Development and Validation of UV Spectrophotometric Method for Estimation of Amlodipine Besylate in Tablet Dosage Form

 

Garima Singh, Amrita Mishra, Anurag Verma, Arun K. Mishra*

Central Facility of Instrumentation, College of Pharmacy, IFTM Lodipur-Rajput, Moradabad-244001 India

ABSTRACT:

The present work was undertaken with an aim to develop a simple validated and cost effective UV spectroscopic method for estimation of Amlodipine besylate (AB) from different pharmaceutical formulations. The analysis was performed on wavelength maxima (λmax) 364nm and 0.1N HCl was used solvent for blank and diluents. The present method was validated as per guidelines of the International Conference on Harmonization (ICH) including parameters as linearity, accuracy, precision, reproducibility and specificity. Using this method, quantification of AB was done from different marketed formulations. Beers Lambert law was obeyed in concentration range of 1-5µg/ml and the line equation was y = 0.025X+0.0044. The mean accuracy was found to be 98.92±0.52% and 99.12±0.22%. The precision in terms of interday and intraday was evaluated and relative standard deviation (RSD) was less than 2%. The proposed method was suitable and specific to the analysis of AB even in the presence of common formulation excipients. The method was applied on two different marketed brands and AB content were 99.5±0.26 and 99.03±.61 respectively of labeled claim. The proposed method was validated as per ICH guidelines and statistically good results were obtained. The results suggest that this method can be employed for routine analysis of AB in bulk and commercial formulations

 

 

INTRODUCTION:

Chemically Amlodipine besylate is 2-[(2-Aminoethoxy) methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxy carbonyl-6-methyl-1,4-dihydropyridinebenzenesulfonate [Figure 1] ^[1]. It is used in the management of hypertension, and coronary artery disease. These medications block the transport of calcium into the smooth muscle cells lining the coronary arteries and other arteries of the body. Since calcium is important in muscle contraction, blocking calcium transport relaxes artery muscles and dilates coronary arteries and other arteries of the body. By relaxing coronary arteries, amlodipine is useful in preventing chest pain (angina) resulting from coronary artery spasm ^[2]. Like other calcium channel blockers, amlodipine acts by relaxing the smooth muscle in the arterial wall, decreasing peripheral resistance and hence reducing blood pressure ^[3].

 

Cost effective, rapid and reliable methods are essential for routine quality control aspects in pharmaceutical industry. Therefore, keeping in view this objective, the present work was taken to develop and validate simple UV Spectrophotometric method for estimation of Amlodipine besylate in dosage formulations.

 

Figure 1: Structure of Amlodipine besylate

 

Earlier methods including spectroscopy ^[4], HPLC ^[5], HPTLC^[6], Chiral LC-MS^[7], LC-MS/MS^[8] for quantification of AB in pharmaceutical dosage forms as well as plasma are reported. Some of the methods are applicable for estimation of AB in combination form. These reported methods involve tedious sample preparation process, costly solvents as well as time consuming steps.

 

Upon literature survey and as per our present knowledge, there is no simple, reliable, rapid and cost effective method for estimation of AB from pharmaceutical dosage forms as well as in bulk formulation. In this study, a simple UV spectrophotometric method was developed and validated as per International Conference on Harmonization (ICH) guidelines ^[9].  The method was also employed to determine the content of AB in two marketed Amlodipine besylate (AB) formulations from Indian market.

 

MATERIALS AND METHODS:

Instruments and materials:

Shimadzu 1800 double beam UV/Vis spectrophotometer, digital balance (Citizen Co. India), micropipette (Modern scientific Co, India) were used in this study. Amlodipine besylate was obtained as a gift sample from Triveni Chemicals, Vapi India. The other chemicals and reagents were of analytical grade.

 

Standard stock solution:

Standard stock solution of AB was prepared by dissolving 25mg of Amlodipine besylate in 10ml 0.1N HCl in a 25ml volumetric flask, shaked well and finally volume was adjusted to get a solution of concentration of 1mg/ml. The 1mg/ml solution was used as a stock solution.

 

Calibration curve:

10ml of 1mg/ml aliquot solution was further diluted up to 50ml by 0.1N HCl in 100ml volumetric flask and final volume was adjusted up to 100ml. This prepared dilution was subjected for wavelength scanning in double beam UV spectrophotometer, in the wavelength region 190 to 700 nm to determine the wavelength of maximum (λmax) of absorption. The λmax was found to be 364nm against the blank. From 1mg/ml stock solution, the serial dilution pattern was followed to obtain aliquots of 1-5µg/ml concentration. The calibration curve was plotted between concentration and absorbance. The optical characteristics of different aliquots are presented in Table 1.

 

Table 1: Calibration curve data of Amlodipine besylate in 0.1N HCl
Concentration (µg/ml)	Mean Absorbance
1	0.032
2	0.052
3	0.085
4	0.102
5	0.135

 

Sample solution preparation:

The proposed method was applied on two different commercial brands of Amlodipine besylate. Twenty tablets of each brand were weighed and powdered. 10mg of Amlodipine besylate powder equivalent was weighed and transferred in a 10ml volumetric flask by diluting it with 10ml 0.1N HCl. After a continuous shaking of 10-15min, the dilution was filtered through Whatman filter paper separately. The filtrate was further suitably diluted in order to get 5µg/ml concentration. Against a blank solution, the absorbance was measured at 364nm wavelength.

Finally employing the calibration curve and linear equation, the concentration was calculated. Determination of accuracy was done by % addition method and presented in table 2.  Amount of drug estimated through this method is presented in table 3.

 

Fig. 2. Calibration curve of Amlodipine besylate:

 

RESULT AND DISCUSSION:

Linearity:

The linearity of the drug was obtained between 1- 5µg/ml concentration ranges. The calibration curve was obtained by plotting absorbance verses concentration and linear regression analysis was performed to get line equation. The line equation found was y = 0.025X+0.0044 and r² was 0.990. The calibration curve was found to be linear in stated concentration [Figure 2]

 

Accuracy:

Accuracy of the method was estimated by standard addition-recovery method. For this, known amount of standard Amlodipine besylate was added in pre-analyzed sample. This was done for 1 µg/ml, 2µg/ml and performed in triplicate. The accuracy values for 1µg/ml and 2µg/ml concentration of Amlodipine besylate were found to be 98.92±0.52% and 99.12±0.22% respectively [Table 2].

 

Precision:

The precision of the assay was determined by repeatability (intraday) and intermediate precision (inter-day) and results were reported as RSD %.  For this, absorbances of 1µg/ml and 2µg/ml concentration solution were measured three times in day and same dilutions were subjected to absorbance measurement in next three days. The RSD% was calculated [Table 2].

 

 

Table 2: Determination of Accuracy (by percentage recovery method) and Precision

	Tablet amount (µg/ml)	%Addition	Amount added (µg/ml)	Amount recovered (µg/ml)	% Recovery	Precision %RSD*
						Interday	Intraday
Amlodipine besylate	1	100	1	0.0989	98.92±0.52	1.88	1.27
	2	100	2	0.1984	99.12±0.22	1.51	1.52

Limit of Detection (LOD) and Limit of quantification (LOQ):

The limit of detection (LOD) and limit of quantification (LOQ) for Amlodipine besylate was determined by using standard deviation of response (S) and slope (M). The LOD and LOQ values are presented in [Table 3].

 

Table 3. Validation Parameters
Parameters	Result
Absorption maxima (λmax)	364nm
Linearity (µg/ml)	1- 5
Linear equation	y=0.025X+0.0044
Correlation coefficient (r2)	0.990
Limit of Detection (LOD) (µg/ml)	0. 27
Limit of Quantification (LOQ) (µg/ml)	0.81

 

Determination of active ingredients in different brands of tablets:

The proposed and validated method was applied to estimate the amount of active ingredient- Amlodipine besylate in two different brands of tablets using twenty tablets in each batch. Results of analysis are presented in table 4. The results of analysis suggest that both the marketed formulations fulfilled the % amount requirement (98-102%) with respect to labeled claim.

 

Table 4: Determination of active ingredients (%)
Sample	Labeled claim (mg/tablet)	The amount found per tablet	% Active ingredient of labeled claim
Brand 1	5	4.975	99.50±.26
Brand 2	10	9.903	99.03±0.61

 

CONCLUSION:

The developed UV spectroscopic method was simple, rapid, cost effective and reproducible with high reproducibility and precision. The parameters were validated as per ICH guidelines. The findings of the work suggest that the method may be applied for quantitative estimation of Amlodipine besylate from bulk and pharmaceutical dosage formulations. This method may also be used in the routine work of quality control aspects.

 

ACKNOWLEDGEMENT:

The authors would like thanks to Prof. R.M. Dubey (Honorable Vice-Chancellor of IFTM University) for his kind support to complete this work with great ease.

 

REFERENCES:

1.     Stoiljkovic ZZ, Avramov ML, Petrović SD, Mijin DZ, Stevanović SI, Lacnjevac UC, Marinkovic AD. Voltammetric and Square-Wave Anodic Stripping Determination of Amlodipine Besylate on Gold Electrode. International Journal of Electrochemical Sciences 7; 2012: 2288 – 2303.

2.       Crea F, Lanza GA. Angina pectoris and normal coronary arteries: cardiac syndrome X. Heart. 90(4); 2004:457-463.

3.       Chrysant SG. Amlodipine besylate/olmesartan medoximil fixed combination for the treatment of hypertension. Expert Review of Cardiovascular Therapy. 7(8); 2009: 887-95.

4.       Sridhar K, Sastry CSP, Reddy MN, Sankar DG, Srinivas KR. Spectrophotometric Determination of Amlodipine Besylate in Pure Forms and Tablets. Analytical Letters. 30(1); 1997: 121-133.

5.       Zarghi A, Foroutan SM, Shafaati A, Khoddam A. Validated HPLC method for determination of amlodipine in human plasma and its application to pharmacokinetic studies. Il Farmaco. 60(9); 2005: 789-792.

6.       Rote AR, Kande SK.  HPTLC method for the simultaneous determination of amlodipine and benazepril in their formulations. Journal of Chromatographic Science. 43(2); 2005: 73-5.

7.       Streel B, Lainé C, Zimmer C, Sibenaler R, Ceccato A. Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry. Journal of Biochemical and Biophysical Methods. 54(1-3); 2002: 357-368.

8.       Bhatt J, Singh S, Subbaiah G, Shah B, Kambli S, Ameta S. A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the estimation of amlodipine in human plasma^. Biomedical Chromatography. 21(2); 2007: 169-175.

 9.      International Conference on Harmonization, Draft Guideline on Validation Procedure, Definition and Terminology Federal Register, 60; 1995: 11260.

 

 

Accepted on 19.06.2012      © RJPT All right reserved