Development and Validation of Simultaneous Estimation of Enalapril Maleate and Amlodipine Besylate in Combined Dosage Forms
Simple, sensitive and specific spectrophotometric methods were developed and validated for quantization of enalapril and amlodipine in tablet dosage form. Three new analytical methods were developed based on the simultaneous estimation of drugs in a binary mixture without prior separation. In simultaneous equation method, the drugs were determined by using the absorptivity values of enalapril and amlodipine at selected wavelengths, viz., 209 and 238 nm, respectively. Second method is based on the determination of graphical absorbance ratio at two selected wavelengths; one being the isoabsorptive point for the drugs (219 nm) and the other being the absorption maximum of amlodipine (238 nm), in this method both the drugs obeyed the Beer-Lambert`s law in the concentration range of 6-18 Î¼g mL-1. The third method is based on the derivative spectrophotometric method at zero crossing wavelengths. These methods are simple, accurate and rapid and they require no preliminary separation and can therefore be used for routine analysis of both drugs in quality control laboratories.
Enalapril maleate is used as an anti hypertensive drug, chemically it is 1-[N-[(S)-1-Carboxy-3-phenylpropyl]- L-alanyl]- L-proline 1¢-ethyl ester, maleate. Official methods for the quantitative estimation of enalapril maleate is, UV-spectrophotometric (Prasad et al., 1999; Walily et al., 1995; Carlucci et al., 1993) and capillary electrophoresis (Zhi et al., 1992) has been reported. Amlodipine besylate is calcium antagonist and chemically, it is 3-ethyl-5-methyl-(4 RS)-2-[(2-amino ethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate benzene sulphonate. The British Pharmacopoeia examines amlodipine besylate by liquid chromatography. Reversed phase HPLC (Zarghi et al., 2005; Naidu et al., 2005; Bahrami and Mirzaeei, 2004; Tatar and Atmaca, 2001; Shang, 1996), HPTLC (Meyyanathan and Suresh, 2005) and UV-spectrophotometric method (Rahman and Nasrul Hoda, 2003; Basavaiah et al., 2003; Sridhar et al., 1997) are few of the methods reported in literature for the simultaneous analysis of amlodipine besylate with various drugs from their respective formulations. Although enalapril and amlodipine are commonly used in dual drug therapy as a potent anti hypertensive drug, yet no method is so far reported for their simultaneous estimation. A successful attempt has been made to estimate these two drugs simultaneously by spectrophotometric analysis.
MATERIALS AND METHODS
Shimadzu 1700 Pharmaspec UV-visible spectrophotometer with a matched pair of
10 nm quartz cells was used. The chemicals used were of analytical grade. The
commercially available tablets of enalapril and amlodipine were procured from
local market. Enalapril maleate and amlodipine besylate received as gift sample
from Ranbaxy Labs. Dewas, were used as such without further purification.
Preparation of Standard Solutions
Solutions of enalapril and amlodipine were prepared by dissolving accurately
weighed 100 mg each of standard enalapril and standard amlodipine in 100 mL
methanol separately. Working standard solutions (A) and (B) were further prepared
by taking 1 mL of stock solution of enalapril and amlodipine in 10 mL volumetric
flasks and made up the volume with methanol.
Methods of Analysis Method I: (Based on Simultaneous Equation Method)
In Fig. 1 Enalapril shows absorption maxima at 209 nm
and amlodipine shows at 238 nm. The calibration curves for enalapril and amlodipine
were prepared in the concentration range of 8-26 μg mL-1 (Fig.
2) and 5-40 μg mL-1 (Fig. 3), respectively
at both the wavelengths i.e., 209 and 238 nm. The absorptivity coefficients
were determined for both the drugs at both the wavelengths and following equations
|| Overlain spectra of enalapril maleate and amlodipine besylate
|| Calibration curve of enalapril at 209 nm
|| Calibration curve of amlodipine at 238 nm
|| Calibration curve of amlodipine and enalapril at 219 nm
|| First order derivative spectra of enalapril maleate
A1 and A2 are absorbances at 209 nm and 238 nm, respectively
and Cena and Camlo are concentrations of enalapril maleate
and amlodipine besylate, respectively. The concentrations of both the drugs
in the mixture were determined by Eq. (1 and 2).
Method II: Graphical Absorbance Ratio Method
This method is based on the method used by Ghanem and his colleques which
makes use of the iso-absorptive point of the two drugs i.e. the wavelength of
equal absorptivity of the two components of the mixture.
|| First order derivative spectra of amlodipine besylate
|| Calibration curve of enalapril at 227 nm
The iso-absorptive point was 219 nm in this case. The other wavelength selected
is the absorption maximum of one of the components. In this case it was 238
nm, the absorption maximum of amlodipine. The concentrations of the two components
are related to the ratio of the absorbance at these two wavelengths. The absorbance
of the mixture was noted at 219 and 238 nm. Calibration curves of enalapril
and amlodipine were plotted in the concentration range 6-18 μg mL-1
(Fig. 4) (range for which Beer-Lamberts law followed).
The absorptivity coefficients were determined for both the drugs and the average
value was taken. These values and the absorbance ratio were used to develop
equations as given:
Where, Qm is A2/A1 and A1, A2 are the absorbances at 219 and 238 nm, respectively. Cx and Cy are concentrations of enalapril and amlodipine resprectively.
Method III: Derivative Spectrophotometric Method
Upon examining the first-derivative spectra of the two drugs, it can be
noticed that enalapril maleate can be determined at 227 nm (Fig.
5) where amlodipine has no contribution and amlodipine can be determined
at 327.5 nm (Fig. 6) where enalapril shows a zero crossing.
Calibration curves for enalapril and amlodipine were prepared in the concentration
range of 8-22 μg mL-1 (Fig. 7) and 4-32 μg
mL-1 (Fig. 8), respectively at wavelengths i.e.,
227 and 327.5 nm.
|| Calibration curve of amlodipine at 327.5 nm
|| Compilation of results of statistical analysis of commercial
|*: Average of nine determinations; SD = Standard Deviation,
%RSD = Relative Standard Deviation and SE = Standard Error, Statistical
calculations were carried out by SPSS (official soft ware)
|| Compilation of results of drug recovery study
|*: Readings are average of nine determinations, statistical
calculations were carried out by SPSS (official soft ware)
Where, x is the concentration in μg mL-1, y is the peak amplitude of the first-derivative curves at 227 and 327.5 nm for enalapril and amlodipine, respectively.
Estimation from Tablets
Twenty tablets were weighed and average weight determined. Powder equivalent
to 5 mg of enalapril and 5 mg amlodipine was extracted quantitatively with small
amount of methanol. Insoluble excipients were separated by filtration. The supernatant
liquid was transferred to 100 mL volumetric flask and the volume made up with
methanol. The solution so obtained was suitably diluted with methanol so that
the concentration (10 μg mL-1) can be read directly from the
calibration curve and the absorbances were taken at different wavelengths as
stated above. Using the Eq. (1-5) the concentrations
were determined (Table 1).
RESULTS AND DISCUSSION
In the first method, the content of enalapril and amlodipine was directly found
from the Eq. 1 and 2. Two wavelengths of
respective absorbance maxima i.e., 209 nm for enalapril and 238 nm for amlodipine
were used for the analysis of the drugs. In the second method, the absorbance
ratio and the absorptivity coefficients were determined and the values were
substituted in the Eq. 3 to give the results. In this method
the primary requirement for developing a method for analysis is that the entire
spectra should follow the beers law at all the wavelength, which was fulfilled
in case of both these drugs. The two wavelength used for analysis of both the
drugs were 219 nm (isoabsorptive point) and 238 nm (wavelength maxima of amlodipine).
In the third method the absorbance of the one drug was taken at the zero crossing
point of the other drug and the values were substituted in the Eq.
4 and 5. The validation parameters were studied at all
the wavelengths for all the methods. Accuracy was determined by calculating
the recovery and the mean was determined (Table 2). The value
of recovery is very close to 100% show the accuracy of the methods. Precision
was calculated as repeatability (standard deviation and relative standard deviation)
for both the drugs. The percent recovery obtained indicates non-interference
from the excipients like starch, magnesium stearate etc. if used in the formulations.
The value of confidence level was under the standard value show the significance
of data. By observing the validation parameters, all the methods were found
to be specific, accurate and precise. Hence these methods can be employed for
routine analysis of these two drugs in combinations.
The main advantage of the proposed methods is its suitability for routine determination of amlodipine and enalapril from their marketed formulations. The proposed methods are economic, simple, sensitive, precise and reproducible and do not require any expensive or sophisticated apparatus, in contrast with the reported chromatographic methods.
Thanks are extended to The Director, Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (C.G.) for providing necessary facilities for research work and AICTE New Delhi for financial assistance under the scheme RPS and MODROB. We are also grateful to Ranbaxy Labs. Dewas for providing gift samples of enalapril and amlodipine.
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