Analytical Method Development and Validation for the Simultaneous Estimation of Candesartan and Simvastatin by UV- Spectrophotometry Method

Kuldeep Chourasiya*, Anup Kumar Chakraborty

Department of Pharmacy, Oriental University, Indore, Madhya Pradesh 453555.

*Corresponding Author E-mail: kuldeep12pharma@gmail.com

ABSTRACT:

Efforts were made in the current work to create a method for the simultaneous estimation of Candesartan and Simvastatin by UV-spectrophotometry method employing simultaneous equation approach. In this approach ethanol was used as a solvent to check the solubility of both the drugs. At 200-400nm wavelengths, 16μg/mL prepared diluted stock solution each of both the drugs were scanned and estimated using methanol as blank. From the overlain spectra, by the observation of spectral characteristics of Candesartan and simvastatin were selected for simultaneous estimation. For simvastatin and candesartan 239nm as well as 262nm wavelength has been chosen respectively. As per ICH guidelines all analytical methods were validated. In this approach, the linearity value of both the drugs (Candesartan and Simvastatin) were found in the concentration range of 5-17.5 μg/mL and 3 – 10.5μg/ml respectively. The correlation coefficient (r2) value was obtained 0.999 for both the drugs. Candesartan and Simvastatin percentage recovery was found to be 99.26% and 97.48%. For intra and interday precision, percentage RSD value was found to be 0.99 and 1.7 (Intraday) as well as 1.0 and 1.78 (Interday). The intraday and interday relative standard deviation (RSD) values obtained by the proposed method are within 2% relative standard deviations. The LOD for candesartan and Simvastatin were found to be 0.258 μg/ml and 0.278μg/ml, and LOQ 0.784μg/ml and 0.84μg/ml respectively. Through the study of the candesartan and simvastatin solution in 0.1 NHCl using various glassware, the robustness of the established approach demonstrates a non-significant influence of the absorption level and the developed method's robustness was proven when the results were tested at various temperatures and when the results did not significantly alter when the analyst was switched.

KEYWORDS: Candesartan, Simvastatin and UV-spectrophotometry, Method development, Simultaneous estimation.

INTRODUCTION:

Simultaneous estimation is significant in the pharmaceutical industry. It is a tool for saving time. Different methods were used for estimation such as chromatographic methods and spectroscopic methods. The research of medications and the creation of new ones have made modest global advances in the health sciences.

The technique of drug molecule detection and pharmaceutical investigation or evaluation of the formulation greatly increases the delivery of safety and therapeutic impact. In order to effectively predict a system's future states, precise estimation of the system's current state is both intrinsically desired and essential. To enable mixture resolution, the raw overlapping spectral data underwent a number of modifications, such as the use of various order derivatives, derivatives of the ratio spectrum, and ratio subtraction algorithms. A breakthrough method for calculating ratio differences has been created with the advantages of being less expensive, more precise, and more time-efficient while still producing results to a high degree. When there are no formal methods available, new methods are created for new drugs and for their composition. Sometimes alternative methods are also used for other existing products for the purpose of saving time and cost and to get better or improved precision and raggedness.¹

For this trial runs are performed, and the approach is validated and optimized. Comparative laboratory data with advantages and demerits are made available an alternative method to replace the current procedure.²

MATERIALS AND METHODS:

Instrumentation:

In present work spectroscopic analysis has been carried in UV- Visible double beam spectrophotometer having path length matched quartz cells of 10mm^3-5.

Choice of Solvent:

Solubility serves as the basis for choosing the solvent. We tested the solubility of Candesartan and Simvastatin in a number of solvent systems among that ethanol was selected for the purpose of method development.

Selection of wavelength:

Using ethanol as a blank, the solutions were scanned at different wavelengths (200 and 400nm) for the estimation of Candesartan and Simvastatin .The stock solutions were diluted to contain 16mg of each drug and by examining the spectral properties of Candesartan and Simvastatin, which were chosen for simultaneous estimate from the overlaid spectra. The chosen wavelengths were found 239nm for Simvastatin and 262 nm for Candesartan.

Linearity and calibration:

Simultaneous Equation method:

The linearity of both the drugs was measured individually as well as the absorbance of the solutions was also done at different wavelengths such as 239 and 262nm. It entails determining the integrated absorbance value with respect to wavelength between the two chosen wavelengths (λ1 and λ2). The region bounded by the curve and the horizontal axis is determined by the processing item for area computation. The horizontal axis is selected by providing the wavelength range over which the area must be measured. The wavelength range was selected based on repeated observation in order to assess the linearity between the area under the curve and concentration. The wavelength ranges of the components are indicated by λ1 and λ2 in combination drugs. The integrated value of absorbance over the wavelength ranges of both drugs is substituted into the simultaneous equation to determine the concentration of the drugs.

Where, X is Simvastatin and Y is Candesartan:

Pipette 0.5 to 1.75ml of the working stock solution of candesartan into a set of six 10ml volumetric flasks, and then make them up to the mark with 0.1N HCl to produce a concentration range of 5 to 17.5μg/ml. Likewise, Pipette 0.3 to 1.5ml of the working stock solution of simvastatin into a series of six 10ml volumetric flasks, and then make up to the marked level with 0.1 N HCl to obtain a concentration range of 3 to 10.5μg/ ml.

Developing a working solution and stock solution (standard):

10mg of pure Candesartan and Simvastatin were accurately weighed and then transferred to a 100ml volumetric flask, where the volume was adjusted with ethanol to the proper amount. To generate the working solution, a standard stock solution with a concentration of 100µg/ml was obtained.⁶

Calibration Curve Preparation:

For Candesartan from the working solution, 0.5, 0.75, 1.0, 1.25, 1.5 and amp; 1.75ml solution were transferred into a series of calibrated 10ml volumetric flasks and later on the volume was make up by the help of ethanol and 0.1N HCl. After that the prepared solutions were scanned at wavelength ranging between 200-400 nm against blank. Likewise candesartan, in case of simvastatin, from the working solution, 0.3, 0.45, 0.6, 0.75, 0.9 and amp; 1.05 solution were transferred into a series of calibrated 10ml volumetric flasks and later on the volume was make up by the help of ethanol and 0.1 N HCl. After that the prepared solutions were scanned at wavelength ranging between 200-400 nm against blank.⁷

Validation of developed method:

Linearity:

Concentration and absorbance were plotted on a calibration curve. Simvastatin demonstrated linearity range of 3 - 10.5μg/ml and candesartan exhibited linearity with the concentration range of 5 - 17.5μg/ml for both the drugs.⁸

Recovery Analysis:

Recovery studies were conducted in order to validate the proposed method's truthfulness. A specific concentration of 2.5, 5 and 7.5µg/ml standard solutions of Candesartan as well as 1.5, 3 and 4.5µg/ml standard solutions of Simvastatin was added to the 50% of pre-analyzed sample solution, and then its recovery was examined. At the appropriate wavelengths, the absorbance of the resultant solutions was measured, and the % recovery was computed.^9-10

Precision:

The method's accuracy was proven by repeatability tests. In order to conduct repeatability experiments, the sample solution was then examined six times. By repeating the determination on the same day and on other days, respectively, the intraday and interday precision were established.

Ruggedness:

The formulation analysis conducted by many analysts proved the method's Ruggedness. The sum and percent RSD were computed.

Limit of Detection and Limit of Quantification:

Six times the linearity studies were conducted. The average slope and standard deviation of the intercept were used to establish the limit of detection and the limit of quantification¹¹.

RESULT:

Method Validation:

The proposed method's linearity, accuracy, precision, robustness, the limit of detection, the limit of quantification, and ruggedness were all validated in accordance with ICH requirements. Table 1 contains the general findings of the validation parameters.

Recovery analysis:

By using the method's recovery percentage at the three-level of percentage addition, the proposed method's accuracy was estimated ¹². Table 2 displays the percentage recovery of Candesartan and Simvastatin, which were determined to be in the range of 96.4 to 102 for Candesartan and 95.33 to 99.11 for Simvastatin. The outcomes of the recovery experiments unquestionably show how accurate the suggested strategy is.

Table 1: Determination of Candesartan and Simvastatin in 0.1 N HCl and its validation limit

Parameter

Values

Absorption Maxima (nm)

Candesartan

Simvastatin

262 nm

239 nm

Linearity Range (μg/ ml)

Candesartan

Simvastatin

5-17.5 (μg/ml)

3-10.5 (μg/ml)

Standard Regressed equation

Candesartan

Simvastatin

Y = 0.1026x + 0.018

Y = 0.0713x - 0.0032

Correlation Coefficient

Candesartan

Simvastatin

0.999

Recovery study (% recovery ± SD)

Candesartan

Simvastatin

99.26%

97.48%

Precision (% RSD)

Candesartan

Intraday- 0.99%

Inter day- 1%

Simvastatin

Intraday – 1.7%

Inter day- 1.78%

LOD (μg/ml)

Candesartan

Simvastatin

0.258 (μg/ml)

0.278 (μg/ml)

LOQ (μg/ml)

Candesartan

Simvastatin

0.784 (μg/ml)

0.84 (μg/ml)

Precision ^13-17

The Intraday and Interday Precision at the three-level of percentage addition were used to estimate the suggested method's precision. The repeatability findings show the accuracy over a brief period of time and during interday assessment. According to data in Table 3, the % RSD of candesartan and simvastatin was 0.99 and 1.7 for intraday precision and 1.0 and 1.78 for interday precision. The proposed method yielded intraday and interday relative standard deviation (RSD) values that are within 2% relative standard deviations.

Table 2: Estimation of Accuracy percentage recovery

Si. No.	Conc. 0f pure Simvastatin μg/ml	Conc. Of pure Candesartanμg/ml	Conc. Of Simvastatin Test μg/ml	Conc. Of candesartan Test μg/ml	Abs. at 239nm	Abs. at 262 nm	conc. Of simvastatin obtained μg/ml	conc. Of candesartan obtained μg/ml	simvastatin recover μg/ml	candesartan recover μg/ml	% simvastatin recover	% candesartan recover
1	3	5	1.5	2.5	1.055	1.093	4.43	7.5	1.43	2.55	95.33	102
2	3	5	3	5	1.399	1.449	5.94	9.97	2.94	4.97	98	99.4
3	3	5	4.5	7.5	1.729	1.79	7.46	12.23	4.46	7.23	99.11	96.4

Table 4: Analysis of Robustness

Concentration μg/ml	With different glassware				At different temperature
	Candesartan		Simvastatin		candesartan		Simvastatin
	Mean	% RSD	Mean	% RSD	Mean	% RSD	Mean	% RSD
10 + 6 μg/ml	98.7	0.98	102.27	0.99	97.93	1.04	103.77	1.92

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

The proposed developed method's sensitivity was examined by the LOD and LOQ testing. The LOD for candesartan and Simvastatin were found to be 0.258 μg/ml and 0.278μg/ml, and LOQ 0.784μg/ml and 0.84 μg/ml respectively. From the data, it is clear that this UV approach is extremely sensitive for the analysis of candesartan and Simvastatin.

Robustness^18-23

The robustness of the provided approach indicates a non-significant influence on the absorption level through measurement of the candesartan and Simvastatin solution in 0.1 N HCl using different glassware and temperatures. Table 4 displays the robustness study's findings.

Ruggedness:

As shown in Table 5, by switching analysts, it was seen that the results did not vary significantly, proving the ruggedness of the created procedure.

Table 5: Ruggedness Study

RUGGEDNESS
Conc. (μg/ml)	Candesartan			Simvastatin
Conc. (μg/ml)	Abs.	Mean	% RSD	Abs.	Mean	% RSD
10 + 6	1.453	99.88	0.74	1.404	99.86	1.71

Assay of synthetic mixture:

The created approach was used to examine the physical combination of candesartan and simvastatin with various excipients. The assay values for all of these formulations were determined to be 101.81% for Simvastatin and 95.85% for Candesartan, in accordance with ICH criteria.

CONCLUSION:

For the simultaneous estimation of candesartan and simvastatin using UV-Spectrophotometry, a new approach has been developed. Simvastatin was chosen at a wavelength of 239nm, and candesartan at a wavelength of 262nm. The analytical technique was approved in accordance with ICH standards. In this approach, the linearity value of both the drugs (Candesartan and Simvastatin) were found in the concentration range of 5-17.5μg/mL and 3 – 10.5μg/ml respectively. The correlation coefficient (r2) value was obtained 0.999 for both the drugs. Candesartan and Simvastatin percentage recovery was found to be 99.26 % and 97.48%. For intra and interday precision, percentage RSD value was found to be 0.99 and 1.7 (Intraday) as well as 1.0 and 1.78 (Interday). The intraday and interday relative standard deviation (RSD) values obtained by the proposed method are within 2 % relative standard deviations. The LOD for candesartan and Simvastatin were found to be 0.258μg/ml and 0.278 μg/ml, and LOQ 0.784μg/ml and 0.84μg/ml respectively. Through the study of the candesartan and simvastatin solution in 0.1 NHCl using various glassware, the robustness of the established approach demonstrates a non-significant influence of the absorption level and the developed method's robustness was proven when the results were tested at various temperatures and when the results did not significantly alter when the analyst was switched. From the above study it was concluded that the above developed method for the simultaneous estimation of both the drug Candesartan and Simvastatin is reliable and can be use for future analysis of different formulations.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Department of Pharmacy, Oriental University for their kind support during research studies

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Received on 30.05.2023 Modified on 28.12.2023

Research J. Pharm. and Tech 2024; 17(7):3151-3155.

DOI: 10.52711/0974-360X.2024.00492

Concentration μg/ml	Intraday Precision				Interday Precision
	Candesartan		Simvastatin		Candesartan		Simvastatin
	Mean±SD	%RSD	Mean±SD	%RSD	Mean±SD	%RSD	Mean±SD	%RSD
10 + 6 μg/ml	101.02	0.99	98.44	1.7	99.49	1	99.99	1.78