ISSN: 23207205

Novus International Journal of

Chemistry
2013, Vol. 2, No. 1
www.novusscientia.org

Accepted on: March 14, 2013

Simultaneous estimation of Spironolactone and Hydroflumethiazide in

pharmaceutical dosage form by second order derivative
UV- spectrophotometry method
Laveena. R. Khanchandani*, Kashyap. K. Bhatt, Dimal A. Shah, Usmangani. K. Chhalotiya
Department of Pharmaceutical Chemistry, Indukaka Ipcowala College of Pharmacy,
Gujarat Technological University, New V.V.Nagar-388121, Gujarat, India

ABSTRACT
A simple, specific, accurate and precise Second order derivative Spectroscopy method was
developed and validated for the estimation of Spironolactone and Hydroflumethiazide in
pharmaceutical dosage form. Spironolactone and Hydroflumethiazide has the absorbance maxima
at 238 nm and 273nm respectively. Beer's law was found to be obeyed in the concentration range of
4-24 g /ml for Spironolactone and Hydroflumethiazide. The method was validated for linearity,
accuracy, precision, limit of detection, limit of quantitation and robustness. The method was found
to be specific, precise and accurate, can be successfully applied for the routine analysis of
Spironolactone and Hydroflumethiazide in bulk, and combined dosage form without any
interference by the excipients. The method was validated according to ICH guidelines.
KEYWORDS: Spironolactone, Hydroflumethiazide, Second order derivative, Spectroscopy
method
*Corresponding author: Laveena. R. Khanchandani
Department of Pharmaceutical Chemistry,
Indukaka Ipcowala College of Pharmacy,
Gujarat Technological University,
New V.V.Nagar-388121, Gujarat, India
Email:klaveena13@gmail.com
INTRODUCTION
Spironolactone (SPI) is chemically 7a-Acetylsulfanyl-3-oxo-17a-pregn-4-ene-21,17
bcarbolactone and the structural formula is shown in Fig: 1. The molecular formula is
C22H32O4S and molecular weight is 416.573g/mol. It stable at room temperature, practically
insoluble in water,soluble in alcohol and freely soluble in benzene and chloroform.
Spironolactone is a potassium-sparing diuretic (water pill) that prevents the body from
absorbing too much salt and keeps the potassium levels from getting too low. Spironolactone
inhibits the effect of aldosterone by competing for intracellular aldosterone receptors in the
distal tubule cells[1-2] .Hydroflumethiazide(HFT) is chemically 1,1-Dioxo-6-(trifluoromethyl)-
3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide and the structural formula is shown in
Fig:2. The molecular formula is C8H8F3N3O4S2 and molecular weight is 331.3g/mol. It is
stable at room temperature, practically insoluble in water, soluble in ethanol (96%);
practically insoluble in ether. Hydroflumethiazide is an oral thiazide used to treat
hypertension and edema. Like other thiazides, hydroflumethiazide promotes water loss from
the body (diuretics).

Novus International Journal of Chemistry 2013, 2(1) 7

 Figure: 1 Structure of Spironolactone

As a diuretic, hydroflumethiazide inhibits active chloride reabsorption at the early distal

tubule via the Na-Cl cotransporter, resulting in an increase in the excretion of sodium,
chloride, and water. Thiazides like hydroflumethiazide also inhibit sodium ion transport
across the renal tubular epithelium through binding to the thiazide sensitive sodium-chloride
transporter. This results in an increase in potassium excretion via the sodium-potassium
exchange mechanism. The antihypertensive mechanism of hydroflumethiazide is less well
understood although it may be mediated through its action on carbonic anhydrases in the
smooth muscle or through its action on the large-conductance calcium-activated potassium
(KCa) channel, also found in the smooth muscle. [1-2]

Figure: 2 Structure of Hydroflumethiazide

Literature survey revealed that a number of methods have been reported for estimation of SPI
and HFT individually or in combination with other drugs. [3-11] However, there is no
Derivative Spectroscopy method reported for the simultaneous estimation of SPI and HFT in
a combined dosage formulation. Present work describes simple, accurate, reproducible and
economical method for simultaneous estimation of spironolactone and hydroflumethiazide in
tablet formulation.
MATERIALS AND METHODS
Instrument
A double-beam Shimadzu UV/Vis spectrophotometer 1700, Pharma spec, wavelength
accuracy of 0.5 nm and a pair of 1-cm matched quartz cells, was used to measure
absorbance of the resulting solution. All weighing were done on electronic balance (Model
Shimadzu BL-220H).
Chemicals and reagents
AR grade of methanol was used.
A gift sample of Spironolactone was obtained from RPG Life Sciences, Ankleshwar, Gujarat,
India and Hydroflumethiazide purchased from SIGMA-ALDRICH Co., 3050 Spruce Street,
St.Louis, MO 63103 USA. Spironolactone and Hydroflumethiazide combination tablets

Novus International Journal of Chemistry 2013, 2(1) 8

(ALDACTIDE, 25 mg Spironolactone and 25 mg Hydroflumethiazide; is manufactured by
Pfizer Limited,UK), were procured from the KATZ DRUGS, NEW YORK, USA.
Preparation of standard stock solutions (1000 g/ml)
SPI and HFT (10mg each) were weighed separately and dissolved in separate 10ml
volumetric flasks each, add few ml of methanol. The volume was made up to the mark with
methanol to obtain stock solution (1000 g/ml) of each drug.
Selection of wavelengths for estimation of SPI and HFT
Working standard solutions of SPI and HFT were diluted appropriately with methanol to
obtain solution containing 10 g/ ml of SPI and 10 g/ ml of HFT. Spectra of above solutions
were scanned in the spectrum mode between 400- 200 nm, with a bandwidth of 2 nm. These
zero order spectra of SPI and HFT were treated to obtain corresponding second order
derivative spectra with an interpoint distance of 8 nm and scaling factor of 100.
The second order derivative spectra were overlapped using memory channels. The Zero
crossing point (ZCPs) values of SPI at which the HFT showed derivative response were
recorded. The wavelength 287.72nm was selected for the quantification of SPI (where the
derivative response for was HFT zero). Similarly, 261.61 nm was selected for quantification
of HFT (where the derivative response for SPI was zero). Characteristic wavelengths (ZCPs)
for SPI and HFT were confirmed by varying the concentration of both the drugs.
Preparation of Calibration curve of SPI and HFT
The standard stock solution (1000 g/ml) of SPI and HFT were appropriately diluted with
methanol to obtain a series of solution (4, 8, 12, 16, 20, 24 g/ml) and (4, 8, 12, 16, 20, 24
g/ml) of SPI and HFT respectively. The standard solution of both drug were scanned in the
spectrum mode from 400 nm to 200 nm against solvent methanol as blank and spectra was
recorded.

Figure: 3 Overlay second derivative spectra of Spironolacotone & Hydroflumethiazide

Procedure
For the purpose of spectral analysis in order to relate chemical structure to electronic
transitions, and for analytical situations in which mixture contribute interfering absorption, a
method of manipulating the spectral data is called derivative spectroscopy.
Derivative spectrophotometry involves the conversions of a normal spectrum to its first,
second or higher derivative spectrum. In the context of derivative spectrophotometry, the
normal absorption spectrum is referred to as the fundamental, zero order, or D0 spectrum.

Novus International Journal of Chemistry 2013, 2(1) 9

The first derivative D1 spectrum is a plot of the rate of change of absorbance with
wavelength against wavelength i.e. a plot of the slope of the fundamental spectrum against
wavelength or a plot of dA/d vs. . The second derivative D2 spectrum is a plot of the
curvature of the D spectrum against wavelength or a plot of d2A/ d2 vs. . [12]
Validation data for this method are given in Table-1.

SPI
0.35
0.3
0.25
0.2
0.15 ABS
0.1 Linear (ABS)
0.05
0
0 10 20 30

Graph: 1 Calibration curve of SPI by UV

HFT
0.8

0.6

0.4 ABS

0.2 Linear (ABS)

0
0 10 20 30

Graph: 2 Calibration curve of HFT by UV

Table 1: Validation Parameters for Second Order Derivative Spectroscopy method

Parameters SPI HFT
Beers law range 4-24 g/ml 4-24 g/ml
Wavelength (nm) 238nm 273nm
Correlation Coefficient 0.998 0.996
Slope 0.0108 0.0266
Intercept 0.0374 0.0606
LOD (g/ml) 1.212 1.098
LOQ (g/ml) 4 4
Intraday precision %RSD 0.63-1.33 0.22-1.84
Interday precision %RSD 0.24-1.32 0.24-0.625

Novus International Journal of Chemistry 2013, 2(1) 10

Procedure for the Analysis of Tablet formulation
A total of 20 tablets were accurately weighed and powdered. An amount equivalent to one
tablet (containing 25 mg of SPI and 25 mg of HFT) was taken and dissolved in few ml
methanol by sonicating it for three minutes. Then the solution was filtered through a
Whatman filter paper (No.40) into 25ml volumetric flask. The residue was washed with few
ml of methanol. The filtrate and washings were combined and volume was made up to the
mark with methanol. The aliquot was appropriately diluted to obtain final solution that
contains 8 g/ml of each drug, SPI and HFT.

Table 2: Recovery studies of SPI and HFT

Level of Amount Amount Total amount % Recovery
Recovery taken(g/ml) added(g/ml) found(g/ml)
SPI HFT SPI HFT SPI HFT SPI HFT
0% 8 8 0 0 7.99 8.01 99.87 100.12
50% 8 8 4 4 12.1 11.89 100.83 99.08
100% 8 8 8 8 16.0 16.1 100 100.62
150% 8 8 12 12 19.99 20.1 99.95 100.50

CONCLUSION
These validated methods are new, rapid, accurate, precise, sensitive, reproducible and
economical and can be employed for routine analysis for simultaneous estimation of
Spironolactone and Hydroflumethiazide in combined dosage form.
ACKNOWLEDGEMENT
The authors are thankful to RPG Life Sciences, Ankleshwar, Gujarat, India for providing gift
sample of Spironolactone and Hydroflumethiazide purchased from SIGMA-ALDRICH Co.,
3050 Spruce Street, St.Louis, MO 63103 USA.
REFERENCES
1. The Merck index, Merck Research Laboratories Division of Merck & Co.,Inc., 13 th
edition, USA, 2006, pp 856,1562.
2. Hardman JG, Limbird LE and Gilman AG, Goodmann & Gilmans The
Pharmacological Basic of Therapeutics, 10th Edi; Mc Graw- Hill Medical Publishing
Division., pp 773,779 .
3. Parimoo P, Bharathi A and Padma K, Simultaneous Determination of Spironolactone
with Hydroflumethiazide and Spironolactone with Furosemide in Combination
Formulations by UV Absorption Method. Indian J. Pharm.Sci., 1995, 57(3), 126-129.
4. Umadevi B and Vetrichelvan T, Simultaneous Estimation of Metolazone and
Spironolactone in Combined Tablet Dosage Form BY UV Spectroscopy,
International Journal of PharmTech Research; 2011, 3(4), 2068-2074.
5. Laxman R and Acharya A, Development and Validation of RP-HPLC and Ultraviolet
Spectrophotometric Methods for simultaneous Determination of Spironolactone and
Torsemide in Pharmaceutical Dosage Form, International Journal of Research in
Ayurveda and Pharmacy; 2010, 1(2), 459-467.

Novus International Journal of Chemistry 2013, 2(1) 11

 6. Bhojani M, Dadhania K and Faldu S, Development and Validation of RP-HPLC
Method for Simultaneous Estimation of Furosemide and Spironolactone in their
Combined Tablet Dosage Form, Journal of Pharmaceutical Science and Bioscientific
Research, 2012, 2(3), 144-147.
7. Sharma MC, Sharm S, Kohlib DV and Sharma AD, Validated TLC Densitometric
method for the quantification of Torsemide and Spironolactone in bulk drug and in
tablet dosage form, Der Pharma Chemica., 2010, 2(1), 121-126.
8. Davit P, Capra P and Vincenti M, Fast gas chromatographic/mass spectrometric
determination of diuretics and masking agents in human urine: Development and
validation of a productive screening protocol for antidoping analysis, Journal of
Chromatography; 2007, 1135(2), 219-29.
9. Jacobsen S and Arnesen E , Fluorometric determination of hydroflumethiazide in
human plasma and urine after its oral administration, European Journal of Clinical
Pharmacology; 1977, 11 (2), 149-154.
10. Patel M, Shah N, Shah V and Deka S, Review on Simultaneous Determination of
Antihypertensive Analytes in Pharmaceutical Dosage Form by Different Analytical
Methods, International Journal of Pharmacy Research and Technology; 2012, 2(2),
09-15.
11. Sharma S, Sharma MC and Kohli DV, Conventional and Micellar Liquid
Chromatography Method with Validation for Torsemide and Spironolactone in Tablet
Combined Dosage Form, Der Pharmacia Lettre; 2010, 2(1), 374-381.
12. Patel R, An Introduction to Analytical Method Development for Pharmaceutical
Formulations, www.pharmainfo.net/review s/introduction-analytical-method-
development-pharmaceutical-formulations.

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