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Simultaneous determination of metolazone and

spironolactone in raw materials, combined tablets and
Cite this: Anal. Methods, 2013, 5, 5644
human urine by high performance liquid
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chromatography
M. I. Walash, N. El-Enany, M. I. Eid and M. E. Fathy*

Received 5th July 2013
Accepted 11th August 2013
DOI: 10.1039/c3ay41110a
www.rsc.org/methods
A new, specic and sensitive reversed-phase high performance liquid chromatographic method was
developed for the simultaneous determination of two diuretic drugs; metolazone (MET) and
spironolactone (SPL). Good chromatographic separation was achieved within 5.0 min on 150 mm
4.6
mm i.d., 5 mm particle size Spherisorb-ODS 2 C18 column. A mobile phase containing a mixture of
methanol and 0.02 M phosphate buer (70 : 30) v/v at pH 3.0 was used. The analysis was performed at a
ow rate of 1 mL min1 with UV detection at 235 nm. Xipamide (XPM) was used as an internal standard
(IS). The proposed method was rectilinear over the ranges of 0.051.0 mg mL1 and 0.510.0 mg mL1
with limits of detection (LOD) of 0.009, 0.04 ng mL1 and limits of quantication (LOQ) of 0.03,
0.11 mg mL1 for MET and SPL, respectively. The suggested method was successfully applied for the
simultaneous analysis of the studied drugs in their laboratory prepared mixtures, single tablets and co-
formulated tablets. The method was further extended to the determination of both drugs in spiked
human urine. The mean percentage recoveries of MET and SPL in spiked human urine were 99.33  2.37
and 99.72  3.27, respectively. The proposed method was also applied for the determination of the
studied drugs in the presence of some co-administered or co-formulated drugs without any interference.
Statistical evaluation and comparison of the data obtained by the proposed and comparison methods
revealed no signicant dierence between the two methods regarding accuracy and precision.

Introduction Metolazone and spironolactone are ocial drugs in the

United States Pharmacopoeia (USP),3 the British Pharmaco-
Metolazone (Fig. 1a), 7-chloro-1,2,3,4-tetrahydro-2-methyl-3-(2- poeia (BP),4 and in the Europium Pharmacopoeia.5
methylphenyl)-4-oxo-6-quinazolinesulfonamide,1 is a diuretic Reviewing the literature revealed that several methods;
with similar actions and uses to those of the thiazide diuretics. such as spectrophotometric,610 HPTLC,11,12 and liquid
It is orally administered for treatment of edema associated with chromatography3,1315 were used for the determination of
heart failure and for management of hypertension.2 Spi- metolazone in pharmaceutical preparations either alone68,14
ronolactone (Fig. 1b), (7a,17a)-7-(acetylthio)-17-hydroxy-3-oxo- or in combination with losartan,13 spironolactone7,9 or
pregn-4-ene-21-carboxylic acid g-lactone,1 is an aldosterone
antagonist. It acts as a potassium-sparing diuretic, increasing
sodium and water excretion and reducing potassium excretion.
Spironolactone is used in the management of heart failure, and
for refractory edema associated with liver cirrhosis. It is
frequently given thiazide diuretics such as furosemide, or
similar diuretics, where it adds to their natriuretic but dimin-
ishes their kaliuretic eects. Hence, potassium is conserved in
those who are at risk from hypokalemia.2
A new combination dosage form of metolazone and spi-
ronolactone is indicated for the treatment and management of
oedema and hypertension.

Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, Fig. 1 The structural formulae of the studied drugs, (a) metolazone, (b)
35516, Mansoura, Egypt. E-mail: mefathy@yahoo.com; Fax: +20 502247496 spironolactone.

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Fig. 2 Typical chromatogram of laboratory prepared mixture; (A) metolazone (1.0 mg mL1) and spironolactone (10.0 mg mL1) (in 1 : 10 ratio); (B) metolazone
(0.5 mg mL1) and spironolactone (10.0 mg mL1) (in 1 : 20 ratio). Under the described chromatographic conditions: a: solvent front; b: metolazone; c: xipamide
(2 mg mL1); d: spironolactone.

ramipril.12,15 On the other hand, few methods have been
reported for the determination of metolazone in biological
samples. These methods include: HPLC for the determination
of metolazone either alone,1619 or with furosemide.20 Also,
liquid chromatography-tandem mass spectrometry (LC-
MS),2124 has been reported for the analysis of metolazone in
human plasma or blood.
Regarding spironolactone, a good guide to the work pub-
lished is presented as the comprehensive monographs in the
series of Analytical Proles of Drug Substances25 and Excipi-
ents.26 The most recently published articles about SPL include:
spectrophotometry,4,7,9,27 TLC,28 liquid chromatography,3,2833
and LC-MS/MS.34
Metolazone and spironolactone are co-formulated in
medicinally recommended ratios of 1 : 10 and 1 : 20,
respectively.
Up to now, only two spectrophotometric methods7,9 were
published concerning the simultaneous determination of both
drugs in pharmaceutical preparations. However, nothing has
been published for simultaneous analysis of MET and SPL in
human urine. Umadevi and Vetrichelvan7 determined MET
and SPL over concentration ranges of 0.52.5 mg mL1 and

Table 1 Optimization of the chromatographic conditions for separation of metolazone and spironolactone mixture by the proposed HPLC methoda

No. of theoretical Mass distribution

plates (N) ratio (Dm)
Resolution Relative retention
Parameter MET SPL MET SPL (Rs) (a)

pH of the mobile phase 2.6 1171 2275 0.377 2.347 8.645 6.225
3.0 1456 2594 0.363 2.137 8.903 6.100
3.5 1371 2496 0.365 2.095 7.916 5.739
4.0 1281 2450 0.366 2.148 8.582 5.869
5.0 1287 2456 0.298 1.664 7.665 5.584
6.0 1347 2972 0.379 1.984 8.866 5.235
7.0 1299 2678 0.359 1.786 8.296 4.975
Ratio of organic modier A/B 25/75 1433 2643 0.243 1.185 6.388 4.876
30/70 1472 2567 0.362 2.129 8.889 5.881
35/65 1490 6540 0.504 2.890 13.932 5.734
40/60 1445 8505 0.803 5.741 20.706 7.149
Ionic strength of phosphate buer, M 0.01 1436 2339 0.325 2.012 8.695 6.191
0.02 1452 2585 0.367 2.154 8.946 5.869
0.04 1433 2456 0.359 2.063 8.716 5.746
0.06 1429 2527 0.362 2.019 8.592 5.577
0.1 1483 2575 0.349 2.076 9.03 5.948
Flow rate (mL min1) 0.6 1586 2873 0.367 2.104 9.345 5.733
0.8 1439 2656 0.363 2.089 8.925 5.755
1.0 1460 2544 0.365 2.116 8.906 5.797
1.2 1202 2314 0.363 2.083 8.292 5.738
1.4 1022 2040 0.362 2.079 7.696 5.743
 2
tR 2DtR
a
A: phosphate buer. B: methanol, where: number of theoretical plates N 5:54 . Resolution R .
Wh=2 W1 W2
tR  tm Dm2
Mass distribution ratio Dm . Relative retention a .
tm Dm1

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Table 2 Analytical performance data for the determination of the studied drugs by the proposed method

Parameter MET SPL

Linearity range (mg mL1) 0.051.0 0.510.0

Intercept (a) 3.80
103 3.20
103
Slope (b) 1.115 0.373
Correlation coecient (r) 0.9999 0.9999
S.D. of residuals (Sy/x) 3.80
103 6.60
103
S.D. of intercept (Sa) 2.90
103 4.20
103
S.D. of slope (Sb) 4.70
103 8.10
104
Percentage relative standard deviation, % RSD 0.843 0.570
Percentage relative error, % error 0.343 0.233
Limit of detection, LOD (mg mL1) 0.009 0.04
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Limit of quantitation, LOQ (mg mL1) 0.03 0.11

525 mg mL1, respectively. Also, Chaudhary9 estimated MET
and SPL over concentration ranges of 15 mg mL1 and 5
25 mg mL1, respectively. Therefore, the proposed method
(which assayed MET and SPL over concentration ranges of
0.051.0 mg mL1 and 0.510.0 mg mL1, respectively) is
considered 10 times more sensitive than the previously
reported methods.7,9 In addition, the proposed method allows
the simultaneous determination of both drugs in their labo-
ratory prepared mixtures, co-formulated tablets considering
two pharmaceutical ratios 1 : 10 and 1 : 20; unlike the reported
methods7,9 which determine both drugs taking only one
pharmaceutical ratio; 1 : 10 (ref. 7) or 1 : 20.9 Moreover, the
proposed method was applied to the analysis of both drugs in
spiked human urine. These facts added to the inherent
advantages of HPLC over spectrophotometry regarding high
resolution and high sensitivity.
In the present work, an ecient HPLC method with UV
detection was utilized for the simultaneous analysis of MET and
SPL with good resolution and in a short chromatographic run;
less than 6 min. This method could be applied for the quanti-
tative determination of the studied drugs in single and co-
formulated tablets, as well as in human urine. No interference
was encountered from other co-administered and co-formu-
lated drugs such as furosemide, hydrochlorothiazide,
propranolol, losartan, ramipril, bumetanide, fosinopril, lisino-
pril, enalapril, captopril and aspirin.

Table 3 Assay results for the determination of the studied drugs in pure form by the proposed and comparison methodsa

Proposed method
Comparison method7
Amount taken Amount found
Compound (mg mL1) (mg mL1) % Found % Found

MET 0.05 0.049 98.20 100.00

0.20 0.199 99.65 101.85
0.40 0.399 99.83 99.38
0.60 0.603 100.43 100.66
0.80 0.804 100.54
1.00 0.995 99.55
Mean 99.70 100.47
S.D. 0.84 1.06
t-Test 1.290
(2.306)
F-Test 1.551
(5.409)
SPL 40.0 40.50 101.25 99.18
80.0 79.60 99.50 100.55
160.0 161.50 100.94 101.21
320.0 316.30 98.84 99.77
400.0 401.60 100.40
800.0 800.30 100.04
Mean 100.02 100.18
S.D. 0.57 0.89
t-Test 0.349
(2.306)
F-Test 2.430
(5.409)
a
N.B. Each result is the average of three separate determinations. The gures between parentheses are the tabulated t and F values at P 0.05.35

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Table 4 Precision data for the determination of the studied drugs by the proposed methoda

MET concentration (mg mL1) SPL concentration (mg mL1)

Parameters 0.20 0.40 0.60 2.0 4.0 6.0

Intraday % Found 98.87 97.72 101.08 99.95 99.14 101.08

100.02 100.45 100.27 101.78 99.76 99.94
100.44 98.98 98.67 99.93 98.44 99.64
(x)  S.D. 99.78  0.81 99.05  1.37 100.01  1.23 100.55  1.06 99.11  0.66 100.22  0.76
% RSD 0.82 1.38 1.23 1.06 0.67 0.76
% Error 0.47 0.80 0.71 0.61 0.39 0.44
Inter-day % Found 101.95 100.61 99.02 101.95 98.66 99.58
96.84 102.55 98.00 102.74 100.29 103.14
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100.85 101.27 100.19 99.94 100.77 100.82

(x)  S.D. 99.88  2.69 101.48  0.99 99.07  1.10 101.54  1.44 99.91  1.11 101.18  1.81
% RSD 2.69 0.97 1.11 1.42 1.11 1.79
% Error 1.56 0.56 0.64 0.82 0.64 1.03
a
N.B. Each result is the average of three separate determinations.

Table 5 Robustness of the proposed method using metolazone (0.5 mg mL1) lters (Millipore, Cork, Ireland) and degassed using a promi-
and spironolactone (5.0 mg mL1)a nence degasser DGU-20A5.
Syringe 0.45 mm lters were used for human urine ltration.
Amount found
(mg mL1) % Found A Consort NV P-901 calibrated pH-meter (Belgium) was used
for pH measurements.
Parameter MET SPL MET SPL Ultrasonic bath (Model: SS 101H 230, USA), vortex mixer
(Model: VM-300P, Gemmy Industrial Corp., Taiwan) and
Methanol ratio, %
69 0.505 5.017 100.91 100.34 centrifuge (Model: 2-16P, Sigma Laborzentrifugen, Germany)
70 0.494 5.090 98.79 101.80 were used for human urine sample preparation.
71 0.511 4.942 102.22 98.83
(x) 100.64 100.32
S.D. 1.73 1.49
Materials and reagents
% RSD 1.72 1.48 All the chemicals and pharmaceuticals used were of analytical
% Error 0.99 0.85 reagent grade and pharmaceutical grade, and the solvents were
pH
of HPLC grade.
2.9 0.494 5.037 98.81 100.74 Metolazone was kindly provided by Pharmaceutical Div.,
3.0 0.492 4.940 98.49 98.80 Pennwalt Corp., Rochester, N.Y. Spironolactone was kindly
3.1 0.504 4.988 100.73 99.75 provided by Memphis for PHARM. & CHEM. IND. CO., Cairo,
(x) 99.34 99.76 Egypt. The purity percentages of MET; 100.45%, and SPL;
S.D. 1.21 0.97
% RSD 1.22 0.97
99.88%, were established by applying the USP3 and BP4
% Error 0.70 0.56 methods, respectively.
Xipamide, used as the internal standard (IS), was provided by
Buer strength, M Egyptian INT. Pharmaceutical Industries CO. (EIPICO), Egypt.
0.01 0.496 5.067 99.28 101.33 Metenix tablets (Sano-aventis S.A.E, Egypt) and aldactone
0.02 0.503 5.021 100.66 100.42
0.04 0.499 4.950 99.80 99.00 tablets (KAHIRA PHARM. and CHEM. IND. CO., Cairo, Egypt)
(x) 99.91 100.25 were purchased from commercial sources in the local
S.D. 0.71 1.17 pharmacy.
% RSD 0.71 1.17 Metenix tablets; batch # 099536, labeled to contain 5 mg
% Error 0.40 0.68 metolazone per tablet.
a
N.B. Each result is the average of three separate determinations. Aldactone 25 mg tablets; batch # 1110433-L and aldacto-
ne 100 mg; batch # 1110556-L.E. tablets labeled to contain 25
and 100 mg spironolactone, respectively.
Experimental Laboratory prepared Co-formulated tablets were prepared
according to their pharmaceutical ratios, by mixing accu-
Apparatus rately weighed quantities (equivalent to either 2.5 or 5.0 mg
Chromatographic separation was carried out using a Shimadzu MET) of the mixed contents of 10 powdered metenix tablets
LC-20AD Prominence liquid chromatogram equipped with a with accurately weighed quantities (equivalent to 50.0 mg
Rheodyne injector valve with a 20 mL loop and a SPD-20A UV SPL) of the mixed contents of 10 powdered aldactone
detector. Mobile phases were ltered using 0.45 mm membrane tablets.

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Table 6 Results of MET and SPL solutions stability and mobile phase stability standard solutions were prepared by appropriate dilution of
using metolazone (0.5 mg mL1) and spironolactone (5.0 mg mL1) the stock solutions with methanol. Standard laboratory
prepared mixture solutions were prepared by mixing appro-
Amount found
(mg mL1) % Found priate volumes of MET and SPL stock solutions in 50.0 mL
volumetric asks and completing to the volume with methanol
Parameter MET SPL MET SPL keeping the pharmaceutical ratios of 1 : 10 and 1 : 20 for MET
and SPL, respectively. Solutions of MET and SPL should be
Stock solution prepared
Fresh 0.504 4.913 100.71 98.25 stored in light resistant containers;3 this was fullled by
2 days ago 0.499 4.974 99.80 99.47 covering the asks with aluminium foil. All solutions were
4 days ago 0.500 5.062 100.01 101.23 stored in the refrigerator at 2  C and found to be stable for at
6 days ago 0.492 5.031 98.38 100.61 least 10 days without alteration.
10 days ago 0.499 5.002 99.91 100.04
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(x) 99.76 99.92

S.D. 0.85 1.14
General recommended procedures
% RSD 0.85 1.14
% Error 0.38 0.51 Construction of calibration graphs. Accurately measured
aliquot volumes of the suitable drug working standard solu-
Mobile phase prepared
tions were transferred into a series of 10.0 mL volumetric asks
Fresh 0.507 5.045 101.38 100.89
3 days ago 0.499 4.960 99.88 99.20 so that the nal concentrations were over the range of 0.051.0
5 days ago 0.504 5.012 100.74 100.23 mg mL1 for MET and 0.510.0 mg mL1 for SPL. To each ask,
7 days ago 0.508 4.952 101.51 99.03 0.2 mL of XPM working standard solution was added as internal
10 days ago 0.492 5.040 98.42 100.80 standard so that its nal concentration was 2.0 mg mL1. Then,
(x) 100.39 100.03
the solutions were completed to the volume with the mobile
S.D. 1.28 0.88
% RSD 1.272 0.87 phase at pH 3.0 and mixed well. Aliquots of 20 mL were injected
% Error 0.57 0.39 (triplicate) and eluted with the mobile phase under the
optimum chromatographic conditions. The average peak area
ratios (drug/I.S.) versus the nal concentration of the drugs in mg
Orthophosphoric acid 85% (Riedel-deHa en, Sleeze, mL1 were plotted. Alternatively, the corresponding regression
Germany). equations were derived.
Acetonitrile, ethanol, n-propanol and 2-propanol (Sigma- Analysis of bulk substances. The method mentioned above
Aldrich, Germany). was applied to the determination of the purity of raw material
Methanol (Tedia, USA). for each drug. The percentage recoveries were calculated by
Sodium dihydrogen phosphate mono hydrate, sodium referring to the previously prepared calibration graphs or using
hydroxide (El-Nasr Pharmaceutical Chemicals Company the corresponding regression equations.
(ADWIC), Egypt). Analysis of MET/SPL laboratory prepared mixtures. Aliquots
Human urine was obtained from healthy volunteers. of MET and SPL standard laboratory prepared mixture solu-
tions were transferred into a series of 10.0 mL volumetric
Chromatographic conditions asks. The solutions were diluted to the volume with the
mobile phase and mixed well. The above procedure described
Column: 150 mm
4.6 mm i.d., 5 mm particle size Spherisorb-
under Construction of calibration graphs was then applied.
ODS 2 C18 column.
The percentage recoveries were calculated by referring to the
Mobile phase: a solution consists of a mixture of methanol
calibration graphs, or using the corresponding regression
and 0.02 M sodium dihydrogen phosphate (70 : 30) v/v and the
equations.
apparent pH was adjusted to 3.0 using orthophosphoric acid.
Analysis of the studied drugs in their single tablets. Ten
The mobile phase was ltered through a 0.45 mm membrane
tablets (Metenix, Aldactone 25, or Aldactone 100) were
lter (Millipore, Cork, Ireland).
accurately weighed, nely pulverized, and thoroughly mixed.
Flow rate: 1 mL min1.
Accurately weighed quantities of pulverized tablets equivalent
UV detector wavelength: 235 nm.
to 5.0 mg of MET or 20.0 mg of SPL were transferred into 50 mL
Internal standard: xipamide (standard solution containing
volumetric asks and about 40.0 mL of methanol were added.
400 mg mL1 of xipamide was prepared in methanol and further
The contents of the ask were sonicated for 30 min, completed
diluted with methanol to get the appropriate working standard
to the volume with the same solvent and ltered. Aliquots
solution).
containing suitable concentrations of the studied drugs were
Temperature: room temperature.
analyzed as described under Construction of calibration
graphs. The nominal contents were calculated either from
Standard solutions previously plotted calibration graphs or using the correspond-
Stock solutions of 400 mg mL1 MET and 400 mg mL1 SPL ing regression equations.
were prepared by dissolving 20.0 mg of MET or SPL in 50.0 mL Analysis of the studied drugs in their laboratory prepared co-
of methanol with the aid of an ultrasonic bath. Working formulated tablets. Accurately weighed quantities of the mixed

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Table 7 Assay results for the determination of the studied drugs in their single tablets by the proposed methoda

Proposed method

Amount taken Amount found

Compound (mg mL1) (mg mL1) % Found Comparison method7

Metenix 5 mg tablets 0.40 0.389 97.47 101.48

0.60 0.597 99.59 98.00
0.80 0.788 98.52 100.74
1.00 1.001 100.12 100.32
Mean 98.53 100.14
S.D. 1.05 1.50
% RSD 1.07
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% Error 0.62
t-Test 1.268 (2.447)
F-Test 1.630 (9.277)
Aldactone 25 mg tablets 4.0 3.936 98.39 99.00
6.0 6.085 101.42 100.50
8.0 7.922 99.03 102.00
10.0 9.844 98.44 99.50
Mean 99.32 100.25
S.D. 1.43 1.32
% RSD 1.44
% Error 0.72
t-Test 0.955 (2.447)
F-Test 1.168 (9.277)
Aldactone 100 mg 4.0 3.951 98.77 100.20
tablets 6.0 6.055 100.91 101.00
8.0 7.891 98.64 101.67
10.0 10.055 100.55 100.75
Mean 99.72 100.91
S.D. 1.18 0.61
% RSD 1.18
% Error 0.59
t-Test 1.788 (2.447)
F-Test 3.742 (9.277)
a
N.B. Each result is the average of three separate determinations. The gures between parentheses are the tabulated t and F values at P 0.05.35

contents of 10 prepared co-formulated tablets equivalent to 2.5 Results and discussion

or 5.0 mg MET and 50.0 mg SPL were transferred into 100 mL
volumetric asks and about 80 mL of methanol were added. The
contents of the ask were sonicated for 30 min, completed to
the volume with the same solvent and ltered. Aliquots from the
ltrate containing suitable concentrations were taken and
analyzed as described under construction of the calibration
graphs. The nominal contents were calculated either from a
previously plotted calibration graph or using the regression
equations.
Analysis of spiked human urine. In 10.0 mL screw-capped
centrifugation tubes, aliquots of human urine (1 mL) were
spiked with aliquot volumes of MET and SPL working standard
solutions and mixed. Methanol was added to each tube so that
the nal volume was 5 mL in each tube. Aer vortex mixing for
10 s, the mixtures were centrifuged at 3500 rpm for 30 min at
room temperature and the supernatants were ltered through
syringe lters. Aliquots of the supernatants (1 mL) were care-
fully aspirated, quantitatively transferred into 10 mL volumetric
asks and analyzed as described under Construction of cali-
bration graphs. A blank experiment was carried out simulta-
neously. The peak area ratios were plotted versus the
concentrations of the drugs in mg mL1.
An HPLC method with UV detection was developed and fully
validated for the simultaneous determination of MET and SPL.
The proposed method permitted the separation of the two drugs
with resolution factors (Rs) 3.80 (MET in respect to XPM) and
5.44 (SPL in respect to XPM) and selectivity factors (a) 2.64
(MET in respect to XPM) and 2.17 (SPL in respect to XPM) in a
reasonable time less than 5 min. Fig. 2 shows a typical chro-
matogram for a laboratory prepared mixture of the two drugs
under the described chromatographic conditions. The retention
times for MET, XPM and SPL were 2.13, 3.12 and 4.75 min,
respectively. The proposed method oers high sensitivity as 0.05
mg mL1 of MET and 0.5 mg mL1 of SPL could be detected
accurately. It also permitted the quantitation of MET and SPL in
single and co-formulated tablets. Moreover, the method was
extended to determine both drugs in spiked human urine.
Optimization of the chromatographic performance and
system suitability
The dierent parameters aecting the chromatographic
performance of the studied drugs were carefully studied in

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Table 8 Assay results for the determination of the studied drugs in their prepared co-formulated tablets by the proposed methoda

Comparison
Proposed method method7

Amount taken Amount found

(mg mL1) (mg mL1) % Found % Found

Preparation MET SPL MET SPL MET SPL MET SPL

Prepared co-formulated tablet (1/10 ratio) 0.40 4.0 0.403 4.037 100.85 100.92 97.98 102.00
0.60 6.0 0.593 5.896 98.92 98.27 101.35 101.00
0.80 8.0 0.806 8.098 100.78 101.22 99.66 99.50
1.0 10.0 0.998 9.970 99.8 99.7 99.19 101.80
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Mean 100.09 100.03 99.55 101.08

S.D. 0.91 1.34 1.40 1.14
% RSD 0.91 1.34
% Error 0.46 0.67
t-Test 0.650 1.191 (2.447)
F-Test 2.332 1.400 (9.277)
Prepared co-formulated tablet (1/20 ratio) 0.05 1.0 0.0503 1.012 100.55 101.17 100.00 102.78
0.10 2.0 0.0993 2.005 99.30 100.25 99.26 100.56
0.30 6.0 0.3008 5.968 100.25 99.46 102.99 100.37
0.50 10.0 0.5000 10.090 100.85 100.9 99.25 101.67
Mean 100.24 100.45 100.38 101.35
S.D. 0.67 0.76 1.78 1.12
% RSD 0.67 0.76
% Error 0.34 0.38
t-Test 0.145 1.333 (2.447)
F-Test 7.018 2.143 (9.277)
a
N.B. Each result is the average of three separate determinations. The gures between parentheses are the tabulated t and F values at P 0.05.34

Table 9 System suitability test parameters for the developed HPLC method
Parameter MET
No. of theoretical plates, N 1460
Capacity factor, k0 0.36
Selectivity factor, a (in respect to I.S.) 2.64
Resolution factor, Rs (in respect to I.S.) 3.80
% RSD 0.843
Retention time (tR) 2.13
order to achieve the most suitable chromatographic conditions.
The choice was based on the highest number of theoretical
plates and the best resolution. Dierent experimental parame-
ters were changed individually while the others were kept
constant. Well-dened symmetrical peaks were obtained aer
thorough experimental trials that can be summarized as
follows.
Choice of column. Three dierent columns were used for
performance investigations, including: 150 mm
4.6 mm i.d.,
5 mm particle size Spherisorb-ODS 2 C18 column; Shim-pack VP-
ODS column (250 mm
4.6 mm i.d., 5 mm particle size) and
Shim-Pack (150 mm
4.6 mm i.d) CLC-Cyanopropyl-bonded
stationary phase. The experimental studies revealed that the
rst column was the most suitable one since it produced
symmetrical, well-dened peaks with high resolution and high
sensitivity within a reasonable analysis time. The second
column was not suitable as it showed delayed peaks. The third
column produced small, overlapped peaks with low sensitivity.
Choice of appropriate wavelength. The UV absorption
SPL spectra of the methanolic solution of the studied drugs
exhibited maxima at 237 for SPL and 235, 273 and 338 nm for
2572
2.13 MET. Both drugs show reasonable absorbance at about 235 nm.
2.17 However, three wavelengths (230, 235, 240) were tried to detect
5.44 the peaks of both drugs showing the highest sensitivity with a
0.570 reasonable response. Therefore, The UV detector response was
4.75
set at 235 nm permitting the determination of both drugs in the
recommended ratio.
Mobile phase composition. Several modications in the
mobile phase composition were performed in order to improve
the performance of the chromatographic system. These modi-
cations included: the change of the type and ratio of the
organic modier, the pH of the mobile phase and the ionic
strength of phosphate buer. The results obtained are shown in
Table 1.
Type of organic modier. Methanol was replaced by either
acetonitrile, ethanol, n-propanol or 2-propanol. Acetonitrile
resulted in good resolution of the two drugs but MET was
overlapped with the solvent front. Upon using ethanol, the
solvent front greatly interfered with the peak of MET, in addi-
tion to the poor resolution of MET peak from SPL peak. n-
Propanol and 2-propanol produced overlapping peaks of MET
and SPL. So, methanol was the organic modier of choice giving
well resolved, highly sensitive peaks within a reasonable time.
Ratio of organic modier. It was observed that the most
critical factor for the separation process is the ratio of methanol
in the mobile phase, where small variations in such ratio

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Table 10 Assay results for the determination of the studied drugs in laboratory prepared mixtures of their pharmaceutical ratiosa

Proposed method Comparison method7

Amount taken Amount found

(mg mL1) (mg mL1) % Found % Found

MET/SPL ratio MET SPL MET SPL MET SPL MET SPL

1/10 ratio 0.40 4.0 0.399 3.998 99.89 99.96 98.06 98.18
0.60 6.0 0.600 5.978 100.05 99.64 100.97 99.09
0.80 8.0 0.791 7.890 98.84 98.63 100.00 99.39
1.0 10.0 0.983 10.041 98.27 100.41 99.27 98.64
Mean 99.26 99.66 99.58 98.83
S.D.
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0.85 0.76 1.23 0.53

% RSD 0.86 0.76
% Error 0.43 0.38
t-Test 0.418 1.810 (2.447)
F-Test 2.073 2.041 (9.277)
1/20 ratio 0.05 1.0 0.049 0.987 98.54 98.71 98.53 101.00
0.10 2.0 0.100 2.004 100.33 100.18 101.47 99.50
0.30 6.0 0.295 6.021 98.22 100.35 99.02 102.00
0.50 10.0 0.497 10.041 99.39 100.41 99.63 100.25
Mean 99.12 99.91 99.66 100.69
S.D. 0.95 0.81 1.29 1.07
% RSD 0.95 0.81
% Error 0.48 0.41
t-Test 0.679 1.157 (2.447)
F-Test 1.849 1.749 (9.277)
a
N.B. Each result is the average of three separate determinations. The gures between parentheses are the tabulated t and F values at P 0.05.35

(70 : 30 v/v) caused signicant changes in the resolution and
sensitivity of the test solutes. This should be taken into
consideration during preparation of the mobile phase.
The eect of changing the ratio of organic modier on the
selectivity and retention times of the test solutes was investi-
gated using mobile phases containing concentrations of 50
80% of methanol. It was found that the retention times of both
MET and SPL decreased upon increasing the ratio of methanol.
However, the decrease in retention time of SPL was more
signicant than that of MET.
The study revealed that the optimum chromatographic
performance was achieved upon using 70% methanol (Table 1).
Although a ratio below 70% gave higher numbers of theoretical
plates and higher resolution values regarding SPL, it gave
unsymmetrical peaks with long unacceptable retention times in
addition to diminished sensitivity. Ratios less than 60% resul-
ted in very small and broad peaks of SPL with retarded retention
time, whereas ratios higher than 75% resulted in complete
overlap of the two drugs and great interference of the solvent
front with the peak of MET.

Fig. 3 Chromatograms of metolazone and spironolactone in their prepared co-formulated tablets: (A) prepared co-formulated tablet (1 : 10 ratio) (0.6 mg mL1 MET
and 6.0 mg mL1 SPL). (B) Prepared co-formulated tablet (1 : 20 ratio) (0.3 mg mL1 MET and 6.0 mg mL1 SPL). a: Solvent front; b: metolazone; c: xipamide (2 mg mL1);
d: spironolactone.

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Fig. 4 Application of the proposed method for the determination of metolazone and spironolactone in spiked human urine. (A) Blank urine under the described
chromatographic conditions. (B) Metolazone (0.4 mg mL1) and spironolactone (0.8 mg mL1) in spiked human urine under the described chromatographic conditions.
a: Metolazone; b: xipamide(1 mg mL1); c: spironolactone.

Table 11 Assay results for the determination of the studied drugs in spiked a ow rate of 1 mL min1 was found to be the optimal one for
human urine by the proposed method good separation in a reasonable time (Table 1).
The nature of internal standard. Dierent internal standards
Amount taken Amount found
Parameter (mg mL1) (mg mL1) % Found such as indapamide, furosemide, hydrochlorothiazide, xipa-
mide were investigated. Xipamide was the internal standard of
MET 0.1 0.097 97.10 choice as it showed good symmetrical and well resolved peaks
0.2 0.196 98.00 from the peaks of the studied drugs under the specied chro-
0.4 0.409 102.48
matographic conditions.
1.0 0.997 99.72
Mean 99.33
S.D. 2.37 Validation of the method
% RSD 2.38
% Error 1.18 Linearity and range. Under the above described experimental
SPL 0.5 0.478 95.70 conditions, a linear relationship was established by plotting the
0.8 0.829 103.66 peak area ratio [drug/I.S.] against the drug concentration. The
1.2 1.203 100.24
concentration ranges were found to be 0.051.0 mg mL1 for MET
1.5 1.489 99.27
Mean 99.72 and 0.510.0 mg mL1 for SPL as cited in Table 2. Linear regres-
S.D. 3.27 sion analysis of the data gave the following equations:
% RSD 3.28
% Error 1.64 P 3.80
103 + 1.115C (r 0.9999) for MET

P 3.20
103 + 0.373C (r 0.9999) for SPL

Apparent pH. The eect of changing the pH of the mobile
phase on the selectivity and retention times of the test solutes
was investigated using mobile phases of pH ranging from 2.6
7.0. The study revealed that the eect of pH of mobile phase was
not critical over the studied pH range. It was found that
changing the pH did not aect the resolution of both drugs.
Also, it was noticed that above pH 3.0, the number of theoretical
plates slightly decreased and became quite constant up to pH 7.
Table 1 shows that pH 3.0 was the most appropriate one
considering dierent chromatographic parameters.
Ionic strength of buer. The eect of changing the ionic
strength of phosphate buer on the selectivity and retention
times of the test solutes was investigated using mobile phases
containing a concentration of 0.010.1 M of phosphate buer. It
was found that changing the ionic strength of phosphate buer
did not aect the selectivity and retention times of the test
solutes (Table 1). However, 0.02 M phosphate buer was
selected to be used throughout the work.
Flow rate. The eect of ow rate on the formation and
separation of peaks of the studied compounds was studied and
where P is the peak area ratio, C is the concentration of the drug
in mg mL1 and r is the correlation coecient.
Statistical analysis35 of the data gave high value of the corre-
lation coecient (r) of the regression equation, small values of
the standard deviation of residuals (Sy/x), of intercept (Sa), and of
slope (Sb), and small values of the percentage relative standard
deviation and the percentage relative error (Table 2). These
data proved the linearity of the calibration graphs.
Limit of quantitation (LOQ) and limit of detection (LOD).
The limit of quantitation (LOQ) was determined by establishing
the lowest concentration that can be measured according to
ICH Q2R1 recommendations36 below which the calibration
graph is non linear. The limit of detection (LOD) was deter-
mined by establishing the minimum level at which the analyte
can be reliably detected.
LOQ 10Sa/b LOD 3.3Sa/b
where Sa standard deviation of the intercept of the calibration
curve and b slope of the calibration curve.

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Fig. 5 Chromatograms showing metolazone and spironolactone with co-administered or co-formulated drugs: a: solvent front; b: metolazone (1 mg mL1); c: spi-
ronolactone (10 mg mL1); BUM: 5 mg mL1 bumetanide; LOS: 4 mg mL1 losartan potassium; HCT: 4 mg mL1 hydrochlorothiazide; LIS: 20 mg mL1 lisinopril dihydrate;
ENP: 5 mg mL1 enalapril; PRP: 3 mg mL1 propranolol hydrochloride; FUR: 2 mg mL1 furosemide; ASP: 6 mg mL1 aspirin.
LOQ and LOD values for MET and SPL by the proposed
method are presented in Table 2. LOQ values were found to be
0.03 and 0.11 mg mL1 while LOD values were found to be 0.009
and 0.04 mg mL1 for MET and SPL, respectively.
Accuracy and precision. To prove the accuracy of the
proposed method, the results of the proposed method were
compared with those obtained using the comparison method.7
Statistical analysis of the results obtained using Student's t-test
and variance ratio F-test35 revealed no signicant dierence
between the performance of the two methods regarding accu-
racy and precision, respectively (Table 3).
The comparison method7 described three UV spectroscopic
methods for simultaneous estimation of metolazone and spi-
ronolactone in combined dosage form. It involves rst deriva-
tive spectroscopy using 266 nm and 289 nm as zero crossing
points for MET and SPL, respectively.
Intra-day precision. Intra-day precision was assessed
through replicate analysis of three concentrations of the studied
drugs at three successive times within the same day. The results
are abridged in (Table 4).
Inter-day precision. Inter-day precision was carried out
through replicate analysis of three concentrations of the studied
drugs on three successive days. The results are summarized in
(Table 4).
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Analytical Methods
The relative standard deviations were found to be very small
indicating reasonable repeatability and intermediate precision
of the proposed method.
Robustness of the method. The robustness of the proposed
method was evaluated by the maintenance of the peak area with
the deliberated changes in the experimental parameters; these
parameters include (pH 3.0  0.1), methanol concentration
70  1% (v/v) and buer strength (0.02  0.01). These minor
changes did not greatly aect the peak area of both drugs. The
results are abridged in (Table 5).
Solution stability and mobile phase stability. The stability of
the stock solutions was determined by quantitation of both
MET and SPL and comparing the results to freshly prepared
solutions. It was found that no signicant change was observed
in standard solution response, relative to freshly prepared
standard. Similarly, the stability of the mobile phase was
checked. The results obtained in Table 6 prove that the sample
solution and mobile phase used during the assay were stable up
to 10 days.
Selectivity. The selectivity of the method was investigated by
observing any interference encountered from common tablet
excipients such as lactose, starch, magnesium stearate, and talc.
It was shown that these compounds did not interfere with the
results of the proposed method as shown in Tables 7 and 8.
Anal. Methods, 2013, 5, 56445656 | 5653

Analytical Methods
System suitability test (SST)
Evaluation of SST parameters was performed during the devel-
opment and optimization of the method (Table 1). Moreover, to
ascertain the eectiveness of the nal operating system it was
subjected to suitability testing. The test was performed by
injecting the standard mixture in triplicate and the parameters
were calculated as reported by the USP.3 SST parameters include
capacity factor (k0 ), selectivity factor (a), resolution factor (Rs)
and column eciency (number of theoretical plates, N). The
nal SST parameters under the optimum chromatographic
conditions are abridged in Table 9.
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Applications
Analysis of MET/SPL laboratory prepared mixtures. The
proposed method was applied to the simultaneous determina-
tion of MET and SPL in laboratory prepared mixtures that
medicinally recommended in ratios of 1 : 10 and 1 : 20 (Fig. 2).
The concentrations of both drugs in the laboratory prepared
mixtures were calculated according to the linear regression
equations of the calibration graphs. The proposed method was
favorably compared with the comparison method.7 The results
obtained are shown in Table 10. Statistical analysis of the
results obtained by the proposed and comparison methods
proved no signicant dierence in the performance of both
methods regarding accuracy and precision.
Pharmaceutical application
Dosage form analysis. The proposed method was successfully
applied to the assay of the studied drugs in single tablets. The
results of the proposed method were favorably compared with
those obtained using the comparison method.7 The results are
abridged in Table 7. The proposed method was further applied
to the determination of the studied drugs in laboratory
prepared co-formulated tablets. The results shown in (Table 8)
are in good agreement with those obtained with the comparison
method.7 Statistical analysis of the results obtained using Stu-
dent's t-test and variance ratio F-test35 revealed no signicant
dierence between the performance of the two methods
regarding the accuracy and precision, respectively (Tables 7
and 8). Fig. 3 shows chromatograms indicating good resolved
peaks of MET and SPL in their laboratory prepared co-formu-
lated tablets.
Biological application. MET is incompletely but fairly readily
absorbed aer oral administration. About 80% of a dose is
excreted in the urine as unchanged drug in 48 h.37 SPL is rapidly
but incompletely absorbed aer oral administration; it is sub-
jected to extensive rst-pass metabolism and enterohepatic
circulation. The metabolism of spironolactone is very complex
and there are a large number of metabolites. About 25 to 55% of
a dose is excreted in the urine in 6 days.37 SPL is excreted 40%
unaltered and 60% metabolized to canrenone (major metabo-
lite).38 The high sensitivity of the proposed method could allow
the determination of the studied drugs in spiked human urine.
Analysis of MET and SPL in spiked human urine. A simple
precipitation procedure was adopted for the analysis of MET
and SPL in spiked human urine using XPM as an internal
standard.
5654 | Anal. Methods, 2013, 5, 56445656
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Fig. 4 shows MET and SPL peaks obtained from spiked urine
analysis. Table 11 shows the results obtained from spiked urine.
Under the above described experimental conditions, a linear
relationship was established by plotting the peak area ratio
against concentration. Linear regression analysis of the data
gave the following equations:
P 1.47
102 + 1.23C (r 0.9998) for MET
P 2.80
103 + 0.38C (r 0.9988) for SPL
where P is the peak area ratio, C is the concentration of the drug
in mg mL1 and r is the correlation coecient.
The high value of the correlation coecient (r) indicates the
good linearity of the calibration graph.
Co-administered and related drugs. The proposed method
allows the determination of the studied drugs in the presence of
some co-administered or co-formulated drugs such as; furose-
mide, hydrochlorothiazide, propranolol, losartan, ramipril,
bumetanide, fosinopril, lisinopril, enalapril, captopril and
aspirin as shown in Fig. 5. None of the above mentioned drugs
interfered with the HPLC assay of the studied drugs except
furosemide which overwrote MET peak.
Conclusion
New simple, accurate and highly sensitive chromatographic
method with UV detection was explored for the simultaneous
determination of MET and SPL in binary mixtures. The
proposed method was found to have limits of detection of 0.009,
0.04 mg mL1 and limits of quantitation of 0.03, 0.11 mg mL1
for MET and SPL, respectively. In addition, it could be applied to
the analysis of both drugs in their single and co-formulated
dosage forms. The good validation criteria of the proposed
method allow its use in quality control laboratories. It also
oers the possibility to determine the studied drugs in the
presence of the frequently co-administered drugs; furosemide,
hydrochlorothiazide, propranolol, losartan, ramipril, bumeta-
nide, fosinopril, lisinopril, enalapril, captopril and aspirin. The
proposed procedure, by virtue of its high sensitivity, could be
applied to the analysis of MET and SPL in spiked human urine
with simple pretreatment procedures.
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