Scientific Research and Essays Vol. 6(29), pp.

6203-6208, 30 November, 2011

Available online at http://www.academicjournals.org/SRE
DOI: 10.5897/SRE11.1673
ISSN 1992-2248 ©2011 Academic Journals

Full Length Research Paper

Flow injection spectrophotometric and

spectrofluorimetric methods for the determination of
candesartan cilexetil in pharmaceutical formulations
Ayisha Khalid1, Amir Waseem2*, Iftikhar Afzal1, Mohammad Yaqoob3, Abdul Nabi3 and
Mohammad Masoom Yasinzai4
1
Department of Pharmacy, University of Balochistan, Quetta-87300 Pakistan.
2
Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan.
3
Department of Chemistry, University of Balochistan, Quetta-87300 Pakistan.
4
Department of Biochemistry, University of Balochistan, Quetta-87300 Pakistan.
Accepted 21 October, 2011

This article describes the development of flow injection methods for the determination of Candesartan
Cilexetil using spectrophotometery and spectrofluorimetry in pharmaceutical formulations. The first
method is based on the UV absorption at 270 nm in phosphate buffer containing cetyl
trimethylammonium bromide (CTAB) and methanol at pH 6.5. The second method based on the
fluorescence excitation and emission at 260 and 384 nm respectively, in phosphate buffer containing
sodium dodecyl sulphate (SDS) and methanol at pH 4.0. Calibration graphs were linear over the
concentration range of 0.1 to 4.0 µg/ml for UV-method and 0.03 to 2.0 µg/ml for fluorescence method.
The limits of detection (3 s) of 0.01 µg/ml for both methods with sample throughput of 80 and 100/h were
obtained respectively. The relative standard deviations of 1.2 to 3.2% (n = 4) for UV-method and 0.5 to
1.8% for fluorescence method were achieved in the concentration range studied. The developed
methods were applied to pharmaceuticals and the results obtained were compared with HPLC reference
method and no significant difference between these methods was observed at 95% confidence level.

Key words: Candesartan cilexetil, spectrophotometery, spectrofluorimetry, pharmaceuticals, flow injection

analysis.

INTRODUCTION

Candesartan, 1-[[(cyclohexyloxy) carbonyl] oxy] ethyl 2-
ethoxy-1-[[2’-(1H-tetrazol 5-yl) [1, 1’-biphenyl]-4-yl]
methyl]-1H-benzimidazole-7-carboxylate is an
angiotensin II receptor antagonist used for the
management and treatment of hypertension. Chemical
formula of candesartan cilexitil is C33H34 N6O6 with
molecular weight of 610.67. The chemical structure of
Candesartan Cilexetil is given in Figure 1. Candesartan
cilexetil (CC) is a prodrug prepared for oral use, having
candesartan as the active ingredient and cilexetil is ester
*Corresponding author. E-mail: waseemq2000@hotmail.com.
(cyclohexyl 1-hydroxyethyl carbonate) added to the active
constituent to enhance the bioavailability. It is converted
to the active substance, candesartan by ester hydrolysis
during absorption from the gastrointestinal tract
(McClellan et al., 1998; Gohike et al., 1999). Candesartan
is AT1 receptors selective non peptide antagonist, with
tight binding to and slow dissociation from the receptor
(Baver et al., 1995). It shows no agonist activity, shows
slow onset and prolonged action that is after absorption
Cmax is achieved in 2 to 3 h; plasma protein binding is
99% and half life is as long as 9 to 10 h. Clearence
through urine is 60 to 65% and bile almost 35 to 40%
(Gao et al., 1996). Candesartan cilexetil is the drug from
the class of ARA II which is relatively a new class of drug.
6204 Sci. Res. Essays
H 3 CH 2 CO
N N N
O
C C
HN N
O CH 3
Figure 1. Structure of Candesartan cilexetil.
ARA II are considered safe as well as effective for the
treatment of hypertension and cardiovascular disorders.
Major determination studies of Candesartan cilexetil are
done on either combination of Candesartan cilexetil with
other ARA II members or with diuretics like
hydrochlorothiazide. Some other combinations are also
used and most work is done on plasma and urine
samples. Techniques used are HPLC with fluorescence
detector (Stenhoff et al., 1999; Gonzalez et al., 2002;
Miyabayashi et al., 1996), LC-MS (Zhao et al., 1999), LC-
UV (Qutab et al., 2007). All these methods are very
tedious, time consuming and involve complex
procedures, therefore require skilled individuals and
expensive instruments. There are methods for the UV
determination of candesartan cilexetil using HPLC; only a
few uses direct UV absorption includes derivative
spectrophotometry (Tatar et al., 2002; Charoo et al.,
2009).The present work is novel in this regards as few
techniques has been used fluorescence detector (as in
HPLC) but none of the direct analysis with fluorescence
reported so far in flow mode, as major work has been
done on losartan. Flow injection analysis (FIA)
techniques are well-established tools for the automation
and miniaturization of analytical methodologies.
Advantages of using FIA are: increased sample injections
throughputs, high versatility, high robustness, decrease
of the human exposure under hazardous
chemical/physical sample pre-treatments. Due to these
advantages, FIA has been coupled with UV-Vis,
fluorescence and chemiluminescence detectors for the
detection of wide variety of analytes (Rishi et al., 2011;
Asgher et al., 2011; Attiq-ur-Rehman et al., 2008, 2009,
2010; Waseem et al., 2007, 2008, 2010; Yaqoob et al.,
2001). The aim of this study design under discussion is
to establish a rapid, direct, easy and equally sensitive,
accurate and reliable UV/fluorescence
H O
O C O
spectrophotometric method for the determination of CC.
The FI-UV/fluorescence spectroscopic layout is flexible
enough to facilitate adequately the future trends of
introducing variations (enzyme columns, multi channels
etc.) to accommodate the scope of more complex
determinations of candesartan cilexetil, its metabolites or
other ARA II family members.
EXPERIMENTAL
Reagents and materials
All glass/plastic ware used during the experiments and storage of
standards and reagents were precleaned and washed with ultra
high purity (UHP) deionised water (Elgastat, maxima, UK). All
reagents used are of analytical grade and solutions were also
prepared in UHP deionised water. Candesartan cilexitil (CC) is
kindly provided by Pharma Evo (Karachi-75400, Pakistan) in
purified form. Pharmaceutical formulations containing CC were
purchased from local market. Standard stock solution was prepared
by dissolving appropriate amount of the CC in methanol so as to
make the concentration of 100 µg ml-1. Working solutions were
obtained by diluting the stock solution with solvent specially
designed for the experiment. All solutions were stored at room
temperature as the drug is temperature and light resistant.
Instrumentation and procedures
FI-UV system
A simple single channel flow-injection manifold described previously
(Jadoon et al., 2010; Rishi et al., 2009; Waseem et al., 2010) was
used in this study (Figure 2). A peristaltic pump (Ismatec,
Switzerland) was used to propel the buffer carrier (methanol: 0.35%
CTAB in phosphate buffer PH 6.5 in a ratio of 1:9 v/v) at a flow rate
of 3.0 ml/min. A rotary injection valve (Omnifit 1106, UK) was used
to inject sample/standards into a buffer carrier stream. The carrier

Sample/standards
Pump
Carrier solution
Figure 2. Flow injection manifold diagram for CC determination.
solution was then passed in a flow-through cell (100 µl) and
monitored at 270 nm wavelength using a spectrophotometer
(Jenway 6505, UK) connected to a chart recorder (Kipp and Zonen
BD40, Holland).
FI-fluorescence system
A peristaltic pump (Ismatec reglo 100, single channel, Switzerland)
was used to deliver the sample carrier solution to the detector
system at a flow rate of 2.0 ml/min (Figure 2). The tubing used in
the pump to facilitate the solvent flow was Teflon tubing 1.02 mm in
diameter. Rotary injection valve (Omnifit 1106, UK) was used to
inject sample/standards into the buffer stream of 0.2% SDS and
10% methanol dissolved in phosphate buffer of pH 4. The sample
carrier solution stream then passed through the flow cell (100 µl)
present in fluorescence spectrophotometer (Shimadzu RF-5301PC)
and intensity of fluorescence was then measured and noted using
computer software.
RESULTS AND DISCUSSION
Optimization of FI-UV system
In order to establish the optimal conditions for the lowest
possible detection limit of CC, the effects of various
parameters were investigated at a fixed wavelength of
270 nm. These include buffers (phosphate, carbonate,
borate, acetate), surfactants (SDS, CTAB, Triton-X100,
Tween-20, Brij-35), solvents (ethanol, methanol,
acetonitrile), flow rate and sample injection volume. The
optimisation studies were done by employing OVAT
technique (one variable at time) using 2.0 µg/ml standard
concentration of CC. Various buffers including sodium
carbonate (pH range: 10 to 12), sodium tetra borate (pH
range: 9.5 to 11), sodium acetate (pH range: 4 to 6.5)
and sodium phosphate (pH range: 6 to 7.5) were
investigated. Phosphate buffer (pH of 6.5) has shown
better response (Hillaert et al., 2003) and was selected
for further studies. Similarly the effect of different
surfactants including SDS, CTAB, Tween-20, Brij-35 and
Triton-X100 were tested (range 0.1 to 1%). CTAB not
only gave maximum response but also gave smooth flow
Khalid et al. 6205
UV/Vis & Fluorescence
Spectrophotometer Waste
without bubbling and obstruction to FI system and
sedimentation on storage which was seen in SDS.
Tween-20 also caused excessive bubbling and gave no
appreciable results, same applies to the rest. Effect of
CTAB concentration in the range of 0.1 to 1%) was also
studied and best response was obtained at 0.35%.
Therefore, CTAB (0.35%) was used as a surfactant in
further studies. Solvents investigated for maximum
response included methanol, ethanol, acetonitrile, almost
all of them showed similar response. The close range of
absorbance and limitations of working on FIA with
organic solvents led to no more solvents probing and
methanol was selected for further investigations. The
percentage of methanol to buffer was optimized in the
range of 5 to 25% (v/v) and 10% methanol showed better
response and was selected for further work. The effect of
flow rate of sample carrier was calibrated in terms of
sensitivity and reagent consumption. The flow rate was
studied over the range of 0.5 to 4 ml/min, flow rate 3.0
ml/min was selected in the entire method of analysis
because of maximum response and low- medium reagent
consumption. Sample volume was probed over the range
of 60 to 360 µl; and 240 µl was found most appropriate as
above this volume absorbance remained approximately
same and using the larger volume results in unnecessary
usage of sample consumption.
Table 1 summarizes the parameters optimized, ranges
studied and optimum conditions selected for the study.
Optimization of fi-fluorescence system
In order to make the system highly sensitive and to detect
the lowest possible concentration, different parameters
were investigated using an OVAT technique that is, one
parameter at time. The parameters studied includes
buffers, pH range, surfactant, solvents, volume of sample
injection and flow rate of sample carrier stream. All these
investigations were done on CC standard of 0.5 µg/ml at
an excitation and emission of 260 and 384 nm (Stenhoff
et al., 1999; Gonzalez et al., 2002; Cagigal et al., 2001).
6206 Sci. Res. Essays

Table 1. Optimization of parameters for FIA system.

Selected value
Parameter Range studied
FI-UV system FI-fluorescence system
Sodium phosphate
Sodium borate
Buffer Sodium phosphate Sodium phosphate
Sodium carbonate
Sodium acetate

Phosphate buffer (pH) 3.5 – 7.5 6.5 4

Methanol
Solvents Ethanol Methanol Methanol
Acetonitrile

Solvents ratio with buffer (%, v/v) 5 – 25 10 10

SDS
CTAB
Surfactants Triton-x100 CTAB SDS
BRIJ-35
Tween-20

Concentration of surfactant (%) 0.1–1.0 0.35 0.2

Flow rate (ml/min) 0.5 – 4.0 3.0 2.0

Injection volume 60 – 360 240 300

The effect of buffers in acidic and basic pH ranges on the
determination of CC is studied; only phosphate buffer (pH
4.0) was found the best suitable buffer for the study.
Similarly, the effect of different solvents was observed
and as methanol, ethanol showed no appreciable
difference and due to working restriction with organic
solvents on FI tubing, methanol was selected but again
after analysing various ratios only 10% methanol was
selected as a solvent. The selected concentration of
methanol gave an appropriate strength of signal and
found harmless for the equipment. Surfactants analysed
includes: SDS, CTAB, Triton-X100, Tween-20 and Brij-
35. Tween-20 and Triton-X100 were rejected due to
much pronounced background signal and off the rest only
SDS gave enhanced fluorescence, less bubbling and
obstruction in smooth flow. So moving forward with SDS
different percentage concentrations were studied ranging
from 0.1 to 1.0%. At the concentration of 0.2%, the signal
had best strength and reproducibility. Therefore, 0.2%
SDS was selected for future use. In physical parameters,
the effect of flow rate of sample carrier and sample
injection volume was studied keeping in view the
sensitivity, speed and reagent consumption. Flow rate of
sample carrier was studied over the range of 0.5 to 3.5
-1
ml/min . Maximum intensity of fluorescence was noted at
2.0 ml/min with strong and reproducible peak height. It
also provides advantage of low reagent consumption, as
well as, less flow obstructions in system.
In the same pattern sample injection volume was
investigated in the range of 60 to 360 µl and it was
observed that the fluorescence intensity showed steady
increase with increase in volume and at 300 µl maximum
response was obtained and was maintained for further
studies (Table 1).
Analytical figures of merit
Under selected conditions, calibration graphs were linear
over the concentration range of 0.1 to 4.0 µg/ml for UV-
method and 0.03 to 2.0 µg/ml for fluorescence method.
The regression equations of A = 0.0283c + 0.0006 (A =
absorbance, c = concentration in µg/ml) and I = 67.95c +
2.36 (I = Fl. intensity; c = concentration in µg/ml) were
obtained for FI-UV-method and FI-fluorescence method
2
respectively. The coefficients of determination (r ) were
0.9997 and 0.9943 respectively. The limits of detection (3
s) of 0.01 for both methods with sample throughput of 80

Table 2. Maximum tolerable concentrations of interferences for CC (0.5 µg/ml) determination (n = 4).
Species added
Nitrate, nitrite, zinc, copper, manganese, chromium and iron.
Sodium, potassium, calcium, magnesium, chloride, sulfate, sucrose, starch, cellulose and gum acacia.
Mannitol, polyethylene glycol, urea and uric acid.
Table 3. Determination of candesartan cilexetil (CC) in different pharmaceutical formulations.
Sample (mg) CC quoted FI-UV system
Tablet-I 8 8.12
Tablet-II 8 7.65
Tablet-III 8 7.82
and 100/h were obtained respectively. The relative
standard deviations of 1.2 to 3.2% (n = 4) for UV-method
and 0.5 to 1.8% for fluorescence were achieved in the
concentration range studied. The developed methods
were applied to pharmaceuticals and the results obtained
were compared with HPLC method and no significant
difference between these methods was observed at 95%
confidence level.
Interference studies
A working concentration of 0.5 µg/ml of CC was selected
for checking the interference of different excipients and
ions on the blank solvent system and on CC
determination for both methods. The response of some
common excipients in drugs including lactose, mannitol,
maltose, sucrose, starch, gum acacia, urea, uric acid,
polyethylene glycol and cellulose, inorganic ions for
example, sodium, potassium, calcium, magnesium, zinc,
copper, manganese, chromium, nitrate, nitrite, chloride
and sulfate ions on CC determination were investigated
under the established conditions. The tolerance limit was
taken as the maximum concentration of the foreign
substances which caused an approximately ±5% relative
error in the determination with respect to CC
concentration (0.5 µg/ml). Table 2 shows that no
significant interference was observed for these foreign
substances for CC determination.
Validation of the proposed method
The proposed FI-UV/fluorescence spectroscopic methods
for the determination of CC in pharmaceutical
formulations were validated by comparing the results with
Khalid et al. 6207
Tolerance ratio
FI-UV system FI-fluorescence system
500 500
300 250
100 100
FI-fluorescence system Reference method
7.91 8.21
7.88 7.81
8.21 7.92
the HPLC reference method (Qutab et al., 2009). The
recovery tests were also performed and the recoveries
found were in the range of 95.45 to 105.6% for
pharmaceutical formulations (Table 4).
Application to real samples
The method discussed earlier was applied on the
commercially available tablets purchased from local
market. Five tablets were weighed and powdered, an
accurate weight of the powder equivalent to one tablet
was mixed with 100 ml of methanol in a 100 ml calibrated
flask, sonicated for about 10 min and filtered to separate
any insoluble matter. The filtrate was collected in a clean
flask. Appropriate aliquots were taken and diluted with
phosphate buffer for each method. The results obtained
for both methods were in complete agreement with the
amount labelled and to the results obtained from HPLC
reference method and thus gives the confidence level of
95%. The data is shown in Table 3.
Conclusions
The developed methods of analysis gave extremely
reliable and efficient results with additional benefits of
reduced reagent consumption and experimental
expenditure. The developed methods are proved to be
user friendly and have markedly increased sample
-1
throughput rate of 80 to 100 h as compared to other
techniques which may even take 30 min for completion of
a single cycle. The design is flexible enough to alter as
the need arise and could be easily installed in lab and
industrial environment with less cost operable by semi
qualified personnel.
6208 Sci. Res. Essays

Table 4. Recovery of candesartan cilexetil from different pharmaceutical

formulations.

Sample Candesartan Cilexetil (µg/ml)

Actual Added Found Recovery (%)
1 0.5 0 0.48 ± 0.02 96 ± 4
0.5 0.97 ± 0.05 97 ± 5
1 1.55 ± 0.05 103±4

2 0.25 0 0.24 ± 0.01 96 ± 4

0.5 0.75 ± 0.03 98 ± 4
1 1.31 ± 0.05 105 ± 4

3 0.5 0 0.49 ± 0.02 98 ± 4

0.5 0.95 ± 0.05 95 ± 5
1 1.48 ± 0.07 98 ± 5

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