INTRODUCTION:

Hypertension and angina are among the most prevalent cardiovascular disorders worldwide, posing a significant burden on healthcare systems and patients' quality of life. Amlodipine Besylate, a third-generation dihydropyridine calcium channel blocker, is widely used in the management of these conditions due to its efficacy in lowering blood pressure and relieving angina symptoms. It works by inhibiting the influx of calcium ions into vascular smooth muscle and cardiac myocytes, resulting in vasodilation and reduced cardiac workload.

The drug’s pharmacokinetics, including its long half-life and high bioavailability, make it a cornerstone in chronic cardiovascular disease management^1,2. Given its importance, ensuring the quality, safety, and efficacy of Amlodipine Besylate in pharmaceutical formulations is critical. This necessitates the development of robust analytical methods for its quantification in bulk drugs and dosage forms.

Analytical methods are integral to the pharmaceutical industry for drug development, quality control, and regulatory compliance. Among the available analytical techniques, UV-visible spectroscopy is recognized for its simplicity, rapidity, cost-effectiveness, and minimal resource requirements³. While various sophisticated methods such as HPLC (High-Performance Liquid Chromatography) and LC-MS (Liquid Chromatography-Mass Spectrometry) exist, these techniques are often time-consuming and expensive.

The developed UV-visible spectroscopic method for the analysis of Amlodipine Besylate offers several significant advantages. It is a simple, rapid, and cost-effective technique, making it ideal for routine quality control in both research and industrial settings. Unlike more complex analytical methods such as HPLC or LC-MS, UV-visible spectroscopy requires minimal equipment and operational expertise, making it accessible for resource-limited laboratories⁴. Additionally, this method aligns with green analytical chemistry principles, as it minimizes the use of organic solvents and reagents, reducing environmental impact. The method’s accuracy, precision, and reliability ensure consistent results, while its validation according to ICH guidelines makes it robust and suitable for regulatory compliance. Overall, this method provides a practical and efficient solution for the quality assurance of Amlodipine Besylate in bulk drugs and pharmaceutical formulations⁵.

The primary objective of this research is to develop and validate a simple, accurate, and precise analytical method for the quantification of Amlodipine Besylate using UV-visible spectroscopy. The study focuses on identifying the maximum absorbance wavelength (λmax) of the drug, establishing a calibration curve over a defined concentration range, and validating the method according to the International Council for Harmonisation (ICH Q2(R1)) guidelines. Key validation parameters include accuracy, precision, linearity, specificity, robustness, limit of detection (LOD), and limit of quantification (LOQ). Additionally, the research aims to demonstrate the applicability of the developed method for analyzing pharmaceutical formulations, ensuring its reliability for routine quality control and regulatory compliance.

MATERIALS AND METHODS:

Chemicals and Reagents:

The analytical grade reagents and solvents used in this study included Amlodipine Besylate reference standard (Provided by Vidisha Analytical Laboratory, Nashik), methanol (analytical grade), and distilled water. All reagents were of high purity, ensuring minimal interference in the analysis. Methanol was selected as the solvent due to its compatibility with UV spectroscopy and its ability to dissolve Amlodipine Besylate efficiently⁶.

Instrumentation:

A UV-Visible spectrophotometer (Shimadzu UV-1800) was used for this study. The instrument had a wavelength range of 200–800nm, an accuracy of ±1nm, and a 1cm quartz cuvette for sample analysis. The spectrophotometer was calibrated before use to ensure accuracy and reliability of results⁷.

Method Development:

Preparation of Standard Solutions:

A stock solution of Amlodipine Besylate (100µg/mL) was prepared by dissolving 100mg of the Amlodipine Besylate in 100mL of methanol. Working solutions of varying concentrations (2µg/mL to 10µg/mL) were then prepared by diluting the stock solution with methanol⁸.

Wavelength Selection:

The λmax of Amlodipine Besylate was determined by scanning the prepared standard solution in the UV range of 200–400nm. The wavelength corresponding to the maximum absorbance was selected for further analysis. This ensures optimal sensitivity and reproducibility of the method⁹.

Linearity Range:

A calibration curve was prepared by plotting absorbance against concentrations of Amlodipine Besylate in the range of 2–10µg/mL. The linear regression equation and correlation coefficient (R²) were determined to assess linearity¹⁰.

Validation Parameters (as per ICH Q2(R1) Guidelines):

Linearity:

The linearity of the method was assessed by preparing and analyzing five different concentrations of Amlodipine Besylate within the range of 2µg/mL to 10 µg/mL. A series of standard solutions were prepared by diluting the stock solution of Amlodipine Besylate with methanol. The absorbance was measured at the selected wavelength (239nm), and a calibration curve was plotted using the concentration versus absorbance data. The linearity was confirmed by calculating the R² value, which indicates the goodness of fit for the calibration curve. A value close to 1.0 demonstrates good linearity, validating the method's ability to quantify Amlodipine Besylate across the specified concentration range¹¹.

Accuracy:

Accuracy was evaluated through recovery studies by spiking known amounts (80%, 100%, and 120%) of Amlodipine Besylate reference standard into a pre-analyzed sample. The samples were then analyzed using the developed method. The percentage recovery was calculated by comparing the measured drug content with the expected drug content. Acceptable recovery values within the range of 98–102% indicate that the method is accurate and can consistently measure Amlodipine Besylate in the presence of potential matrix components¹².

Precision:

Precision was evaluated by performing both intra-day and inter-day studies. Intra-day precision (repeatability) was determined by analyzing the same concentration of Amlodipine Besylate multiple times within the same day. Inter-day precision (intermediate precision) was determined by analyzing the same concentration on three different days. The results for both intra-day and inter-day precision were expressed as the percentage relative standard deviation (%RSD). A %RSD value below 2% is considered acceptable and confirms the method's consistency and reproducibility¹³.

Specificity:

Specificity was assessed by analyzing blank solutions, standard solutions, and sample solutions containing potential excipients and impurities. The objective was to ensure that there were no interferences or significant changes in the absorbance that could be attributed to the excipients, impurities, or other components of the tablet formulation. The method's specificity was confirmed if no significant absorption peaks were detected at the selected wavelength (239 nm), indicating that the absorbance measurements were solely due to Amlodipine Besylate¹⁴.

LOD and LOQ:

The limit of detection (LOD) and limit of quantification (LOQ) were calculated using the formulae:

LOD = (3.3 × SD) / S

LOQ = (10 × SD) / S.

Where, SD represents the standard deviation of the response and S is the slope of the calibration curve. The LOD indicates the smallest amount of Amlodipine Besylate that can be reliably detected, while the LOQ represents the smallest amount that can be quantified with acceptable precision and accuracy. The lower the LOD and LOQ values, the more sensitive the method is for detecting low concentrations of the drug¹⁵.

Robustness:

Robustness was evaluated by making small deliberate changes in experimental conditions, such as varying the wavelength by ±2nm and altering the solvent composition (methanol: water ratio). The consistency of the method was assessed by measuring the absorbance of a standard solution of Amlodipine Besylate under these altered conditions. The absorbance values were compared to those obtained under the standard conditions to determine whether these variations affected the results. If the method demonstrated minimal deviation, it was considered robust, indicating that the method could tolerate small changes in the experimental setup without significant loss of accuracy or precision.

Sample Analysis:

For the analysis of Amlip-5 Tablets (Cipla), 20 tablets were accurately weighed, and the average weight of a single tablet was calculated. The tablets were then crushed into a fine powder, and an amount equivalent to 5 mg of Amlodipine Besylate was weighed. This powdered sample was dissolved in 50mL of methanol in a volumetric flask, ensuring complete dissolution. The solution was filtered using Whatman filter paper to remove excipients and undissolved particles, and the filtrate was diluted to a final volume of 100mL with methanol, resulting in the stock solution. For the standard solution, 5mg of Amlodipine Besylate reference standard was accurately weighed and dissolved in methanol, and the volume was made up to 100mL to prepare a 50µg/mL stock solution. A working standard solution of 6µg/mL was prepared by further dilution of the stock solution with methanol¹⁶. The absorbance of both the sample and standard solutions was measured at 239nm using a UV-Visible spectrophotometer, the λmax of Amlodipine Besylate. The drug content in the tablet formulation was determined by comparing the absorbance of the sample to the standard solution. The assay value was calculated using the formula:

Labeled Claim (mg)

Assay Value (%) = ------------------------------------- × 100

Measured Drug Content (mg)

RESULTS AND DISCUSSION:

Method Development:

To establish a robust analytical method for Amlodipine Besylate, we conducted a UV-Visible spectrum analysis of the drug within the range of 200–400nm using different solvents. This systematic approach aimed to identify the solvent that provides the most distinct and reliable absorbance peak, ensuring suitability for quantitative analysis. Through the evaluation of various solvents, methanol was identified as the most suitable, as it yielded a well-defined and distinct absorbance peak.

Determination of Wavelength:

In methanol, Amlodipine Besylate exhibited a distinct absorbance peak at 239nm, which corresponds to its λmax (Figure 1). This wavelength was selected as the basis for quantitative analysis due to its optimal sensitivity and minimal spectral interference. The λmax is critical as it represents the wavelength at which the drug demonstrates maximum absorbance, ensuring precise detection and measurement.

The selection of 239nm as the analytical wavelength was based on its precision, reproducibility, and linearity, ensuring accurate quantification even at low drug concentrations. This wavelength provides a strong foundation for the development of a sensitive and reliable method, suitable for detecting and measuring Amlodipine Besylate in a variety of pharmaceutical formulations¹⁷.

Figure 1: UV spectra of Amlodipine besylate at 200-400nm

Calibration Curve and Linearity:

The calibration curve for Amlodipine Besylate was constructed (Table 1 and Figure 2) in the concentration range of 2–10µg/mL. The correlation coefficient (R²) was found to be 0.9995, which indicates a very strong linear relationship between absorbance and concentration within this range. The excellent linearity confirms the method’s ability to accurately measure concentrations of Amlodipine Besylate and provides a reliable basis for quantification in routine analyses.

Table 1: Linearity data of Amlodipine besylate

Sr. No.	Concentration (µg/mL)	Absorbance (at 239 nm)
1	2	0.200
2	4	0.391
3	6	0.590
4	8	0.781
5	10	0.972

Figure 2: Calibration curve of Amlodipine besylate

Accuracy:

The accuracy of the method was assessed by performing recovery studies at three different spiked levels (80%, 100%, and 120% of the target concentration). The recovery results were excellent, indicating the method's ability to accurately measure Amlodipine Besylate in the presence of other components in the formulation (Table 2).

Table 2: Data of recovery study

Spiked Level (%)	Amount Added (µg/mL)	Amount Recovered (µg/mL)	Recovery (%)
80	4.8	4.79	99.79
100	6.0	6.02	100.33
120	7.2	7.19	99.86

The recovery study shows excellent accuracy of the method for Amlodipine Besylate. At 80%, 100%, and 120% spiked levels, the recoveries were 99.79%, 100.33%, and 99.86%, respectively. These results confirm the method's high accuracy and reliability for quantifying Amlodipine Besylate in pharmaceutical formulations. This consistency in recovery rates at different concentrations shows the method’s robustness in handling variations in sample concentrations¹⁸.

Precision:

The precision of the method was evaluated by performing intra-day and inter-day studies. Precision refers to the consistency and reproducibility of results when the method is applied under the same or different conditions. Result of precision analysis of Amlodipine Besylate (Intra-day and Inter-day) was shown in Table 3 and 4.

Table 3: Precision Analysis of Amlodipine Besylate (Intra-day)

Conc. (µg/mL)	Absorbance	Mean	SD	%RSD
2	0.122	0.1233	0.00195	1.58%
	0.126
	0.123
4	0.247	0.2467	0.00252	1.02%
	0.244
	0.249
6	0.372	0.3730	0.00265	0.71%
	0.376
	0.371
8	0.495	0.4947	0.00252	0.51%
	0.497
	0.492
10	0.624	0.6243	0.00252	0.40%
	0.622
	0.627
Mean %RSD				0.84%

Table 4: Precision Analysis of Amlodipine Besylate (Inter-day)

Conc. (µg/mL)	Absorbance	Mean	SD	%RSD
2	0.123	0.1243	0.0015	1.20%
	0.124
	0.126
4	0.245	0.2447	0.0026	1.10%
	0.242
	0.247
6	0.371	0.3730	0.0020	0.50%
	0.375
	0.373
8	0.493	0.4940	0.0026	0.50%
	0.497
	0.492
10	0.625	0.620	0.0041	0.66%
	0.622
	0.618
Mean %RSD				0.79%

Intra-day Precision:

The intra-day precision, assessed by performing multiple measurements within a single day, resulted in a %RSD of 0.84%, indicating minimal variation in repeated measurements.

Inter-day Precision:

The inter-day precision, which assesses variation across different days, yielded a %RSD of 0.79%, further confirming the method's reliability across different testing days.

Both intra-day and inter-day precision values were well below the acceptable limit of 2%, indicating the method's high reproducibility and suitability for routine analysis¹⁹.

Specificity:

The specificity of the method was tested by analyzing Amlodipine Besylate in the presence of potential excipients that may be present in tablet formulations. No significant interference was observed at the selected wavelength (239nm), ensuring that the absorbance readings were attributable solely to Amlodipine Besylate. This confirms that the developed method is specific for Amlodipine Besylate and can be used to analyze formulations without concern for interference from other components. Result of the specificity analysis of amlodipine besylate shown in Table 5.

Table 5: Specificity Analysis of Amlodipine Besylate

Sample	Absorbance at 239 nm	Observation
Amlodipine Besylate Standard	0.375	Absorbance consistent with pure drug
Placebo (Excipients only)	0.002	No significant absorbance at 239 nm
Amlodipine Besylate + Excipients	0.374	No interference; absorbance consistent with pure drug

Sensitivity:

The sensitivity of the method was determined by calculating the Limit of Detection (LOD) and Limit of Quantification (LOQ), which are crucial parameters for assessing the method’s ability to detect trace amounts of the drug. Result of sensitivity analysis shown in Table 6.

Table 6: Sensitivity Analysis of Amlodipine Besylate

Parameter	Value (µg/mL)	Interpretation
LOD	0.096	Indicates the smallest amount of Amlodipine Besylate that can be reliably detected.
LOQ	0.291	Represents the smallest amount of Amlodipine Besylate that can be quantified with accuracy.

LOD (Limit of Detection): 0.096µg/mL

LOQ (Limit of Quantification): 0.291 µg/mL

The LOD and LOQ values are low, suggesting that the method is highly sensitive and capable of detecting even very small quantities of Amlodipine Besylate. This sensitivity is particularly important for detecting the drug in low-concentration formulations and for stability studies where trace levels may be present.

Robustness:

The robustness of the method was assessed by making small deliberate variations in experimental conditions, such as changing the wavelength by ±2nm and altering the solvent composition (methanol: water ratio). The results showed minimal deviation in absorbance values, confirming that the method is robust and that small variations in the experimental setup do not significantly affect the outcome. This characteristic is important for ensuring consistent performance under slightly varying laboratory conditions^20,21. Result of robustness analysis shown in Table 7.

Table 7: Robustness Analysis of Amlodipine Besylate

Condition	Variation	Observed Absorbance	% Deviation
Standard Condition	Wavelength: 239 nm	0.375	0.00%
Wavelength Variation	237 nm (-2 nm)	0.372	0.80%
Wavelength Variation	241 nm (+2 nm)	0.378	0.80%
Solvent Ratio Variation	Methanol:Water (48:52)	0.374	0.27%
Solvent Ratio Variation	Methanol:Water (52:48)	0.376	0.27%

Sample Analysis:

The developed and validated method was applied to analyze Amlodipine Besylate tablets labeled to contain 5 mg of the drug. The results showed that the actual drug content in the tablets was 4.99mg, corresponding to an assay value of 99.87% of the labeled claim. This confirms that the method is suitable for the quantitative analysis of Amlodipine Besylate in pharmaceutical dosage forms, demonstrating its practical applicability in routine quality control testing.

CONCLUSION:

The UV-Visible spectrophotometric method developed for the analysis of Amlodipine Besylate has been successfully validated in accordance with ICH Q2(R1) guidelines. The method demonstrated excellent linearity, accuracy, precision, and specificity, making it highly suitable for routine pharmaceutical analysis. With a linearity range of 2–10µg/mL, the method showed a strong correlation (R² = 0.9995), indicating reliable quantification. The recovery studies revealed a mean recovery of 99.85%, confirming the accuracy of the method for Amlodipine Besylate analysis. Furthermore, the method’s precision was validated through intra-day and inter-day studies, with %RSD values of 0.84% and 0.79%, respectively. The sensitivity, demonstrated by the low LOD (0.096µg/mL) and LOQ (0.291µg/mL), further supports its capability to detect trace amounts of the drug. Additionally, the robustness of the method ensures consistent performance under slightly varied experimental conditions. The method's application to analyze Amlodipine Besylate tablets resulted in a 99.87% assay, confirming its effectiveness in pharmaceutical quality control. This method offers a simple, accurate, precise, and cost-effective approach for the quantitative analysis of Amlodipine Besylate in pharmaceutical formulations. It is suitable for routine quality control and can be adopted by the pharmaceutical industry for the analysis of Amlodipine Besylate in both bulk and formulated products.

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Received on 03.03.2025 Revised on 20.03.2025

Accepted on 02.04.2025 Published on 14.04.2025

Available online from April 18, 2025

Asian J. Research Chem.2025; 18(2):71-76.

DOI: 10.52711/0974-4150.2025.00011

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