INTRODUCTION:

Amlodipine besylate, firstly approved by the FDA in 1987, is a common antihypertensive drug belonging to the group of drugs called dihydropyridine calcium channel blockers that prevent the transmembrane influx of calcium ions into vascular smooth muscles and cardiac muscles, which in turn affects their contractile process and consequences in reduced blood pressure. Amlodipine besylate is usually used in the treatment of high blood pressure and angina. Amlodipine besylate has antioxidant properties and an ability to improve the production of nitric oxide (NO), an essential vasodilator that reduces blood pressure. The option for single daily dosing of amlodipine is an attractive feature of this drug.

Chemically Amlodipine besylate is benzenesulfonic acid;3-O-ethyl 5-O-methyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate

Fig. 1: Structure of Amlodipine besylate

Amlodipine besylate may be used alone or in combination with other antihypertensive and antianginal agents for the treatment of the following situations

· Hypertension

· Coronary artery disease

· Chronic stable angina

· Vasospastic angina (Prinzmetal’s or Variant angina)

· Angiographically documented coronary artery disease in patients without heart failure or an ejection fraction < 40%.^1-8

MATERIALS AND METHODS:

Instrument:

Using a Single beam UV spectrophotometer (Aglient Tech. Carry 60 Uv-Vis, Model GS2281) and Digital weighing balance, Spectrophotometric analysis was carried out.

Chemicals and reagents:

Reference standard of Amlodipine besylate was a munificent gift from – pharma company. The marketed formulations of Amlodipine besylate each of 10mg, Amlovas -10 and Amlopres -10 were purchased from local market. Pure ethanol was purchased from Central Drug House Pvt. Ltd. Dehradun, India. All other chemicals and solvents employed for the study were of analytical grade.

Characterization and identification of Amlodipine besylate:

Melting point study:

Melting point of the compound was determined by melting point apparatus. The compound was positioned in one –ended-sealed capillary. The capillary and thermometer were placed in the respective caves in the apparatus. Temperature was set by the regulator and temperature at which the compound melted was measured by the thermometer.

Solubility study:

1gm of Amlodipine besylate was dissolved in 10 ml of ethanol in stoppered vessel and stirred for few minutes at room temperature and examined visually for the presence of any residue.

Method development for estimation of Amlodipine besylate by UV spectroscopy:

Preparation of standard stock solution of Amlodipine besylate:

50mg of the drug was accurately weighed and transferred to 50ml of volumetric flask and dissolved in 20ml of solvent ethanol. Then volume was made up to the mark with ethanol.

Determination of wavelength of maximum absorbance (λ_max):

1ml of standard stock solution was pipette out and transferred to a 10ml volumetric flask, marked as A and the volume was made up to the mark with ethanol. Then from A (having conc.100μg/ml), 1ml of the solution is taken in 10ml volumetric flask and then volume was made upto the mark with ethanol. The absorbance of this solution was scanned in the U.V range of 200-400nm against ethanol as a blank. The λ_maxobtained was 360nm. ⁹

Preparation of calibration curve for Amlodipine besylate at 360nm:

0.5-4ml of solution from A (100μg/ml) were pipette out into a series of 10ml volumetric flask. Then the volumes were made upto the mark with ethanol to obtain the solution in the concentration range of 5-40μg/ml of drug. The absorbance of these resultant solutions were measured at 360nm against ethanol as blank and a graph was plotted between absorbance obtained and the concentrations of the solution.¹⁰

Method validation:

Linearity and Range:

A linear relationship was observed between absorbance and concentration in the range of 5-40μg/ml of drug in the solution.

Precision:

Precision of the methods was studied as intra-day, inter-day.

· Intra-day Precision- Intra-day study was carried out by examining a 15μg/ml, 20μg/ml and 25μg/ml concentration of drug for three times in the same day.

· Inter-day Precision– Inter-day precision was carried out by examining a 15μg/ml, 20μg/ml and 25μg/ml concentration of drug for three times in next day.^11,12

· Repeatability- Repeatability study was carried out by examining 20μg/ml concentration of drug for six times.

Accuracy:

The Accuracy of the method was evaluated by recovery studies at three different levels i.e. 80%, 100%, and 120%. The recovery studies were carried out by the addition of a known amount of standard solution of Amlodipine besylate to pre-analysed solutions. The resulting solutions then re-analysed by the proposed method.^13,14

Limits of Detection and Limit of Quantification:

The Sensitivity of the method was determined in terms of LOD and LOQ. The limit of detection and limit of quantification of Amlodipine besylate were calculated by using following equation as per ICH guidelines.

LOD = 3.3 x σ/S

LOQ = 10 x σ/S

Robustness:

The robustness of an analytical method is a measure of its ability stay unaffected by minor, but intentional variations in method parameters and gives an indication of its reliability during normal use. The robustness of the proposed method involves the 20μg/ml sample solution of Amloidipine besylate and is evaluated by a change in wavelength.^15,16

Ruggedness:

The ruggedness is a degree of reproducibility of test result under verification of condition such as a different analyst, different instruments, and different days. To determine ruggedness of the proposed method, the sample solutions of 20μg/ml of Amlodipine besylate was prepared by different analysts and analyzed.^16,17

Estimation of Amlodipine besylate in tablet dosage form:

Ten tablets were weighed and crushed to get a fine powder. The powder sample equivalent to 50mg Amlodipine besylate was transferred to a 100ml volumetric flask and was dissolved in 100ml ethanol. The solution was sonicated for 10 min. Then it was filtered through whatman filter paper. After filtration a final concentration of 10μg/ml solution was prepared and the absorbance was measured at 360nm.^18-20

RESULT AND DISCUSSION:

Characterization and identification of Amlodipine besylate:

Melting point study:

The melting range of Amlodipine besylate was found to be 195-201⁰C (using melting point apparatus, observed melting point was 201⁰C).

Solubility study:

Ethanol was selected for the study of Amlodipine besylate, as the drug was freely soluble in it.

Determination of wavelength of maximum absorbance (λ_max):

The absorbance of 10μg/ml solution was scanned in the U.V range of 200-400nm against ethanol as a blank. The λ_maxobtained was 360nm (fig.2).

Fig.2: Scan of Amlodipine besylate at 200-400nm

Preparation of calibration curve for Amlodipine besylate at 360nm:

The calibration curve of Amlodipine besylate was prepared in the range of 5-40μg/ml at 360nm, represented in table 1 and figure 3.

Table 1: Data for calibration curve of Amlodipine besylate

Concentration	Absorbance
5	0.0655
10	0.1296
15	0.2105
20	0.2799
25	0.3474
30	0.4001
35	0.4747
40	0.5417

Fig. 3: Calibration curve of Amlodipine besylate

Method validation:

Linearity and Range:

The response of the drug was found to be linear in the range of 5-40μg/ml concentrations. The calibration curve was obtained by plotting the absorbance v/s concentration data. The calibration equation for Amlodipine besylate obtained was y = 0.013x + 0.001 with the calibration coefficient R² = 0.998.

Precision:

The precision (measurement of intra-day, inter-day, repeatability) results showed good reproducibility, %RSD was below 2% as represented in table 2, 3 and 4. This indicated that the method was highly precise.

(a) Intra-day precision

Table 2: Intra-day precision results of Amlodipine besylate

Conc.	Absorbance			Mean	SD	% RSD	Mean % RSD
Conc.	0 hr	3 hrs	6 hrs	Mean	SD	% RSD	Mean % RSD
15μg/ml	0.19656	0.19343	0.20026	0.19675	0.0005948	0.3028	0.2035
20μg/ml	0.2553	0.2668	0.2655	0.2704	0.0004545	0.1729
25μg/ml	0.3610	0.3569	0.3549	0.3576	0.0004848	0.135

(b) Inter-day precision:

Table 3: Inter-day precision results of Amlodipine besylate

Conc.	Absorbance			Mean	SD	%RSD	Mean %RSD
Conc.	0 hr	3 hrs	6 hrs	Mean	SD	%RSD	Mean %RSD
15μg/ml	0.1957	0.2019	0.1976	0.1984	0.0003252	0.1639	0.3682
20μg/ml	0.267	0.2713	0.2735	0.2706	0.0004295	0.1587
25μg/ml	0.36536	0.3670	0.3602	0.3641	0.0004984	0.1368

(c) Repeatability

Table 4: Data for repeatability.

Concentration	Absorbance
20μg/ml	0.2713 0.2712 0.2715 0.2716 0.2717 0.2712
Average	0.2716
SD	0.000214
%RSD	0.0789

Table 5: Evaluation of accuracy study of pure drug Amlodipine besylate.

Drug	Conc. (µg/ ml)	Spiked level	Amount added (µg/ml)	Amount found	% Recovery
Amlodipine	10	80	8	18.1	100%
Amlodipine	10	100	10	20.5	102%
Amlodipine	10	120	12	21.8	99%

Accuracy:

The accuracy study was performed at three levels 80%, 100% and 120%. The results are shown in table no. 5

Limits of detection and Quantitation (LOD and LOQ):

Determination of the limits of detection and quantitation was carried out based on the standard deviations of the y-intercept and slope of the calibration curve as defined in International Conference on Harmonization (ICH) guidelines. The LOD and LOQ were 0.08123µg/ml and 0.2461µg/ml respectively.

Robustness:

In Robustness study the 20µg/ml concentration of pure drug was scanned at different wavelengths viz. 355nm, 360nm, 365nm presented in table 6.

Table 6: The evaluation data for Robustness study at 20μg/ml concentration for pure drug.

Concentration	355 nm	360 nm	365 nm
20μg/ml	0.2552 0.2555 0.2553	0.2779 0.2764 0.2763	0.2737 0.2731 0.2719
Mean	0.2553	0.2768	0.2729
SD	0.000153	0.000896	0.000017
%RSD	0.0599	0.323	0.336

Ruggedness:

Ruggedness of the proposed method was determined by analysis of sample solution (20µg/ml) prepared by different analysts presented in table 7.

Table 7: The evaluation data for ruggedness study at 20 μg/ml concentration for pure.

Analyst 1		Analyst 2
Concentration	Absorbance	Concentration	Absorbance
20 μg/ml	0.2669 0.2665 0.2661	20 μg/ml	0.2892 0.2894 0.2894
Mean	0.2665	Mean	0.2893
SD	0.0004	SD	0.000115
%RSD	0.1500	%RSD	0.0397

Estimation of Amlodipine besylate in tablet dosage form:

This is clear from the study that the estimated and claimed amounts are in good agreement. The % assay were 98.3 and 99.2 for amlovas and amlopres respectively (Table 8).

Table 8: Result for estimation of Amlodipine besylate in tablet dosage form:

Brand	Labelled claim	Absorbance	%Assay
Amlovas-10 (A)	10mg	0.1332	98.3%
Amlopres-10 (B)	10mg	0.1344	99.2%

CONCLUSION:

The developed method for quantification of Amlodipine besylate in API and tablet dosage form was easy, accurate, selective, sensitive, reproducible and reliable. The method was validated for several parameters like accuracy, precision, robustness and ruggedness as per ICH guidelines. On the basis of the results obtained, it can be concluded that the developed U.V spectroscopic method can be employed in routine analysis of Amlodipine besylate.

ACKNOWLEDGEMENT:

The authors are grateful to Shri Guru Ram Rai University Patel Nagar, Dehradun for providing all the laboratory facilities to carry out the work.

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Received on 23.11.2021 Modified on 30.12.2021

Asian J. Pharm. Ana. 2022; 12(2):94-98.

DOI: 10.52711/2231-5675.2022.00017