INTRODUCTION:

Molecular structure:

Amlodipine besylate is chemically, 3-Ethyl 5-methyl (4RS)-2-[(2- aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1,4- dihydropyridine-3,5 dicarboxylate benzenesulphonate. The molecular structure of amlodipine besylate is given in Fig. 01. Amlodipine besylate is a long-acting calcium channel blocker used as an anti-hypertensive and in the treatment of angina¹. Amlodipine besylate is slightly soluble in water; sparingly soluble in ethanol; soluble in O-phosphoric acid, dichloro methane, dimethyl sulphoxide and dimethyl formamide. The molecular structure of amlodipine besylate is shown in Fig. 01. Literature survey reveals that very few analytical methods have been reported for the estimation of amlodipine besylate includes HPLC^2-11, UPLC¹², HPTLC^13,14, TLC¹⁵, LC-MS/MS¹⁶ and some UV/Vis spectrophotometric methods^17-20. Estimations were carried out either using single drug or in combination with other drugs. Most of the methods have been reported for the quantification of amlodipine besylate in dosage forms and in biological fluids. The main objective of the work was to develop simple, fast, inexpensive, sensitive and accurate method which could be applied to analyse amlodipine besylate in pure form and in pharmaceutical dosage form.

Fig. 01: Structure of Amlodipine besylate

MATERIALS AND METHODS:

An analytical UV/Vis double beam spectrophotometer (model T 60) with 1 cm matched quartz cells was used for all spectral measurements. All the chemicals used in the investigation were of analytical grade. Authentic drug sample of amlodipine besylate was given as a gift sample by Hetero drugs Ltd., Hyderabad. Tablets of amlodipine besylate are procured from local market.

Method development:

Preparation of standard stock solution:

Standard stock solution was prepared by dissolving accurately weighed 100 mg of amlodipine besylate in dimethyl sulphoxide and the volume was made up to 100 ml with dimethyl sulphoxide. (Stock solution-I, 1000 mcg/ml). 10 ml of stock solution-I was diluted to 100 ml with dimethyl sulphoxide. (Stock solution-II, 100 mcg/ml). 1.5 ml of stock solution-II was transferred to a 10ml volumetric flask. To the flask 1ml of 5% w/v potassium hydroxide was added and the final volume was diluted to 10 ml with dimethyl sulphoxide, so that to produce the concentration 10 mcg/ml. The absorbance of yellow chromogen obtained was measured against respective blank solution in the visible region of 400-600 nm, which shows maximum absorbance at 456 nm. The absorption spectrum of amlodipine besylate is shown in Fig. 02. The optical characteristics such as Beer’s law limit, Molar extinction coefficient and Sandell’s sensitivity are expressed in Table 01.

Fig. 02- Absorption spectrum of Amlodipine besylate in Dimethyl sulphoxide and 5%w/v Potassium hydroxide

Table: 01 Optimum conditions, Optical characteristics and Statistical data of the regression equation in Visible spectrophotometry:

Parameter	Visible spectrophotometry
l_max (nm)	456
Beer’s law limits (mcg/ml)	3-30
Molar extinction coefficient (mol^-1cm^-1)	0.0068 X10⁴
Sandell’s sensitivity (mcg/cm²-0.001 absorbance units)	0.0102
Regression equation (Y*)	Y=0.0222C + 0.0034
Slope (b)	0.0222
Intercept (a)	0.0034
Correlation coefficient(r²)	0.9995
% RSD**	0.86
Limit of detection (mcg/ml)	0.104
Limit of quantitation (mcg/ml)	0.322

*Y= bC + a where C is the concentration of amlodipine besylate in mcg/ml and Y is the absorbance at the respective l_max.

**Average of five determinations.

Preparation of standard curve:

Aliquots of standard solution of amlodipine besylate ranging from 0.3-3.0 ml (1 ml = 100 mcg) were transferred into a series of 10 ml volumetric flasks. To each flask 1ml of 5% w/v Potassium Hydroxide was added and the volume in each flask was made up to 10 ml with dimethyl sulphoxide and the absorbances of yellow coloured chromogens were measured at 456 nm against solvent blank. The obtained absorbance values when plotted against the concentration of amlodipine besylate gives the calibration graph. The concentration of the unknown sample was determined from the calibration graph. The calibration curve of amlodipine besylate is shown in Fig. 03

Fig. 03- Calibration curve of Amlodipine besylate in Dimethyl sulphoxide and 5%w/v Potassium hydroxide

Sample preparation of Amlodipine besylate:

20 tablets of two different brands of amlodipine besylate were taken, and all the tablets were crushed to fine powder by using pestle and mortor. Powder equivalent to 25 mg of amlodipine besylate was weighed accurately and transferred into a 25 ml standard volumetric flask. The contents were dissolved in dimethyl sulphoxide and sonicated for five minutes. This solution was filtered through 0.45µ whatmann filter paper. 5 ml of the filtrate was diluted to 50 ml with dimethyl sulphoxide to get the solution of 100 mcg/ml. An aliquot of 1.5 ml of test solution was transferred to a 10ml volumetric flask. To the flask 1ml of 5% w/v potassium hydroxide was added and the final volume was diluted to 10 ml with dimethyl sulphoxide, so that to produce the concentration 10 mcg/ml. The absorbance of yellow chromogen obtained was measured against respective blank solution in the visible region of 400-600 nm, which shows maximum absorbance at 456 nm. The amount of amlodipine besylate present in the sample solution was computed from the respective calibration curve. The results are expressed in Table 02.

Table: 02: Evaluation of amlodipine besylate in pharmaceutical formulations by visible spectrophotometry:

Brand used	Label claimed (mg)	Amount found by proposed method(mg)	% label claim	% RSD*
Tab-a	10	9.962	99.62	0.334
Tab-b	10	9.940	99.40	0.545

*Average of five determinations.

Validation of spectrophotometric method:

Accuracy:

Accuracy is the closeness of the test results obtained by the method to the true value. The recovery technique was performed to judge the accuracy of the proposed method. For this, known quantities of the amlodipine besylate solution were mixed with definite amounts of pre-analyzed formulations and the mixtures were analyzed. The total amount of amlodipine besylate was determined by using the proposed method and the amount of added drug was calculated by the difference. The results are expressed in Table 03.

Table: 03: Results of recovery studies of amlodipine besylate estimation by visible spectrophotometry:

Brand used	Label claimed (mg)	Mean assay value	Known amount of amlodipine besylate added	Mean % recovery ±%RSD*
Tab-a	10	99.62	2mg	99.90±1.09
			4mg	99.93±1.88
Tab-b	10	99.40	2mg	99.48±0.89
			4mg	99.73±0.65

*Average of five determinations.

Precision:

The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples. It provides an indication of random error results and was expressed as coefficient of variation (CV).

Intra and inter-day precision:

A variation of results within the same day (intra-day), variation of results between days (inter-day) was analyzed. Intra-day precision was determined by analyzing amlodipine besylate for five times in the same day at 456 nm. Inter-day precision was determined by analyzing the drug daily once for five days at 456 nm. The results are expressed in Table 04 and 05.

Table: 04: Results of intraday precision studies of amlodipine besylate estimation by visible spectrophotometry:

Brand used	Label claimed (mg)	Amount found by proposed method(mg)	% label claim	% RSD*
Tab-a	10	9.958	99.58	0.334
Tab-b	10	9.934	99.34	0.745

*Average of five determinations.

Table: 05: Results of inter day precision studies of amlodipine besylate estimation by visible spectrophotometry:

Brand used	Label claimed (mg)	Amount found by proposed method(mg)	% label claim	% RSD*
Tab-a	10	9.951	99.51	0.306
Tab-b	10	9.931	99.31	0.72

*Average of five determinations.

Linearity:

The linearity of the method was demonstrated over the concentration range of 3-30 mcg/ml of the target concentration. Accurately weighed 100 mg of pure drug was taken in clean, dry 100 ml volumetric flask and dissolved in small volume of dimethyl sulphoxide and made up the volume to 100 ml with dimethyl sulphoxide. This gave 1000 mcg/ml of drug concentration (Stock solution-I). From this 10 ml of solution was pipetted out into 100 ml volumetric flask and volume was made upto the mark with dimethyl sulphoxide (Stock solution-II, 100 mcg/ml).

Concentrations of 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 mcg/ml were prepared from above prepared Stock solution-II, calibration curve was plotted and the correlation coefficient was calculated.

Ruggedness and Robustness:

The solutions were prepared and analyzed with change in the analytical conditions like different laboratory conditions and different analysts. The results are expressed in Table 06 and 07.

Table: 06: Results of ruggedness studies of amlodipine besylate estimation by visible spectrophotometry:

Brand used

Label claimed (mg)

Normal condition

(Mean assay value)

Changed condition

(Mean assay value) ±%RSD*

Tab-a

99.62

99.34±0.76

Tab-b

99.40

99.17±0.36

*Average of five determinations.

Table: 07: Results of robustness studies of amlodipine besylate estimation by visible spectrophotometry:

Brand used

Label claimed (mg)

Normal condition

(Mean assay value)

Changed condition

(Mean assay value) ±%RSD*

Tab-a

99.62

99.24±0.736

Tab-b

99.40

99.10±0.96

*Average of five determinations.

RESULTS AND DISCUSSION:

In the present investigation the λmax of the amlodipine besylate was found to be 456.0nm. Amlodipine besylate follows linearity in the concentration range of 3-30mcg/ml. Two brands of tablets were analyzed and amount of drug were determined by proposed method; it was in good agreement with the label claim. The proposed method was validated as per the ICH guidelines. The recovery studies were carried out by adding a known amount of drug to pre analysed sample at two different levels and the % recoveries were ranges from 99.48-99.93%, which shows the accuracy of method. Intra-day and Inter-day precision of the assay was determined by analyzing the drug sample using same concentration. The intra-day and inter-day % RSD values were calculated and lying in the range of 0.306-0.745%. Ruggedness of proposed method was studied with the help of two different analysts and results were evaluated by calculating the % RSD values; lying within the range of 0.36-0.76%. For robustness the % RSD values were found to be in the range of 0.736-0.96%.

CONCLUSION:

The proposed method is economic, sensitive, accurate, reproducible and useful for the routine determination of amlodipine besylate in tablet formulation.

ACKNOWLEDGMENT:

The authors are grateful to the management of Vagdevi College of Pharmacy for providing the necessary facilities and support to carry out this work.

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Received on 10.06.2011 Modified on 17.06.2011

Research J. Pharm. and Tech. 4(9): Sept. 2011; Page 1432-1435