Int J Pharm Pharm Sci, Vol 7, Issue 11, 360-364Original Article


A RAPID RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTITATIVE ESTIMATION OF EPLERENONE IN TABLETS

D. VIJAY KUMAR, S. SRINIVAS, 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: 11 Feb 2015 Revised and Accepted: 05 Oct 2015


ABSTRACT

Objective: To develop a rapid, sensitive, accurate, precise, linear and rugged Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method and validate as per ICH guidelines for the quantitative estimation of Eplerenone in tablets.

Methods: The optimized method uses a reverse phase column, Waters Symmetry C18 (250 X 4.6 mm; 5μ), a mobile phase of triethylammonium phosphate buffer (pH 2.3):acetonitrile in the proportion of 40:60 v/v, flow rate of 1.0 ml/min and a detection wavelength of 240 nm using a UV detector.

Results: The developed method resulted in Eplerenone eluting at 3.63 min. Eplerenone exhibited linearity in the range 15-45μg/ml. The precision is exemplified by relative standard deviation of 0.34%. Percentage Mean recovery was found to be in the range of 98‐102, during accuracy studies. The limit of detection (LOD) and limit of quantitiation (LOQ) was found to be 39.16μg/ml and 118.66μg/ml respectively.

Conclusion: A sensitive, rapid, accurate, precise, linear and rugged RP-HPLC method was developed and validated for the quantitative estimation of Eplerenone in tablets as per ICH guidelines and hence it can be used for routine analysis in various pharmaceutical industries.

Keywords: RP‐HPLC, 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 d 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.

Fig. 1: Structure of Eplerenone

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]. Literature reveals use of potassium dihydrogen orthophosphate as buffer (pH 3.2) and ammonium acetate buffer (pH 7.0) along with organic modifier as mobile phase for assay methods in formulations using RP-HPLC. As there is no literature reported on using triethyl ammonium phosphate as aqueous media along with organic modifier, we here report a new and a rapid RP-HPLC validated method for the quantitative estimation of Eplerenone in tablets using triethyl ammonium phosphate buffer (pH 2.3) along with acetonitrile as mobile phase.

MATERIALS AND METHODS

Chemicals and reagents

Analytically pure sample of Eplerenone with purities greater than 95% was obtained as the gift sample from Chandra labs, Hyderabad, India and tablet formulation [PLANEP] was procured from Apollo Pharmacy, Hyderabad, India with labelled amount 25 mg of Eplerenone. Acetonitrile (HPLC grade), water (HPLC grade), Triethylamine (AR Grade) and ortho phosphoric acid (AR Grade) were obtained from SD Fine chemicals (Hyderabad, India), 0.45 and 0.22μm Nylon membrane filters were obtained from Spincotech Private Limited, Hyderabad, India.

Instrument

HPLC analysis was performed on Shimadzu LC-20AD Prominence Liquid Chromatograph comprising a LC-20AD pump, Shimadzu SPD-20A Prominence UV-VISIBLE detector and a reverse phase C18 column, Waters Symmetry (250 X 4.6 mm; 5μ). A manually operating Rheodyne injector with 20 μL sample loop was equipped with the HPLC system. The HPLC system was controlled with “Lab solutions lite” software. A double beam UV-visible spectrophotometer (Shimadzu, model UV-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), a sonicator (sonica, model 2200 MH).

Method

Selection of wavelength

Suitable wavelength for the HPLC analysis was determined by recording UV spectrum in the range of 200-400 nm for Eplerenone. Suitable wavelength selected was 240 nm (fig. 2).

Fig. 2: UV spectrum of eplerenone

Chromatographic conditions

The developed method uses a reverse phase C18 column, Waters Symmetry C18 (250 X 4.6 mm; 5μ), mobile phase of triethylammonium phosphate buffer (pH 2.3): acetonitrile in the proportion of 40:60 v/v. The mobile phase was set at a flow rate of 1.0 ml/min and the volume injected was 20μl for every injection. The detection wavelength was set at 240 nm.

Buffer preparation

The buffer solution was prepared by adding 5 ml of triethylamine to 1000 ml of HPLC grade water and later pH was adjusted to 2.3 using 30% v/v of ortho phosphoric acid in water. The buffer was then filtered through 0.45 μm nylon membrane filter.

Mobile phase preparation

The mobile phase was prepared by mixing buffer and acetonitrile in the ratio of 40:60 v/v and later it was sonicated for 10 min for the removal of air bubbles.

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 50 ml ofdiluent (same as mobile phase) and then sonicated for 2 min to dissolve. Later the solution was made up to the mark using the mobile phase. This is considered as the stock standard solution (100µg/ml). From the stock solution, 3 ml was pipetted out and to 10 ml using the mobile phase to get a concentration of 30µg/ml, treated as 100% target concentration.

Preparation of stock and working sample solution

Not less than 20 tablets were weighed and taken into a mortar, crushed and then uniformly mixed. Test stock solution of Eplerenone (500μg/ml) was prepared by transferring weight equivalent to 25 mg of Eplerenone to 40 ml of a mobile phase which is sonicated for 4 min and later made up to 50 ml with the mobile phase. This solution was filtered using 0.22micron syringe filter. From the above stock solution 0.6 ml was pipetted out and made up to 10 ml to get working sample solution equivalent to a concentration of 30µg/ml for Eplerenone, concentration equal to 100% target concentration.

RESULTS AND DISCUSSION

Method development

A Reverse phase HPLC method was developed keeping in mind the system suitability parameters i.e. tailing factor (T), number of theoretical plates (N), runtime and the cost effectiveness. The optimized method developed resulted in the elution of Eplerenone at 3.63 min. Fig. 3 and 4 represent chromatograms of blank solution and the standard solution (30µg/ml) respectively. The total run time is 6 min. System suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (RT), number of theoretical plates (N) and peak Tailing factor (T) were evaluated for injection of the standard at the working concentration. The results are given in table 1.

In order to test the applicability of the developed method to a commercial formulation, PLANEP was chromatographed at working concentration (30µg/ml) and it is shown in fig. 5. The sample peak was identified by comparing the retention time with the standard drug fig. 4. System suitability parameters were within the acceptance limits, ideal for the chromatographed sample. Integration of separated peak area was done and drug concentration was determined by using the peak area concentration relationship obtained in the standardization step. The protocol affords reproducible assay of the drug in the sample ranging between 98 and 102%, which is the standard level in any pharmaceutical quality control.

Fig. 3: Typical chromatogram of blank solution


Table 1: System suitabilitystudies results

Parameters

Eplerenone

Retention time (min)

3.63

Number Of Theoretical plates (N)

5954

Tailing factor (T)

1.82


Fig. 4: Typical chromatogram of the standard solution


Fig. 5: Typical chromatogram for the tablet formulation.

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. RP-HPLC method developed was validated according to International Conference on Harmonization (ICH) guidelines [13] for validation of analytical procedures. The method was validated for the parameters like system suitability, specificity, linearity, accuracy, precision, and ruggedness, limit of detection (LOD) and limit of quantitiation (LOQ).

Specificity

Fig. 3-5 for blank, standard drug solution and sample chromatogram reveal that the peaks obtained in the standard solution and the sample solution at working concentrations are only because of the drug as blank has no peak at the retention time of Eplerenone. Accordingly it can be concluded that, the method developed is said to be specific.

Precision

System precision

Six replicate injections of the standard solution at the working concentration showed % RSD(Relative Standard Deviation) less than 2 concerning peak area for the drug, which indicates the acceptable reproducibility and thereby the precision of the system. System precision results are tabulated in table 2.

Method precision

Method precision was determined by performing assay of sample under the tests of repeatability (Intraday precision) at working concentration.

Repeatability (Intraday precision)

Six consecutive injections of the sample from the same homogeneous mixture at working concentration showed % RSDless than 2 concerning % assay for the drug 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 3).

Linearity

Standard solutions of Eplerenone at different concentrations level (50%, 75%, 100%, 125% and 150%) were prepared. Calibration curve was constructed by plotting the concentration level of drug versus the corresponding peak area. The results show an excellent correlation between peak area and concentration level of drug within the concentration range (15-45µg/ml) for the drug and the results are given in table 4 and fig. 6. The correlation coefficient of Eplerenone is 0.998 and hence the method is said to be linear.

Accuracy

Accuracy was determined by means of recovery experiments, by the determination of % mean recovery of sample 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.

Sensitivity

The sensitivity of measurement of Eplerenone by use of the proposed method was estimated in terms of the limit of quantitation (LOQ) and the limit of detection (LOD). LOQ and LOD were calculated by the use of the equations LOD = 3.3s/S and LOQ = 10s/S where s is the standard deviation of response of calibration plot and S is the slope of the corresponding calibration plot. The limit of detection (LOD) and limit of quantitiation (LOQ) was found to be 39.16μg/ml and 118.66μg/ml respectively.

Table 2: System precision results of Eplerenone

Injection no. (n)

RT

Peak Area

1

3.631

1728619

2

3.642

1712266

3

3.634

1724394

4

3.634

1722646

5

3.648

1720596

Average

3.637

1721704

Standard Deviation

0.007

6046.5

%RSD

0.192

0.351


Table 3: Intraday precision results of Eplerenone

n

RT

Peak area

%Assay

1

3.632

1746464

101.43

2

3.624

1751581

101.73

3

3.628

1743313

101.25

4

3.632

1755407

101.95

5

3.631

1752102

101.76

6

3.628

1739400

101.02

Average

3.628

1755378

101.52

Standard Deviation

0.0028

12136.2

0.35

%RSD

0.077

0.691

0.344


Table 4: Calibration data for Eplerenone

% Level

Concentration (mg/ml)

Peak area

50

15

838943

75

22.5

1221643

100

30

1631265

125

37.5

2059254

150

45

2525331

Regression coefficient

0.998

Regression equation

y=56138.5x-28868


Fig. 6: Linearity graph of Eplerenone


Table 5: Results of Accuracy studies for Eplerenone

% Level

Peak area

% Recovery

% Mean recovery

%RSD

50

873273

862248

870870

101.443

100.162

101.164

100.92

0.66

100

1751581

1743313

1755407

101.735

101.255

101.958

101.649

0.35

150

2538435

2553430

2541449

98.281

98.862

98.398

98.51

0.31


Table 6: Ruggedness results of Eplerenone

n

RT

Peak area

%Assay

1

3.627

1740565

101.095

2

3.629

1736765

100.875

3

3.637

1745625

101.389

4

3.641

1735499

100.801

5

3.641

1740967

101.119

6

3.641

1750437

101.669

Average

3.636

1741643

101.15

Standard deviation

0.005

5104.79

0.322

%RSD

0.161

0.293

0.318


Ruggedness

Ruggedness was evaluated by performing assay of the formulations by different analyst by injecting six consecutive injections of the sample at the working concentration from the same homogeneous mixture of tablets. This study showed % RSDless than 2 concerning % assay for both the drugs which indicate that the method developed is rugged and hence can be understood that the method gives reproducible results irrespective of analyst (table 6).

CONCLUSION

A reverse phase HPLC isocratic method developed has been validated as per ICH guidelines in terms of specificity, accuracy, precision, linearity, and ruggedness, limit of detection and limit of quantitation, for the quantitative estimation of Eplerenone in tablets. The precision is exemplified by relative standard deviation of 0.34%. A good linear relationship was observed for the drug between concentration ranges of 15 and 45µg/ml. Accuracy studies revealed that mean recoveries were between 98 and 102%, an indicative of accurate method. The limit of detection (LOD) and limit of quantitiation (LOQ) was found to be 39.16μg/ml and 118.66μg/ml respectively. Accordingly it can be concluded that the developed reverse phase isocratic HPLC method is sensitive, accurate, precise, linear and rugged and therefore the method can be used for the routine analysis of Eplerenone in tablets.

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 gift sample of Eplerenone.

REFERENCES

  1. Brajesh M, Naina K, Shashank S. Quantitative determination of eplerenone in bulk drug and tablet dosage form by TLC/densitometry. Int J Pharm Life Sci 2011;2;502-5.
  2. Charles T, Stier Jr. Eplerenone: a selective aldosterone blocker. Cardiovasc Drug Rev 2003;21:169-84.
  3. Naresh Chandra Reddy M, Chandra sekhar KB. Development and validation of gradient RP-HPLC for estimation of impurities in Eplerenone tablet dosage form. Int Res J Pharm App Sci 2012;2:58-75.
  4. Weinberger MH, Roniker B, Krause SL, Weiss RJ. Eplerenone, a selective Aldosterone blocker, in mild-to-moderate hypertension. Am J Hypertens 2002;15:709-16.
  5. White WB, Duprez D, St Hillaire R, Krause S, Roniker B, Kuse-Hamilton J, et al. Effects of the selective aldosterone blocker Eplerenone versus the calcium antagonist Amlodipine in systolic hypertension. Hypertension 2003;41:1021-6.
  6. Moore TD, Nawarskas JJ, Anderson JR. Eplerenone: a selective aldosterone receptor antagonist for hypertension and heart failure. Heart Dis 2003;5:354-63.
  7. Chatterjee S, Moeller C, Shah N, Bolorunduro O, Lichstein E, Moskovits N, et al. Eplerenone is not superior to older and less expensive aldosterone antagonists. Am J Med 2011;125:817-25.
  8. Zhang JY, Fast DM, Breau AP. Development and validation of a liquid chromatography-tandem mass spectrometric assay for Eplerenone and its hydrolyzed metabolite in human plasma. J Chromatogr B: Anal Technol Biomed Life Sci 2003;25;787:333-44.
  9. Zhang JiY, Douglas MF, Breau AP. A validated SPE–LC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine. J Pharm Biomed Anal 2003;31:103-15.
  10. Naina K, Brajesh M, Shashank S. Development and validation of UV-Spectrophotometric determination of eplerenone in bulk and tablets. Int J Pharm Life Sci 2010;1:231-3.
  11. Banode VS, Khedekar PB, Tarte PS. Spectrophotometric estimation of Eplerenone in bulk drug and tablets. Int J Chem Tech Res 2011;3:398-402.
  12. Rane K, Patil R, Sangshetti JN, Yeole RD, Shinde DB. Stability-indicating RP-HPLC method for analysis of Eplerenone in the bulk drug and in a pharmaceutical dosage form. Acta Chromatogr 2009;21:619-9.
  13. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use. Validation of Analytical Procedures: Text and Methodology ICH Q2 (R1); 2005.


About this article

Title

A RAPID RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTITATIVE ESTIMATION OF EPLERENONE IN TABLETS

Keywords

RP‐HPLC, Eplerenone, Method development, Validation

Date

05-10-2015

Additional Links

Manuscript Submission

Journal

International Journal of Pharmacy and Pharmaceutical Sciences
Vol 7, Issue 11, 2015 Page: 360-364

Online ISSN

0975-1491

Statistics

217 Views | Downloads

Authors & Affiliations

D. Vijay Kumar
Vijaya College of Pharmacy
India

S. Srinivas
Vijaya College of Pharmacy
India

A. Ashok Kumar
Department of Pharmaceutical Analysis and Quality Assurance, Vijaya College of Pharmacy, Munaganur (Village), Hayathnagar (Mandal), Hyderabad 501511, India
India


Article Tools



Refbacks

  • There are currently no refbacks.