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 chromatography2,3, in urine
by gas chromatography- mass spectrometry4 and, simultaneously with
its active metabolite in biological fluids, by HPLC5- 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 anti-hypertensive
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 besylate11
in pure form as well as in pharmaceutical formulations.
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 spectrometry28 and fluorimetry,29
derivative spectroscopy,30,31 simultaneous multicomponent mode of
analysis and difference spectrophotometry32-34.
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 reported35 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 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 μg/ mL
each of LP and AB in two different 50 ml volumetric flasks.
Determination
of Absorption Maxima:
By appropriate dilution of two standard drug solutions
with methanol, solutions containing 10 μg ml-1 of LP and 10
μg 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 (λ1) and 237.5 nm (λ
2) respectively. The overlain spectra showed λmax 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 μg /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-
A1 ay2 – A2 a
y1
Cx = …………Eq.
(i)
ax1ay2-ax2ay1
A1 a x2 – A2
a x1
Cy = a
y1ax2 - ay2a x1 …………Eq.
(ii)
Where, A1 and A2 are absorbances
of mixture at 208 nm and 237.5 nm respectively, ax1 and ax2
are absorptivities of LP at λ1 and λ2
respectively and ay1 and ay2 are absorptivities of AB at
λ1 and λ2 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 λmax 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
Q0 – Q2
a1
C1 = ×
Q1–Q2
A
For
AB
Q0 – Q1
a2
C2 = ×
Q2–Q1
A
Where,
Absorbance of sample at 237.5 nm
Q0
=
Absorbance of sample at 242.5 nm
Absorptivity of LP at 237.5 nm
Q1
=
Absorptivity of LP at 242.5nm
Absorptivity of AB at 237.5 nm
Q2
=
Absorptivity of AB at 242.5nm
A = Absorbance of sample at isoabsorptive point,
a1 and a2 = 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
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 μg ml-1of
LP and 100 μg 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 μg/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 calculated which should be less
than 2 %. The results are tabulated in Table 4.
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 λmax 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
respectively. We are also thankful to Mr. Shreeniwas Mohite sir for his moral
support and guidance.
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