SPIRONOLACTONE

',
Badraddin M.H. Al-Hadiya* Fathalla Bela12,

Yousif A. Asiri', and Othman A. Gubara'

(1) Department of Clinical Pharmacy

College of Pharmacy
King Saud University,
P.O. Box 2457
Riyadh11451
Kingdom of Saudi Arabia

(2) Department of Pharmaceutical Chemistry

College of Pharmacy
King Saud University,
P.O. Box 2457
Riyadh 1 1451
Kingdom of Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES 26 1 Copyright B 2002 Elsevier Science (USA)

AND EXCIPIWTS - VOLUME 29 AU rights reserved.
1075-6280/02$35.00
262 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

Contents

1. Description
1.1 Nomenclature
I. 1.1 Systematic Chemical Names
I. 1.2 Nonproprietary Names
I. 1.3 Proprietary Names
1.2 Formulae
1.2.1 Empirical Formula, Molecular Weight, CAS
Number
1.2.2 Structural Formula
1.3 Elemental Analysis
1.4 Appearance
1.5 Uses and Applications

2. Methods of Preparation
2.1 Method (1)
2.2 Method (2)
2.3 Method (3)

3. Physical Properties
3.1 Ionization Constants
3.2 Solubility Characteristics
3.3 Partition Coefficient
3.4 Specific Rotation
3.5 X-Ray Powder Diffraction
3.6 Thermal Methods of analysis
3.6.1 Melting Behavior
3.6.2 Differential Scanning Calorimetry
3.7 Spectroscopy
3.7.1 UVNIS Spectroscopy
3.7.2 Vibrational Spectroscopy
3.7.3 Nuclear Magnetic Resonance Spectrometry
[Link] 'H-NMR Spectrum
[Link] 13C-NMRSpectrum
3.8 Mass Spectrometry
 SPIRONOLACTONE 263

4. Methods of Analysis
4.1 Identification
4.1.1 Palladium Chloride Test
4.1.2 Sulfuric Acid Test
4.1.3 Lead Acetate Test
4.2 Compendia1 Tests
4.3 Spectroscopic Analysis
4.4. Polarography
4.5 Chromatographic Methods of Analysis
4.5.1 Thin Layer Chromatography
4.5.2 Gas Chromatography
4.5.3 High Performance Liquid Chromatography
4.5.4 Capillary Zone Electrophoresis
4.5.5 Flow Injection Analysis
4.6 Thermal Analysis
4.7 Protein Binding Studies

5. Clinical Applications
5.1 Pharmacological Action
5.2 Mechanisms of Action

6. Pharmacokinetic Profile
6.1 Absorption
6.2 Distribution
6.3 Metabolism and Elimination

7. Drug Interactions
7.1 Drugs Increasing Serum Potassium Concentrations
7.2 Other Drugs
7.3 Precautions
7.3.1 Carcinogenicity/ Tumorigenicity
7.3.2 Pregnancy
7.3.3 Breast-Feeding
7.3.4 Pediatrics
7.3.5 Geriatrics
264 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

8. Toxicity and Adverse Effects

8.1 Those More Frequently Occurring
8.2 Those Less Frequently Occurring

9. Acknowledgement

10. References
 SPIRONOLACTONE 265

1. Description
1.1 Nomenclature [6]
1.1.1 Systematic Chemical Names
(7a, 17a)-7-(acetylthio)-17-hydroxy-3-oxo-pregn-4-ene-21-
carboxylic acid y-lactone
17-hydroxy-7a-mercapto-3-oxo- 17a-pregn-4-ene-2 1-carboxylic acid
y-lactone, acetate
3 -(3-oxo-7a-acetylthio- 17p-hydroxy-4-androsten-17a-yl)
propionic acid y-lactone

1.1.2 Nonproprietary Names [7]

Generic: Spironolactone
Synonyms: Espironolactona

1.1.3 Proprietary Names [9]

Aldactone, Aldopur, Altex, Diatensec, laractone, Sincornen,
Spiretic, Spiroctan, Spirolang, Spirolone, Spirotone.

1.2 Formulae [S-121

1.2.1 Empirical Formula, Molecular Weight, CAS Number
C24H3204S [MW = 416.5741
CAS number = 52-0 1-7

1.2.2 Structural Formula

H3c-s 0
266 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

1.3 Elemental Analysis

The calculated elemental composition is as follows:
carbon: 69.20%
hydrogen: 7.74%
oxygen: 15.36%
sulfur: 7.70%

1.4 Appearance [9]

Spironolactone is a white or yellowish white crystalline powder that has a
slight characteristic odor.

1.5 Uses and Applications [9]

The synthesis and subsequent widespread therapeutic use of
spironolactone has been a logical consequence of the discovery of the most
potent mineralocoticoid hormone in man, aldosterone. The latter
compound was isolated in 1952 from dog and monkey adrenal venous
blood [ 11, which also was shown to be present in human urine [2].

Soon after the discovery of aldosterone, it became apparent that its

excessive production played an important role in the physiopathology of a
variety of disorders, such as COWSsyndrome, congestive heart failure,
and nephritic syndrome [3]. It therefore seemed valuable to develop
compounds with aldosterone-antagonist properties. This approach had its
beginning in the work of Landau et a1 [4].

In 1957, Cella and Kagawa [5] reported about the preparation of two
steroidal 17-spirolactones, which were capable of blocking the urinary
sodiudpotassium action of aldosterone and desoxycorticosterone-acetate
in adrenalectomized rats. These compounds were closely related in
structure to aldosterone and were assigned the code names SC 5233 and
SC 8 109, respectively. Structures of these three compounds are shown in
Figure 1.
 SPIRONOLACTONE 267

Aldosterone SC 5233

SC 8109

Figure 1. Structures of aldosterone, SC 5233, and SC 8109.

268 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

Spironolactone is a steroid with a structure resembling that of the natural

adrenocortical hormone, aldosterone. It acts on the distal portion of the
renal tubule as a competitive antagonist of aldosterone. It also acts as a
potassium-sparing diuretic, increasing sodium and water excretion, and
reducing potassium excretion. The substance is reported to have a slow
onset of action (requiring 2-3 days for maximum effect), and a similarly
slow diminishment of action (over 2-5 days on discontinuation). It is used
for the treatment of refractory edema (associated with heart failure),
cirrhosis of the liver (or the nephrotic syndrome), and in ascites associated
with malignancy. It is frequently given with the thiazides, furosemide, and
the similar diuretics. It has been also used for the treatment of essential
hypertension.

2. Methods of Preparation
Three methods for the preparation of spironolactone have been reported,
and the routes are illustrated in Schemes 1-3.

2.1 Method (1) [15]

The first method for the preparation of spironolactone is outlined in
Scheme 1. Carbonation of the Grignard reagent of 17a-ethynyl-5-
androstene-3P,17P-diol (I) yielded an acetylenic acid (11). Selective
reduction of the acetylenic bond was accomplished by catalytic
hydrogenation over palladium on calcium carbonate, using dioxane and
pyridine as solvents. Treatment of the product with mineral acid yielded
the unsaturated lactone, 3-(3 0, 17P-dihydroxy-5-androstene-17a-yl)-
propenoic acid lactone (111), which was easily reduced to the saturated
lactone (IV) by hydrogen over palladium on charcoal. Oppenauer
oxidation of the product afforded 3-(3-0~0-17P-hydroxy-4-androsten-
17a-yl) propionic acid lactone (V). Unsaturation at Cg was then
introduced by treatment with chloranil. Finally, the resulting 17-hydroxy-
3-0x0- 17a-pregna-4,6-diene-2 1-carboxylic acid-y-lactone (VI) was
reacted with thioacetic acid, yielding 17-hydroxy-7a-mercapto-3-oxo-
17a-pregn-4-ene-2 1-carboxylic acid y-lactone-7-acetate, spironolactone
(VII) [13].
 SPIRONOLACTONE 269

Scheme 1
270 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, A N D 0 . A . GUBARA

Scbeme 2
 SPIRONOLACTONE 27 1

Scheme 3

I( H O V
111

Pyi-idine. HBr
BIZ/ CrO,
bmmination, followed by
oxidation.

DHFLiBr-LizCOs

IV
Androstadienone Bmmoketone derivative

ACSH thiol acetic acid will be added in 1,6 addition

to the double bond, acidic media will remove the
ethylene acctal (protecting group and C103 will
oxidize aldehydic group into COOH with
spontaneous cyclization.

I
272 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

2.2 Method (2) [14]

The second method of preparation (shown in Scheme 2) depends on
treating dehydroepiandrosterone (prepared from cholestrol or sitosterol)
with acetylene to form the l7a-ethnyl-l7P-hydroxy derivative, which is
carbonated to the 17a-propionic acid. Reduction of the unsaturated acid in
alkaline solution yields the saturated acid, which cyclizes to the lactone on
acidification. Bromination to the 5,6-dibromo-compound, followed by
oxidation of the hydroxyl group to the ketone, and then dehydro-
bromination to the 7a-hydroxyl derivative, produces spironolactone when
esterified with thiolacetic acid.
2.3 Method (3) [ 151
In the third method (illustrated in Scheme 3), spironolactone (I) was
prepared from dehyrdoepiandrosterone (11). This starting material was
treated with 3-chloropropionaldehyde ethylene acetal and lithium. The
resulting acetal(II1) was treated with a pyridine-HBr-Brz solution,
followed by Cr03 oxidation to give the bromo ketone. This compound
was refluxed in dimethyl formamide containing LiBr-Liz C 0 3 to give the
androstadienone (IV). The addition of AcSH to IV, followed by Cr03
oxidation, gave spironolactone in 23% overall yield.

3. Physical Properties
3.1 Ionization Constants
Spironolactone does not contain any potentially ionizable groups, and hence
has no ionization constants.

3.2 Solubility Characteristics

The following solubility values have been reported for spironolactone in
different solvents at room temperature (25°C) [lo]:
Solvent Solubility (mg/mL)
Water 0.028
Methanol 6.9
Ethanol 27.9
Chloroform 50.0
Heptane 0.24
 SPIRONOLACTONE 273

3.3 Partition Coefficient

Spironolactone is preferentially extracted from water into heptane. The
partition coefficient has been reported as 3.5 at 25°C [lo].

3.4 Specific Rotation

The specific rotation of a 10 mg/mL solution of spironolactone in
chloroform is between -33" and -37" [8].

3.5 X-Ray Powder Diffraction

The X-ray powder diffraction pattern of spironolactone has been measured
using a Philips PW- 1710 diffractometer, equipped with a single crystal
monochromator and using a copper Ka radiation. The pattern obtained is
shown in Figure 2, and the scattering angles, interplanar d-spacings, and
relative intensities are found in Table 1.

3.6 Thermal Methods of analysis

3.6.1 Melting Behavior
The melting range of spironolactone is between 198 and 207"C,
accompanied by decomposition. Occasionally materials may show
preliminary melting at about 135"C, followed by re-solidification [8], and
this behavior indicates the existence of another, metastable, crystal form.

3.6.2 Differential Scanning Calorimetry

The DSC thermogram of spironolactone was obtained using a Du Pont
TA-9900 thermal analyzer system, interfaced to the Du Pont data
collection system. The thermogram shown in Figure 3 was collected over
a range of 50 to 250"C, using a heating rate of lO"C/minute. It was found
that the compound melted at 210.5"C, with an enthalpy of fusion equal to
43.40 J/g.
274 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

I t I I I 1

Scattcring Anplr (deg2-01

Figure 2. X-ray powder diffraction pattern of spironolactone.

 SPIRONOLACTONE 275

Table 1
Scattering Angles, Interplanar d-Spacings, and Relative
Intensities in the X-Ray Powder Diffraction of Spironolactone

Scattering Angle
d-Spacing (A) Relative Intensity
(degrees 2-8)
9.124 9.5899 17.21
11.53 7.6683 14.63
12.419 7.1216 3 [Link]
14.821 5.9721 0.56
16.021 5.5274 49.03
16.663 5.3160 84.45
17.30 5.1215 100.00
18.131 4.8887 4.55
18.520 4.7870 22.02
19.077 4.6485 8.43
20.357 4.3589 38.89
20.740 4.2793 2.91
21.945 4.0468 8.50
22.912 3.8782 4.85
23.224 3.8268 8.71
24.772 3.591 1 4.74
26.893 3.3125 0.91
28.467 3.1328 0.94
29.949 2.9821 5.97
30.943 2.8875 0.57
3 1.826 2.8095 0.73
32.822 2.7264 1.16
33.963 2.6374 0.40
35.405 2.5332 0.93
36.577 2.4547 0.36
38.672 2.3264 1.11
39.544 2.2770 0.89
41.223 2.1881 1.36
44.487 2.0349 0.63
46.708 1.9431 0.29
60.71 1.5242 0.22
276 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

I I I 1 I I 1
75, 1000 125.0 Isao 175.0 200.0 225.0
Ttmperat urt ( *C 1

Figure 3. Differential scanning calorimetry thermogram pattern of

spironolactone.
 SPIRONOLACTONE 277

3.7 Spectroscopy
3.7.1 UVNIS Spectroscopy
UV absorption spectra of spironolactone were recorded using a Shimadzu
UV-VIS spectrophotometer, model 1601 PC. Spectra were obtained in
methanol, 0.1M HC1, and 0.1M NaOH, and these are shown in Figure 4.
The values of Al%,lcrn,and the molar absorptivity values at their
corresponding Laare shown in the following table:

Solvent Wavelength Al%,lcm Molar Absorptivit

System Maximum (nm) (L-Mol-'-cm-')

Methanol 237.8 458 19055

0.1M HCl 242.4 461 19180

0.1M NaOH 293.6 78 3245

246.6 430 17890
215.8 66 1 27500

3.7.2 Vibrational Spectroscopy

The infrared absorption spectrum of spironolactone was obtained in a KBr
pellet using a Perkin-Elmer infrared spectrophotometer. The principal
absorption peaks of the spectrum shown in Figure 5 were noted at 1765,
1693,1673,1240, 1178,1135,1123 and 1193 cm-'.

3.7.3 Nuclear Magnetic Resonance Spectrometry

The 'H-NMR and 13C-NMRspectra of spironolactone were obtained using
a Jeol400 MHZ Eclipse FT-NMR Summit Spectrometer

[Link] 'H-NMR Spectrum

The full 'H-NMR spectrum of spironolactone in CDC13 is shown in Figure
6, with various regions being expanded in Figures 7A through Figure 7H
for easier viewing. Assignments for the resonance bands are given in
Table 2.
 278 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

A
b
I

Figure 4. Ultraviolet absorption spectrum of spironolactone dissolved

as 22 pg/mL in methanol (- ), 0.1M HCl (- - * - .),
and 0.1M NaOH (- - - -).
 SPIRONOLACTONE 279

WAVENUMBER ( EM")

Figure 5. Infrared absorption spectrum of spironolactone.

280 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

Figure 6. Complete 'H-NMR spectrum of spironolactone.

 SPIRONOLACTONE 28 1

Table 2

Assignments for the Resonance Bands observed

in the 'H-NMR Spectrum of Spironolactone
282 [Link]-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. G U B A M

1
R
I
!
I
I
I
I

I
i

-- a
I

1
Figure 7A. H-NMR Spectrum of Spironolactone, 0.0 - 8.0ppm.
 .

-.
3.42 I

247
N
m
w
284 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

I__

i
I

Ij

Figure 7C. 'H-NMR Spectrum of Spironolactone, 0.9 - 1.05 ppm.

 SPIRONOLACTONE 285

//

I
if
i
I
i
!
I

Figure 7D. ‘H-NMR Spectrum of Spironolactone, 0.8 - 2.1 ppm.

286 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

- " . . . ._".

li'
.I
I
i

Figure 7E. 'H-NMR Spectrum of Spironolactone, 1.1 - 2.6 ppm.

 SPIRONOLACTONE 287

Figure 7F. 'H-NMR Spectrum of Spironolactone, 2.13 - 2.9 ppm.

288 B.M.H. AL-HADIYA, [Link], Y.A. ASIRI, AND O.A. GUBARA

I
X 3 put. pr MUllon i 1H

Figure 7G. 'H-NMR Spectrum of Spironolactone,3.9 - 4.0 ppm.

 SPIRONOLACTONE 289

, s

Figure 7H. 'H-NMR Spectrum of Spironolactone, 3.8 - 7.4 ppm.

290 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

[Link] I3C-NMR Spectrum

The I3C-NMR spectrum of spironolactone was obtained in CDC13, and the
spectra are shown in Figures 8 and 9. The assignments for the observed
resonance bands are provided in Table 3.

3.8 Mass Spectrometry

The mass spectrum of spironolactone was obtained utilizing a Shimadzu
PQ-5000 Mass Spectrometer, where the parent ion was collided with
helium carrier gas. Figure 10 shows the detailed mass fragmentation
pattern, where a base peak was noted at m/z = 34 1, along with a molecular
ion peak at m/z = 416. The mass fragmentation pattern of the compound
is detailed in Table 5. The peak that appeared at m/z = 267 is due to loss
of CH3CS0, COz and two CH3, while the peak at m/z = 325 is due to
splitting of CH3CS0, 'CH3, and 'H from spironolactone.

4. Methods of Analysis
4.1 Identification
4.1.1 Palladium Chloride Test [7]
The reagent is prepared by dissolving 0.1 g of PdClz in 5 mL of 2M HC1,
diluting to 100 mL with water, and then mixing with an equal volume of
2M NaOH. The reagent gives brown color with spironolactone.

4.1.2 Sulfuric Acid Test [7]

Shake about 10 mg of spironolactone with 2 mL of 50% sulfuric acid,
whereupon an orange solution with an intense yellowish green
fluorescence is produced. Heat the solution gently, and the color becomes
deep red with the evolution of H2S. Add the solution to 10 mL of water,
and a greenish yellow solution is produced which shows opalescence or a
precipitate.

4.1.3 Lead Acetate Test [ 111

Add 100 mg of spironolactone to a mixture of 10 mL of water and 2 mL of
1N NaOH, boil for 3 minutes, and cool. Add 1 mL of glacial acetic acid
and 1 mL of lead acetate solution; a brown to black precipitate of lead
sulfide is produced.
 SPIRONOLACTONE 29 1

._.. . . .._. . . .

Figure 8. Full I3C nuclear magnetic resonance spectrum of

Spironolactone.
292 B.M.H. AL-HADIYA,F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

TabIe 4

Assignments for the Resonance Bands observed in the

13C-NMRSpectrum of Spironolactone

23 24
S-C-CH3
H

Assignment
shift

14.65 45.13
17.84 c
19 45.51
20.56 c
16 46.03 ClO
22.37 CIS 49.57 C8
c
14

31.21 c
6 77.14 c
7

31.40 c11 95.61 c

17

33.97 c
12 127.03 c4
35.25 c
21 165.77 c5
35.69 c
22 176.63 c
20

38.53 c2 194.45 c
23
 SPIRONOLACTONE 293

i
i

Figure 9. Expanded I3C nuclear magnetic resonance spectrum of

Spironolactone.
294 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. CUBARA

mlz

Figure 10. Mass spectrum of Spironolactone.

 SPIRONOLACTONE 295

Table 4

Mass Fragmentation pattern of Spironolactone

Relative intensity Fragment

L
I
Po)

43.10 100.00 CH3-CEO’

44.44

79.10 19.91

105.10 21.58 0
0

107.15 13.80
0
0

131.20 9.15

a.
I

143.20 7.94 0

L 145.20 7.93
0 H
296 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

Table 4 (continued)

Mass Fragmentation pattern of Spironolactone

8.92
0 H

10.43

I
 SPIRONOLACTONE 297

4.2 Compendia1 Tests

Spironolactone is the subject of monographs in the United States
Pharmacopoeia (USP), as well as in the European, Chinese, Poland,
Japanese, and International Pharmacopoeias.

Both The European Pharmacopoeia [ 123 and the British Pharmacopoeia

[ 1 11 describe a spectrophotometric method for spironolactone. The
method is based on measurement of the absorbance of a 10 pg/mL
methanolic solution at 238 nm. The content is calculated taking 470 as the
value of

The USP [8] recommends use of an HPLC method. The mobile phase is a
55:45 mixture of acetonitrile and aqueous 0.02M dibasic ammonium
phosphate, eluted at a flow rate of 1 mL/min. The column is ODS (4 mm
x 30 cm), and detection is on the basis of the UV absorbance at 254 nm.

4.3 Spectroscopic Analysis

A ratio-spectra, zero-crossing, derivative spectrophotometric method was
described for the analysis of spironolactone in presence of
hydrochlorothiazide [ 161. After extracting the drugs from their tablets
with 1 : 1 0.1 N HCl - methanol, the first derivative of the ratio of their
absorption spectra to that of standard solution is computed at 270.7 and
269.9 nm.

Univariate and multivariate spectroscopy was applied to the analysis of

spironolactone in presence of chlorthalidone [ 171. Satisfactory results
were obtained by partial least squares regression, with the calibration
curve being linear over the range 2.92 - 14.6 &mL. A kinetic-
spectrophotometric method was described for the determination of
spironolactone and canrenone in urine that also used a partial least-squares
regression method [ 181. After the compounds were extracted from urine,
the spectra were recorded at 400 - 520 nm for 10 minutes at 30 second
intervals. The relative error was less than 5%.

First-derivative spectroscopy was applied to the analysis of mixture of

spironolactone and frusemide in combined dosage forms [ 191. Calibration
curves were linear up to 20 pg/mL. The same mixture has been analyzed
by extraction with methanol, and subsequent measurement in either 0.1 N
HCl or in 0.1 N NaOH [20]. The relative standard deviation was in the
298 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

range of 1.4 - 7.8%, and recoveries were 98.6 - 99.9%.

Wahbi reported on the use of second-derivative spectroscopy for the

determination of canrenone in spironolactone, and found that the method
is stability-indicating for spironolactone [211. First and second-derivative
spectroscopy were also utilized for the simultaneous analysis of
spironolactone in combination with either hydrochlorothiazide or
frusemide [22]. The drugs are extracted with ethanol and analyzed
through the use of the zero-crossing method. Nowakowska [23] analyzed
mixtures of spironolactone and hydrochlorothiazide in tablets by
measuring the absorbance of all species at 239 nm, and that of
hydrochlorothiazide at 3 18 nm. The recoveries for spironolactone were
found to be 97.9 to 101.8%, with a coefficient of variation of less than
0.1Yo.

Several calorimetric methods were reported for spironolactone using

different derivatization reagents, such as thiosemicarbazide at 440 nm
[24], 4-dinitrobenzene at 400 nm [25], isoniazide at 375 nm [26], acetic
acid-acetic anhydride then H2S04 at 500 nm [27], 2,3,5-triphenyl-
tetrazolium chloride and tetramethylammonium hydroxide at 480 nm [28];
sodium nitroprusside at 700 nm, and phosphotungestic acid at 41 0 nm
~91.

Other spectrophotometric techniques have been reported for the analysis of

spironolactone. Near infrared diffuse reflectance first-derivative
spectroscopy was used for determination of spironolactone in
pharmaceutical dosage forms [30]. Readings were taken at 15 nm
intervals, and then 81 absorbance readings were imput into a computer for
principal component analysis.

Fourier-transform Raman and Fourier-transform infrared spectroscopic

methods were adapted for the afialysis of spironolactone and
differentiation of its polymorphic forms [3 1, 321.

Neubert and Koch described a fluorimetric method for the determination

of spironolactone metabolites in serum. The method used two extraction
steps with dichloroethane at different pH values, followed by treatment
with 65% sulfuric acid, and final measurement of the fluorescence at
436/525 nm [33].
 SPIRONOLACTONE 299

4.4. Polarography
Belal described a direct-current (DC) and differential pulse (DPP)
polarographic method for spironolactone in dosage forms, using pH 10
Britton-Robinson buffer containing 40% methanol as a supporting
electrolyte [34]. The current-concentration plot was found to be linear
over the ranges of 0.005-1.O mM (DC mode) and 0.0025-1 .O mM (DPP
mode). A similar polarographic method based on the measurement of the
cathodic current in 0.005 M HzSOdmethanol (1:l) was described [35].

4.5 Chromatographic Methods of Analysis

4.5.1 Thin Layer Chromatography
Three TLC systems have been described for the identification of
spironolactone [36], all of which are based on the use of silica gel G as the
adsorbent. Location of eluted sport was effected using either acidified
iodoplatinate, or acidified potassium permanganate:

System A: chloroform-acetone (4: 1): Rf = 66

System B: ethyl acetate-methanol-strong ammonia solution (85: 10:5):
Rf =79
System C: ethyl acetate: Rf = 51

Another TLC method involving also the use of silica gel G as an

adsorbent, and a solvent system consisting of: benzene-ethyl acetate-
methanol ([Link]) [lo]. The Rfvalue was 67, and detection was
accomplished using phosphomolybdic acid at 80°C for 10 minutes.

4.5.2 Gas Chromatography

Spironolactone is thermally stable, so therefore GC systems reported for
its determination are relatively few. The reported methods are compiled in
Table 5.
300 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

Table 5

Gas Chromatographic Methods of Analysis of Spironolactone

Source Column Carrier Flow Temp. Detection Ref

gas rate

Bulk SE-30 column, Nitrogen 45 330°C Flame [37]

drug containing mL/min. ionization
2.5% SE 30 on
800- 100 mesh
chromosorb G ,
2mx4mm
internal
diameter

Human HP-5 (0.33 pm) Helium 0.6-0.8 Program- Mass 1381

Urine Column 17 m x mL/min med spectrum
0.2 mm i.d.

1
Human SE-30 column, Nitrogen 60 230°C Flame [39]
Urine packed with mL/min ionization
1% SE 30 on
diatomite CQ
80- 100 mesh,
1.5 mx4mm
i.d.

Human Glass column Nitrogen 120 250°C Electron- [40]

plasma packed with mL/min Capture
2% OV-1 on
CQ 80-100
mesh (1.5 m x
4 mm i.d.)
 SPIRONOLACTONE 301

4.5.3 High Performance Liquid Chromatography

HPLC is the most frequently used technique for the analysis of
spironolactone as the bulk drug, in its dosage forms, or in biological fluids.
The reported HPLC methods are summarized in Table 6.

4.5.4 Capillary Zone Electrophoresis

A CZE method has been described for the determination of spironolactone
[59]. The analysis was conducted at pH 8, with UV detection (244 nm), at
a constant voltage of 20 kV. The migration time was 11.9 minutes.

4.5.5 Flow Injection Analysis

A flow-injection method was reported for simultaneous determination of
spironolactone and hydrochlorothiazide [60]. Samples are injected into a
carrier stream of pH 5 acetate buffer, and spectra recorded from 220 - 350
nm at 1-second intervals and at an integration time of 0.4 seconds [60].

4.6 Thermal Analysis

Thermal analysis (TGA and DSC) of spironolactone and its ethanol,
methanol, acetonitrile, ethyl acetate, and benzene solvates has been used to
characterize the materials [61]. The analysis was carried out under dry
nitrogen ( 50 mL/min), with the samples being contained in aluminum
sample pans. Thermograms were obtained at a heating rate of
1O"C/minute, with the final temperature being 230°C. Spironolactone and
its methanol and ethanol solvates exhibited small exothermic transitions in
addition to the anticipated endotherms.

4.7 Protein Binding Studies

Protein binding of spironolactone and canrenone was determined by both
ultrafiltration (room temperature) [63] and equilibrium dialysis (overnight
at 37°C) [64]. Both spironolactone and canrenone were extensively
(>89%) bound to plasma proteins, and their blood-to-plasma concentration
ratio was about 0.5, indicating that in blood these steroids were largely
confined to plasma.
302 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

Table 6

HPLC Methods for the Analysis of Spironolactone

Material Column Mobile Phase and Detector Ref

(Flow Rate)

Human urine Lichro- H20 / acetonitrile / Diode

sphere 100 H3P04 / triethylamine array
RP-18, 5 (530: 470: 0.65: 0.145
Clm pH 3.3 or 800: 200:
0.38: 0.145 pH 6
(1 mL/min)

Aqueous 4 Pm Aqueous 21.5% THF 250 nm

formulations Nova-pak (1 mL/min)
and rat serum phenyl,
150x 3.9
mm i.d.

Human S5 ODS 2 Acetonitrile/ H3P04 254 nm [431

plasma Kontron (89:ll) pH 3.4
columns (1 mL/min)
250 x 4.6
mm i.d.

Human urine Hypersil Aqueous Acetonitrile 238 nm

ODS 30 (1 mL/min)
pm 125 x
4 mm

Urine Spherisorb Aqueous 42 mM-SDS 254 nm

ODs2 C18, containing 4%
5 pm, 120 propanol pH 4.5 with
x 4.6 10 mM phosphate
mm,i.d. buffer (1 mL/min)
 SPIRONOLACTONE 303

Table 6 (continued)

HPLC Methods for the Analysis of Spironolactone

Materir Column Mobile Phase and Detector

(Flow Rate)

Tablets Spherisorb 0.07M SDS containing 254 nm

ODS 2 , 5 0.5% pentanol, pH
pm. 120 x 6.9 (1 mL/min)
4.6 mm,
i.d.

Urine HP- Na H2PO4 / propyl- 230 nm

Hypersil ammonium chloride
ODs, (25 buffer pH 3 or acetate
cmx4 buffer pH 4 (1
mm) mL/min)

Dosage Nucleosil Methanol-acetonitrile- 254 nm;

forms CIS, 10 H2O-acetic acid (50: 280 nm
pm, 25 40: 10: 1) or
cmx4 methanol- acetonitrile-
mm, i.d. H20 -triethylamine
(550: [Link])
(1.5 mL/min)

Stability i: Micro- 63% methanol in 238 nm;

study bondapak 0.01M KH2P04
c18, 30 (2 mL/min)
cm x 3.9
mm, i.d.

ii: Micro- 39% acetonitrile in 254 nm

bondapak 0.01M KH2P04
phenyl, 30 (1.2 mL/min)
cm x 3.9
mm
304 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIlU, AND O.A. GUBARA

Table 6 (continued)

HPLC Methods for the Analysis of Spironolactone

Material Column Mobile Phase and

(Flow Rate)
_______~

I
Biological Spherisorb Aqueous 65%
fluids S5 ODS 1, methanol adjusted to
5 pm,12.5 pH 3.4 with H3P04
cm x 4.9 (2 mL/min)
mm, i.d.

Dosage form Excalibar 0.02 M NaH2P04 /

Spherisorb methanol (3:2)
NH2,5 (1.5 mWmin)
pm, 25 cm
x 4.6 mm,
i.d.
I
Tablets Apex Methanol - 0.1M Ampero- [52]
Cyan0 RP NaHZP04 (9: 1 1) metric
5 pm, 15 (1 mL/min)
cm x 4.6
mm, i.d.

Plasma Micro- Water- methanol- 254nm [53]

Bondapak acetonitrile (82: 65:
C18, 10 53) (6 mL/min)
Pm

Dosage Micro- Chloroform: methanol 254nm [54]

forms bondapak (9: 1 1) (1 mL/min)
NH2

Plasma 65- 100% methanol in 254nm [55]

water (gradient
elution) (1 mL/min)
 SPIRONOLACTONE 305

Table 6 (continued)

HPLC Methods for the Analysis of Spironolactone

Material Column Mobile Phase and Detecta Ref

(Flow Rate)
~~

Tablets Lichro- Aqueous 50% 271 run

sorb RP, acetonitrile
C18, 5 pm (1 mL/min)

Serum Partisil, 5 Isopropyl ether- 240 nm

pm, 150 x methanol (393:7)
4.6 mm, (2.2 mWmin)
i.d.

Plasma and Spherisorb Aqueous 65% 285 nm

urine ODS 2, methanol
125 x 4.6 (1 mL/min)
mm, i d .
306 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

5. Clinical Apdications
5.1 Pharmacological Action
Spironolactone competitively inhibits the physiologic effects of the
adrenocortical hormone aldosterone on the distal tubules, thereby
producing increased excretion of sodium chloride and water, and
decreased excretion of potassium, ammonium, titratable acid, and
phosphate. Spironolactone is a potassium-sparing diuretic that has diuretic
activity only in the presence of aldosterone, and its effects are most
pronounced in patients with aldosteronism. Spironolactone does not
interfere with renal tubular transport mechanisms, and does not inhibit
carbonic anhydrase.

Renal plasma flow and glomerular filtration rate are usually unaffected,
but free water clearance may increase. Because most sodium is re-
absorbed in the proximal renal tubules, spironolactone is relatively
ineffective when administered alone. Concomitant administration of a
diuretic which blocks re-absorption of sodium proximal to the distal
portion of the nephron (such as a thiazide or loop diuretic) is required for
maximum diuretic effects. When administered with other diuretics,
spironolactone produces an additive or synergistic diuretic response and
decreases potassium excretion caused by the other diuretic [65].

Spironolactone reportedly has hypotensive activity when given to

hypertensive patients, by blocking the effect of aldosterone on arteriolar
smooth muscle by altering the extracellular-intracellularsodium gradient.

Spironolactone exhibits antiandrogenic effects in males and females. It

decreases testosterone biosynthesis by inhibiting steroid 17a-
monooxygenase (1 7a-hydroxylase) activity, possibly secondary to
destruction of microsomal cytochrome P-450 in tissues with high steroid
17a-monooxygenase activity (testes, adrenals) [65].

5.2 Mechanisms of Action

Spironolactone inhibits aldosterone effects by competing with aldosterone
for intracellular mineralocorticoid receptors. When spironolactone (or its
active metabolites) binds to these receptors, the complex fails to
translocate into the nucleus a n d or bind to the nuclear chromatin. All of
the subsequent phases of the effects of aldosterone are therefore inhibited,
 SPIRONOLACTONE 307

clinically manifested as a natriuresis and potassium retaining action [66].

By competing with aldosterone for receptor sites, spironolactone is

effective in reducing blood pressure, edema, and ascites in conditions of
primary or secondary hyperaldosteronism. Spironolactone is also effective
in managing essential hypertension, although aldosterone secretion may be
within normal limits. The precise mechanism of hypotensive activity
action has not been determined, but it has been suggested that the drug
may act by blocking the effect of aldosterone on arteriolar smooth muscle
or by altering the extracellular-intracellularsodium gradient [67].

Spironolactone induced a marked and statistically significant inhibitory

effect on the cardiovascular reactivity to both the adrenergic and the
rennin-angiotensin-aldosteronesystems. This may play a major role in the
vascular and antihypertensive properties of the drug [68].

The mechanism of antiandrogenic activity of spironolactone is complex,

and appears to involve several effects of the drug. Hirsutism, an
androgen-related increase in growth of facial and body hair, may occur in
women with increased androgen production or with hypersensitivity of the
hair follicle to androgenic stimulation [69]. The identification of the
antiandrogenic activity of spironolactone has led to its use in the treatment
of hirsutism. Spironolactone interferes with testosterone biosynthesis by
reducing 17-hydroxylase activity, possibly secondary to destruction of
microsomal cytochrome P-450 in tissues with high steroid 17a-
monooxygenase activity (e.g.,testes, adrenals), and thus lowers plasma
testosterone levels [70].

It has also been shown that spironolactone inhibits the binding of

dihydrotestosterone to cytosol protein receptor. The latter mechanism
accounts for a direct anti-androgenic effect on target tissues (androgen
receptor in human hair follicles) [70, 711. Spironolactone-induced
increases in serum estradiol concentration also may contribute to its anti-
androgenic activity, although such increases may not occur consistently.
Such increases appear to result from increased conversion of testosterone
to estradiol. Spironolactone may have variable effects on serum 17-
hydroxyprogesterone concentrations, possibly decreasing its production by
inhibiting steroid 17a-monooxygenase activity or decreasing its
conversion (with resultant accumulation) to androstenedione by inhibiting
cytochrome P450-dependent 17a-hydroxyprogesterone aldolase (1 7,20-
308 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIFU, AND O.A. GUBARA

desmolase) activity. Serum progesterone concentrations may increase

with the drug secondary to decreased hydroxylation (via steroid 17a-
monooxygenase) to 17-hydroxyprogesterone. In children compensatory
increases in lutropin (luteinizing hormone) and follicle-stimulating
hormone secretion can occur, probably secondary to the drug’s anti-
androgenic effects (i. e . , a feedback response to decreasing serum
testosterone concentrations a n d or peripheral androgenic activity) [65].

6. Pharmacokinetic Profile
6.1 Absorption
Initial formulations of spironolactone were poorly absorbed from
gastrointestinal tract because of less than optimal disintegration and
dissolution [72]. In contrast to these early versions, newer preparations
display enhanced absorption and oral bioavailability of 90% [73], resulting
in a fall by a factor of four in the recommended oral dose of the
medications [74].

Because of its low aqueous solubility, spironolactone has not been

formulated for intravenous administration [72]. The oral absorption of
spironolactone is improved by using micronized drug, or through the use
of inclusion complexes of spironolactone with cyclodextrins.

Concomitant intake of food enhances drug absorption, most likely by

increasing the degree of gastric dissolution, and decreasing the first-pass
effect of spironolactone. The latter effect is possibly due to the secretion
of bile acids in response to a meal serving that serves to enhance
dissolution of the lipophilic compound [74].

Following a single oral dose of 100 mg of spironolactone, peak serum

concentrations of the drug occur within 1-2 hours, and peak serum
concentrations of its principal metabolites are attained within 2-4 hours
[65,67,75-771. When administered alone, spironolactone has a gradual
onset of diuretic action, with the maximum effect being reached on the
third day of therapy [65]. The delay in onset may result from the time
required for adequate concentrations of the drug or its metabolites to
accumulate [65]. When a thiazide diuretic is used concomitantly with
spironolactone, diuresis usually occurs on the first day of therapy [65].
 SPIRONOLACTONE 309

6.2 Distribution
Spironolactone and canrenone (a major metabolite of the drug) are both
more than 90% bound to plasma proteins. Spironolactone or its
metabolites may cross the placental barrier, and canrenone is distributed
into breast milk [67,78,79].

6.3 Metabolism and Elimination

Spironolactone is rapidly and extensively metabolized in the body
(particularly in liver) into a large number of metabolites (see Figure 10 for
a metabolic map), with no unchanged drug appearing in the urine. It has
been difficult to determine the half-life of the parent compound in humans.
At least 17 metabolites have been isolated, of which 7a-thiomethyl-
spironolactone, 6~-hydroxy-7a-thiomethylspironolactone, and canrenone,
the principal metabolites, are the most pharmacologically active [65,80].
Canrenone is a dethioacetylated (non-sulfur containing) metabolite.
This metabolite is thought to be primarily responsible for the
drug’s therapeutic effects. Approximately 25% to 30% of an
administered dose is converted to canrenone, which accounts for
one-tenth and one-fourth the anti-mineralocorticoid activity, after
single and multiple doses, respectively [81,82].
7a-thiomethylspironolactone is a sulfur-containing metabolite.
This metabolite undergoes further metabolism to produce a
sulfoxidized metabolite [75,76,8 1,821.
~-hydroxy-7a-thiomethylspironolactoneis a sulfur-containing
metabolite that undergoes further metabolism to produce a
sulfoxidized metabolite [8 1,821.
It is uncertain to what extent the actions of spironolactone are dependant
on the parent compound or its metabolites.

The elimination half-life of canrenone ranges from approximately 12 to 20

hours, depending upon the spironolactone dose administered [ 831.
Metabolites of spironolactone are primarily eliminated renally, with only
minimal biliary excretion. Little to no parent drug is recoverable in the
urine, reflecting the complete biotransformation of the compound
[72,74,83].
3 10 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

'SOCOCH3 'SH , - Canrenoate

ester
glucuronide

Spironolactone ,/7a-thio-spironolactone\ Canrenoate

0 'SCH3
- Reduced
derivatives
OH

7a-thiomethylspirolactone GP-OH-7a-thiomethyl- Canrenone

spirolactone

c:"
0 'SCH3 0
OH
0 dO
15a-OH-canrenone and
7a-methylsulfinylspirolactone G!3-0H-7a-methy1- possible other hydroxylated
sulfinylspirolactone derivatives

7a-rnethylsulfonylspirolactone 6!3-OH-7a-methyl-
sulfonylspirolactone

Figure 1 1. Metabolic pathway of spironolactone in humans,

 SPIRONOLACTONE 311

7. Drug Interactions
7.1 Drugs Increasing Serum Potassium Concentrations
Spironolactone should not be used concurrently with another potassium-
sparing agent (e.g.,amiloride, triamterene), or potassium-containing
medications, or potassium supplements, or salt substitutes containing
substantial amounts of potassium. Concomitant therapy with these drugs
may increase the risk of hyperkalemia compared with spironolactone alone
[65, 841.

Because indomethacin may increase serum potassium concentrations,

indomethacin and spironolactone should be administered concomitantly
with caution. Potassium-sparing diuretics should be used with caution,
and serum potassium should be determined frequently in patients receiving
an angiotensin-converting enzyme (ACE) inhibitor (e.g.,captopril).
Concomitant administration with an ACE inhibitor may increase the risk
of hyperkalemia. The dosage of spironolactone should be reduced, or the
drug discontinued, as necessary. Patients with renal impairment may be at
increased risk of hyperkalemia [65].

7.2 Other Drugs

When used in conjunction with other diuretics or hypotensive agents,
spironolactone may be additive with, or may potentiate, the action of these
drugs. Therefore, dosage of these drugs, particularly ganglionic blocking
agents, may need to be reduced by at least 50% when concomitant
spironolactone therapy is instituted.

Spironolactone reportedly reduces vascular responsiveness to

norepinephrine, so regional or general anesthesia should be used with
caution in patients receiving spironolactone.

Aspirin has been shown to slightly reduce the nattiuretic effect of

spironolactone in healthy individuals, possibly by reducing active renal
tubular secretion of canrenone, the active metabolite of spironolactone.
However, the hypotensive effect of spironolactone and its effect on urinary
potassium excretion in hypertensive patients is apparently not affected.
Until more clinical data are available on this potential interaction, patients
receiving both drugs should be monitored for signs and symptoms of
decreased clinical response to spironolactone [65].
312 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIR1,ANDO.A. GUBARA

Concurrent use of lithium with spironolactone is not recommended, as the

drug may provoke lithium toxicity by reducing renal clearance [841.

Spironolactone may increase the half-life of digoxin, so dosage reduction

or increased dosing intervals of digoxin may be necessary, and careful
monitoring is recommended.

7.3 Precautions [84]

7.3.1 Carcinogenicity/ Tumorigenicity
Breast carcinoma has been reported in men and women taking
spironolactone, but a direct causal relationship has not been established.

Spironolactone has been found to be tumorgenic in rats, mainly in

endocrine organs and the liver. A statistically significant dose-related
increase in benign adenomas of the thyroid and testes was found in male
rats given spironolactone in doses up to 250 times the usual daily human
dose of 2 mg/kg of body weight. In addition, a dose-related increase in
proliferative liver changes was revealed in male rats. Hepatocytomegaly,
hyperplastic nodules, and hepatocellular carcinoma were evident at the
highest dosage level of 500 mgkg. In female rats, a statistically
significant increase in malignant mammary tumors was seen at the mid-
dose level.

7.3.2 Pregnancy
Pregnant women should be advised to contact their physician before taking
spironolactone, since routine use of diuretics during normal pregnancy is
inappropriate and exposes the mother and her fetus to an unnecessary
hazard. Diuretics do not prevent development of toxemia of pregnancy,
and there is no satisfactory evidence that they are useful in the treatment of
toxemia. Diuretics are indicated only in the treatment of edema due to
pathologic causes, or as a short course of treatment in patients with severe
hypervolemia.

Spironolactone may cross the placenta, however, problems in humans have

not been documented.
 SPIRONOLACTONE 313

7.3.3 Breast-Feeding
Problems associated with spironolactone distribution into human breast
milk have not been documented. However, canrenone (an active
metabolite of spironolactone) is distributed into breast milk.

7.3.4 Pediatrics
Studies performed to date have not demonstrated pediatric-specific
problems that would limit the usefulness of potassium-sparing diuretics in
children.

7.3.5 Geriatrics
Although appropriate studies on the relationship of age to the effects of
potassium-sparing diuretics have not been performed in the geriatric
population, the elderly may be at an increased risk of developing
hyperkalemia. In addition, elderly patients are more likely to have age-
related renal function impairments, which may require caution in patients-
receiving potassium-sparing diuretics.

8. Toxicity and Adverse Effects

The following side/ adverse effects have been selected on the basis of their
potential clinical significance. Possible signs and symptoms were stated
where appropriate [841.

8.1 Those More Frequently Occurring

Hyperkalemia: May occur in up to 26% of patients receiving
spironolactone, even when combined with thiazide diuretics. Irregular
heartbeat is usually the earliest clinical indication of hyperkalemia, and is
readily detected by ECG. Other signs and symptoms include confusion,
nervousness, numbness or tingling in extremities (hands, feet, or lips),
shortness of breath or difficult breathing, unusual tiredness or weakness, or
weakness or heaviness of legs.

Gastrointestinal irritation: nausea or vomiting, stomach cramps and

diarrhea.
3]4 B.M.H. AL-HADIYA, F. BELAL, Y.A. ASIRI, AND O.A. GUBARA

8.2 Those Less Frequently Occurring

Effects related to dose and/or duration of therapy:
1. Antiandrogenic or endocrine effects. These include breast
tenderness in females, deepening of voice in females,
enlargement of breasts in males, inability to have or keep an
erection, increased hair growth in females, irregular menstrual
periods, and sweating.
2. Central nervous system effects, such as clumsiness or headache.

Effects not related to dose and/or duration of therapy:

1. Decreased sexual ability.
2. Dizziness.
3 . Hyponatremia (drowsiness, dryness of mouth, increased thirst,
lack of energy).

Effects rarely occurring:

I . Allergic reaction or anaphylaxis (shortness of breath, skin rash
or itching).
2. Agranulocytosis (fever or chills, cough or hoarseness, lower
back or side pain, painfull or difficult urination).

9. Acknowledgement
The authors sincerely and appreciably thank Professor [Link]-Badr for his
valuable suggestions, support, and advice throughout the work, and for his
review of this publication. The authors also wish to express their gratitude
to Professor H.A. El-Obeid for his valuable technical help, and Mr. Tanvir
A. Butt (Department of Pharmaceutical Chemistry, College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia) for his helpful assistance in
typing this manuscript.
 SPIRONOLACTONE 315

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