Analytical Method Development and Validation
for Simultaneous Estimation of Candesartan Cilexetil and Hydrochlorothiazide in
Tablet Dosage form
R.M. Gaurkhede*,
A.V. Chandewar.
P.W.College of Pharmacy, Dhamangaon road, Yavatmal. 445001
(M.S.)
*Corresponding Author E-mail:
rahulgaurkhede@gmail.com
ABSTRACT:
A simple, precise and reproducible stability
indicating HPLC method has been developed and validated for simultaneous determination
of Candesartan and Hydrochlorothiazide on an Hypersil BDS C18 column (150*4.6*5um)
using a mobile phase consisting of phosphate buffer pH 5.4 and acetonitrile (65:35)
at flow 1.0 ml/min. Detection was carried out at 232nm . The method was validated
with respect to specificity, accuracy linearity, precession, ruggedness and robustness
parameters as per ICH guidelines. Linearity of the method was found to be 0.999;
%RSD of precession was below 2.0%, recovery of added drug within 98-102 %, System
suitability parameters were within limit for Candesartan and Hydrochlorothiazide.
This method is simple, accurate and precise, sensitive and applicable for analysis
of Candesartan and Hydrochlorothiazide in tablet formulation. In Stress degradation
studies were also carried out under stress testing conditions of acidic, basic,
oxidative, photolytic, thermal and neutral degradation.
KEYWORDS: Validation, ICH, RP-HPLC, Stress degradation study
INTRODUCTION:
Candesartan Cilexetil is chemically known as
(±)-1-Hydroxyethyl2-ethoxy-1-[p-(o-1H-tetrazol-5ylphenyl)benzyl]-7-benzimidazolecarboxylate,
cyclohexyl carbonate (ester), It is a prodrug and is hydrolysed to Candesartan during
absorption from the gastrointestinal tract. Candesartan is a selective AT1 subtype
angiotensin II receptor antagonist1, 2. Hydrochlorothiazide is chemically
known as 6-chloro-1,1-dioxo-3,4-dihydro-2H-1λ,2,4-benzothiadiazine-7-sulfonamide.It
is diuretic medication used
to treat high blood
pressure and swelling due to
fluid buildup. Other uses include diabetes
insipid us, renal tubular acidosis, and to decrease the risk of kidney stones in those
with high calcium
level in the urine3, 4.
Various spectrophotometric methods have been
reported for the determination of Candesartan and HTZ with other combination in
pharmaceutical tablets using different reagents; UV spectrophotometric and HPLC
method were developed5-14. To the best of our knowledge, no official
method has been described for the simultaneous determination of this combination
in pharmaceutical formulation by RPHPLC method. Hence the aim of the present investigation
was to develop simultaneous method for estimation of Candesartan and HCTZ for its
Pharmaceutical Preparation. Therefore it is desirable to develop simple and reproducible
analytical methods For chromatographic method used in analytical application, there
should be more consistency in validation practice with key analytical parameter,
including. System Suitability, accuracy, precision, specificity, limit
of detection, limit of quantization, linearity, range, ruggedness, robustness, stability15.
MATERIALS AND METHODS:
Candesartan and Hydrochlorothiazide working standard were
received as gift sample from Spectrum Pharma Research Centre Hyderabad. Commercially
available Candesar H 16-12.5 was purchased from local market. HPLC grade Acetonitrile
Methanol (Merck) and AR grade Potassium Hydrogen Phosphate, Orthophosphoric acid
, Hydrochloric acid , Sodium Hydroxide , Hydrogen Peroxide (RANKEM) was used. Mili
Q water used in mobile phase and diluent preparation. Chromatographic separation
performed on Waters® liquid chromatographic system equipped with PDA detector, with
a 20ul injection loop volume by using M power software .
Preparation of mobile phase:
The mobile phase was prepared by
using 25mM potassium phosphate buffer solution adjusted pH to 5.4 by using diluted
Orthophosphoric acid and Acetonitrile in ratio 65:35% v/v.
Preparation of diluent:
Mixture of Water: Acetonitrile
in ratio of 50:50%v/v was prepared.
Chromatographic conditions:
The Chromatographic separation were achieved by using Hypersil
BDS C18 column (150*4.6*5um) analytical column. The mobile phase consisting of phosphate
buffer :Acetonitrile in (65:35) at flow 1.0 ml/min. Detection was carried out at
232nm and with isocratic program run time was 6 min. The column Oven temperature
was 30°C and ambient sampler temperature.
Preparation of standard solution:
Weighed accurately and transferred 16 mg of Candesartan and 12.5mg of Hydrochlorothiazide
standard into a 50ml volumetric to this added diluent and sonicated to dissolved
and made up to the final volume with diluents. Further diluted 1ml of above solution
in to a 10ml volumetric flask and then made up to the final volume with diluent
to get a concentration 32ug/ml and 25ug/ml respectively.
Preparation of sample solution:
Ten tablets (Candesar H) were weighed and crushed to fine
powder, uniformly mixed. The powder equivalent to one tablet weight was taken in
to 50 ml volumetric flask and added diluent, sonicated for 30 min with intermediate
shaking and made up to mark with diluent. Pipetted out 1ml of this solution in to
10 ml volumetric flask and made to mark with diluent, mixed well.
RESULTS AND DISCUSSION:
Optimisation of method by changing mobile phase, column,
flow rate, column temperature, and buffer composition. Colum were selected based
on the column chemistry and get good peak shape, resolution between two peak and
pass system suitability parameter. The mobile were optimised so that there were
no interferences of diluent and excipients. The drug 10 μg/ml solution of the
drugs in methanol were scanned using UV-Visible spectrophotometer (SHIMADZU 2000)
within the wavelength region of 200–400 nm against methanol as blank. The absorption
maxima for Candesartan was 254 and for Hydrochlorothiazide was 270 nm and in the
combination was 232nm.The diluent (Blank) and standard were injected to HPLC system,
it was observed that there was no interferences of blank with standard mixture of
two peak as shown in fig 1. The retention time obtained Hydrochlorothiazide (HCTZ)
and Candesartan was 2.31min and 2.915 respectively
Fig 1. System suitability of Candesartan
and Hydrochlorothiazide
VALIDATION PARAMETERS:
The method was validated for method precision,
linearity, accuracy, precision, and specificity study as per ICH norms. All the
validation studies were carried out by replicate injection of the sample and standard
solutions.
System Suitability
Six replicates injection of standard solution was injected
into the chromatograph as per methodology. The results of system suitability of
Candesartan and Hydrochlorothiazide are as follows Table no1.
Table 1. System Suitability results of Candesartan and
HCTZ
|
Parameters
|
Observation
|
|
|
Candesartan
|
Hydrochlorothiazide
|
|
Theoretical plate
|
10471
|
3228
|
|
Tailing Factor
|
1.14
|
1.02
|
|
% RSD of the area
of replicate injection
|
0.37
|
0.59
|
Accuracy (Recovery):
Accuracy at three different concentration levels
50%, 100% and 150% by replicate analysis (n=3). The result of accuracy study was
reported in Table 2. From the recovery study it was clear that the method was very
accurate for quantitative estimation of Candesartan and HCTZ . All the statistical
results were within the range of acceptance i.e. %RSD<2.0.
Table 2 Accuracy study data of Candesartan and HCTZ
|
% Recovery of Level
|
Amount added (ug/ml)
|
Amount Recovered (ug/ml)
|
% Recovery
|
Mean recovery
|
% RSD
|
|
Candesartan
|
|
|
50%
|
1
|
16.00
|
16.31
|
101.97
|
101.31
|
0.72
|
|
2
|
16.00
|
16.08
|
100.52
|
|
3
|
16.00
|
16.23
|
101.45
|
|
100%
|
1
|
32.00
|
32.28
|
100.88
|
100.28
|
0.67
|
|
2
|
32.00
|
31.86
|
99.56
|
|
3
|
32.00
|
32.13
|
100.41
|
|
150%
|
1
|
48.00
|
47.82
|
99.63
|
100.30
|
0.62
|
|
2
|
48.00
|
48.42
|
100.87
|
|
3
|
48.00
|
48.19
|
100.40
|
|
HCTZ
|
|
|
50%
|
1
|
12.50
|
12.74
|
101.95
|
100.67
|
1.12
|
|
2
|
12.50
|
12.48
|
99.83
|
|
3
|
12.50
|
12.53
|
100.23
|
|
100%
|
1
|
25.00
|
25.43
|
101.710
|
100.62
|
1.42
|
|
2
|
25.00
|
24.75
|
99.00
|
|
3
|
25.00
|
25.29
|
101.15
|
|
150%
|
1
|
37.50
|
37.14
|
99.04
|
100.22
|
1.59
|
|
2
|
37.50
|
37.34
|
99.59
|
|
3
|
37.50
|
38.26
|
102.04
|
Linearity:
Linearity of method was determined from 8
- 48 μgmL-1 for Candesartan and 6.25 – 37.5 μgmL-1 for HCTZ. The correlation
coefficient was obtained 0.9998 and 0.9999 for Candesartan and HCTZ respectively.
Precision, Limit of Detection, and Limit of
Quantitation:
The concentrations of both the drugs were measured
on the same day with different six preparation and with different day, different
column and another six preparation for inter day study. The limits of detection
and quantitation, LOD and LOQ, were calculated by use of the equations LOD = 3.3σ/S
and LOQ = 10σ/S, where σ is the standard deviation and S is the slope
of the calibration curve. The results are reported in Table no 3.
Table 3. Precision and Inter Day Precision,
LOD and LOQ Studies
|
Drug
|
Method
Precision %RSD (n=6)
|
Inter
day Precision %RSD (n=6)
|
LOD(ppm)
|
LOQ(ppm)
|
|
Candesartan
|
0.46
|
0.46
|
0.01
|
0.02
|
|
HCTZ
|
0.76
|
0.76
|
0.03
|
0.09
|
Robustness:
Robustness of the method was demonstrated by deliberately
varying the chromatographic condition such as Flow rate changed by ± 10% ( i.e.
0.9mLmin and 1.1mL/min)Column temperature changed by ± 50C (300C
i.e.250C and 350C).Mobile phase composition changed by absolute
±2% variation ( i.e. Buffer : Acetonitrile 670:330 and 630:370). The results were
tabulated as in Table 4
Table 4. Robustness data for system suitability
of Candesartan and HCTZ
|
|
Candesartan
|
HCTZ
|
|
Robustness
Parameter
|
Theoretical
plates
|
Tailing
Factor
|
%RSD
|
Theoretical
plates
|
Tailing
Factor
|
%RSD
|
|
Limits
|
NLT
2000
|
NMT
2.0
|
NMT
2.0
|
NLT
2000
|
NMT
2.0
|
NMT
2.0
|
|
Low Flow
|
10847
|
1.09
|
0.55
|
6157
|
1.02
|
0.61
|
|
High Flow
|
9568
|
1.08
|
1.14
|
6016
|
1.05
|
0.20
|
|
Low Organic
|
9909
|
1.11
|
1.80
|
6064
|
1.02
|
0.84
|
|
High Organic
|
9562
|
1.08
|
1.90
|
6069
|
1.02
|
1.90
|
|
Low Temperature
|
9910
|
1.02
|
0.37
|
6161
|
1.02
|
0.37
|
|
High Temperature
|
10487
|
1.09
|
0.56
|
6414
|
1.02
|
0.28
|
Specificity:
Specificity of the method was assessed by comparing the
chromatograms obtained from standard drugs, with the chromatogram obtained from
tablet solutions. Also Specificity covers the degradation of the drug substances
and drug product in Acid degradation, Base degradation, Peroxide degradation, Neutral
(reflux condition) Thermal degradation and Photo degradation. All degradation conditions
with % degradation and peak purity were mention in table no.5
Table.5 Forced degradation data
|
Degradation
condition
|
%Assay
|
% Total degradation
|
Purity Angle
|
Purity
Threshold
|
|
|
Candesartan
|
HCTZ
|
Candesartan
|
HCTZ
|
Candesartan
|
HCTZ
|
Candesartan
|
HCTZ
|
|
AS such
|
100.00
|
99.40
|
NA
|
NA
|
0.195
|
0.285
|
0.355
|
0.451
|
|
Acid degradation
(2N_HCl/60ºC/30min.)
|
97.9
|
97.9
|
2.1
|
1.5
|
0.197
|
0.265
|
0.486
|
0.462
|
|
Base degradation
(2N_NaOH/600C/30min.)
|
98.3
|
98.3
|
1.7
|
1.1
|
0.197
|
0.507
|
0.503
|
0.518
|
|
Peroxide
degradation (20%_H2O2/600C/30min.)
|
97.6
|
96.9
|
2.4
|
2.5
|
0.191
|
0.403
|
0.495
|
0.407
|
|
Neutral
degradation (H2O_60ºC /6 hrs.)
|
99.5
|
98.9
|
0.5
|
0.5
|
0.242
|
0.374
|
0.547
|
0.558
|
|
Thermal
degradation (105 ºC /6hrs.)
|
99.0
|
99.0
|
1.0
|
0.4
|
0.202
|
0.431
|
0.947
|
0.513
|
|
Photo degradation
(1.2million lux hours)
|
99.1
|
98.8
|
0.9
|
0.6
|
0.199
|
0.108
|
0.482
|
0.480
|
For purity: Purity Angle < Purity Threshold
The developed method was found specific and selective,
as there was no interference of blank and individual drug. Also method was found
stability indicating based on degradation study, Candesartan was found to be sensitive
to acidic, basic, peroxide conditions. No significant degradation was obtained for
candesartan under any of above mentioned stress conditions. Hydrochlorothiazide
was found to be sensitive to acidic, basic, peroxide conditions. No significant
degradation was obtained for hydrochlorothiazide under any of above mentioned stress
conditions.
CONCLUSION:
A new, reversed-phase HPLC method has been developed for
simultaneous analysis Candesartan and Hydrochlorothiazide in a tablet formulation.
It was shown above that, the method was linear, accurate, reproducible, repeatable,
precise, robust, specific and stability indicating proving the reliability of the
method. The run time is relatively short, as method is stability indicating which
enable rapid determination of many samples in routine and quality control analysis
of tablet formulations. Hence, the proposed method was successfully applied to analyze
preparation containing Candesartan and HCTZ.
ACKNOWLEDGEMENT:
The authors are thankful to Spectrum Lab Hyderabad for
providing the gift sample of Candesartan and Hydrochlorothiazide.
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