INTRODUCTION:

Amlodipine (AB), 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 besylate¹ in pure form as well as in pharmaceutical formulations. They include high performance liquid chromatography^2-7reversed phase high performance liquid chromatography^8-11 high performance thin layer chromatography^12-15 gas chromatography¹⁶gas chromatography–mass spectrometry¹⁷ liquid chromatography with tandem mass spectrometry¹⁸ and fluorimetry¹⁹ derivative spectroscopy,^20,21 simultaneous multicomponent mode of analysis and difference spectrophotometry^22-24.

Lisinopril (LN), (2S)-1-[(2S)-6-amino-2[[(1S)-1- carboxy-3-phenylpropyl]amino] hexanoyl]pyrrole- 2-carboxylic acid, is a synthetic dipeptide (lysyl–proline) used for the treatment of hypertension,for congestive heart failure and after myocardial infraction ^25-27.

It reduces both angiotensin and aldosterone plasma concentrations through the inhibition of angiotensin converting enzyme^25-27. Several methods have been presented in the literature for the determination of LN in pharmaceutical preparations.^28–40 These include HPLC,^{29,33,37,39,40} GC,^34,35 spectrophotometry^29,36,39, derivative spectrophoto-metry,^{28,32 33,37}spectrofluorimetry,^29,

³⁶ capillary electrophoresis^30,31 and HPTLC-densitometry.²⁸

By these two methods no UV spectrophotometric study on Amlodipine and Lisinopril in tablet dosage form in pharmaceutical preparations has been found in literature survey. There was only one method has been reported²² for estimation of Amlodipine and Lisinopril in tablet by derivative spectroscopy 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 Amlodipine Besylate and Lisinopril in tablet dosage form. This communication forms the first report of two simple, sensitive and eproducible methods for the simultaneous estimation of Amlodipine Besylate and Lisinopril 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. Glassware 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 and Lisinopril reference standard was kindly provided by Biochem Pharmaceutical Industries LTD, Daman. The pharmaceutical preparations of combination of Amlodipine besylate and Lisinopril that is Amlosafe – LS* 5/5 tablet (Aristo, Mumbai). Triple distilled water is used as solvent. All the solutions were protected for light and were analyzed on the day of preparations.

Selection of common solvent:

Triple distilled water 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 Amlodipine besylate (AB) and Lisinopril (LN) were repared individually by dissolving quantity of AB equivalent to Amlodipine base 10 mg and 10 mg of LN separately in 20 ml of triple distilled water. It was then sonicated for 10 minutes and the final volume of both the solutions were made up to 100 ml with triple distilled water to get stock solutions containing 100μg/ mL each of AB and LN in two different 100 m volumetric flasks.

Determination of Absorption Maxima:

By appropriate dilution of two standard drug solutions with triple distilled water, solutions containing 10 μg ml^-1 of AB and 10 μg ml^-1 of LN were scanned separately in the range of 200- 600 nm to determine the wavelength of maximum absorption for both the drugs. AB and LN showed absorbance maxima at 239 nm (λ₁) and 207 nm (λ₂) respectively. The overlain spectra showed λ max of both drugs and also isoabsorptive points at 212.5 nm (Fig. 1).

Method I (Simultaneous equation method):

Two wavelengths selected for the method are 239 nm and 207nm that are absorption maximas of AB and LN respectively in triple distilled water. The stock solutions of both the drugs were further diluted separately with triple distilled water to get a series of standard solutions of 5-30μg /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 six 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 = ----------------------- ………Eq. (ii)

a y1ax2 - ay2a x1

Where, A1 and A2 are absorbances of mixture at 239 nm and 207 nm respectively, ax1 and ax2 are absorptivities of AB at λ₁ and λ₂ respectively and ay1 and ay2 are absorptivities of LN at λ₁ and λ₂ respectively. Cx and Cy are concentrations of AB and LN respectively.

Fig.1. Overlain spectra of Amlodipine besylate (AB) and Lisinopril (LN) in triple distilled water.

Method II (Absorbance ratio or Q-analysis method):

From the overlain spectrum of AB and LN, two wavelengths were selected one at 212.5 nm which is the isoabsorptive point for both the drugs and the other at 239 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 AB

Q0 – Q2 a1

C1 = ----------------- × -------

Q1–Q2 A

For LN

Q0 – Q1 a2

C2 = ------------- × ---------

Q2–Q1 A

Where,

Absorbance of sample at 239 nm

Q0 = -------------------------------------------

Absorbance of sample at 212.5 nm

Absorptivity of AB at 239 nm

Q1 = -------------------------------------------

Absorptivity of AB at 212.5nm

Absorptivity of LN at 239 nm

Q2 = -------------------------------------------

Absorptivity of LN at 212.5 nm

A = Absorbance of sample at isoabsorptive point, a1 and a2 = Absorptivities of AB and LN respectively at isoabsorptive point.

Table 2: Results of analysis of tablet samples.

Method	Drug	Label Claim	*% Label Claim estimated ± R. S. D.**
I	AB	5	99.78 ± 0.7529
I	LN	5	100.18 ± 0 .8964
II	AB	5	100.02 ± 0.7245
II	LN	5	100.08 ± 0.2046

Application of the proposed method for the determination of AB and LN in tablets:

Twenty tablets of marketed formulation Amlosafe – LS* 5/5 tablet (ARISTO, Mumbai) containing AB equivalent to Amlodipine base 5 mg and LN 5 mg were weighted, and finely powdered. For analysis of drug, quantity of powder equivalent to 5 mg of Amlodipine base and 5 mg of LN was weighed and dissolved in 40mL of triple distilled water 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 triple distilled water to get a stock solution containing 100 μg ml-¹of AB and 100 μg ml^-1 LN. Appropriate aliquots of AB and LN within the Beer’s law limit were taken. In Method I, the concentration of both AB and LN were determined by measuring the absorbance of the sample at 207 nm and 239 nm. Values were substituted in the respective formula to obtain concentrations.

For Method II, the concentration of both AB and LN were determined by measuring absorbance of the sample at 212.5 nm and 239 nm and values were substituted in the respective formula to obtain concentrations. Results of tablet analysis are shown in Table 2.

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 at three different levels (80%, 100% and 120%). Percent recovery for AB and LN , by both the methods, was found in the range of 99.27 % to101.49 %.

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solution of AB and LN. For simultaneous equation method and Q analysis, the Beer- Lambert’s concentration range was found to be 5-10 μg/ml for AB and LN.

Table 3: Recovery study

Method	Drug	Label Claim	*% Recovery (Mean ± R. S. D)**
I	AB	5	101.49 ± 0.8804
I	LN	5	99.27 ± 0.1308
II	AB	5	100.76 ± 0.4401
II	LN	5	99.73 ± 0.3437

RESULTS AND DISCUSSION:

The overlain spectra of AB and LN exhibit λmax of 239 nm and 207 nm for AB and LN respectively which are quite separated from each other. Additionally one isoabsorptive point was observed at 212.5 nm. This wavelength was selected for simultaneous estimation of AB and LN for Q value analysis and it is assume to be sensitive wavelength. Standard calibration curves for AB and LN were linear with correlation coefficients (r) values in the range of 0.9995- 0.9996 at all the selected wavelengths and the values were average of six readings with standard deviation in the range of 0.2046 – 0 .8964. The calibration curves were repeated three times in a day and the average % RSD was found to be 0.645 for AB and 1.31 for LN. The accuracy of the method was conformed by recovery studies from tablet at three different levels of 80 % , 100 %, 120 % ; recovery in the range of 95 – 110% justifies the accuracy of method.

CONCLUSION:

The most striking feature of this method is its simplicity, economy 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 Amlodipine besylate and Lisinopril 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 Biochem Pharmaceutical Industries LTD, Daman for providing the gift sample of Amlodipine Besylate and Lisinopril.

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Received on 23.11.2009 Modified on 20.12.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 201-204

Parameter	Method I		Method II
Parameter	AB	LN	AB	LN
Beer’s law limit (_g ml-1)	5-30	5-30	5-30	5-30
Correlation coefficient (r)	0.9996	0.9995	0.9996	0.9995
Molar absorptivity (lit/mole/cm)	17458.66	15935.45	18358.75	16925.55
Sandell's sensitivity (mcg/Sq.cm/0.001)	0.03248	0.027707	0.03325	0.028045
Slope	0.0311	0.0366	0.0313	0.0359
Intercept	0.007533	0.0104	0.007825	0.0110