptivity values.
Parameter
Method I
Method II
LP AB LP AB
Beer’s law limit (g ml -1 ) 2-20 2-20 2-20 2-20
Correlation coefficient (r) 0.9988 0.9991 0.9993 0.9970
Molar absorptivity (lit/mole/cm) 42942.37 19435.23 21396.44 18694.30
Sandell's sensitivity
0.0107 0.0210 0.021546 0.021872
(mcg/Sq.cm/0.001)
Slope 0.0955 0.0468 0.04602 0.04325
Intercept -0.0063 -0.0158 -0.017533 -0.02353
Table 2: Results of analysis of laboratory samples.
Analyte
Method I
Method II
LP AB LP AB
% Conc. Estimated (Mean ± S.D.) 99.79 ± 0.13 99.74 ± 0.15 99.85 ± 0.03 101.09 ± 0.82
Coefficient of variance 0.0008 0.0002 0.0001 0.0004
Average of three determinations; R.S.D.; Relative Standard Deviation.
Table 3: Results of analysis of tablet samples.
Method Drug Label Claim % Label Claim ± R. S. D. Coefficient of variance % Recovery* (Mean ±
R. S. D)
LP 5 103.39 ± 1.224 0.0155 99.95± 0.97
I AB 50 105.06± 3.25 0.111 99.33± 0.72
LP 5 103.93± 2.66 0.072 102.93± 0.21
II AB 50 105.05± 3.25 0.111 101.02± 0.37
Average of three determinations; R.S.D.; Relative Standard Deviation
Table 4: Results of intermediate precisions.
.
Method I
Method II
Day
% Label claim estimated
(Mean ± % R.S.D.)
% Label claim estimated
(Mean ± % R.S.D.)
LP AB LP AB
Intraday 105.83
± 0.73
100.04
± 0.43
100.87
± 0.56
112.5
± 2.2
Interday 106.86
± 1.27
108.05
± 1.71
99.25
± 1.76
100.6
± 0.36
was filtered through whatman filter paper no. 41 and then
final volume of the solution was made up to 50 ml with
methanol to get a stock solution containing 100 g ml -1 of LP
and 100 g ml -1 AB. Appropriate aliquots of LP and AB
within the Beer’s law limit were taken. In Method I, the
concentration of both LP and AB were determined by
measuring the absorbance of the sample at 208 nm and 237.5
nm. Values were substituted in the respective formula to
obtain concentrations.
For Method II, the concentration of both LP and AB
were determined by measuring absorbance of the sample
at 242.5 nm and 237.5 nm and values were substituted in
the respective formula to obtain concentrations. Results
of tablet analysis are shown in Table 3.
VALIDATION:
The method was validated according to ICH Q2B
guidelines for validation of analytical procedures in order to
determine the linearity, sensitivity, precision and accuracy
for the analyte.
Accuracy:
To ascertain the accuracy of the proposed methods,
recovery studies were carried out by standard addition
method at three different levels (80%, 100% and 120%).
Percent recovery for LP and AB, by both the methods,
was found in the range of 101.44% to 100.17%.
Linearity:
The linearity of measurement was evaluated by
analyzing different concentration of the standard
solution of LP and AB. For simultaneous equation
method and Q analysis, the Beer- Lambert’s
concentration range was found to be 2-20 g/ml for LP
and AB.
Precision:
Precision was studied to find out intra and inter-day
variations in the test method of LP and AB. Calibration
curves prepared in medium were run in triplicate in same day
and for three days. %RSD (relative standard deviation) were
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calculated which should be less than 2 %. The results are
tabulated in Table 4.
Asian J. Research Chem. 2(1): Jan.-March, 2009
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respectively. We are also thankful to Mr. Shreeniwas
Mohite sir for his moral support and guidance.
Fig.1: Overlain spectra of LP and AB. Overlain spectra of
Losartan potassium (RM) and Amlodipine besylate (AB) in
methanol.
RESULTS AND DISCUSSION:
The overlain spectra of LP and AB exhibit max of 208
nm and 237.5 nm for LP and AB respectively which are
quite separated from each other. Additionally one
isoabsorptive point was observed at 242.5 nm. This
wavelength was selected for simultaneous estimation of
LP and AB for Q value analysis and it is assume to be
sensitive wavelength. Standard calibration curves for LP
and AB were linear with correlation coefficients (r)
values in the range of 0.9970- 0.9993 at all the selected
wavelengths and the values were average of three
readings with standard deviation in the range of 0.13 –
0.82. The calibration curves were repeated three times in
a day and the average % RSD was found to be 0.645 for
LP and 1.31 for AB, similarly the method was repeated
for three different days and average % RSD was found
to be 1.51 for LP and 1.03 for AB. The accuracy of the
method was conformed by recovery studies from tablet
at three different levels of standard additions; recovery
in the range of 95 – 110% justifies the accuracy of
method.
CONCLUSION:
The most striking feature of this method is its simplicity
and rapidity, non- requiring- consuming sample
preparations such as extraction of solvents, heating,
degassing which are needed for HPLC procedure. These
are new and novel methods and can be employed for
routine analysis in quality control analysis. The described
methods give accurate and precise results for
determination of Losartan potassium and Amlodipine
besylate mixture in marketed formulation.
ACKNOWLEDGEMENTS:
The authors are thankful to the Principal Dr. S. B. Bhise,
Govt. College of Pharmacy, Karad, Dist. Satara,
Maharashtra for providing necessary facilities and
Shreya Life Sciences Pvt. Ltd. Aurangabad and Lupin
Research Park, Pune (M. S.) for providing the gift
sample of Amlodipine Besylate and Losartan Potassium
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