Acta Chromatographica, no.

19, 2007

SIMPLE AND SENSITIVE LC–UV METHOD

FOR SIMULTANEOUS ANALYSIS
OF HYDROCHLOROTHIAZIDE AND CANDESARTAN
CILEXETIL IN PHARMACEUTICAL FORMULATIONS

S. S. Qutab1, S. N. Razzaq1, M. Ashfaq1, Z. A. Shuja1, and I. U. Khan1,*

1
Schazoo Laboratories (Pvt) Ltd, 45 G.T. Road, Lahore-54000, Pakistan
2
Department of Chemistry, Government College University, Lahore-54000, Pakistan

SUMMARY
A simple, sensitive, and inexpensive high-performance liquid-chroma-
tographic method has been developed for simultaneous determination of hydro-
chlorothiazide and candesartan cilexetil in pharmaceutical formulations. Chro-
matographic separation was achieved on a Phenyl-2 column with a 25:75:0.2
mixture of 0.02 M potassium dihydrogen phosphate, methanol, and triethyl-
amine, final pH 6.0 ± 0.1, as mobile phase. Detection was at 271 nm. Respon-
se was a linear function of concentration in the range 5–45 µg mL−1 for hy-
drochlorothiazide and 12–56 µg mL−1 for candesartan cilexetil; the correlation
coefficients were 0.9993 and 0.9991, respectively. Total elution time for the
two components was less than 5 min.

INTRODUCTION
Cartex-H tablets (Schazoo Laboratories Lahore Pakistan), which con-
tain candesartan cilexetil and hydrochlorothiazide, are one of the most com-
monly used formulations for treatment of high blood pressure when one
medicine (monotherapy) is not sufficiently effective. Hydrochlorothiazide (6-
chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide; Fig.
1) is a diuretic. Several analytical methods, including LC–MS [1,2], voltamet-
ry [3], spectrophotometry [4–9], capillary electrophoresis [10], and HPLC [11
–15], have already been reported for its determination, either alone or in com-
bination with other drugs. Candesartan cilexetil ((±)-1-hydroxyethyl-2-ethoxy-

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1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]-7-benzimidazolecarboxylate cyclohe-
xyl carbonate; Fig. 2) is a non-peptide angiotensin II receptor. The literature
contains very few methods for analysis of candesartan cilexetil; those reported
include HPLC with fluorimetric detection [16,17] and spectrofluorimetry [18].
HPLC and ratio derivative spectrophotometric methods have been used for
simultaneous determination of the two compounds [19,20]. The HPLC me-
thod used diode-array detection for simultaneous quantification of hydrochlo-
rothiazide and candesartan cilexetil. The retention time was more than 6.5
min.

Fig. 1
Chemical structure of hydrochlorothiazide

Fig. 2
Chemical structure of candesartan cilexetil

In this paper we describe a simple, inexpensive, sensitive, and valida-

ted HPLC method with elution time less than five minutes for simultaneous
determination of candesartan cilexetil and hydrochlorothiazide in pharma-

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ceutical formulations. The method has been successfully used for quality-
control analysis of the drugs and for other analytical purposes.

EXPERIMENTAL
Chemicals and Reagents
Reference standards of candesartan cilexetil and hydrochlorothiazide
were obtained from Schazoo Laboratories (Lahore, Pakistan). A pharmaceuti-
cal product (Catex-H tablets) containing the same drugs, obtained from the
same laboratory, was used in the experiments. Cartex-H tablets were reported
to contain 16 mg candesartan cilexetil and 12.5 mg hydrochlorothiazide. Me-
thanol (HPLC grade), potassium dihydrogen phosphate, orthophosphoric acid,
and triethylamine (analytical reagent grade) were purchased from Merck (La-
hore, Pakistan). All excipients used were of pharmaceutical grade. Starch was
purchased from Rafhan (Pakistan), lactose from Borculo (Holland), magne-
sium stearate from Coin Chen (China), and avicel from JRS Pharma (India).
Deionized water was used throughout the experiment. Before use, mobile pha-
se was filtered through 0.45-µm cellulose acetate filters from Millipore (USA).
Whatman no. 41 filter papers (obtained commercially) were used in the prepa-
ration of sample solutions.
Apparatus and Chromatographic Conditions
A Shimadzu LC-10A system comprising a model LC-10AT pump, an
SPD-10A variable-wavelength detector (operated at 271 nm), a CBM-10A in-
terface module with class LC-10 HPLC software, and a Rheodyne injection
valve with a 20-µL loop was used for development and evaluation of the me-
thod. Compounds were separated on a 250 mm × 4.6 mm i.d., 5-µm particle,
Hypersil (UK) Phenyl-2 column. The mobile phase was a 25:75:0.2 mixture
of 0.02 M potassium dihydrogen phosphate, methanol, and triethylamine, final
pH 6.0 ± 0.1 (adjusted by addition of 10% orthophosphoric acid); the flow ra-
te was 1 mL min−1. The mobile phase was degassed by sonication before use.
An external standard method was used. UV detection was performed at 271
nm. HPLC was performed at room temperature. Peak identity was confirmed
by comparison of spectra and retention times with those of standards.

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Preparation of Standard Solutions
A stock solution of candesartan cilexetil and hydrochlorothiazide refe-
rence standards (0.32 and 0.25 mg mL−1, respectively) was prepared in metha-
nol, because both drugs are soluble in this solvent. Working standard solution
(32 µg mL−1 candesartan cilexetil and 25 µg mL−1 hydrochlorothiazide) was
obtained by diluting the stock solution with the mobile phase.
Linearity
The linearity of the method was checked by analyzing five solutions in
the range 5–45 µg mL−1 for hydrochlorothiazide (5, 15, 25, 35, and 45 µg mL−1)
and 12–52 µg mL−1 for candesartan cilexetil (12, 22, 32, 42, 52 µg mL−1). Each
solution was prepared in triplicate.
Limits of Detection and Quantification
The limit of detection (LOD) is defined as the lowest concentration of
an analyte that can be readily detected but not necessarily quantified. It is usu-
ally regarded as the amount for which the signal-to-noise ratio (SNR) is 3:1.
The limit of quantitation (LOQ) is defined as the lowest concentration of an
analyte that can be quantified with acceptable precision and accuracy. It is usu-
ally regarded as the amount for which the SNR is 10:1. Two types of solution,
i.e. blank and spiked with known progressively decreasing concentrations of
each analyte, were prepared and analysed. The limits of detection (LOD) and
quantification (LOQ) were then established by evaluating the minimum levels
at which the analyte could be readily detected or accurately quantified, res-
pectively.
Accuracy
Method accuracy was determined by addition of known amounts of
hydrochlorothiazide and candesartan cilexetil to a sample solution of known
concentration and comparing calculated and measured concentrations. A sam-
ple solution containing hydrochlorothiazide and candesartan cilexetil (0.25 and
0.32 mg mL−1, respectively) was prepared by dissolving 12.5 mg hydrochlo-
rothiazide and 16 mg candesartan cilexetil in 50 mL methanol. Samples (3 mL)
of the filtered solution were transferred to 50-mL volumetric flasks containing
1.0, 2.0, 3.0, or 4.0 mL hydrochlorothiazide and candesartan cilexetil standard

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solution (0.25 and 0.32 mg mL−1, respectively). The solutions were diluted to vo-
lume with mobile phase so the final concentrations were 20.0, 25.0, 30.0, and
35.0 µg mL−1 for hydrochlorothiazide and 25.6, 32.0, 38.4, and 44.8 µg mL−1
for candesartan cilexetil. Each solution was prepared in triplicate.
Specificity
A synthetic mixture containing 16 mg candesartan cilexetil, 12.5 mg hy-
drochlorothiazide, and 30 mg each of starch, lactose, magnesium stearate, and
avicel, which are present as excipients in the pharmaceutical formulation, was
accurately weighed and transferred to a 50-mL volumetric flask. The mixture
was shaken well with 30 mL methanol and then diluted to volume with me-
thanol. After filtration, 5 mL of the filtrate was transferred to a 50-mL volu-
metric flask and diluted to volume with mobile phase, to furnish a final so-
lution containing 32 µg mL−1 candesartan cilexetil and 25 µg mL−1 hydrochlo-
rothiazide.
Application of the Method to Tablets
Twenty tablets were weighed to obtain the average weight. They were
then were ground manually using a pestle and mortar made of china clay. An
amount of powder equivalent to 16 mg candesartan cilexetil and 12.5 mg hy-
drochlorothiazide was transferred to a 50-mL volumetric flask with 30 mL
methanol, shaken for 5 min, then diluted to volume with methanol to furnish a
solution containing 0.32 mg mL−1 candesartan cilexetil and 0.25 mg mL−1 hy-
drochlorothiazide. After filtration the solution was diluted with mobile phase
to give a final concentration of 32 µg mL−1 candesartan cilexetil and 25 µg mL−1
hydrochlorothiazide.

RESULTS AND DISCUSSION

Method Optimization
Conditions were optimized for simple, isocratic, accurate, and sensiti-
ve simultaneous HPLC determination of hydrochlorothiazide and candesartan
cilexetil in tablet formulations. A large number of HPLC methods have been
developed for analysis of hydrochlorothiazide but very few for candesartan
cilexetil.

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 Method development was started with 80% methanol in water, but no
peaks were observed. The mobile phase was then adjusted by mixing po-
tassium dihydrogen phosphate (0.02 M) with methanol in the ratio 20:80. This
resulted in distorted signals that were not well defined. Addition of 0.2 mL
triethylamine and subsequent adjustment of the pH with phosphoric acid
resulted in good separation and symmetrical peaks. Addition of triethylamine
improves the separation by masking polar silanol groups on the stationary
phase, thus enabling analyte molecules to move through the column without
interference from the stationary phase.
The optimum mobile phase was, therefore, 0.02 M potassium dihydro-
gen phosphate–methanol–triethylamine 25:75:0.2 (v/v), final pH 6.0 ± 0.1. Un-
der these experimental conditions sharp peaks were obtained for hydrochloro-
thiazide and candesartan cilexetil at the retention times 2.8 and 4.9 min, res-
pectively, as shown in Fig. 3.

Fig. 3
Chromatogram obtained from hydrochlorothiazide and candesartan reference substances

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Method Validation
The method was validated for linearity, accuracy, precision, limits of
detection and quantification, and specificity.
Linearity
The linearity of the method was evaluated by analyzing five solutions
in the concentration ranges 5–45 µg mL−1 for hydrochlorothiazide and 12–52
µg mL−1 for candesartan cilexetil. The peak areas obtained from different
concentrations of the drugs were used to calculate linear regression equations.
These were Y = 1.36X − 0.085 and Y = 1.20X + 0.041, with correlation coeffi-
cients of 0.9993 and 0.9991 for hydrochlorothiazide and candesartan, respec-
tively The high values of the correlation coefficients were indicative of linear
relationships between analyte concentration and peak area.
Limits of Detection and Quantification
The limits of detection (LOD) and quantification (LOQ) were establi-
shed by evaluating the minimum level at which the analyte could be readily
detected and quantified with accuracy, respectively. The LOD was 0.08 and
0.13 µg mL−1 for hydrochlorothiazide and candesartan cilexetil, respectively
(SNR 3:1) and the LOQ was 0.19 and 0.22 µg mL−1 for hydrochlorothiazide and
candesartan cilexetil, respectively (SNR 10:1).
Accuracy
Method accuracy was checked by preparing synthetic mixtures contai-
ning different amounts of hydrochlorothiazide and candesartan cilexetil and
analyzing the mixtures by use of the method. Percentage recovery, relative
standard deviation, and relative percentage error were then calculated. The re-
sults obtained (Table I) indicate that recoveries were excellent, not less than
99% and that relative standard deviations and relative percentage error were
less than 2%.
Precision
Intra-day precision was calculated from results obtained from fivefold
replicate analysis of samples at three different concentrations on the same
day. Inter-day precision was calculated from results from the same samples
analyzed on five consecutive days. The results obtained are listed in Table II.

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Table I
Accuracy of the method

Concentration Amount recovered Recovery RSD

Drug RPEa
(µg mL−1) (µg mL−1) (%) (%)
20.0 19.90 99.50 1.59 0.50
25.0 25.18 100.72 1.98 0.72
Hydrochlorothiazide
30.0 30.21 100.70 1.85 0.70
35.0 34.51 98.60 1.86 1.40
25.6 25.86 101.02 1.30 1.02
32.0 31.82 99.44 0.56 0.56
Candesartan cilexetil
38.4 38.77 100.96 0.38 0.96
44.8 45.23 100.96 1.40 0.96
a
Relative percentage error

Table II
Intra-day and inter-day precision of the method

Concn Intra-day precision Inter-day precision

Compound n
(µg mL−1) Mean RSD (%) Mean RSD (%)
6.25 5 6.24 1.60 6.26 2.08
Hydrochlorothiazide 12.5 5 12.46 1.04 12.49 1.60
25.0 5 25.16 0.91 25.21 0.71
8.0 5 8.03 1.00 7.98 1.50
Candesartan cilexetil 16.0 5 16.12 1.12 16.09 1.37
32.0 5 31.92 0.47 32.08 0.97

Specificity
The specificity of the method was tested by calculating the percentage
recovery of each component in the presence of the other component and in the
presence of possible interfering materials such as starch, lactose, magnesium
stearate, and avicel. The results are presented in Table III, which shows sepa-
ration of analytes from the excipients was complete.

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Table III
Specificity of the method

Hydrochlorothiazidea Candesartan cilexetila

Added Recovered Recovery Added Recovered Recovery
(µg mL−1) (µg mL−1) (%) (µg mL−1) (µg mL−1) (%)
25.00 25.28 101.12 32.00 32.32 101.0
25.00 25.56 102.24 32.00 32.61 101.91
25.00 25.44 101.76 32.00 31.75 99.22
25.00 25.12 100.48 32.00 32.13 100.41
Mean 101.4 100.63
RSD 0.76% 1.12%
a
Each sample contained 60 µg mL−1 each of starch, lactose, magnesium stearate, and avicel

Stability
The stability of candesartan cilexetil and hydrochlorothiazide in solution
was checked by determining the percentage deviation of the amounts present
in solution after 72 h at room temperature in comparison with the amount at
zero time. The results obtained after 72 h showed no significant variation; the
percentage deviation was less than 2% of the initial amount. This is indicative
of good stability of each component in the mixture over a period of 72 h.
Application of the Method to Tablets
The method was used for determination of hydrochlorothiazide and can-
desartan cilexetil in tablet formulations. The results obtained (Table IV) sho-
wed percentage recoveries were high and RSD (%) values were low, which
confirms the method is suitable for routine determination of these components
in their pharmaceutical preparations. Figure 4 shows a typical chromatogram
obtained from analysis of a tablet formulation.

CONCLUSION
A simple and accurate reversed-phase HPLC method has been establi-
shed for simultaneous determination of candesartan cilexetil and hydrochloro-
thiazide. The method was validated by testing its linearity, accuracy, preci-

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Table IV
Results from analysis of hydrochlorothiazide and candesartan cilexetil in tablets

Hydrochlorothiazide Candesartan cilexetil

Added Recovered Recovery Added Recovered Recovery
(µg mL−1) (µg mL−1) (%) (µg mL−1) (µg mL−1) (%)
25.00 25.45 101.80 32.00 32.18 100.56
25.00 25.52 102.08 32.00 32.56 101.75
25.00 25.13 101.52 32.00 32.48 101.50
Mean 101.47 101.27
RSD 0.82% 0.62%

Fig. 4
Chromatogram obtained from hydrochlorothiazide and candesartan cilexetil in tablets

sion, limits of detection and quantitation, and specificity. The run time of less
than five minutes enables its application for routine analysis of candesartan
cilexetil and hydrochlorothiazide in pharmaceutical formulations.

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