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Simultaneous determination of metolazone and
spironolactone in raw materials, combined tablets and
human urine by high performance liquid
chromatography
M. I. Walash, N. El-Enany, M. I. Eid and M. E. Fathy*
A new, specific 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 buffer (70 : 30) v/v at pH 3.0 was used. The analysis was performed at a
flow 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 0.05–1.0 mg mL1 and 0.5–10.0 mg mL1
with limits of detection (LOD) of 0.009, 0.04 ng mL1 and limits of quantification (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 coformulated tablets. The method was further extended to the determination of both drugs in spiked
Received 5th July 2013
Accepted 11th August 2013
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.
DOI: 10.1039/c3ay41110a
www.rsc.org/methods
Statistical evaluation and comparison of the data obtained by the proposed and comparison methods
revealed no significant difference between the two methods regarding accuracy and precision.
Introduction
Metolazone (Fig. 1a), 7-chloro-1,2,3,4-tetrahydro-2-methyl-3-(2methylphenyl)-4-oxo-6-quinazolinesulfonamide,1 is a diuretic
with similar actions and uses to those of the thiazide diuretics.
It is orally administered for treatment of edema associated with
heart failure and for management of hypertension.2 Spironolactone (Fig. 1b), (7a,17a)-7-(acetylthio)-17-hydroxy-3-oxopregn-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 diminishes their kaliuretic effects. Hence, potassium is conserved in
those who are at risk from hypokalemia.2
A new combination dosage form of metolazone and spironolactone is indicated for the treatment and management of
oedema and hypertension.
Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura,
35516, Mansoura, Egypt. E-mail: [email protected]; Fax: +20 502247496
5644 | Anal. Methods, 2013, 5, 5644–5656
Metolazone and spironolactone are official drugs in the
United States Pharmacopoeia (USP),3 the British Pharmacopoeia (BP),4 and in the Europium Pharmacopoeia.5
Reviewing the literature revealed that several methods;
such as spectrophotometric,6–10 HPTLC,11,12 and liquid
chromatography3,13–15 were used for the determination of
metolazone in pharmaceutical preparations either alone6–8,14
or in combination with losartan,13 spironolactone7,9 or
Fig. 1 The structural formulae of the studied drugs, (a) metolazone, (b)
spironolactone.
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Paper
Analytical Methods
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,16–19 or with furosemide.20 Also,
liquid chromatography-tandem mass spectrometry (LCMS),21–24 has been reported for the analysis of metolazone in
human plasma or blood.
Regarding spironolactone, a good guide to the work published is presented as the comprehensive monographs in the
series of Analytical Proles of Drug Substances25 and Excipients.26 The most recently published articles about SPL include:
Table 1
Optimization of the chromatographic conditions for separation of metolazone and spironolactone mixture by the proposed HPLC methoda
Parameter
pH of the mobile phase
Ratio of organic modier A/B
Ionic strength of phosphate buffer, M
Flow rate (mL min1)
a
A:
spectrophotometry,4,7,9,27 TLC,28 liquid chromatography,3,28–33
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 0.5–2.5 mg mL1 and
phosphate
buffer.
B:
Mass distribution ratio ðDm Þ ¼
methanol,
2.6
3.0
3.5
4.0
5.0
6.0
7.0
25/75
30/70
35/65
40/60
0.01
0.02
0.04
0.06
0.1
0.6
0.8
1.0
1.2
1.4
where:
No. of theoretical
plates (N)
Mass distribution
ratio (Dm)
MET
SPL
MET
SPL
1171
1456
1371
1281
1287
1347
1299
1433
1472
1490
1445
1436
1452
1433
1429
1483
1586
1439
1460
1202
1022
2275
2594
2496
2450
2456
2972
2678
2643
2567
6540
8505
2339
2585
2456
2527
2575
2873
2656
2544
2314
2040
0.377
0.363
0.365
0.366
0.298
0.379
0.359
0.243
0.362
0.504
0.803
0.325
0.367
0.359
0.362
0.349
0.367
0.363
0.365
0.363
0.362
2.347
2.137
2.095
2.148
1.664
1.984
1.786
1.185
2.129
2.890
5.741
2.012
2.154
2.063
2.019
2.076
2.104
2.089
2.116
2.083
2.079
number
of
tR tm
Dm2
. Relative retention ðaÞ ¼
.
tm
Dm1
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theoretical
plates
Resolution
(Rs)
Relative retention
(a)
8.645
6.225
8.903
6.100
7.916
5.739
8.582
5.869
7.665
5.584
8.866
5.235
8.296
4.975
6.388
4.876
8.889
5.881
13.932
5.734
20.706
7.149
8.695
6.191
8.946
5.869
8.716
5.746
8.592
5.577
9.03
5.948
9.345
5.733
8.925
5.755
8.906
5.797
8.292
5.738
7.696
5.743
2
tR
2DtR
ðNÞ ¼ 5:54
. Resolution ðRÞ ¼
.
Wh=2
W1 þ W2
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Table 2
Paper
Analytical performance data for the determination of the studied drugs by the proposed method
Parameter
MET
SPL
Linearity range (mg mL1)
Intercept (a)
Slope (b)
Correlation coefficient (r)
S.D. of residuals (Sy/x)
S.D. of intercept (Sa)
S.D. of slope (Sb)
Percentage relative standard deviation, % RSD
Percentage relative error, % error
Limit of detection, LOD (mg mL1)
Limit of quantitation, LOQ (mg mL1)
0.05–1.0
3.80 103
1.115
0.9999
3.80 103
2.90 103
4.70 103
0.843
0.343
0.009
0.03
0.5–10.0
3.20 103
0.373
0.9999
6.60 103
4.20 103
8.10 104
0.570
0.233
0.04
0.11
5–25 mg mL1, respectively. Also, Chaudhary9 estimated MET
and SPL over concentration ranges of 1–5 mg mL1 and 5–
25 mg mL1, respectively. Therefore, the proposed method
(which assayed MET and SPL over concentration ranges of
0.05–1.0 mg mL1 and 0.5–10.0 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 laboratory 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
Table 3
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 efficient 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 quantitative determination of the studied drugs in single and coformulated tablets, as well as in human urine. No interference
was encountered from other co-administered and co-formulated drugs such as furosemide, hydrochlorothiazide,
propranolol, losartan, ramipril, bumetanide, fosinopril, lisinopril, enalapril, captopril and aspirin.
Assay results for the determination of the studied drugs in pure form by the proposed and comparison methodsa
Proposed method
Compound
MET
Comparison method7
Amount taken
(mg mL1)
Amount found
(mg mL1)
0.05
0.20
0.40
0.60
0.80
1.00
0.049
0.199
0.399
0.603
0.804
0.995
Mean
S.D.
t-Test
F-Test
SPL
40.0
80.0
160.0
320.0
400.0
800.0
Mean
S.D.
t-Test
F-Test
a
40.50
79.60
161.50
316.30
401.60
800.30
% Found
% Found
98.20
99.65
99.83
100.43
100.54
99.55
99.70
0.84
1.290
(2.306)
1.551
(5.409)
101.25
99.50
100.94
98.84
100.40
100.04
100.02
0.57
0.349
(2.306)
2.430
(5.409)
100.00
101.85
99.38
100.66
100.47
1.06
99.18
100.55
101.21
99.77
100.18
0.89
N.B. Each result is the average of three separate determinations. The gures between parentheses are the tabulated t and F values at P ¼ 0.05.35
5646 | Anal. Methods, 2013, 5, 5644–5656
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Table 4
Analytical Methods
Precision data for the determination of the studied drugs by the proposed methoda
MET concentration (mg mL1)
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Parameters
Intraday
% Found
Inter-day
(x) S.D.
% RSD
% Error
% Found
(x) S.D.
% RSD
% Error
a
SPL concentration (mg mL1)
0.20
0.40
0.60
2.0
4.0
6.0
98.87
100.02
100.44
99.78 0.81
0.82
0.47
101.95
96.84
100.85
99.88 2.69
2.69
1.56
97.72
100.45
98.98
99.05 1.37
1.38
0.80
100.61
102.55
101.27
101.48 0.99
0.97
0.56
101.08
100.27
98.67
100.01 1.23
1.23
0.71
99.02
98.00
100.19
99.07 1.10
1.11
0.64
99.95
101.78
99.93
100.55 1.06
1.06
0.61
101.95
102.74
99.94
101.54 1.44
1.42
0.82
99.14
99.76
98.44
99.11 0.66
0.67
0.39
98.66
100.29
100.77
99.91 1.11
1.11
0.64
101.08
99.94
99.64
100.22 0.76
0.76
0.44
99.58
103.14
100.82
101.18 1.81
1.79
1.03
N.B. Each result is the average of three separate determinations.
Table 5 Robustness of the proposed method using metolazone (0.5 mg mL1)
and spironolactone (5.0 mg mL1)a
Parameter
Amount found
(mg mL1)
% Found
MET
SPL
MET
SPL
5.017
5.090
4.942
100.91
98.79
102.22
100.64
1.73
1.72
0.99
100.34
101.80
98.83
100.32
1.49
1.48
0.85
5.037
4.940
4.988
98.81
98.49
100.73
99.34
1.21
1.22
0.70
100.74
98.80
99.75
99.76
0.97
0.97
0.56
5.067
5.021
4.950
99.28
100.66
99.80
99.91
0.71
0.71
0.40
101.33
100.42
99.00
100.25
1.17
1.17
0.68
Methanol ratio, %
69
0.505
70
0.494
71
0.511
(x)
S.D.
% RSD
% Error
pH
2.9
3.0
3.1
(x)
S.D.
% RSD
% Error
0.494
0.492
0.504
Buffer strength, M
0.01
0.496
0.02
0.503
0.04
0.499
(x)
S.D.
% RSD
% Error
a
N.B. Each result is the average of three separate determinations.
Experimental
Apparatus
Chromatographic separation was carried out using a Shimadzu
LC-20AD Prominence liquid chromatogram equipped with a
Rheodyne injector valve with a 20 mL loop and a SPD-20A UV
detector. Mobile phases were ltered using 0.45 mm membrane
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lters (Millipore, Cork, Ireland) and degassed using a prominence degasser DGU-20A5.
Syringe 0.45 mm lters were used for human urine ltration.
A Consort NV P-901 calibrated pH-meter (Belgium) was used
for pH measurements.
Ultrasonic bath (Model: SS 101H 230, USA), vortex mixer
(Model: VM-300P, Gemmy Industrial Corp., Taiwan) and
centrifuge (Model: 2-16P, Sigma Laborzentrifugen, Germany)
were used for human urine sample preparation.
Materials and reagents
All the chemicals and pharmaceuticals used were of analytical
reagent grade and pharmaceutical grade, and the solvents were
of HPLC grade.
Metolazone was kindly provided by Pharmaceutical Div.,
Pennwalt Corp., Rochester, N.Y. Spironolactone was kindly
provided by Memphis for PHARM. & CHEM. IND. CO., Cairo,
Egypt. The purity percentages of MET; 100.45%, and SPL;
99.88%, were established by applying the USP3 and BP4
methods, respectively.
Xipamide, used as the internal standard (IS), was provided by
Egyptian INT. Pharmaceutical Industries CO. (EIPICO), Egypt.
Metenix tablets (Sano-aventis S.A.E, Egypt) and aldactone
tablets (KAHIRA PHARM. and CHEM. IND. CO., Cairo, Egypt)
were purchased from commercial sources in the local
pharmacy.
Metenix tablets; batch # 099536, labeled to contain 5 mg
metolazone per tablet.
Aldactone 25 mg tablets; batch # 1110433-L and aldactone 100 mg; batch # 1110556-L.E. tablets labeled to contain 25
and 100 mg spironolactone, respectively.
Laboratory prepared Co-formulated tablets were prepared
according to their pharmaceutical ratios, by mixing accurately weighed quantities (equivalent to either 2.5 or 5.0 mg
MET) of the mixed contents of 10 powdered metenix tablets
with accurately weighed quantities (equivalent to 50.0 mg
SPL) of the mixed contents of 10 powdered aldactone
tablets.
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Table 6 Results of MET and SPL solutions stability and mobile phase stability
using metolazone (0.5 mg mL1) and spironolactone (5.0 mg mL1)
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Parameter
Amount found
(mg mL1)
% Found
MET
SPL
MET
SPL
4.913
4.974
5.062
5.031
5.002
100.71
99.80
100.01
98.38
99.91
99.76
0.85
0.85
0.38
98.25
99.47
101.23
100.61
100.04
99.92
1.14
1.14
0.51
101.38
99.88
100.74
101.51
98.42
100.39
1.28
1.272
0.57
100.89
99.20
100.23
99.03
100.80
100.03
0.88
0.87
0.39
Stock solution prepared
Fresh
0.504
2 days ago
0.499
4 days ago
0.500
6 days ago
0.492
10 days ago
0.499
(x)
S.D.
% RSD
% Error
Mobile phase prepared
Fresh
0.507
3 days ago
0.499
5 days ago
0.504
7 days ago
0.508
10 days ago
0.492
(x)
S.D.
% RSD
% Error
5.045
4.960
5.012
4.952
5.040
Orthophosphoric acid 85% (Riedel-deHa¨
en, Sleeze,
Germany).
Acetonitrile, ethanol, n-propanol and 2-propanol (SigmaAldrich, Germany).
Methanol (Tedia, USA).
Sodium dihydrogen phosphate mono hydrate, sodium
hydroxide (El-Nasr Pharmaceutical Chemicals Company
(ADWIC), Egypt).
Human urine was obtained from healthy volunteers.
Chromatographic conditions
Column: 150 mm 4.6 mm i.d., 5 mm particle size SpherisorbODS 2 C18 column.
Mobile phase: a solution consists of a mixture of methanol
and 0.02 M sodium dihydrogen phosphate (70 : 30) v/v and the
apparent pH was adjusted to 3.0 using orthophosphoric acid.
The mobile phase was ltered through a 0.45 mm membrane
lter (Millipore, Cork, Ireland).
Flow rate: 1 mL min1.
UV detector wavelength: 235 nm.
Internal standard: xipamide (standard solution containing
400 mg mL1 of xipamide was prepared in methanol and further
diluted with methanol to get the appropriate working standard
solution).
Temperature: room temperature.
Standard solutions
Stock solutions of 400 mg mL1 MET and 400 mg mL1 SPL
were prepared by dissolving 20.0 mg of MET or SPL in 50.0 mL
of methanol with the aid of an ultrasonic bath. Working
5648 | Anal. Methods, 2013, 5, 5644–5656
standard solutions were prepared by appropriate dilution of
the stock solutions with methanol. Standard laboratory
prepared mixture solutions were prepared by mixing appropriate volumes of MET and SPL stock solutions in 50.0 mL
volumetric asks and completing to the volume with methanol
keeping the pharmaceutical ratios of 1 : 10 and 1 : 20 for MET
and SPL, respectively. Solutions of MET and SPL should be
stored in light resistant containers;3 this was fullled by
covering the asks with aluminium foil. All solutions were
stored in the refrigerator at 2 C and found to be stable for at
least 10 days without alteration.
General recommended procedures
Construction of calibration graphs. Accurately measured
aliquot volumes of the suitable drug working standard solutions were transferred into a series of 10.0 mL volumetric asks
so that the nal concentrations were over the range of 0.05–1.0
mg mL1 for MET and 0.5–10.0 mg mL1 for SPL. To each ask,
0.2 mL of XPM working standard solution was added as internal
standard so that its nal concentration was 2.0 mg mL1. Then,
the solutions were completed to the volume with the mobile
phase at pH 3.0 and mixed well. Aliquots of 20 mL were injected
(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
mL1 were plotted. Alternatively, the corresponding regression
equations were derived.
Analysis of bulk substances. The method mentioned above
was applied to the determination of the purity of raw material
for each drug. The percentage recoveries were calculated by
referring to the previously prepared calibration graphs or using
the corresponding regression equations.
Analysis of MET/SPL laboratory prepared mixtures. Aliquots
of MET and SPL standard laboratory prepared mixture solutions were transferred into a series of 10.0 mL volumetric
asks. The solutions were diluted to the volume with the
mobile phase and mixed well. The above procedure described
under “Construction of calibration graphs” was then applied.
The percentage recoveries were calculated by referring to the
calibration graphs, or using the corresponding regression
equations.
Analysis of the studied drugs in their single tablets. Ten
tablets (Metenix, Aldactone 25, or Aldactone 100) were
accurately weighed, nely pulverized, and thoroughly mixed.
Accurately weighed quantities of pulverized tablets equivalent
to 5.0 mg of MET or 20.0 mg of SPL were transferred into 50 mL
volumetric asks and about 40.0 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
containing suitable concentrations of the studied drugs were
analyzed as described under “Construction of calibration
graphs”. The nominal contents were calculated either from
previously plotted calibration graphs or using the corresponding regression equations.
Analysis of the studied drugs in their laboratory prepared coformulated tablets. Accurately weighed quantities of the mixed
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Table 7
Analytical Methods
Assay results for the determination of the studied drugs in their single tablets by the proposed methoda
Proposed method
Compound
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Metenix 5 mg tablets
Mean
S.D.
% RSD
% Error
t-Test
F-Test
Aldactone 25 mg tablets
Mean
S.D.
% RSD
% Error
t-Test
F-Test
Aldactone 100 mg
tablets
Amount taken
(mg mL1)
Amount found
(mg mL1)
0.40
0.60
0.80
1.00
0.389
0.597
0.788
1.001
4.0
6.0
8.0
10.0
4.0
6.0
8.0
10.0
3.936
6.085
7.922
9.844
3.951
6.055
7.891
10.055
Mean
S.D.
% RSD
% Error
t-Test
F-Test
a
% Found
Comparison method7
97.47
99.59
98.52
100.12
98.53
1.05
1.07
0.62
1.268
1.630
98.39
101.42
99.03
98.44
99.32
1.43
1.44
0.72
0.955
1.168
98.77
100.91
98.64
100.55
99.72
1.18
1.18
0.59
1.788
3.742
101.48
98.00
100.74
100.32
100.14
1.50
(2.447)
(9.277)
99.00
100.50
102.00
99.50
100.25
1.32
(2.447)
(9.277)
100.20
101.00
101.67
100.75
100.91
0.61
(2.447)
(9.277)
N.B. Each result is the average of three separate determinations. The gures between parentheses are the tabulated t and F values at P ¼ 0.05.35
contents of 10 prepared co-formulated tablets equivalent to 2.5
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. Aer 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 carefully aspirated, quantitatively transferred into 10 mL volumetric
asks and analyzed as described under “Construction of calibration graphs”. A blank experiment was carried out simultaneously. The peak area ratios were plotted versus the
concentrations of the drugs in mg mL1.
This journal is ª The Royal Society of Chemistry 2013
Results and discussion
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) ¼ 3.80 (MET in respect to XPM) and
5.44 (SPL in respect to XPM) and selectivity factors (a) ¼ 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 chromatogram 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 offers 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 different parameters affecting the chromatographic
performance of the studied drugs were carefully studied in
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Analytical Methods
Table 8
Paper
Assay results for the determination of the studied drugs in their prepared co-formulated tablets by the proposed methoda
Comparison
method7
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Proposed method
Amount taken
(mg mL1)
Amount found
(mg mL1)
% Found
Preparation
MET
SPL
MET
MET
SPL
MET
SPL
Prepared co-formulated tablet (1/10 ratio)
0.40
0.60
0.80
1.0
4.0
6.0
8.0
10.0
0.403
0.593
0.806
0.998
100.85
98.92
100.78
99.8
100.09
0.91
0.91
0.46
0.650
2.332
100.55
99.30
100.25
100.85
100.24
0.67
0.67
0.34
0.145
7.018
100.92
98.27
101.22
99.7
100.03
1.34
1.34
0.67
1.191
1.400
101.17
100.25
99.46
100.9
100.45
0.76
0.76
0.38
1.333
2.143
97.98
101.35
99.66
99.19
99.55
1.40
102.00
101.00
99.50
101.80
101.08
1.14
100.00
99.26
102.99
99.25
100.38
1.78
(2.447)
(9.277)
102.78
100.56
100.37
101.67
101.35
1.12
Mean
S.D.
% RSD
% Error
t-Test
F-Test
Prepared co-formulated tablet (1/20 ratio)
0.05
0.10
0.30
0.50
1.0
2.0
6.0
10.0
0.0503
0.0993
0.3008
0.5000
Mean
S.D.
% RSD
% Error
t-Test
F-Test
a
SPL
4.037
5.896
8.098
9.970
1.012
2.005
5.968
10.090
% Found
(2.447)
(9.277)
N.B. Each result is the average of three separate determinations. The gures between parentheses are the tabulated t and F values at P ¼ 0.05.34
Table 9
System suitability test parameters for the developed HPLC method
Parameter
MET
SPL
No. of theoretical plates, N
Capacity factor, k0
Selectivity factor, a (in respect to I.S.)
Resolution factor, Rs (in respect to I.S.)
% RSD
Retention time (tR)
1460
0.36
2.64
3.80
0.843
2.13
2572
2.13
2.17
5.44
0.570
4.75
order to achieve the most suitable chromatographic conditions.
The choice was based on the highest number of theoretical
plates and the best resolution. Different experimental parameters were changed individually while the others were kept
constant. Well-dened symmetrical peaks were obtained aer
thorough experimental trials that can be summarized as
follows.
Choice of column. Three different 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 VPODS 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-dened 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.
5650 | Anal. Methods, 2013, 5, 5644–5656
Choice of appropriate wavelength. The UV absorption
spectra of the methanolic solution of the studied drugs
exhibited maxima at 237 for SPL and 235, 273 and 338 nm for
MET. Both drugs show reasonable absorbance at about 235 nm.
However, three wavelengths (230, 235, 240) were tried to detect
the peaks of both drugs showing the highest sensitivity with a
reasonable response. Therefore, The UV detector response was
set at 235 nm permitting the determination of both drugs in the
recommended ratio.
Mobile phase composition. Several modications 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 modier, the pH of the mobile phase and the ionic
strength of phosphate buffer. The results obtained are shown in
Table 1.
Type of organic modier. 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 addition to the poor resolution of MET peak from SPL peak. nPropanol and 2-propanol produced overlapping peaks of MET
and SPL. So, methanol was the organic modier of choice giving
well resolved, highly sensitive peaks within a reasonable time.
Ratio of organic modier. 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
Analytical Methods
Assay results for the determination of the studied drugs in laboratory prepared mixtures of their pharmaceutical ratiosa
Comparison method7
Proposed method
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Amount taken
(mg mL1)
Amount found
(mg mL1)
% Found
% Found
MET/SPL ratio
MET
SPL
MET
SPL
MET
SPL
MET
SPL
1/10 ratio
0.40
0.60
0.80
1.0
4.0
6.0
8.0
10.0
0.399
0.600
0.791
0.983
3.998
5.978
7.890
10.041
99.89
100.05
98.84
98.27
99.26
0.85
0.86
0.43
0.418
2.073
98.54
100.33
98.22
99.39
99.12
0.95
0.95
0.48
0.679
1.849
99.96
99.64
98.63
100.41
99.66
0.76
0.76
0.38
1.810
2.041
98.71
100.18
100.35
100.41
99.91
0.81
0.81
0.41
1.157
1.749
98.06
100.97
100.00
99.27
99.58
1.23
98.18
99.09
99.39
98.64
98.83
0.53
98.53
101.47
99.02
99.63
99.66
1.29
(2.447)
(9.277)
101.00
99.50
102.00
100.25
100.69
1.07
Mean
S.D.
% RSD
% Error
t-Test
F-Test
1/20 ratio
0.05
0.10
0.30
0.50
1.0
2.0
6.0
10.0
0.049
0.100
0.295
0.497
0.987
2.004
6.021
10.041
Mean
S.D.
% RSD
% Error
t-Test
F-Test
a
(2.447)
(9.277)
N.B. Each result is the average of three separate determinations. The gures between parentheses are the tabulated t and F values at P ¼ 0.05.35
(70 : 30 v/v) caused signicant changes in the resolution and
sensitivity of the test solutes. This should be taken into
consideration during preparation of the mobile phase.
The effect of changing the ratio of organic modier on the
selectivity and retention times of the test solutes was investigated 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
signicant 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% resulted 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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Paper
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
human urine by the proposed method
Parameter
MET
Mean
S.D.
% RSD
% Error
SPL
Amount taken
(mg mL1)
Amount found
(mg mL1)
0.1
0.2
0.4
1.0
0.097
0.196
0.409
0.997
0.5
0.8
1.2
1.5
0.478
0.829
1.203
1.489
Mean
S.D.
% RSD
% Error
% Found
97.10
98.00
102.48
99.72
99.33
2.37
2.38
1.18
95.70
103.66
100.24
99.27
99.72
3.27
3.28
1.64
a ow rate of 1 mL min1 was found to be the optimal one for
good separation in a reasonable time (Table 1).
The nature of internal standard. Different internal standards
such as indapamide, furosemide, hydrochlorothiazide, xipamide were investigated. Xipamide was the internal standard of
choice as it showed good symmetrical and well resolved peaks
from the peaks of the studied drugs under the specied chromatographic conditions.
Validation of the method
Linearity and range. Under the above described experimental
conditions, a linear relationship was established by plotting the
peak area ratio [drug/I.S.] against the drug concentration. The
concentration ranges were found to be 0.05–1.0 mg mL1 for MET
and 0.5–10.0 mg mL1 for SPL as cited in Table 2. Linear regression analysis of the data gave the following equations:
P ¼ 3.80 103 + 1.115C (r ¼ 0.9999) for MET
P ¼ 3.20 103 + 0.373C (r ¼ 0.9999) for SPL
Apparent pH. The effect 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 effect of pH of mobile phase was
not critical over the studied pH range. It was found that
changing the pH did not affect 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 different chromatographic parameters.
Ionic strength of buffer. The effect of changing the ionic
strength of phosphate buffer on the selectivity and retention
times of the test solutes was investigated using mobile phases
containing a concentration of 0.01–0.1 M of phosphate buffer. It
was found that changing the ionic strength of phosphate buffer
did not affect the selectivity and retention times of the test
solutes (Table 1). However, 0.02 M phosphate buffer was
selected to be used throughout the work.
Flow rate. The effect of ow rate on the formation and
separation of peaks of the studied compounds was studied and
5652 | Anal. Methods, 2013, 5, 5644–5656
where P is the peak area ratio, C is the concentration of the drug
in mg mL1 and r is the correlation coefficient.
Statistical analysis35 of the data gave high value of the correlation coefficient (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 determined by establishing the minimum level at which the analyte
can be reliably detected.
LOQ ¼ 10Sa/b LOD ¼ 3.3Sa/b
where Sa ¼ standard deviation of the intercept of the calibration
curve and b ¼ slope of the calibration curve.
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Analytical Methods
Fig. 5 Chromatograms showing metolazone and spironolactone with co-administered or co-formulated drugs: a: solvent front; b: metolazone (1 mg mL1); c: spironolactone (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 signicant difference
between the performance of the two methods regarding accuracy and precision, respectively (Table 3).
The comparison method7 described three UV spectroscopic
methods for simultaneous estimation of metolazone and spironolactone in combined dosage form. It involves rst derivative 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).
This journal is ª The Royal Society of Chemistry 2013
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 buffer strength (0.02 0.01). These minor
changes did not greatly affect 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 signicant 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.
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Analytical Methods
System suitability test (SST)
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Evaluation of SST parameters was performed during the development and optimization of the method (Table 1). Moreover, to
ascertain the effectiveness 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 efficiency (number of theoretical plates, N). The
nal SST parameters under the optimum chromatographic
conditions are abridged in Table 9.
Applications
Analysis of MET/SPL laboratory prepared mixtures. The
proposed method was applied to the simultaneous determination 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 signicant difference 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 Student's t-test and variance ratio F-test35 revealed no signicant
difference 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-formulated tablets.
Biological application. MET is incompletely but fairly readily
absorbed aer 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 aer oral administration; it is subjected 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 metabolite).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, 5644–5656
Paper
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 ¼ 1.47 102 + 1.23C (r ¼ 0.9998) for MET
P ¼ 2.80 103 + 0.38C (r ¼ 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 coefficient.
The high value of the correlation coefficient (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; furosemide, 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
offers the possibility to determine the studied drugs in the
presence of the frequently co-administered drugs; furosemide,
hydrochlorothiazide, propranolol, losartan, ramipril, bumetanide, 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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