“RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION
OF MOXONIDINE AND HYDROCHLOROTHIAZIDE IN BULK
DRUG AND TABLET FORMULATION”
SHILPA KORTI*, CHANNABASAVARAJ K P, SOMASHEKAR P L
Department of Pharmaceutical Chemistry,
Government College of Pharmacy,
Bengaluru, Karnataka, India.
Address for correspondence *
Contact no- 09980676409
Email ID- Ksuhaani@gmail.com
ABSTRACT
A new, simple, sensitive, selective, precise, rapid, economic and isocratic reverse
phase high performance liquid chromatography (RP-HPLC) method was developed and
validated for simultaneous estimation of Moxonidine (MOX) and Hydrochlorothiazide
(HCTZ) in bulk drug and tablet dosage forms. The separation was achieved by using C8
Phenomenex Luna (250 x 4.6mm, 5µm) column with a mobile phase consisting of
acetonitrile and formic acid solution (0.2%v/v) in the ratio 50:50. The mobile phase
was delivered at a flow rate of 0.8 mL/min. Analysis was performed at ambient
temperature with detection wavelength at 245 nm. The retention times of MOX and
HCTZ were found to be 3.0 and 4.8 min and the calibration curves were linear
(r2=0.999) over a concentration range from 1-35µg/mL for MOX and HCTZ
respectively. Limit of detection (LOD) and Limit of quantitation (LOQ) were
0.08µg/mL and 0.1µg/mL for MOX and 0.2µg/mL and 0.4µg/mL for HCTZ
respectively. The developed method was validated for parameters like system
suitability, specificity, linearity, accuracy, precision, ruggedness and robustness as per
ICH guidelines and the results were found to be within the limits. So it can be used for
the routine quality control of MOX and HCTZ in bulk sample and tablet dosage forms.
KEY WORDS: Moxonidine, Hydrochlorothiazide, RP-HPLC.
INTRODUCTION
Moxonidine, chemically
4-Chloro-N-(imidazolidin-2-ylidene)-6-methoxy-2
methylpyrimidin-5-amine. It has empirical formula C9H12ClO5 shown in Fig 1.
Moxonidine is a new generation centrally acting antihypertensive drug used for the
treatment of mild to moderate essential hypertension. It is a selective agonist at
the imidazoline receptor subtype 1 (I1). This receptor subtype is found in both the
rostral ventro-lateral pressor and ventromedial depressor areas of the medulla
oblongata. Moxonidine causes a decrease in sympathetic nervous system activity and
therefore a decrease in blood pressure. [1, 2] Hydrochlorothiazide, chemically 6-chloro3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide1,1-dioxide. It has molecular
formula C7H8ClN3O4S2 shown in Fig 2. Hydrochlorothiazide belongs to
the thiazide class of diuretics. It reduces blood volume by acting on the kidneys to
reduce sodium (Na+) reabsorption in the distal convoluted tubule.[3,4] The combination
of Moxonidine (2mg) and Hydrochlorothiazide (12.5mg) is widely used to treat the
patients suffering from essential hypertention. This combination also permits effective
antihypertensive treatment over a longer period.
On literature survey it was found that several analytical methods like Ultra Violet
Spectrometer (UV), [5,6,7,8,9,10,11] High Performance Thin Liquid Chromatography
(HPTLC), [12,13] High Performance Liquid Chromatography (HPLC), [14] Reverse Phase
High performance Liquid Chromatography (RP-HPLC), [15,16,17,18,19,20] Ultra Performace
Liquid Chromatography (UPLC), [21,22,23] Liquid Chromatography- ElectroSpray
Ionisation - Mass Spectroscopy (LC-ESI-MS) [24,25,26,27,28] have been reported for the
determination and estimation of Moxonidine and Hydrochlorothiazide individually.
The extensive literature review revealed that no analytical methods were reported for
simultaneous estimation of Moxonidine and Hydrochlorothiazide in pharmaceutical
formulations. Hence new modified method was developed for simultaneous estimation
of Moxonidine and Hydrochlorothiazide by RP-HPLC in pharmaceutical dosage forms.
The proposed method was validated as per ICH guidelines. [29, 30]
HN
N
HN
H3C
O
Cl
H
N
S
S
Cl
N
N
CH3
Fig. 1. Structure of Moxonidine
H2N
O
NH
O
O
O
Fig.2. Structure of Hydrochlorothiazide
MATERIALS AND METHODS
Chemicals and Reagents:
HPLC grades, acetonitrile and all other analytical grade reagents were purchased from
Rankem and Loba Chemie India. HPLC grade water was prepared using Milli-Q water
purification system. Class A glassware is used throughout the experiment. Reference
Standard MOX and HCTZ are gift samples were obtained from Micro labs, Bengaluru.
MOXAVAS-H, Macleodes tablets were purchased from local market.
Instrument & Chromatographic conditions:
The chromatographic system consists of LC-20AT SHIMADZU -SPD-20A equipped
with UV detector and Autosampler injector with 20 µl loop volume. Chromatographic
separations were carried out using C8 Phenomenex Luna column (250x4.6mm, 5µ).
The mobile phase consisting of acetonitrile and formic acid solution (0.2%v/v) in the
ratio 50:50. The flow rate was 0.8mL/min and detection was carried out at 245nm using
UV detector.
Preparation of standard stock solutions of MOX and HCTZ:
Accurately 10 mg of MOX and HCTZ were weighed separately and transferred into a
clean and dry 10 ml volumetric flask, dissolved with sufficient volume of diluent
consists of ACN and Water (50:50). The volume was made up to 10 ml with diluent to
get a concentration of 1000 µg/ml (Stock I). 1 ml of resulting stock solution was further
diluted to 10 ml volumetric flask with diluent to get a concentration of 100µg/ml for
both MOX and HCTZ respectively (Stock II). From stock II, 1 ml was transferred into
a 10 ml volumetric flask and final volume was then made up to 10 ml with diluent to
get a concentration of 10µg/ml of MOX and HCTZ respectively.
Preparation of sample stock solution:
Twenty tablets of MOXAVAS-H each containing 0.2 mg of MOX and 12.5 mg of
HCTZ were weighed and finely powered. Powder equivalent to 0.2 mg of MOX and
12.5 mg of HCTZ were taken together and transferred into a clean, dry 100 ml
volumetric flask. The powder was first dissolved in diluent and sonicated for 10 mins.
The resulting mixture was then filtered through whatmann filter no 0.45μ. The final
volume of filtrate was made up to 100 ml with diluent. 1ml of the resulting solution
was diluted to 10ml with diluent to get a concentration of 2µg/ml and 125µg/ml of
MOX and HCTZ respectively.
METHOD DEVELOPMENT
A variety of mobile phases were investigated in the development of a RP-HPLC
method for the analysis of MOX and HCTZ. A mixture of Acetonitrile and 0.2% v/v
Formic acid solution (50:50v/v) was found to be the most suitable mobile phase for
ideal separation of MOX and HCTZ. The solvent mixture was filtered through a 0.45 μ
whatmann filter paper and sonicated before use. It was pumped through the column at a
flow rate of 0.8 mL/min. The column was maintained at an ambient temperature. The
detection of the drug was monitored at 245 nm. Under these optimized
chromatographic conditions the retention time obtained for the drugs MOX and HCTZ
was 3.0 min and 4.8 min respectively. A typical chromatogram showing the separation
of the drugs is as shown in Fig: 3.
Fig: 3 Chromatogram of Moxonidine and Hydrochlorothiazide
METHOD VALIDATION
The validation of developed method was done as per ICH guidelines [29, 30] which
include Linearity, LOD and LOQ System suitability, Precision (system precision,
method precision), Accuracy, Ruggedness, Robustness (flow rate, wavelength, and
mobile phase ratio) and Assay.
Linearity:
Linearity is the ability of the method to produce results that is directly proportional to
the concentration of the analyte in samples with given range. The various concentration
of working standard solutions of MOX and HCTZ were made by pipetting 0.1-3.5 ml
from stock (II) into a series of 10 ml volumetric flask and diluted to 10ml with diluent
to get the final concentration of 1 to 35µg/ml solutions. 20 µl of each of these working
standard solutions of MOX and HCTZ ranging from 1 to 35 µg/ml were injected into a
chromatograph at flow rate of 0.8 mL/min. Retention time and peak area obtained were
recorded and standard calibration curve was plotted for MOX and HCTZ and linearity
equations were derived. The calibration curves were shown in Fig: 4 and Table 1
Sl.
No
1
2
3
4
5
6
7
8
9
10
11
12
13
Table 1: Linearity data for MOX and HCTZ
Volume of MOX
Final
Peak
and HCTZ
Concentration
Volume
area* for
working standard
(µg/ml)
(ml)
MOX
solution(ml)
36215
0.1
10
1
0.2
10
2
65703
129582
0.4
10
4
194637
0.6
10
6
253032
0.8
10
8
320829
1.0
10
10
383460
1.2
10
12
1.4
1.5
2.0
2.5
3.0
3.5
10
10
10
10
10
10
Peak
area* for
HCTZ
12249
24925
46927
73248
94323
120017
146652
449716
172148
14
15
483285
184672
628706
245927
20
777387
293683
25
937368
358572
30
1072093
414494
35
*Average of five determinations
1200000
MOX
y = 30719x + 10857
R² = 0.9996
1000000
HCTZ
y = 11873x + 2076.7
R² = 0.9993
Area
800000
600000
400000
200000
0
0
5
10
15
20
25
30
35
40
Concentration µg/ml
Fig: 4 Linearity plot for Moxonidine and Hydrochlorothiazide
Limit of Detection and limit of Quantification:
The limit of detection (LOD) is the smallest concentration that can be detected but not
necessarily quantified as an exact value. The limit of quantitation (LOQ) is the lowest
amount of analyte in the sample that can be quantitatively determined with precision
and accuracy.
For estimation of LOD, LOQ and visualization method was followed. In visualization
method lower dilutions of the standard drugs of MOX and HCTZ were successively
prepared, injected in to the chromatograph and response obtained was recorded and
presented in Table 1.
Table: 2 Result of LOD and LOQ
Drug
MOX
HCTZ
LOD
(µg/ml)
0.08
0.2
LOQ
(µg/ml)
0.1
0.4
System suitability parameter:
A Standard solution of MOX and HCTZ working standard was prepared as per
procedure and was injected six times into the HPLC system. The system suitability
parameters were evaluated from standard Chromatograms obtained by calculating the
% RSD of retention times, tailing factor, theoretical plates and peak areas from six
replicate injections. From the system suitability studies it was observed that all the
parameters were within limit. Hence it was concluded that the Instrument, Reagents and
Column were suitable to perform the assay. The Fig: 3 and Table 3 shows the results.
Table: 3 System Suitability Parameters
System Suitability Factor
MOX
HCTZ
Acceptance Criteria
Theoretical plates
3044
6603
>2000
HETP (mm)
Tailing factor
49
1.2
23
1.2
<2
Resolution
7.83
>2
Specificity:
Specificity is a procedure to detect quantitatively the analyte in presence of component
that may be expected to be present in the sample matrix. Commonly used excipients in
tablet preparation were spiked in a pre-weighed quantity of drugs and then area was
measured and calculations done to determine the quantity of the drugs.
Precision:
The precision of an analytical procedure expresses the closeness of agreement (degree
of scatter) between a series of measurements obtained from multiple sampling of the
same homogeneous sample under the prescribed conditions.
System precision:
Successive six injections of standard solution (six replicates) were injected into a
HPLC chromatograph, the peak area and chromatograms obtained were recorded. The
% relative standard deviation was calculated for peak areas of replicates.
Method precision:
Intraday precision
Intraday precision of test method is demonstrated by 5 injections of the same batch
(same conc) of samples at 0, 1, 2, 3, 4, 5, 6 and 12hrs on same day.
Inter day Precision
Inter day precision of test method is demonstrated by 5 injections of the same batch
(same conc) of samples on 3 successive days.
The amounts of MOX and HCTZ in sample were calculated from their respective
standard linearity equations. The results obtained were presented in Table 4-8
Table: 4 System Precision Data for MOX and HCTZ
Replicates
MOX Peak Area*
HCTZ Peak Area*
1
311076
119728
2
310856
121910
3
320829
120017
4
310933
119188
5
316886
123317
6
321166
119470
315291
120605
Average
4983.11
1641.28
Standard Deviation
1.58
1.36
%RSD
Table: 5 Method Precision Data for MOX and HCTZ
Replicates
MOX
Concentration
Peak Area*
(µg/ml)
HCTZ
Concentration
Peak Area*
(µg/ml)
1
320886
10.09
121817
10.09
2
3
4
;5
6
320163
316575
320876
319933
320651
10.07
9.95
10.09
10.06
10..08
10.06
0.05
0.53
120178
119470
119188
121820
120986
9.95
9.89
9.86
10.09
10.02
9.98
0.10
0.97
Mean
Standard Deviation
%RSD
Table: 6 Intraday Precision Data of MOX and HCTZ
Replicate
1
2
3
4
5
6
7
Time
Interval
MOX
Peak Area* Conc.
(μg/ml)
1hr
320642
10.08
2hr
310946
9.77
3hr
320184
10.07
4hr
320817
10.09
5hr
320676
10.09
6hr
319945
10.06
12hr
318966
10.03
10.02
Mean
0.12
Standard Deviation
1.24
% RSD
HCTZ
Peak Area * Conc.
(μg/ml)
120953
10.01
119467
9.89
119152
9.86
119698
9.91
121900
10.09
121836
10.09
120477
9.97
9.97
0.10
1.02
Table: 7 Interday Precision Data of MOX and HCTZ
Replicate
1
2
3
Date
Interval
Day 1
Day 2
Day 3
MOX
Peak Area*
Conc.
(μg/ml)
320765
10.09
318958
10.03
319844
10.06
HCTZ
Peak Area*
Conc.
(μg/ml)
121260
10.04
120810
10.00
119880
9.92
Mean
10.06
9.99
Standard Deviation
% RSD
0.03
0.29
0.06
0.59
Table: 8 Results of Precision parameters
Precision
Parameters
System Precision
MOX
% RSD
1.58
HCTZ
% RSD
1.36
Acceptance
Criteria
< 2.0%
Method Precision
0.53
0.97
< 2.0%
Intra day
Inter day
1.24
0.29
1.02
0.59
< 2.0%
< 2.0%
Ruggedness:
Ruggedness was determined by the assay of sample sets by different analysts, the peak
area and the chromatograms were recorded. The percentage assay of standard drug was
calculated from the peak areas of replicates. The results obtained were mentioned in
Table 9-11.
Table: 9 Intermediate Precision Data of Analyst 1
MOX
HCTZ
Replicates
Peak Area*
%Assay
Peak Area*
%Assay
319666
100.53
120280
99.56
1
318998
100.31
120781
99.98
2
319886
100.60
119964
99.29
3
320895
100.93
119188
98.64
4
320157
100.69
121317
100.04
5
317813
99.92
119778
99.13
6
100.50
99.50
Mean
Table: 10 Intermediate precision data Analyst 2
MOX
HCTZ
Replicates
Peak Area*
%Assay
Peak Area*
%Assay
1
2
3
4
5
6
316574
320854
310878
320719
319058
319847
99.52
100.91
97.67
100.87
100.33
100.59
99.98
120840
119823
121498
119994
119979
123116
100,03
99.17
100.58
99.32
99.30
101.95
100.06
Mean
Table: 11 Results of Ruggedness
Drug
Analyst 1
Analyst 2
Acceptance Criteria
MOX
100.50%
99.96%
90-110%
HCTZ
99.50%
100.06%
90-110%
Accuracy:
Accuracy is expressed as percentage recovery by the assay of known added amounts of
analyte carried out at three different levels (80%, 100%, and 120%). Excellent recovery
and low relative standard deviation value showed that the method was suitably accurate
for potency assay of Moxonidine and Hydrochlorothiazide simultaneously in the drug
substances. The results were tabulated in the Table 12-13.
Table: 12 Results of Accuracy of MOX
Level
80%
100%
120%
Stand
Conc.
(µg/ml)
Sample
Conc.
(µg/ml)
Peak
Area*
Total
Conc.
(µg/ml)
I
II
8
2
312891
9.83
Amt of
standard
recovered
(µg/ml)
7.83
8
2
311678
9.79
7.79
97.41
III
I
8
10
2
2
320072
378432
10.07
11.97
8.07
9.97
100.82
99.66
II
III
I
II
III
10
10
12
12
12
2
2
2
2
2
367854
377987
425767
430987
430871
11.62
11.95
13.51
13.68
13.67
9.62
9.95
11.51
11.68
11.67
96.21
99.51
95.89
97.30
97.27
Replicate
%
Recovery
97.90
Table 13: Recovery study data for HCTZ
Replicate
Level
80%
100%
120%
Stand Sample
Conc.
Conc.
(µg/ml) (µg/ml)
I
8
II
III
I
II
III
I
II
III
8
8
10
10
10
12
12
12
125
125
125
125
125
125
125
125
125
Peak
Area*
Total
Conc.
(µg/ml)
1580106
132.91
Amt of
standard
recovere
d
(µg/ml)
7.91
1580278
1579912
1600446
1601566
1602871
1626975
1627766
1625962
132.92
132.89
134.62
134.72
134.83
136.86
136.92
136.77
7.92
7.89
9.62
9.72
9.83
11.86
11.92
11.77
%
Recover
y
98.86
99.04
98.66
96.22
97.17
98.26
98.80
99.36
98.09
Robustness:
The robustness of an analytical procedure is a measure of its capacity to remain
unaffected by small, but deliberate variations in method parameters and provides an
indication of its reliability during normal usage. Robustness of the method was
investigated under a variety of conditions like change in flow rate (± 0.1 ml/min),
wavelength (± 2 nm) and mobile phase composition (± 5 from absolute value). % assay
for all the robustness studies is as shown in Table 14.
Table: 14 Results of Robustness
Parameters
Area
Change in flow
rate
% Assay of
MOX
104.51
Area
117993
% Assay of
HCTZ
104.38
0.7ml
331888
0.8ml
320895
100.93
121900
100.92
0.9ml
309524
97.23
119059
96.05
Mobile phase
ratio (ACN:
Buffer)
(55:45)
(50:50)
(45:55)
304913
320092
302891
95.72
100.67
95.07
114914
120900
115981
95.04
100.92
96.05
Wavelength in
nm
243
314957
96.56
117993
97.63
245
247
320895
310524
100.93
97.55
121900
119059
100.92
98.53
Assay:
To provide an exact result which allows an accurate statement on the content or
potency of the analyte in a sample. The proposed method was also evaluated by the
assay of MOX and HCTZ in their combined dosage formulation. The results were
mentioned in the Table 15.
Table: 15 Assay results of Tablet formulation
Peak
Peak
area*for area*for
MOX
HCTZ
71962
1449741
Amount of
drug recovered
MOX (µg/ml)
Amount of
Drug recovered
HCTZ (µg/ml)
% Assay
for
MOX
% Assay
for
HCTZ
1.99
121.93
99.46
97.54
RESULTS AND DISCUSSION
Literature review reveals only few UV and HPLC methods have been reported for the
estimation of MOX and HCTZ alone in bulk and tablet dosage formulation till so far.
So, new simple, rapid and accurate RP-HPLC method has been developed and
validated for the simultaneous estimation of MOX and HCTZ in bulk and tablet dosage
formulation. The separation was carried out by using C8 Phenomenex Luna (250 x
4.6mm, 5µm) column with a mobile phase consisting of acetonitrile: buffer (0.2%v/v
formic acid solution) in the ratio 50:50v/v. The mobile phase was delivered at a flow
rate of 0.8 ml/min with detection at 245 nm. The retention times of MOX and HCTZ
were found to be 3.0 and 4.8 min and the calibration curves were linear (R2=0.999)
over a concentration range from 1-35µg/ml for MOX and HCTZ respectively. Limit of
detection (LOD) and Limit of quantitation (LOQ) were 0.08µg/ml and 0.1µg/ml for
MOX and 0.2µg/ml and 0.4µg/ml for HCTZ respectively. The developed method was
validated for parameters like system suitability, specificity, linearity, accuracy,
precision, ruggedness and robustness as per ICH guidelines and the results were found
to be within the limits.
CONCLUSION
The reverse phase high performance liquid chromatographic method for the
determination of Moxonidine and Hydrochlorothiazide in bulk drug and tablet dosage
form was found to be accurate and precise. The elution of the analytes by thus
developed and validated isocratic method is shorter than the gradient method reported
so far. Thus, the proposed RP-HPLC method can be successfully applied for the routine
quality control analysis of Moxonidine and Hydrochlorothiazide in bulk and tablet
dosage form.
ACKNOWLEDGMENT:
The authors are thankful to Micro Labs, Bengaluru, for providing gift samples of MOX
and HCTZ for the research work.
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