Development and validation of a dissolution test for Candesartan ...

J Pharm Educ Res Vol. 2, Issue No. 2, December 2011
Development and validation of a dissolution test for Candesartan cilexetil in
tablet forms using reverse phase – High performance liquid
chromatography
R Revathi* 1 , T Ethiraj 2 , Jhansi L. Marreddy 1 , V Ganeshan 2
1 Department of Pharmaceutical Analysis,
2 Department of Pharmaceutics,
The Erode College of Pharmacy and Research Institute, Erode - 638112, Tamil Nadu, India.
*E-mail: revathethiraj@gmail.com
Received: October 08, 2011; Accepted: November 15, 2011
ABSTRACT
The aim of this study was to develop and validate a dissolution test for the quality control of candesartan cilexetil
tablets, labeled as containing 8 mg of active pharmaceutical ingredient (API) , using an reverse phase – high
performance liquid chromatography (RP-HPLC) method. After the determination of solubility, the conditions
selected were paddle at 100 rpm, with 1000 ml of 1 % sodium lauryl sulphate (SLS) in water, pH adjusted to 6.8
with 3N hydrochloric acid at 37°C ± 0.5°C. Under these conditions, the in vitro release profiles of candesartan
cilexetil uncoated 8 mg tablets shown good results. The drug release was evaluated by reverse phase – high
performance liquid chromatography method using 0.02M mono potassium phosphate: acetonitrile: triethylamine
in the ratio of 40:60:02 and adjusted pH to 6 with phosphoric acid at flow rate of 1 ml/min. The method was
validated for specificity, linearity, accuracy, precision and solution stability as per ICH guidelines to meet
requirements for a global regulatory filing.
Keywords: Dissolution testing, Paddle method, Validation, Reverse phase – High performance liquid chromatography,
Sodium lauryl sulphate, Candesartan cilexetil tablets.
INTRODUCTION
Dissolution testing has emerged in the pharmaceutical
field as a very important tool to characterize drug product
performance. Dissolution tests are used not only for quality
control of finished products, but also to assess several
stages of formulation development, for screening and
proper assessment of different formulations 1 .
Candesartan cilexetil (Fig. 1) is chemically, 2-Ethoxy-
3-[2-(1H-tetrazol-5-yl)-4-yl methyl]-3H- benzoimidazole-
4-carboxylic acid 1-cyclohexyloxy carbonyl oxy ethyl
ester. It has a molecular formula of C 33 H 34 N 6 O 6 and a
molecular weight of 610.67. It is practically insoluble in
water and sparingly soluble in methanol 2 .
Fig. 1. Chemical Structure of Candesartan Cilexetil.
The drug is an angiotensin II receptor (type AT1)
antagonist and it acts by blocking the vasoconstrictor and
aldosterone secreting effects of angiotensin II by
selectively blocking the binding of angiotensin II to the
AT1 receptor in many tissues such as vascular smooth
muscle and the adrenal gland 3 . It is used to treat
hypertension either alone or in combination with other
antihypertensive agents.
Few literatures revealed that the RP-HPLC and High
Performance Thin Layer Chromatography (HPTLC)
method for the estimation of candesartan cilexetil in solid
dosage forms 4-11 . Quantitation and dissolution studies
were validated with RP-HPLC method for some
pharmaceutical dosage forms 12-16 .
The method for determination of candesartan cilexetil
in solid dosage forms and its dissolution profiles are
available only in United States Food and Drug
Administration (USFDA) guidelines and United States
Pharmacopoeia (USP) 35, National Formulary (NF 30).
When compared to USP method, the preparation of
dissolution medium in the proposed method is simple and
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J Pharm Educ Res Vol. 2, Issue No. 2, December 2011
less expensive. USFDA guidelines also recommended that
the use of surfactant like SLS is suitable for dissolution of
water insoluble or sparingly water soluble drug products.
No other method validations have been reported for
dissolution of candasartan cilexetil in solid dosage forms,
so following experiment was performed.
This present study describes the development and
validation of a simple dissolution test for 8 mg candesartan
cilexetil tablets, which was optimized on the basis of
solubility and stability considerations.
Material and Methods
Instrumentation
Dissolution test was conducted using an Electro lab
TDT-14L dissolution tester using USP Apparatus at a
temperature of 37°C ± 0.5°C. A model Agilent
Technology-1100 series liquid chromatography equipped
with G1311A Quat pump, G1329A Auto injector, G1379A
Degasser, G1316A Column oven, G1314 A multi
wavelength detector, SCL-10AVP system controller and
chem. station manager system software were used.
Reagents and materials
Milli-Q water was used for preparing both dissolution
media and HPLC mobile phase. All other reagents and
chemicals were of analytical or HPLC grade. Candesartan
cilexetil reference substance (assigned purity, 99.8 %).
Tablets containing candesartan cilexetil (8 mg) of brand
Ranbaxy Laboratories Ltd., were purchased from the
local market.
Chromatographic conditions
Chromatography was achieved on a C 18 (Prodigy,
150!4.6 mm, 5µ) column. The mobile phase was a mixture
of 0.02 M mono potassium phosphate buffer: acetonitrile:
triethyl amine in the ratio of 40:60:0.2 and adjusted the
pH to 6.0 with phosphoric acid which was filtered (0.45
µm) and degassed before use. All analysis was performed
at room temperature at a flow rate of 1 ml/min. Detection
was made at 254 nm. Triplicate of 20 µl injections were
utilized for each analysis.
Dissolution Test Conditions
The solubility study and percentage drug release was
determined in 1000 ml of 0.1 M hydrochloric acid, sodium
phosphate buffer pH 7.0 and 1 % aqueous SLS solution,
(pH adjusted to 6.8 with 3N hydrochloric acid). Drug
release tests were carried out with paddle method (USP
72
apparatus II) at 75 rpm and 100 rpm. The temperature of
the cell was maintained at 37°C ± 0.5°C by using a
thermostatic bath. Sampling aliquots of 5.0 ml were
withdrawn at 5, 10, 15, 20 and 30 min and replaced with
an equal volume of the fresh medium to maintain a constant
total volume. After the end of each test time, sample
aliquots were filtered and quantified.
The percentage content was calculated by validated
RP-HPLC method and these contents results were used
to calculate the percentage release on each time of
dissolution profile. The cumulative percentage of drug
released was plotted against time in order to obtain the
release profile.
Method Validation
The dissolution tests were validated to candesartan
cilexetil tablets through the determination of specificity,
linearity, precision, accuracy and solution stability 17,18 .
Prior to injecting sample solutions, the column was
equilibrated for at least 30 min with the mobile phase
flowing through the system. System suitability tests were
carried out by making six replicate injections of a standard
solution containing 8 µg/ml of candesartan cilexetil and
analyzing the chromatograms for candesartan cilexetil
peak area, theoretical plates and tailing factor.
Specificity
The dissolution test specificity was evaluated by
preparing sample placebo of the commercial formulation
of tablets. These samples were transferred to separate
vessels with 1000 ml of dissolution medium at 37°C ±
0.5°C and stirred for 45 min at 100 rpm using a paddle
(USP apparatus 2). Aliquots of these solutions were
withdrawn, filtered through 0.45 µ membrane filters and
analyzed by the RP-HPLC method using candesartan
cilexetil standard solution of 8 µg/ml.
Linearity
To assess the linearity, 50 % - 150 % level of
concentrated solutions were prepared and standard curve
of candesartan cilexetil were constructed, by plotting the
concentration (µg/ml) against peak area. The calculation
of regression line was employed by the method of least
squares.
Precision
The precision of the method was determined by
measuring the intra-day precision and the inter-day

J Pharm Educ Res Vol. 2, Issue No. 2, December 2011
precision, both expressed as % RSD. Candesartan cilexetil
tablets were subjected to dissolution test conditions (1000
ml of dissolution medium pre-heated at 37°C ± 0.5°C,
paddle with stirring rate of 100 rpm, 45 min) in the same
day (intra-day precision) and in two different days by
different analysts (inter-day precision).
Accuracy
The accuracy was evaluated for the proposed method
by adding known amount of candesartan cilexetil standard
drug (50%, 100%, 150% level) to the tablet powder, which
were subjected to dissolution test conditions described
above. Each solution was analysed in triplicate. The
accuracy was calculated as the percentage of the drug
recovered from the formulation matrix.
Solution stability
The solution stability was analyzed over a specified
period of time, verifying the response of the sample
solution stored at bench top condition (25°C) and
refrigeration (5°C). The chromatograms obtained by the
RP-HPLC method from freshly prepared solution were
compared.
Results and Discussion
Solubility determination and dissolution test condition
When dissolution test is not defined in the monograph
of the dosage form, comparison of drug dissolution profiles
is recommended on three different dissolution media, in
the pH range of 1-7.5. The selection of a dissolution
medium may be based on the solubility data and dosage
range of the drug product. Hydrochloric acid, phosphate
buffer and purified water are typical mediums used for
dissolution test and these mediums were evaluated.
Candesartan cilexetil was insoluble in aqueous medium.
For poorly soluble drugs, a percentage of surfactant can
be used to enhance drug solubility and it is also
recommended by USFDA. Then different concentrations
of SLS (0.5 %, 0.75 % and 1 %) were prepared in purified
water and used for dissolution study.
At 75 rpm, the cumulative percentage drug release
was considerably less than that at 100 rpm in above said
dissolution medium. It was observed that less than 75 %
of drug was dissolved at 30 min in hydrochloric acid and
phosphate buffer at a speed of both 75 rpm and 100 rpm
(Fig. 2).
73
Fig. 2. Dissolution profiles of candesartan cilexetil in different
dissolution medium at 75 rpm and 100 rpm.
In 0.1 N hydrochloric acid medium, the drug was not
completely soluble; some of the drug particles were
detected at the bottom of the dissolution vessel. But at
the end of the 45 min, the drug was completely soluble in
phosphate buffer pH 7. The cumulative percentage drug
release obtained was low when compared to 1 % SLS
dissolution medium. At the end of 45 min the 1 % SLS
medium showed 100 % cumulative drug release at 75
rpm as well as 100 rpm level. But the candesartan cilexetil
drug was completely released from its formulation at the
end of the 30 min at a speed of 100 rpm.
In 0.5 %, 0.75 %, and 1 % SLS solutions showed
more than 87 % of drug release with in 30 min (Fig. 3).
But in 1 % SLS medium, % drug dissolved was nearly
100 % with in 20 min at stirring rate of 100 rpm. The
cumulative percentages of drug in all the above said
solutions were tabulated in Tables 1 and 2. The analysis
of variance showed no significant difference between the
results obtained at 75 rpm and 100 rpm (p < (or) > 0.05).
Fig. 3. Dissolution profiles of candesartan cilexetil in different
concentrations of SLS solution at 75 rpm and 100 rpm.

J Pharm Educ Res Vol. 2, Issue No. 2, December 2011
Table. 1. Cumulative percentage drug release in different media.
Time, 75 rpm 100 rpm
min 0.1 N HCl Buffer pH 7 1 % SLS solution 0.1 N HCl Buffer pH 7 1 % SLS solution
0.0 0.0 0.0 0.0 0.0 0.0 0.0
5.0 32.5 34.4 40.5 34.2 34.1 48.5
10.0 38.2 42.0 49.2 42.4 46.5 73.7
15.0 44.6 49.2 62.5 56.4 52.1 97.9
20.0 52.4 58.5 79.4 68.4 62.5 100.2
30.0 64.5 71.5 98.2 70.2 76.3 100.3
0.1N HCl = 0.1 Normality Hydro Chloric Acid, SLS = Sodium Lauryl Sulphate, rpm = Revolution per minute, min = minute
Table. 2. Cumulative percentage drug release in different media.
Time, 75 rpm 100 rpm
min 0.5% SLS 0.75% SLS 1% SLS 0.5% SLS 0.75% SLS 1% SLS
0.0 0.0 0.0 0.0 0.0 0.0 0.0
5.0 36.4 38.1 41.2 38.2 41.4 48.6
10.0 42.5 46.4 49.6 43.5 52.5 74.1
15.0 55.6 59.2 62.9 56.4 64.3 97.4
20.0 72.5 74.5 80.1 74.1 82.1 99.5
30.0 87.4 88.9 98.4 89.5 98.9 100.4
0.1N HCl = 0.1 Normality Hydro Chloric Acid, SLS = Sodium Lauryl Sulphate, rpm = Revolution per minute, min = minute
However, it was observed that % drug release was
high in 1 % SLS solution at 100 rpm. Based on these
results, the selected conditions for dissolution test of
candesartan cilexetil tablets were 1000 ml of 1 % SLS
solution in water, pH adjusted to 6.8 with 3 N hydrochloric
acid using paddle apparatus at stirring rate of 100 rpm. In
the present study, the % drug dissolved for candesartan
cilexetil tablet was > 60 % in 30 min in all the condition.
But % drug release showed > 85 % within 30 min for 0.5
%, 0.75 %, and 1 % SLS and the suggested acceptance
criteria could be 85 %.
74
The specificity of the dissolution test was evaluated
through the analysis of placebo tablets from a dissolution
test using the HPLC method (Fig. 4). The specificity test
by HPLC demonstrated that the excipients from tablets
do not interfere in the drug peak. Thus, the HPLC method
is useful to quantify candesartan cilexetil in pharmaceutical
formulation by comparing standard drug with sample (Fig.
5 and 6).
The stability of candesartan cilexetil was satisfied up
to 24 hours at bench top condition (25°C) and refrigeration
(5°C) and results were shown in Table 3.
Fig. 4. Chromatogram for placebo (Specificity study) Fig. 5. Chromatogram for candesartan cilexetil standard drug

J Pharm Educ Res Vol. 2, Issue No. 2, December 2011
Fig. 6. Chromatogram for sample preparation
Table. 3. Stability study data.
Time,h Mean % Assay
Bench top (at 25ºC) Refrigerator (at 5ºC)
Initial 98.7 % 96.3 %
12 98.9 % 96.5 %
24 98.5 % 96.1 %
h = hour
Linearity of the method was evaluated at five
concentration levels by diluting the standard stock solution
to give solutions in the range of 4.0–12 ìg/ml. The
calibration curve for candesartan cilexetil was prepared
by plotting the graph with area versus concentration.
Calibration data for candesartan cilexetil was shown in
Table 4. The representative linear equation was
Y=321.1X–10.31 and correlation coefficient 0.999 for
candesartan cilexetil. Linearity observed in the expected
concentration range demonstrated the suitability of the
method for analysis. This indicated that the method is
linear in the specified range for the analysis of candesartan
cilexetil in solid dosage form.
Table. 4. Regression analysis of calibration graphs for candesartan
cilexetil.
Parameters Candesartan cilexetil
Linearity range (µg/ml) 4-12
Correlation co-efficient (R 2 ) 0.999
Regression equation Y=321.1X-10.31
Slope 31005
Intercept 9.437
µg/ml = micro gram/milli litre
75
The precision results of the dissolution method were
evaluated by analyzing intra-day precision and inter-day
precision (Table 5). The % RSD values were obtained at
0.3 % for intra-day precision and 0.5 % for inter-day
precision. % RSD values not more than 2 % indicated
the good precision of the method.
Table. 5. Precision of the assay method: intra-day precision and interday
precision.
S.No Intra-day precision Inter-day precision
% Assay % Assay
1 98.77 100.82
2 99.05 100.85
3 99.50 100.52
4 98.73 100.63
5 98.96 101.77
6 99.21 101.29
Mean 99.04 100.98
%RSD 0.3 0.5
The recovery experiments were carried out by the
standard addition method (Fig.7 and 8). The method was
found to be accurate with % recovery of 98.71%–
101.20% and has found with acceptable % RSD of not
more than 2% at each level. The recoveries obtained by
the dissolution method for candesartan cilexetil were
shown in Table 6.
Fig. 7. Accuracy – 50 % level

J Pharm Educ Res Vol. 2, Issue No. 2, December 2011
Table 6. Accuracy data for the dissolution method.
Concentration of Amountadded Amountfound % Recovery* % RSD
spike level (mg/ml) (mg/ml)
Fig. 8. Accuracy – 150 % level
0.004026 0.004093
50 % 0.004026 0.004084 101.2% 0.466
0.004026 0.004056
0.008052 0.008030
100 % 0.008052 0.008042 99.74% 0.121
0.008052 0.008023
0.012080 0.011916
150 % 0.012080 0.011894 98.71% 0.289
0.012080 0.011962
*(n=3), mg/ml = milli gram/milli litre, %RSD= Percentage Relative Standard Deviation
Conclusions
The dissolution test developed and validated for
candesartan cilexetil tablets was considered satisfactory.
The conditions that allowed the dissolution determination
were 1000 ml of 0.1 % SLS (pH 6.8) at 37°C ± 0.5°C,
paddle apparatus, 100 rpm stirring speed and filtration
with quantitative filter. In these conditions, the candesartan
cilexetil was more stable. It can be concluded that the
proposed method was fully validated and it was found to
be simple, sensitive, accurate, precise, reproducible and
relatively inexpensive and they gave an acceptable
recovery of the analyte. Hence, the developed method
can be recommended for routine quality control analysis
of candesartan cilexetil in tablet formulation.
76
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