Chandrakanth.Vadloori et al.

/ Journal of Pharmacy Research 2012,5(8),3998-4000

Research Article Available online through
ISSN: 0974-6943 http://jprsolutions.info
Development and Validation of RP-HPLC Method for Simultaneous Estimation of
Spironolactone and Frusemide in Bulk and Pharmaceutical Dosage Forms
Chandrakanth.Vadloori*,Venkat Tallada**.
Department of Pharmaceutical Analysis ,vikas college of pharmacy, (V)Rayanigudem, (M)Suryapet, Nalgonda(District),A.P,508376, India
Received on:09-05-2012; Revised on: 14-06-2012; Accepted on:22-07-2012

ABSTRACT
A simple, precise, specific and accurate Reverse phase HPLC method has been developed for the determination of Spironolactone And Frusemide in bulk
and pharmaceutical dosage forms. Chromatography was performed on a Azilent Zobax Rx C8 column. (4.6 x 150mm, 5 ) , with potassium dihydrogen
phosphate buffer (pH 7.51) and methanol in the ratio of 60:40 v/v as a mobile phase at a flow rate of 1.0 ml/ min. Detection was performed at 215nm. The
retention time of Spironolactone and frusemide was found to be 2.391 min and4.602. By adoption of this procedure is Spironolactone And Frusemide
eluted completely. Linear calibration plots for Spironolactone and Frusemide were obtained between 250-750g/ml and 100-300g/ml. The method of
analysis was used for quantification Spironolactone And Frusemide for in pharmaceutical preparations with a coefficient of variation < 2%. Results of
analysis were validated statistically and by recovery studies. The method was validated according to the ICH guidelines with respect to specificity,
linearity, accuracy, precision, ruggedness and robustness.

Key words: Spironolactone, Frusemide, potassium dihydrogen phosphate, Methanol, Coefficient variation.

INTRODUCTION
Spironolactone is chemically 17-hydroxy-7-mercapto-3-oxo-17-pregn-4- calcium, and chloride ions, and enhancing water excretion. Therapeutic uses
ene-21-carboxylic acid lactone-7-acetate and the structural formula is shown include treatments for hypertension, severe hypercalcemia, and edema. From
in Fig: 1. The molecular formula is C22H32O4S and molecular weight is the literature survey, it was found that Spironolactone and Frusemide was
416.573g/mol. It stable at room temperature, practically insoluble in water, estimated by analytical methods such as few UV-Visible methods high-
soluble in alcohol and freely soluble in benzene and chloroform. performance liquid chromatographic (HPLC) method and gas chromatogra-
Spironolactone is a potassium-sparing diuretic (water pill) that prevents phy. The present developed method was simple, precise, specific and accu-
your body from absorbing too much salt and keeps your potassium levels rate.
from getting too low. Spironolactone inhibits the effect of aldosterone by
competing for intracellular aldosterone receptors in the distal tubule cells. EXPERIMENTAL

MATERIALS AND METHODS

For HPLC, Waters 2690 series model equipped with Auto sample,
PDA(2998) detector with Empower 2 software was employed for the in-
vestigation. All the chemicals used in the investigation were of HPLC grade.
The chromatographic analysis was performed on a Azilent Zobax Rx C8
column. (4.6 x 150mm, 5 ). The mobile phase consists of potassium
dihydrogen phosphate buffer (pH 7.51) and methanol in the ratio of 60:40
v/v. The optimized chromatographic conditions are summarized in Table 1.
Fig: 1. Chemical Structure of Fig: 2. Chemical Structure of
Spironolactone. Preparation of mobile phase
Frusemide.
400 ml of HPLC grade methanol was mixed with 600 ml of potassium
Frusemide is chemically 4-chloro-2-(furan-2-ylmethylamino)- 5- dihydrogen phosphate buffer prepared in double distilled water and its pH
sulfamoylbenzoic acid.and structural formula shown in Fig:2 The molecular was adjusted to 7.51 using 0.1 sodiumhydroxide. Then it was ultra soni-
formula is C H ClN O S And molecular weight is 330.745g/mol. This cated for 20 minutes and then filtered through 0.4m membrane filter paper.
Frusemode is12soluble
11 2 5
in acetone , methanol , DMSO, and alkali hydroxides.
It is slightly soluble in ethanol. Furosemide is practically insoluble in water. Preparation of standard stock solution
Furosemide is a potent diruetic with rapid action. It acts to inhibit electro- 50 mg of Spironolactone And 20 mg of Frusemide standard drug was weighed
lyte absorption in the kidney, increasing excretion of sodium, potassium, accurately and transferred to 100 ml volumetric flask. The drug was dis-
solved in 50 ml of methanol and sonicate for 20 mins then volume was made
*Corresponding author. up to the mark with methanol.. The concentration of Spironolactone And
V.Chandrakanth, Frusemide was found 500 g/ml and 200?g/ml
M.pharm(IInd year),
Pharmaceutical Analysis Preparation of marketed formulations
and Quality assurance, Twenty tablets are (Aldostix) were weighed crushed into fine powder . the
Vikas college of pharmacy, tablet powder equailent to 50 mg of Spironolactone and 20 mg of Frusemide
(V)Rayanigudem-508376 (M)Suryapet, was transferred to 10 ml volumetric flask And add 5ml methanol sonicated
Nalgonda(District),A.P

Journal of Pharmacy Research Vol.5 Issue 8.August 2012 3998-4000

 Chandrakanth.Vadloori et al. / Journal of Pharmacy Research 2012,5(8),3998-4000
for 10 mins Finally volume make up with methanol. The solution filter Appropriate aliquots were pipetted out from the standard stock solution
through 0.4 m membrane filter paper. and used for calibration graphs to (Frusemide)(200g/ml) into a series of 10 ml volumetric flasks. The volume
give a final concentration within the range of linearity. The solution was was made up to the mark with the mobile phase to get a set of solutions
analyzed for the estimation of drug by proposed method. having the concentration, ranging from 100-300g/ml of the drug. The solu-
tions were injected using a 5l fixed loop into the chromatographic system
Chromatographic condition at the flow rate of 1.0 ml / min and the effluents were monitored at 215 nm,
The mobile phase containing potassium dihydrogen phosphate buffer (pH chromatograms were recorded. The Frusemide was eluted at 4.602 mi .
7.51) and methanol in the ratio of 60:40 v/v was selected as the optimum shown in Fig: 3.The calibration curve was constructed by plotting average
composition of mobile phase, as this solvent system resolved for the com- peak area versus concentration and was presented in Fig: 5.
ponent ideally. The flow rate was set to 1.0 ml/min and UV detection was
carried out at 215 nm. The mobile phase and sample was degassed by
sonication for 20 min and filtered through 0.4 m membrane filter paper. All
determinations were performed at constant column temperature (300C).

Preparation of Calibration curve and Analysis of Spironolactone And

Frusemide
Appropriate aliquots were pipetted out from the standard stock solution
(Spironolactone) (500 g/ml) into a series of 10 ml volumetric flasks. The
volume was made up to the mark with the mobile phase to get a set of
solutions having the concentration, ranging from 250-750g/ml of the drug.
The solutions were injected using a 5l fixed loop into the chromatographic
system at the flow rate of 1.0 ml / min and the effluents were monitored at
215 nm, chromatograms were recorded. The Spironolactone was eluted at
2.391 min as shown in Fig: 3.
Fig: 5. Calibration curve of Frusemide at 215 nm by RP-HPLC method.

Analysis of Spironolactone And Frusemide in Pharmaceutical for-

mulations
The procedure for the preparation of the sample solution remains same as
explained above. From this stock solution, various dilutions of the sample
solution were prepared and analysed. A 5l volume of each sample solution
was injected into the sample injector of HPLC system and their chromato-
grams were recorded under the same chromatographic conditions as de-
scribed above. The area of each peak was determined at 215 nm and the
amount of drug present in the sample was determined.

Method validation
The developed analytical method was subjected to validation with respect
to various parameters such as accuracy, precision, linearity and range, ro-
bustness, ruggedness, LOD and LOQ as per the ICH guidelines.
Fig: 3. Chromatogram of Spironolactone and Frusemide by RP-HPLC method.
The calibration curve was constructed by plotting average peak area versus RESULTS AND DISCUSSION
concentration and was presented in Fig: 4. In this method, the conditions were optimized to obtain complete elution
of Spironolactone and Frusemide. Mobile phase and flow rate selection
was based on peak parameters (height, tailing factor and theoretical plates),
run time, resolution. The system with potassium dihydrogen phosphate
buffer (pH 7.51) and methanol in the ratio of 60:40v/v.

The run time was set at 7 min and the retention time for Spironolactone and
frusemide was found 2.391 and 4.602 and min as shown in Fig: 3.The
sample solution was injected 6 times and the retention times were found to
be same. When the concentrations of Spironolactone and Frusemide and its
respective peak areas were subjected to regression analysis, a good linear
relationship (r 2 =0.9995) was observed between the concentration of
Spironolactone and Frusemide and the respective peak areas in the range
250-750?g/ml and 100-300 ?g/ml. The regression of Spironolactone was
found to be Y = 125268X + 32857, where Y is the peak area and X is the
concentration of Spironolactone).The regression of Frusemide was found
to be Y = 21259X+11469.4, where Y is the peak area and X is the
concentration of Frusemide.

Fig: 4. Calibration curve of Spironolactone at 215 nm by RP-HPLC method. The regression equation was used to estimate the amount of Spironolactone

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 Chandrakanth.Vadloori et al. / Journal of Pharmacy Research 2012,5(8),3998-4000
Table 1: Optimized chromatographic conditions for the proposed method was validated in accordance with ICH parameters and applied for
method for Spironolactone and Frusemide. analysis of the same in marketed formulations.
Parameters Optimized condition

Linear range (g/ml) 250-750 g/ml and 100-300 g/ml

CONCLUSION
Detection wavelength (nm) 215 Thus, it can be concluded that the method developed in the present inves-
Temperature 30 tigation was economical, simple, sensitive, accurate, robust, rapid and pre-
Retention Time (t) (min) 2.391 and 4.602
Run time (min) 7 mins cise. Hence, the above said method can be successfully applied for the
Limit of Detection (g/ml) 0.0002 and 0.0007 estimation of Spironolactone and Frusemide in pharmaceutical dosage forms.
Limit of Quantification (g/ml) 0.0006 and 0.0024
ACKNOWLEDGEMENT
Table 2: System suitability test parameters for the proposed method
We would like to thank Aristopharma,Limited Dhaka, India for providing
for Spironolactone and Frusemide.
reference sample of Spironolactone and Frusemide to facilitate this work.
Parameters Optimized condition

Retention Time (t) (min) 2.391 and 4.602 REFERENCES

Theoretical plates (N) 4025 and 6424 1. Indian Pharmacopiea vol 1, government of india, ministry of wealth
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2. Ram Vijay R, Dave Pragnesh N, Joshi Hitendra S, develop Simul-
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Parameters Optimized condition Formulation.
Linearity range (g/ml) 250-750 g/ml and 100-300g/ml 3. Ram VR, Dave PN, Joshi HS HPLC Assay Method for Simulta-
Regression equation (Y=mx +c) Y = 125268X + 32857& neous Determination of Spironolactone and Furosemide in Tablet
Y = 21259X+11469.4
Slope (m) 125268 and 21259 Formulation.
Intercept (c) 32857 and 11469.4 4. British Pharmacopiea 1998 stationary office books (TSO) pp
Correlation coefficient (r2) 0.9995 and 0.9998 798-800
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Table 4: Summary of validation parameters for the proposed method Method for Simultaneous Determination of Spironolactone and
Furosemide in Tablet Formulation
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Method Retention Time Tailing factor 7. Millership JS et al; reported Ratio spectra derivative spectro-
Parameters
Flow rate (ml/min) Spironolactone Frusemide Spironolactone Frusemide photometry for the determination of Furosemide and Spironolac-
tone in a capsule formulation
0.9 3.181 6.098 1.5 1.4 8. Carda-Broch S et al; reported the determination of Furosemide in
1.0 2.391 4.602 1.5 1.3 urine samples by direct injection in a micellar liquid chromato-
graphic system
1.1 1.922 3.753 1.5 1.3
9. Nagori BP et al; reported the RP-HPLC method for simultaneous
and Frusemide , either in formulations or in validation study (precision and estimation of Furosemide and Amiloride hydrochloride in tablet
accuracy) ruggedness of the proposed method was determined by analysis formulations
of sample by changes in different parameter like flow rate, and analyst 10. Dinc E et al; reported Spectophotometric quantitative resolution
using similar operational and environmental conditions. The proposed of hydrochlorothiazide and Spironolactone in tablets by
chemometric analysis methods.
Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 8.August 2012 3998-4000