Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 2 pp. 357ñ367, 2017
ISSN 0001-6837
Polish Pharmaceutical Society
ANALYSIS
DEVELOPMENT AND VALIDATION OF HPLC ANALYTICAL METHODS
USED FOR DETERMINATION OF ASSAY, CONTENT UNIFORMITY
AND DISSOLUTION OF IMMEDIATE RELEASE CANDESARTAN
CILEXETIL 32 MG TABLETS
MERAL YUCE1 and YILMAZ CAPAN2*
Department of Pharmaceutical Technology, Faculty of Pharmacy, Trakya University,
Balkan Campus, 22030, Edirne, Turkey
2
Department of Pharmaceutical Technology, Faculty of Pharmacy, Hacettepe University,
Sihhiye 06100, Ankara, Turkey
1
Abstract: New analytical methods have been developed and validated on high performance liquid chromatography (HPLC) to assess the assay, content uniformity and dissolution of immediate release candesartan cilexetil 32 mg tablets. Method development studies were performed on cyano column. Mobile phase of assay and
content uniformity test consisted of mixture of 0.05 M phosphate buffer, pH 4.5 and methanol (40 : 60, v/v)
adjusted to pH 4.0 with trifluoroacetic acid, whereas mobile phase of dissolution test consisted of mixture of 1
mM phosphate buffer and acetonitrile (50 : 50, v/v) adjusted to pH 2.0 with trifluoroacetic acid. Mobile
phases were pumped at flow rate of 1.0 mL/min, ultraviolet-visible (UV) detector was operated at 254 nm,
injection volume was set at 20 µL, column temperature was held at 25∞C. Dissolution medium was 0.05 M phosphate buffer, pH 6.5 including 0.70% (w/v) polysorbate 20. Validation studies met acceptance criteria of system suitability, specificity, linearity and range, accuracy, precision, detection limit (LOD), quantitation limit
(LOQ) and robustness parameters.
Keywords: assay, candesartan cilexetil, content uniformity, dissolution, method validation, tablet
Candesartan cilexetil (Fig. 1), ((±)-1-(cyclohexyloxycarbonyloxy)ethyl-2-ethoxy-1-{[2í-(1Htetrazol-5-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylate), is used for treatments of hypertension (high blood pressure) and heart failure (4). It
is practically insoluble in water, sparingly soluble in
methanol and highly hydrophobic (5ñ8). Dissolution
of immediate release candesartan cilexetil 32 mg
tablets is performed in 0.05 M phosphate buffer, pH
6.5 including 0.70% (w/v) polysorbate 20 of 900 mL
volume (9).
Candesartan cilexetil has pharmacopoeial
monograph (10, 11). Development and validation of
a dissolution test for immediate release candesartan
cilexetil 32 mg tablets have not been seen in literature. On the other hand, a common analytical
method with its validation study evaluating both
assay and content uniformity of immediate release
candesartan cilexetil 32 mg tablets has not been
found.
A dissolution test can identify formulation and
manufacturing factors of production batches, in
order to ensure in vitro similarity in relation to clinical batch approved with in vivo study (1). Assay is
the content or potency of drug substance, defining
its existing amount inside dosage form (2).
Uniformity of dosage units is the degree of uniformity of drug substance amount among dosage units.
Content uniformity test is applied to uncoated
tablets, containing ratio of drug substance less than
25 mg or 25% (3).
Figure 1. Candesartan cilexetil (CAS number 145040-37-5)
* Corresponding author: e-mail: [email protected]; phone: + 90 312 305 21 68, fax: + 90 312 310 09 06
357
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MERAL YUCE and YILMAZ CAPAN
For this reason, HPLC analytical method testing assay, content uniformity and dissolution of candesartan cilexetil tablets in 32 mg dose were aimed
for development and validation in this study.
Method validation studies were done according to
system suitability, specificity, linearity and range,
accuracy, precision, LOD, LOQ and robustness
parameters (12-20).
EXPERIMENTAL
Chemicals and reagents
The reagents provided from the following suppliers were all of analytical grade: Polysorbate 20
(Tween 20, Merck-Schuchardt, Germany), trifluoroacetic acid (Uvasol, Merck-Schuchardt, Germany),
methanol, acetonitrile (Merck, Germany), sodium
dihydrogen phosphate dihydrate (NaH2PO4.2H2O,
Emprove, Merck, Germany), potassium dihydrogen
phosphate (KH2PO4) and sodium hydroxide (NaOH,
Riedel-de HaÎn, Germany) and hydrochloric acid
(HCl, J.T. Baker, Holland). Cronus 0.45 µm nylon
25 mm syringe filters were purchased from SMI ñ
LabHut Ltd. (UK). Advantec 0.45 µm cellulose
acetate 25 mm syringe filters were purchased from
Advantec MFS, Inc. (USA). Membrane 0.45 µm
high volume (HV) filters were purchased from
Merck Millipore Corp. (Germany). All aqueous
solutions were prepared with HPLC grade type I
water, obtained in-house, from a Milli ñ Q Gradient
A-10 water purification system (Merck Millipore
Corp., Germany).
The tablet ingredients were all pharmaceutical
grade: Candesartan cilexetil (lot no: 5251-10-017,
XunQiao, LinHai, Zhejiang, China), lactose monohydrate (Pharmatose 90M, DMV - Fonterra
Excipients, Germany), polyethylene glycol 4000
(Merck-Schuchardt, Germany), hydroxypropyl cellulose (Klucel LF ñ Pharm, Ashland, USA), starch
maize (Roquette, France), carboxymethyl cellulose
calcium (Nichirin Chemical Ind., Japan) and magnesium stearate (Peter Greven, Germany).
Certified candesartan cilexetil drug substance
was used as working standard. Immediate release
candesartan cilexetil 32 mg tablets were prepared
with above tablet ingredients. Placebo tablets were
also prepared for validation studies, excluding candesartan cilexetil.
Instrumentation and chromatographic conditions
The chromatographic system used to develop
assay, content uniformity and dissolution methods
was Agilent Technologies HP 1200 series having
UV detector and auto sampler (USA). The chro-
matographic conditions differed only in mobile
phases, whereas the others remained constant.
Chromatographic separations were achieved
on cyano column (250 ◊ 4.6 mm, 5 µm particle;
Clipeus, Higgins Analytical Inc., USA). The mobile
phases were pumped through the column at a flow
rate of 1.0 mL/min, maintaining temperature at
25OC. The injection volume to carry out the chromatography was set at 20 µL. The UV detection
wavelength (λ) was set at 254 nm. Assay and content uniformity test required 13 min and dissolution
test required 12 min for separation.
The mobile phase for assay and content uniformity test consisted of 40% 0.05 M phosphate buffer
solution, pH 4.5 and 60% methanol adjusted to pH
4.0 with trifluoroacetic acid. To prepare 0.05 M
phosphate buffer solution, pH 4.5, 6.8 g KH2PO4
was weighed, diluted to 1000 mL with HPLC grade
water and dissolved for 5 min by mixing.
The mobile phase for dissolution test consisted
of 50% acetonitrile and 50% 1 mM phosphate buffer
solution adjusted to pH 2.0 with trifluoroacetic acid.
To prepare 1 mM phosphate buffer solution, 136.08
mg KH2PO4 was weighed, diluted to 1000 mL with
HPLC grade water and dissolved for 5 min by mixing.
The buffer solutions and mobile phases were
filtered through HV-membrane filter.
The assay of tablet samples was calculated using
equation 1:
Drug amount (mg) = A (sample)/A (standard) ◊
concentration (standard) ◊ volume (sample)/
weight (sample) ◊ mean tablet weight (Eq. 1)
where, A (sample) is the measured peak area of
sample solution, A (standard) is the measured peak
area of standard solution of candesartan cilexetil,
concentration (standard) is the concentration of standard solution of candesartan cilexetil (mg/mL), volume of sample solution is 50 mL, mean tablet
weight is 260 mg, weight of sample is equivalent to
mean tablet weight (mg), taken from mixture of 5
powdered tablets.
The content of tablet samples was calculated
using equation 2:
Drug content (%) = A (sample)/A (standard) ◊
concentration (standard) ◊ volume (sample)/
weight (sample) ◊ mean tablet weight/L
◊ 100
(Eq. 2)
where, L is drug load of 32 mg and weight of sample is the tablet weight (mg).
A calibrated dissolution apparatus (Sotax AG,
Switzerland) was used with paddles at 50 rotations
per minute (rpm) and bath temperature maintained
at 37 ± 0.5OC. Freshly prepared 0.05 M phosphate
Development and validation of HPLC analytical methods used for...
buffer, pH 6.5 (900 mL) including 0.70% (w/v)
polysorbate 20 was used as dissolution medium. To
prepare the dissolution medium, 7.8 g NaH2PO4 ◊
2H2O was weighed, dissolved in about 900 mL of
HPLC grade water and pH was adjusted to 6.5 ±
0.05 with 400 g/L NaOH reference solution. Finally,
it was diluted up to 1000 mL with additional HPLC
grade water. Seven grams polysorbate 20 was added
to prepared buffer solution and mixed for 10 min. In
each dissolution test, 6 tablets were evaluated.
Tablet sample solutions were collected at 10, 20, 30,
45 and 60 min (9). At each time point, 5 mL sample
was removed from each vessel using a glass pipette
and equal volume of fresh medium was added to
each vessel to maintain the volume constant. After
filtration through nylon filter, each sample was
transferred to vial and analyzed.
The dissolved amount of candesartan cilexetil
from tablet samples was calculated using equation 3:
Dissolved (%) = 900L ◊ A (sample)/A (standard)
◊ concentration (standard) ◊ 100
(Eq. 3)
Preparation of working standard solutions
Standard solution of candesartan cilexetil for
assay and content uniformity test was prepared at
0.64 mg/mL amount corresponding to 100% concentration. Candesartan cilexetil (32 mg) was
weighed, placed in a 50 mL volumetric flask and
diluted up to 50 mL with methanol. This solution
was sonicated for 10 min and passed through cellulose acetate filter.
Standard solution of candesartan cilexetil for
dissolution test was prepared at 35.56 µg/mL
amount corresponding to 100% concentration.
Candesartan cilexetil (35.56 mg) was weighed,
placed in a 50 mL volumetric flask and diluted up to
50 mL with methanol. This solution was sonicated
for 10 min. Five mL was transferred using a glass
pipette into 100 mL volumetric flask, diluted up to
100 mL with 0.05 M phosphate buffer, pH 6.5
including 0.70% (w/v) polysorbate 20 and passed
through nylon filter.
Standard solutions were transferred to vials.
Due to required replicates of injection (n) as 3 ≤ n ≤ 6
(13), they were analyzed in 5 replicates.
Preparation of calibration standard solutions
The range in which linearity is evaluated for
assay test is 80-120%, for content uniformity is 60140%, for dissolution test is 60-120% of the test
concentration, including at least 5 levels (12, 17).
Calibration standard solutions for assay and
content uniformity test were prepared in 60% (0.384
mg/mL), 80% (0.512 mg/mL), 90% (0.576 mg/mL),
359
100% (0.64 mg/mL), 110% (0.704 mg/mL), 120%
(0.768 mg/mL), 140% (0.896 mg/mL) of working
standard solution.
Calibration standard solutions for dissolution
test were prepared in 60% (21.336 µg/mL), 70%
(24.892 µg/mL), 80% (28.448 µg/mL), 90% (32.004
µg/mL), 100% (35.56 µg/mL), 110% (39.116
µg/mL), 120% (42.672 µg/mL) of working standard
solution.
Preparation of calibration standard solutions
was done by applying the same analytical method
with working standard solution for assay and content uniformity test or dissolution test, differing in
weighed amount of candesartan cilexetil. Each solution was analyzed in duplicate.
Preparation of placebo solutions
For assay and content uniformity test 10 placebo tablets were crushed with mortar hand, 228 mg
powdered placebo (mean tablet weight-drug load)
was weighed, placed in a 50 mL volumetric flask
and diluted up to 50 mL with methanol. It was sonicated for 10 min. In order to prepare 10% (v/v)
placebo solution in methanol, 10 mL of the solution
was transferred using a glass pipette into 100 mL
volumetric flask, diluted up to 100 mL with
methanol and passed through cellulose acetate filter.
For dissolution test 10 placebo tablets were
crushed with mortar hand, 228 mg powdered placebo was weighed, diluted to 900 mL with dissolution
medium in a volumetric flask. It was held for 60 min
in shaking water bath conditioned to 37 ± 0.5OC and
then passed through nylon filter.
Each placebo solution was transferred to vial
and analyzed in duplicate.
Preparation of sample solutions
In validation study, accuracy is evaluated using
at least 3 levels/3 replicates covering the specified
range (12, 17).
Sample solutions prepared for assay and content uniformity test were in levels of 60% (0.384
mg/mL), 80% (0.512 mg/mL), 100% (0.64 mg/mL),
120% (0.768 mg/mL), 140% (0.896 mg/mL).
Related amount of candesartan cilexetil and 228 mg
placebo were weighed, placed in a 50 mL volumetric flask, diluted up to 50 mL with methanol, sonicated for 10 min and passed through cellulose
acetate filter.
Sample solutions prepared for dissolution test
were in levels of 60% (21.336 µg/mL), 90% (32.004
µg/mL), 120% (42.672 µg/mL). Related amount of
candesartan cilexetil and 228 mg placebo were
weighed, placed in a 50 mL volumetric flask, diluted
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MERAL YUCE and YILMAZ CAPAN
up to 50 mL with methanol and sonicated for 10 min.
Five mL was transferred using a glass pipette into
100 mL volumetric flask, diluted up to 100 mL with
0.05 M phosphate buffer, pH 6.5 including 0.70%
(w/v) polysorbate 20 and passed through nylon filter.
Each sample solution was transferred to vial
and analyzed in duplicate.
Method validation studies
System suitability
System suitability standard solution was working standard solution, prepared in 0.64 mg/mL for
assay and content uniformity test and 35.56 µg/mL
for dissolution test corresponding to 100% concentration. System suitability was determined from 5
replicate injections of the system suitability standard
before sample analyses. The acceptance criteria
were; number of theoretical plates (N) > 2000, tailing factor (symmetry factor, As) of 0.8 < As < 1.5,
relative standard deviation (RSD) ≤ 1.5% for peak
area of assay and content uniformity test and RSD ≤
2.0% for peak area of dissolution test (13, 16).
Specificity
For the specificity study of assay and content
uniformity test, identification of candesartan cilexetil was studied comparing the chromatograms of
system suitability standard solution, 10% (v/v)
placebo solution in methanol, methanol as solvent
and 10% (v/v) tablet sample solution in methanol. In
order to prepare 10% (v/v) tablet sample solution in
methanol, 10 tablets were crushed with mortar hand
and 260 mg was weighed, placed in a 50 mL volumetric flask, diluted up to 50 mL with methanol and
sonicated for 10 min. Ten mL of the solution was
transferred using a glass pipette into 100 mL volumetric flask, diluted up to 100 mL with methanol
and passed through cellulose acetate filter.
For the specificity study of dissolution test,
identification of the candesartan cilexetil was studied comparing the chromatograms of system suitability standard solution, placebo solution, dissolution medium and tablet sample solution taken from
dissolution study.
All chromatograms were examined to check
the absence of interference between candesartan
cilexetil, both with placebo solution (excipients) and
methanol or dissolution medium (12).
Linearity and range
To obtain the calibration line for assay test, 5
levels of calibration standard solutions over 80120% (0.512 mg/mL, 0.576 mg/mL, 0.64 mg/mL,
0.704 mg/mL, 0.768 mg/mL) were prepared.
To obtain the calibration line for content uniformity test, 7 levels of calibration standard solutions over 60-140% (0.384 mg/mL, 0.512 mg/mL,
0.576 mg/mL, 0.64 mg/mL, 0.704 mg/mL, 0.768
mg/mL, 0.896 mg/mL) were prepared.
To obtain the calibration line for dissolution
test, 7 levels of calibration standard solutions over
60-120% (21.336 µg/mL, 24.892 µg/mL, 28.448
µg/mL, 32.004 µg/mL, 35.56 µg/mL, 39.116 µg/mL,
42.672 µg/mL) were prepared.
The analytical ranges were established for
obtaining acceptable linearity, accuracy and precision. The data of peak area versus drug concentration were treated by linear least square regression
analysis. The experimental results were represented
graphically, obtaining a calibration graph and a calibration equation. The acceptance criterion was
regression coefficient (r) ≥ 0.999 (16).
Accuracy
The accuracy of assay test was operated as
described in sample solution preparation at levels of
80% (3 samples), 100% (3 samples) and 120% (3
samples), where a known amount of the drug was
added to a determined amount of placebo and 9 individual samples were prepared. For accuracy testing
of content uniformity, 60% (3 samples) and 140% (3
samples) levels were included and 6 individual samples were additionally prepared.
The accuracy of dissolution test was operated
as described in sample solution preparation at levels
of 60% (3 samples), 90% (3 samples) and 120% (3
samples) and 9 individual samples were prepared.
Recovered candesartan cilexetil amount was calculated in relation to the added amount. The acceptance criteria for each recovery was 98-102% and for
RSD of each level (3 samples) was ≤ 2.0% for assay
and content uniformity test, whereas for dissolution
test the acceptance criteria for each recovery was
95-105% and for RSD of each level (3 samples) was
≤ 3.0% (12, 19).
Precision
In the precision study, repeatability and intermediate precision tests were carried out.
For repeatability testing of assay and content
uniformity, 6 consecutive tablet sample solutions
were prepared. In order to prepare tablet sample
solution in methanol, 10 tablets were crushed with
mortar hand and 260 mg was weighed, placed in a
50 mL volumetric flask, diluted up to 50 mL with
methanol and sonicated for 10 min. Tablet sample
solution was passed through cellulose acetate filter,
transferred to vial and analyzed. For repeatability
Development and validation of HPLC analytical methods used for...
361
Figure 2. Chromatogram of system suitability (a); Chromatogram of 10% (v/v) placebo solution in methanol (b); Chromatogram of
methanol (c); Chromatogram of 10% (v/v) tablet sample solution in methanol (d); for assay and content uniformity test
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MERAL YUCE and YILMAZ CAPAN
testing of dissolution, 6 consecutive tablet sample
solutions were taken from the same vessel at 30 min.
Each tablet sample solution was passed through
nylon filter, transferred to vial and analyzed. The
acceptance criterion for RSD of 6 tablet sample
solutions for repeatability testing was ≤ 2.0%.
For testing the intermediate precision, study of
the variability took place with the same analyst, the
same column and the same equipment on 2 different
working days. The procedure of assay and content
uniformity test was realized following up the
repeatability testing. For testing the intermediate
Figure 3. Chromatogram of system suitability standard (a); Chromatogram of placebo solution (b); Chromatogram of dissolution medium
(c); Chromatogram of tablet sample solution taken from dissolution study (d); for dissolution test
363
Development and validation of HPLC analytical methods used for...
precision of dissolution test, 6 different tablet sample solutions were taken from 6 different vessels at
30 min. The difference between mean recovery % of
2 days (∆) for intermediate precision testing was calculated (12).
Recovery of sample solution (%) =
Cs ◊ 100 / C0
(Eq. 6)
where, C0 is the concentration of analyte at the
beginning and Cs is the concentration of analyte at
sampling time (12, 19).
LOD and LOQ
LOD and LOQ of the tests were calculated
using equations 4 and 5:
LOD = 3.3 σ / S
(Eq. 4)
RESULTS
LOQ = 10 σ / S
(Eq. 5)
where, σ is standard deviation (SD) of the response
and S is the slope of the calibration curve (12).
Robustness
The study of robustness was carried out to
evaluate the influence of variations in the chromatographic conditions and the stability of standard and
sample solutions.
The factors chosen for chromatographic separation and stability study of sample solution preparation were temperature (OC + 10OC), pH (± 0.1), salt
concentration (KH2PO4 % ± 10%), organic compound concentration (methanol % ± 3% or acetonitrile % ± 2.5%) and flow rate (mL/min ± 0.1) of
mobile phase (13, 18). The acceptance criteria of
system suitability parameters for chromatographic
separation were applied.
The stability of standard and sample solutions
was evaluated at 0, 24, 48 h for assay and content
uniformity test and at 0, 12, 24 h for dissolution test
held in amber glass at ambient temperature. The
acceptance criterion for recovery was 98-102% for
assay and content uniformity test and 97-103% for
dissolution test, calculated using equation 6:
System suitability
Candesartan cilexetil peaks appear at 10.1 min
for assay and content uniformity test and at 10.4 min
for dissolution test (Figs. 2, 3). Mean peak area of 20
421, As of 1.339 and N of 12 238 with their RSD of
0.041%, 0.202% and 0%, respectively, were determined for assay and content uniformity test, mean
peak area of 1 115.1, As of 0.851 and N of 57 865
with their RSD of 0.037%, 0.134% and 0.878%,
respectively, were determined for dissolution test.
Specificity
No major peak other than candesartan cilexetil
in both methanol and dissolution medium was
observed, there was no interference between excipients. Candesartan cilexetil was noticed to be eluted
from the ingredients (Figs. 2, 3).
Linearity and range
The calibration lines for assay, content uniformity and dissolution tests are shown in Figure 4. The
equation of calibration line for assay test was found
to be y = 32 277x ñ 14.682, with r of 0.9997; r2 of
0.9994 was obtained for the standard curve within
95% confidence interval (CI). The equation of calibration line for content uniformity test was found to
be y = 32 831x - 152.15, with r of 0.9994; r2 of
0.9988 was obtained for the standard curve within
Table 1. Accuracy study results for assay, content uniformity and dissolution tests.
Concentration
level
Assay and content uniformity test
60%
80%
Sample
100%
120%
Dissolution test
140%
60%
Recovery (%)
90%
120%
Recovery (%)
1
99.37
98.92
99.37
98.41
99.06
99.93
99.54
100.22
2
101.99
100.76
99.47
98.65
99.77
99.87
100.60
99.76
3
101.18
98.77
98.81
100.63
100.90
99.93
100.54
99.18
Mean (%)
100.85
99.48
99.22
99.23
99.91
99.91
100.23
99.72
SD*
1.341
1.108
0.356
1.218
0.928
0.035
0.595
0.521
RSD** (%)
1.330
1.114
0.359
1.228
0.929
0.035
0.594
0.523
CI***
(α**** = 0.05)
± 3.332
± 2.753
± 0.884
± 3.027
± 2.305
± 0.086
± 1.479
± 1.295
*SD: Standard deviation. **RSD: Relative standard deviation. ***CI: Confidence interval. ****α: Level of significance.
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MERAL YUCE and YILMAZ CAPAN
Table 2. Precision study results for assay, content uniformity and dissolution tests.
Assay and content uniformity test
Repeatability
Sample
mg/tablet
Dissolution test
Intermediate precision
Concentration (%)
mg/tablet
Concentration (%)
Repeatability
Intermediate
precision
Concentration (%)
1
31.83
99.47
32.68
102.13
95.09
101.75
2
32.16
100.50
32.82
102.56
94.31
92.51
3
32.38
101.19
32.85
102.66
95.13
103.97
4
32.27
100.84
32.76
102.38
94.37
86.63
5
32.43
101.34
32.59
101.84
95.06
97.96
6
32.38
101.19
32.65
102.03
90.64
105.44
Mean (%)
32.24
100.76
32.73
102.27
94.10
98.04
SD*
0.224
0.699
0.102
0.319
1.735
7.273
RSD** (%)
0.694
0.694
0.311
0.312
1.844
7.418
CI***
(α**** = 0.05)
± 0.235
± 0.734
± 0.107
± 0.335
± 1.821
± 7.632
*SD: Standard deviation. **RSD: Relative standard deviation. ***CI: Confidence interval. ****α: Level of significance.
95% CI. The equation of calibration line for dissolution test was found to be y = 31.828x - 5.2, with r
of 0.9992; r2 of 0.9985 was obtained for the standard
curve within 95% CI.
Accuracy
The recovery results obtained for accuracy of
assay, content uniformity and dissolution tests are
presented in Table 1. The recovery results for assay
test ranged from 98.41 to 100.76% and RSD ranged
from 0.359 to 1.228%. The recovery results for content uniformity test ranged from 99.06 to 101.99%
and RSD ranged from 0.929 to 1.330%. The recovery results for dissolution test ranged from 99.18 to
100.60% and RSD ranged from 0.035 to 0.594%.
Precision
The precision study results are presented in
Table 2. Assay and content uniformity test had mean
recovery of 100.76% and RSD of 0.694% for
repeatability testing corresponding to the first day,
mean recovery of 102.27% and RSD of 0.312% for
intermediate precision corresponding to the second
day.
Dissolution test had mean recovery of 94.10%
and RSD of 1.844% for repeatability testing corresponding to the first day, mean recovery of 98.04%
and RSD of 7.418% for intermediate precision corresponding to the second day.
The RSDs obtained for repeatability testings
were below 2.0%. The difference between mean
recovery results of individual days was obtained to
be 1.51% (∆ = 102.27 - 100.76%) for assay and content uniformity test, whereas for dissolution test it
was obtained to be 3.94% (∆ = 98.04 ñ 94.10%).
RSD of 7.418% for intermediate precision was
obtained to be below 10.0% (20).
LOD and LOQ
LOD result obtained for assay test was 0.009
mg/mL, for content uniformity test was 0.022
mg/mL and for dissolution test was 1.060 µg/mL.
LOQ result obtained for assay test was 0.028
mg/mL, for content uniformity test was 0.066
mg/mL and for dissolution test was 3.211 µg/mL.
Robustness
Small variations of chromatographic conditions were obtained, but all of them met the acceptance criteria of system suitability parameters.
The recovery results at 48 h of working standard and tablet sample solutions for assay and content uniformity test were obtained to be 99.81 and
101.03%, respectively, within acceptable limits.
For dissolution test, the recovery result at 12 h
of working standard solution was obtained to be
97.60% within acceptable limits; where the recovery
result at 12 h of tablet sample solution was 96.31%,
out of limits.
DISCUSSION AND CONCLUSION
The development of analytical methods started
with selecting the chromatographic conditions for
Development and validation of HPLC analytical methods used for...
candesartan cilexetil determination in tablets.
Dissolution method was firstly developed, by mainly
modifying of mobile phase KH2PO4 salt and acetonitrile ratios for sufficient system suitability parameters. The optimum conditions were reached after
evaluation of minumum retention time and good
365
chromatogram separation of candesartan cilexetil.
Chromatographic conditions of dissolution method
were determined to be: mobile phase organic compound concentration 50% acetonitrile, mobile phase
salt concentration 50% 1 mM phosphate buffer solution, mobile phase pH 2.0, flow rate 1.0 mL/min,
Figure 4. Calibration line for assay test (a); Calibration line for content uniformity test (b); Calibration line for dissolution test (c)
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MERAL YUCE and YILMAZ CAPAN
temperature 25OC, injection volume 20 µL, UV
detection wavelength 254 nm. Developed assay and
content uniformity method differed in mobile phase
composition, whereas the other parameters remained
the same. In respect of solubility, methanol was chosen as organic solvent instead of dissolution medium
and as mobile phase organic compound instead of
acetonitrile. The necessity of changing the mobile
phase composition was the lack of chromatogram
separation, when dissolution medium was removed;
for which a new solvent such as methanol in which
candesartan cilexetil is soluble was needed,. The
mobile phase composition for assay and content uniformity method was specified after obtaining sufficient system suitability parameters; thus, mobile
phase organic compound concentration 60%
methanol, mobile phase salt concentration 40% 0.05
M phosphate buffer solution, pH 4.5 and mobile
phase pH 4.0 were set as optimum conditions.
This work has proposed a common assay and
content uniformity method and a dissolution method
used for evaluating immediate release candesartan
cilexetil 32 mg tablets, complying with validation
requirements. It has been proved that the common
assay and content uniformity method was specific,
linear between 60 and 140% of the working concentration (0.64 mg/mL) for candesartan cilexetil with r
higher than 0.999, exact, precise, accurate and robust
regarding the stability of solutions and applied chromatographic conditions across the analytical range.
The dissolution method was also concluded to be:
specific, linear between 60 and 120% of the working
concentration (35.56 µg/mL) for candesartan cilexetil with r higher than 0.999, exact, precise, accurate
and robust regarding the applied chromatographic
conditions across the analytical range. Tablet sample
solution taken from dissolution study was concluded
to be analyzed without waiting.
Thus, the validated methods were successfully
established for quality control purposes of developed tablets.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Acknowledgments
13.
Authors thank to Zentiva Saglik Urunleri
(Turkey) for providing drug substance, Ýlko Ýlac
(Turkey) for providing carboxymethyl cellulose calcium and IMCD (Turkey) for providing Klucel LF ñ
Pharm.
14.
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Received: 28. 01. 2016