INTRODUCTION:

Amlodipine besylate is chemically 3-Ethyl 5-methyl (4RS)-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5- dicarboxylate benzene sulphonate. Amlodipine besylate is used in the management of hypertension¹ and coronary artery disease².

Analysis is an important component in the formulation development of any drug molecule. A suitable and validated method has to be available for the analysis of drug(s) in the bulk, in drug delivery systems, from release dissolution studies and in biological samples. If a suitable method, for specific need, is not available then it becomes essential to develop a simple, sensitive, accurate, precise, reproducible method for the estimation of drug samples.

It is available in several officials in Pharmacopoeia^3-5. Literature survey reveals that, Spectrophotometric methods^6-15, High performance liquid chromatography [HPLC]^16-18, high performance thin layer chromatography [HPTLC]¹⁹,ultra performance liquid chromatography [UPLC]²⁰. In the present study, an attempt has been made to develop UV Spectrophotometric method for the determination of Amlodipine besylate in bulk and marketed formulations using dimethyl sulphoxide.Thus the present study was undertaken to develop and validate a simple, sensitive, accurate, precise, and reproducible U.V method for Amlodipine besylate.

Figure No. 1 Structure of Amlodipine besylate.

MATERIALS AND METHODS:

Instrument and materials:

The present work was carried out on ElicoSL164 UV- Visible spectrophotometer having double beam detector configuration. Amlodipine besylate pure drug was obtain from Spectrum Labs Hyderabad as gift sample with 99.99% w/w assay value and was used without further purification. The absorption spectra of reference and test solution were carried out in a 1 cm quartz cell over the range of 200-400 nm. All chemicals of analytical grade used as it is.

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 distilled water. (Stock solution-II, 100 mcg/ml). 1 ml of stock solution-II was diluted to 10 ml with distilled water, so that to produce the concentration 10 mcg/ml. The absorbance of resulting solution was measured against respective blank solution in the UV region of 200-400 nm, which shows maximum absorbance at 365 nm.

Preparation of calibration curve:

Aliquots of standard solution of amlodipine besylate ranging from 0.5-2.5 ml (1 ml = 100 mcg) were transferred into a series of 10 ml volumetric flasks. The volume in each flask was made up to 10 ml with distilled water and the absorbances were measured at 365 nm against solvent blank. The obtained absorbance values when plotted against the concentration of amlodipine besylate give the calibration graph. The concentration of the unknown sample was determined from the calibration graph.

Figure No 2-Overlay spectrum of amlodipine besylate in Dimethyl sulphoxide (5-25mcg/ml).

Figure No 3- Calibration curve of amlodipine besylate in Dimethyl sulphoxide (5-25mcg/ml).

Preparation of sample solution:

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 0.45 µwhatmann filter paper. 5 ml of the filtrate was diluted to 50 ml with distilled water to get the solution of 100 mcg/ml. An aliquot of 1 ml of test solution was diluted to 10 ml with distilled water so that to produce the concentration 10 mcg/ml.

Table No.1 - Calibration Curve parameter.

S. No.	Concentration ( mg/ml)	Absorbance ±SD	%Relative standard deviation
1	5	0.550 ± 0.03	1.8
2	10	1.251 ± 0.027	1.5
3	15	1.556 ± 0.032	1.7
4	20	1.655 ± 0.047	2.7
5	25	1.855 ± 0.042	2.5

RESULT AND DISCUSSION:

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 365 nm. Inter-day precision was determined by analyzing the drug daily once for five days at 365 nm.The relative standard deviation(RSD) and assay values obtained by two analysts were 0.36, 99.50 and 0.31, 99.60 respectively (Table no.4).

Table.2 - Validation parameters

Sr. No	Parameter	Result
1	Absorption maxima(nm)	365
2	Linearity Range (mg/ml)	5-25
3	Standard Regression Equation	Y=0.0163C - 0.0012
4	Correlation Coefficient (r2 )	0.9999
5	Molar absorptivity	0.0086 X10⁴
6	Accuracy (% recovery ±SD)	100.786±1.586
7	Precision	99.87% (Intra-day precision) and 99.82%(Inter-day precision)
8	Specificity	A 10 mg/ml solution of candidate drug in Dimethyl sulphoxide at UV detection of 365 nm will show an absorbance value of 1.251 ± 0.027
9	Sandell’s Sensitivity (mg/cm2/0.001 absorbance unit)⁸	0.080
10	LOD (mg/ml)	0.108
11	LOQ (mg/ml)	0.340

Accuracy (Recovery Test):

Accuracy is the closeness of the test results obtained by the method to the true value. The recovery experiments were performed by adding known amounts to tablet. The recovery was performed at three levels, 50,100and 150% of amlodipine besylate standard concentration. 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 recovery values for amlodipine besylate ranged from 99.97 ± 0.3969 (Table no.3) was studied by recovery experiments.

Linearity:

The linearity of the response of the drug was verified at 2 to 40 _g/ml concentrations, but linearity was found to be between 5-25 g/ml concentration. The calibration graphs were obtained by plotting the absorbance versus the concentration data and were treated by linear regression analysis (Table no.2). The equation of the calibration curve for amlodipine besylate obtained y = 0.0163C--0.00120,the calibration curve was found to be linear in the a forementioned concentrations. The correlation coefficient (r2) of determination was 0.9999.

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

The LOD and LOQ of amlodipine besylate were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines ²¹.The LOD and LOQ Was found to be as in table no.2.

CONCLUSION:

From the results the method described in this paper for the determination of Amlodipine besylate from tablet formulation is simple, accurate, sensitive and reproducible. The proposed method could be applied for routine analysis in quality control laboratories.

Table No.4 - Determination of Precision

Sample number	Assay of Amlodipine besylate	As % labelled amount
Sample number	Analyst-I (Intra-day precision)	Analyst-II (Inter-day precision)
1	99.96	99.78
2	99.37	98.73
3	99.69	100.12
4	100.13	99.52
5	98.99	101.21
6	101.12	99.56
Mean	99.876	99.82
RSD	0.539	0.673

ACKNOWLEDGEMENTS:

We are thankful to Spectrum Labs at Hyderabad for providing the gift sample of Amlodipine besylate.We would also like to thank Dr. A. Lakshmana Rao, Principle, V.V. Institute of Pharmaceutical Sciences for providing all the facilities to complete our work successfully.

REFERENCES:

1. Wang JG. A combined role of calcium channel blockers and angiotensin receptor blockers in stroke prevention. Vascular health and risk management. (2009); (5): 593–605.

2. Amlodipine Besylate. The American Society of Health-System Pharmacists. 2011. April.

3. British Pharmacopoeia. London: 2008: 137.

4. Norvasc. Online Drug Description and Clinical Pharmacology of amlodipine. The Internet Drug Index. 2008.

5. European Pharmacopoeia: 981-982.

6. Sridhar K, Sastry CSP, Reddy MN. Spectrophotometric determination of amlodipine besylate in pure forms and tablets. Analytical letters.1997;30:121-126.

7. Gohil K, Trivedi P, Molvi KI. Spectrophotometric analysis of amlodipine besylate in bulk and in tablet dosage forms. Indian Journal Pharma.Sci. 2005; 67:376-378.

8. Raman N and Nasrul Hoda M. Validated spectroscopic method for determination of amlodipine besylate in drug formulation using 2,3-dichloro -5,6 dicyno -1,4 benzoquino and ascorbic acid. JPBA. 2003; 31:381-392.

9. Khopade SA and Jain NK. Difference spectrophotometric estimation of amlodipine besylate. Indian Drugs. 2000; 37:351-353.

10. Singh G, Mishra A, Verma A. Development and Validation of UV Spectrophotometric Method for Estimation of Amlodipine Besylate in Tablet Dosage Form. RJPT. 2012; (7):931-933

11. Juya V, Chaudhary M, Kumar P, Gnanarajan G, Yadav PK. Method Development and its Validation for Simultaneous Estimation of Atorvastatin and Amlodipine in Combination in Tablet Dosage Form by UV Spectroscopy, Using in Multi-Component Mode of Analysis.JPR. 2008. Oct-December.

12. Baheti KG, Panigrahi S. Simultaneous UV Spectroscopic Estimation of Amalodipine Besylate and Indpamide in Bulk and Tablet Dosage Form. Int J Adv Pharm Biol Scie. 2012; (2); 155-164.

13. Seema M, Dinesh R, Madhulika T. Validated UV Spectrophotometric Method for Simultaneous Estimation of Metoprolol Succinate and Amlodipine Besylate in Their Combined Tablet Dosage Form. IJABC.2013; 3(3); 82-85.

14. Priyanka RP, Sachin UR, Pandurang ND, Kishor BB. Simultaneous Estimation of Ramipril and Amlodipine by UV Spectrophotometric Method. Research J. Pharm. and Tech. 2009 ; (2).

15. Paramita D, Sandip P, Radhika P, Subramanyam E, Sharbaraya A. Simultaneous Estimation of Aliskiren and Amlodipine in Tablet Dosage form by UV Spectroscopy. Int. J. Drug Dev. & Res. 2012; 4 (2): 265-270.

16. Hamed M, Habib A, Sherin F, Badran I. Development and Validation of Quality by Design Optimised Reversed Phase HPLC Method for Simultaneous Estimation of Telmisartan and Amaldipine. IJBPR. 2013; 4(10): 697-701.

17. Suhas S, Ashok P, Prasad K, Trinath M. Simultaneous Estimation of Telmisartan & Amlodipine in Bulk and Tablets by UV and RP-HPLC Method. JAPHR, 2011; 3.

18. Saurabh KB and Nitin MV. Simultaneous Estimation of Amalodipine and Rosuvastatin in Combined Bulk Forms by RP-HPLC Using Ultraviolet Detection. Bull Pharm Res. 2013;3(1).

19. Dhandapani B, Anjaneyulu N, Venkateshwarlu Y, Rasheed S. HPTLC Method Development and Validation for the Simultaneous Estimation of Amlodipine Besylate and Nebivolol Hydrochloride in tablet dosage form. JPR.2010; (3):332.

20. Shiva NV P B, Prasad K D. Quantitative Determination of Amlodipine in Human Plasma by Ultra Performance Liquid Chromatography-Electro Spray Ionization Mass Spectrometry Application to a Clinical Pharmacokinetic Study. Asian J Pharm Clin Res.2012; 89-93.

21. Text on Validation of Analytical Procedures Q2 (R1) in, I.C.H. Harmonised Tripartite Guidelines; Nov. 1996.

Received on 10.04.2014 Modified on 28.04.2014

Asian J. Research Chem. 7(6): June 2014; Page 596-599

Ingredient	Tablet amount (mg/ml)	Level of addition (%)	Amount added (mg)	Drug found (mg/ml)	% Recovery	Average % recovery
Amlodipine besylate	10	50	2mg	1.988	100.10
	10	100	4mg	3.945	101.39	100.78±1.586
	10	150	6mg	5.948	100.87