Int J Pharm Pharm Sci, Vol 7, Issue 9, 327-330Original Article
A RAPID ASSAY METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF EPLERENONE IN TABLETS BY UV SPECTROPHOTOMETRY
B. SHAILAJA, K. SWARNA, M. AFREEN, A. ASHOK KUMAR*
Department of Pharmaceutical analysis and Quality Assurance, Vijaya college of pharmacy, Munaganur (village), Hayathnagar (mandal), Hyderabad 501511, India
Email: ashok576@gmail.com
Received: 28 Apr 2015 Revised and Accepted: 22 Jul 2015
ABSTRACT
Objective: To develop a rapid, simple and a cheap UV spectrophotometric assay method for the estimation of Eplerenone in tablets and validate as per ICH guidelines.
Methods: The optimized method uses 0.05N HCl as a solvent for the estimation of assay of Eplerenone in tablets at a detection wavelength of 245 nm.
Results: System precision and intra-day precision are exemplified by relative standard deviation of 1.5% and 1.3% respectively. Method was found to be rugged as precision was found to be 1.2%. The developed method resulted in Eplerenone exhibiting linearity in the range 5-15μg/ml. Percentage Mean recovery was found to be in the range of 98‐102, during accuracy studies.
Conclusion: A rapid, simple and a cheap UV spectrophotometric assay method was developed and validated for the estimation of Eplerenone in tablets as per ICH guidelines and hence it can be used for the routine analysis in various pharmaceutical industries.
Keywords: UV, Eplerenone, Method development, Validation.
INTRODUCTION
Eplerenone (fig. 1) is pregen-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3-oxo,γ-lactone, methyl ester (7α,11α,17α)[1,3]. It has a molecular formula of C24H30O6 and a molecular mass of 414.49. Eplerenone is the first highly selective aldosterone receptor antagonist (SARA) to effectively block aldosterone at receptor sites in body tissues, aldosterone being a component of rennin-angiotensin-aldosterone system [1-6]. Eplerenone is used for treatment of hypertension and heart failure [1-6].
Eplerenone is specifically indicated for the reduction of risk of cardiovascular death in people with heart failure and left ventricular dysfunction within 3–14 days of an acute myocardial infarction, in combination with standard therapies and as treatment against hypertension. It appears equivalent to spironolactone but is much more expensive [7]. It is marketed by Pfizer under the trade name Inspra. Eplerenone is a potassium-sparing diuretic, meaning that it helps the body get rid of water but still keep potassium.
Few analytical methods have been reported for the determination of Eplerenone in biological fluids by LCMS [8-9], in bulk and formulations by UV Spectroscopy [10-11], TLC/Densitometry [1] and RP-HPLC [3,12]. There exists two UV assay methods reported [10-11] using methanol (30%) and methanol (70%) in water as solvent respectively. Both the UV methods are costlier due to use of organic solvent methanol.
Fig. 1: Structure of eplerenone
Hence, we here report a simple and a cheap UV spectrometric assay method for the estimation of Eplerenone in tablets using aqueous solution of potassium dihydrogen orthophosphate buffer (pH 2.0).
MATERIALS AND METHODS
Materials
Instrument
A double beam UV-visible spectrophotometer (Shimadzu, model 1800) having two matched quartz cells with 1 cm light path and loaded with UV probe software (version 2.41) was used for recording of spectra and measuring absorbance.
An electronic analytical weighing balance (0.1 mg sensitivity, Shimadzu AY 220), digital pH meter (DELUX model 101) and a sonicator (sonica, model 2200 MH) were used in this study.
Chemicals and reagents
Analytically pure sample of Eplerenone with purities greater than 99% was obtained as gift sample from Chandra labs, Hyderabad, India and tablet formulation [PLANEP] was procured from Medplus pharmacy, Hyderabad, India with labelled claim of 25 mg. Concentrated HCl was obtained from SD Fine chemicals (Hyderabad, India).
Method
Selection of suitable detection wavelength
Suitable wavelength for the total experiment was determined by recording UV spectrum in the range of 200-400 nm for Eplerenone in 0.05N HCl as solvent and suitable wavelength selected was 245 nm (fig. 2).
Preparation of stock and working standard solution
10 mg of Eplerenone was accurately weighed and taken in 100 ml clean and dry volumetric flask containing 80 ml of solvent and then the solution was made up to the mark using the solvent. This is considered as the standard stock solution (100mg/ml). 1 ml of the stock solution was pipetted out and made up to 10 ml to get a concentration 10µg/ml, treated as the working standard, 100% target concentration.
Preparation of stock and working sample solution
Not less than 20 tablets were taken and grinded using pestle and mortar. Test stock solution of 500μg/ml was prepared by transferring weight equivalent to 25 mg of Eplerenone to 80 ml of solvent which is sonicated and shaked intermittently for 5 min and later made up to 100 ml with solvent. This solution was filtered using 0.22micron syringe filter. From the above stock solution 0.4 ml was pipetted out and made up to 10 ml to get working sample solution concentration equivalent to 10µg/ml, 100% target concentration.
Fig. 2: UV spectrum of standard
RESULTS AND DISCUSSION
Method Development
Various solvents were explored including water, potassium dihydrogen orthophosphate solution at various pH’s, Hydrochloric acid at 0.1N and 0.05N and sodium hydroxide at 0.1N and 0.05N. Eplerenone was found to be soluble and stable for the minimum of 1 hour at room temperature using 0.05N Hydrochloric acid and hence this solvent was initiated for the determination of suitable detection wavelength and working concentration of standard. In order to test the applicability of the developed method to a commercial formulation, assay of PLANEP tablets were studied at working concentration. Assay for working concentration of the sample at 245 nm was in acceptance limits (98-102%) using the solvent via intermittent shaking and sonication method for 5 minutes fig. 3 illustrates UV spectrum for the sample. Hence the method is optimized.
Method validation
Validation of the analytical method is the process that establishes by laboratory studies in which the performance characteristics of the method meet the requirements for the intended analytical application. UV spectrophotometric method developed was validated according to International Conference on Harmonization (ICH) guidelines [13]. The method was validated for the parameters like linearity, accuracy, system precision, intra-day precision and inter-day precision/intermediate precision/ruggedness.
Fig. 3: UV spectrum of sample
Precision
System precision
Six replicate recording of absorbances at 245 nm of the standard solution at the working concentration showed % RSD (Relative Standard Deviation) less than 2, which indicates acceptable reproducibility and thereby the precision of the system. System precision results are tabulated in table 1.
Method precision
Method precision was determined by performing the assay of the sample under the tests of (i) repeatability (Intra day precision) and (ii) Intermediate precision (Inter day precision or ruggedness) performed during 2 consecutive days by two different analysts, at working concentration.
Table 1: System precision results of Eplerenone
n |
Absorbance |
1 |
0.392 |
2 |
0.374 |
3 |
0.383 |
4 |
0.380 |
5 |
0.384 |
Average |
0.382 |
SD |
0.006 |
%RSD |
1.5 |
Repeatability (Intra day precision)
Six consecutive recording of absorbance at 245 nm of the sample from the same homogeneous mixture at working concentration showed % RSD less than 2 concerning % assay, which indicate that the method developed is method precise by the test of repeatability and hence can be understood that the method gives consistently reproducible results (table 2).
Table 2: Intraday precision results of Eplerenone
n |
Sample absorbance |
% Assay |
1 |
0.378 |
99 |
2 |
0.379 |
99.2 |
3 |
0.39 |
102 |
4 |
0.379 |
99.2 |
5 |
0.384 |
100 |
6 |
0.39 |
102 |
Average |
100.2 |
|
SD |
0.005 |
|
% RSD |
1.3 |
Intermediate Precision (Inter day precision/Ruggedness)
Assay precision between two consecutive days performed by different analysts of the sample showed % RSD less than 2, which indicate the method developed is inter day precise/rugged (table 3).
Table 3: Inter day precision results
n |
Analyst 1 |
Analyst 2 |
1 |
99 |
98 |
2 |
99.2 |
99.2 |
3 |
102 |
100 |
4 |
99.2 |
101 |
5 |
100 |
101.2 |
6 |
102 |
101.4 |
Average |
100.2 |
100.1 |
SD |
0.005 |
0.005 |
%RSD |
1.3 |
1.2 |
Linearity
Standard solutions of Eplerenone at different concentrations level (50%, 75%, 100%, 125% and 150%) were prepared. Calibration curve (fig. 4) was constructed by plotting the concentration level of drug versus absorbance at 245 nm.
The results show an excellent correlation between absorbance and concentration level of drug within the concentration range (5-15µg/ml) for the drug (table 4). The correlation coefficient was greater than 0.995, which meet the method validation acceptance criteria and hence the method is said to be linear in the range of 5-15µg/ml.
Table 4: Calibration data for Eplerenone
% Level |
Concentration (mg/ml) |
Absorbance |
50 |
5 |
0.235 |
75 |
7.5 |
0.358 |
100 |
10 |
0.462 |
125 |
12.5 |
0.56 |
150 |
15 |
0.68 |
Regression equation: y=0.04368x+0.0222 |
||
Regression coefficient: 0.9984 |
||
Table 5: Results of accuracy studies for Eplerenone
% Level |
Absorbance |
% Recovery |
% Mean recovery |
%RSD |
50 |
0.211 |
99.9 |
||
50 |
0.221 |
102.5 |
101.5 |
1.8 |
50 |
0.201 |
102.3 |
||
100 |
0.378 |
101.4 |
||
100 |
0.379 |
101.7 |
101.7 |
1 |
100 |
0.384 |
102 |
||
150 |
0.622 |
102 |
||
150 |
0.638 |
101.9 |
101.5 |
1.2 |
150 |
0.634 |
100.7 |
Table 6: Optical characteristics and validation parameters of Eplerenone
Parameters |
Results |
Detection wavelength (nm) |
245 |
Beer’s Law limits (mg/ml) |
5-15 |
Regression equation (y = mx+c) |
y=0.04368x+0.0222 |
Correlation coefficient (r2) |
0.9984 |
Slope (m) |
0.04368 |
Intercept (c) |
0.0222 |
% Relative Standard Deviation (% RSD) System precision |
1.5 |
(% RSD) Intra-day precision |
1.3 |
(% RSD) Inter-day precision |
1.2 |
Accuracy (% Mean Recovery) |
|
50 % Level |
101.57 |
100 % Level |
101.7 |
150 % Level |
101.5 |
Fig. 4: Linearity graph of Eplereone
Accuracy
Accuracy was determined by means of recovery experiments, by the determination of % mean recovery of sample by percentage method at three different levels (50-150%). At each level, three determinations were performed. Percent mean recovery was calculated as shown in table 5. The accepted limits of recovery are 98%-102% and all observed data are within the required range which indicates good recovery values and hence the accuracy of the method developed.
CONCLUSION
A simple UV spectrophotometric method was developed and validated for the quantitative estimation of Eplerenone in capsules as per ICH guidelines. The optimized method uses 0.05N HCl as a solvent for the estimation of assay of Eplerenone in tablets at a detection wavelength of 245 nm. System precision and intra-day precision are exemplified by relative standard deviation of 1.5% and 1.3% respectively. Method was found to be rugged as precision was found to be 1.2%. The developed method resulted in Eplerenone exhibiting linearity in the range 5-15μg/ml. Percentage Mean recovery was found to be in the range of 98‐102, during accuracy studies. Accordingly it is concluded that the developed UV spectrophotometric assay method is rapid, cheap, simple, accurate, precise, linear and rugged and therefore the method can be used for the routine analysis of Eplerenone in tablets in various pharmaceutical industries.
ACKNOWLEDGEMENT
The authors would like to thank the management of Vijaya College of pharmacy (VJYH), Hyderabad, for providing the necessary facilities to carry out of this research work. The authors are grateful to Chandra labs, Hyderabad for providing drug in form of gift sample.
CONFLICT OF INTERESTS
Declared None
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