Development and Validation

of a Stability-Indicating LC Method
to Quantify Hydrochlorothiazide
in Oral Suspension for Pediatric Use
2008, 67, 647–652

Monika Piazzon Tagliari&, Hellen Karine Stulzer, Fabio Seigi Murakami, Gislaine Kuminek,
Bruno Valente, Paulo Renato Oliveira, Marcos Antonio Segatto Silva
Centro de Ciências da Saúde, Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina (UFSC),
Campus Universitário Trindade, Bloco K, sala 207, Florianópolis, SC 88040-900, Brazil; E-Mail: monikatag@yahoo.com.br

Received: 8 October 2007 / Revised: 18 December 2007 / Accepted: 9 January 2008

Online publication: 9 February 2008

Most antihypertensive drugs are

administered orally and are only avail-
Abstract able in solid oral dosage forms, therefore
there is a potential need for pediatric
A stability-indicating reversed-phase high-performance liquid chromatography (LC) method extemporaneous solutions [4].
was developed and validated for the determination of hydrochlorothiazide in an oral Primary hypertension in children is
suspension. Isocratic chromatography was performed on a C18 column with 0.1 M sodium related to several metabolic abnormali-
phosphate buffer pH 3.0/acetonitrile (70:30 v/v) as mobile phase, at a flow rate of ties that are associated to overweight,
1.3 mL min 1, and UV detection at 254 nm. The method was linear (r2 = 0.9998), accurate metabolic syndrome, and type 2 diabetes
(mean recovery = 100.3%), and precise (RSD <2%). It was also validated for specificity and [5].
robustness. The method was successfully applied for the quality control analysis of a new Hydrochlorothiazide (HCTZ), chemi-
pharmaceutical formulation of HCTZ for pediatric use. cally known as 6-chloro-3,4-dihydro-2H-
1,2,4-benzothiadiazine-7-sulfonamide-1,
1-dioxide, is a white and odorless crys-
talline powder, slightly soluble in water
Keywords and sparingly soluble in methanol [6]. It
is a thiazide diuretic and antihyperten-
Column liquid chromatography
sive drug used in the treatment of oede-
Stability-indicating
ma associated with congestive hearth
Validation
failure and renal and hepatic disorders
Pediatric oral suspension
Hydrochlorothiazide [7]. Official monographs on HCTZ raw
material and tablets are included in the
European Pharmacopoeia [8] and US
Pharmacopoeia 30 [9]. Analytical meth-
ods have been described for the quanti-
Introduction opened, and its content is administered tative determination of HCTZ in solid
with a palatable drink or mixed with pharmaceutical dosage forms applying
Oral administration of drugs is a fre- solid food [2]. Besides the possibility of electrochemical or spectrophotometric
quently used way of giving medicines to interaction of the active pharmaceutical methods [10] and in association with
children. However, depending on their ingredient with the drink or food, the other active pharmaceutical ingredients,
age, many children are unable to swal- poor reproducibility of the tablet including spectrophotometric [11], LC
low whole tablets or capsules [1]. When breaking could compromise the correct [10, 12], thin layer chromatography [13],
these forms are prescribed, usually the dose administration and the efficiency of and capillary electrophoretic and elec-
tablet is crushed, or the capsule is the treatment [3]. trochromatographic methods [14].

Full Short Communication Chromatographia 2008, 67, April (No. 7/8) 647
DOI: 10.1365/s10337-008-0546-1
0009-5893/08/04
2008 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH
 Although stability data for extempo-
raneous suspensions of some antihyper-
tensive agents like lisinopril, captopril
and quinopril have been published,
[2, 4, 15] such suspensions have inherent
problems, including limited stability,
questionable uniformity, and unknown
bioavailability [16]. There is no com-
mercially available formulation of HCTZ
oral suspension, because of its instability
in aqueous solution and therefore the
development of a liquid formulation is a
significant challenge [17].
A new pharmaceutical formulation
of HCTZ suspension has been developed
and to assure its quality for further
studies in vivo (protocol already
approved CEPSH-UFSC: 259/06), a
stability-indicating analytical method
should be developed and validated. At
the moment, there is no published
method validated for the quantitative
analysis of HCTZ in suspension, and
even with methods published for its
determinations in solid pharmaceutical
formulations, the applicability of the
methodologies to pharmaceutical sus-
pensions must be evaluated demonstrat-
ing the validation parameters, including
the specificity of the methods towards
potential degradation products and for-
mulation excipients. Liquid chromatog-
raphy with UV detection has been used
for quality control of most of the phar-
maceuticals due to its simplicity, high
resolution and significant precision and
accuracy. The aim of the present work
was to develop and validate a stability-
indicating LC method for the determi-
nation of HCTZ in a pharmaceutical
oral suspension.
Experimental
Instruments and Analytical
Conditions
The LC analysis was performed on a
Shimadzu LC-10A system (Kyoto,
Japan) equipped with an LC-10AD
pump, SPD-10AVVP UV detector,
SPD-M10AVP photodiode array (PDA)
detector, SCL-10AVP system controller,
DGU-14A degasser, CTO-10ASVP col-
umn oven, and the sample injection was
performed via a Rheodyne 7125 valve
648
with a 20 lL loop. The detector was set
at 254 nm and peak areas were inte-
grated automatically by computer using
a Shimadzu Class VP V 6.14 software
program. The experiments were carried
out on a reversed-phase Phenomenex
(Torrance, USA) Luna C18 column
(250 mm · 4.6 mm I.D., with a particle
size of 5 lm and pore size of 100 Å),
maintained at 40 ± 1 C. A security
guard holder (4.0 mm · 3.0 mm I.D.)
was used to protect the analytical col-
umn. The mobile phase consisted of
0.1 M sodium phosphate buffer pH 3.0
and acetonitrile (70:30 v/v), and was
eluted isocratically at a flow rate of
1.3 mL min 1.
Chemicals
The HCTZ reference standard
(99H1207) with stated purity of 100.1%
and chlorothiazide (CTZ, 52K1441)
were obtained from Sigma–Aldrich
(Steinheim, Germany). The HCTZ raw
material was donated by LAFESC
(Laboratório Industrial Farmacêutico de
Santa Catarina, Florianópolis, Brazil).
Ultra-pure water was provided by a
Milli-Q purification system (Millipore,
Bedford, USA). Methanol and acetoni-
trile of LC grade were purchased from
Vetec (Rio de Janeiro, Brazil). All
chemicals used were of pharmaceutical
or special analytical grade.
Production of HCTZ
Suspension
HCTZ raw material was crushed in a
Mortar with a pestle to get smaller par-
ticle sizes. As part of this process, the
particles were individually wetted with
glycerin, and a polymeric solution of
carboxymethylcellulose sodium (0.6%)
containing sodium benzoate (0.1%) as a
preservative was added. The final for-
mulation was kept in stability pH 3.2.
Preparation of Sample
Solutions
Before the measurements, each suspen-
sion vial was stirred on a magnetic stir
Chromatographia 2008, 67, April (No. 7/8)
plate during 60 s to assure homogeneity
at the time to take the aliquot. An ali-
quot of 0.3 mL were pipetted from the
suspension and quantitatively trans-
ferred in to a 25 mL volumetric flask
containing 2 mL of methanol, and stir-
red in an ultrasonic bath for 10 min. The
volume was completed with the mobile
phase (30 lg mL 1), and the resulted
solution was filtered through a 0.45 mm
nylon membrane.
Preparation of Standard
Solutions
A stock standard solution of 1,000 lg
mL 1 was prepared by dissolving 25 mg
of the HCTZ reference standard in 2 mL
of acetonitrile and 10 mL of mobile
phase in a 25 mL volumetric flask, and
stirred in an ultrasonic bath for 10 min.
The volume was completed with the
same mobile phase. A stock solution of
CTZ at 1,000 lg mL 1 was also pre-
pared in the mobile phase.
Method Validation
Analytical method development and
validation play a major role in the dis-
covery, development, and manufacture
of pharmaceuticals [18]. The Interna-
tional Conference on Harmonization
(ICH) [19] requires the stress testing to
be carried out to elucidate the inherent
stability characteristics of the active
substance. An ideal stability-indicating
method quantifies the drug per se and
also resolves its degradation products
[20].
The method was validated to quan-
tify the HTCZ in a pediatric suspension
by the determination of the following
parameters according to the ICH [19]:
specificity, linearity, accuracy, precision,
robustness, quantification and detection
limits.
System Suitability
The system suitability was carried out to
evaluate the resolution and reproduci-
bility of the system for the analysis to
be performed, using six replicate ana-
lyses of the drug at a concentration of
Full Short Communication
30 lg mL 1, evaluating the following
parameters: peak area, retention time,
asymmetry and theoretical plates.
Specificity
In order to determine the specificity of
the method, the absence of interference
was checked by the excipients which take
part in the pharmaceutical preparation
(placebo solution). Moreover, the speci-
ficity was determined according to ICH
[19] by subjecting a sample solution
(1,000 lg mL 1) to accelerated degra-
dation by acid, alkaline, neutral, oxida-
tive, photolytic, and thermal stress
conditions to evaluate the interference of
degradation products in the quantitation
of HCTZ. Acid, alkaline and neutral
degradation of the drug were carried out
with sample solutions in 0.1 N HCl,
0.1 N NaOH, and purified water,
refluxed at 60 C for 2 h. The oxidative
degradation was induced by storing the
sample in 3% hydrogen peroxide, at
ambient temperature for 24 h, protected
from light. Photolytic studies were per-
formed after exposition of HCTZ in
solid state and diluted in purified water,
0.1 N HCl and 0.1 N NaOH, in a
photostability chamber for 72 h. To
investigate the stability of the drug under
thermal stress conditions, bulk drug was
spread in a thin layer on a petri plate and
subjected to dry heat at 80 C in an
oven. The samples were analyzed after
72 h. After the procedures, the samples
were diluted in mobile phase to a final
concentration of 30 lg mL 1.
The formation of CTZ as major
degradation product in stress conditions
was verified by spiking with its standard.
The suspicion that CTZ could be formed
during degradation of HCTZ was based
on information in the literature that
CTZ can be present as a synthetic con-
taminant or a degradation product in the
HCTZ [21].
Linearity, and Limits of Detection (LOD)
and Quantitation (LOQ)
Linearity was determined by construct-
ing three calibration curves on three
different days. For the construction of
each calibration curve five standard
concentrations of HCTZ (10, 20, 30, 40
Full Short Communication
Fig. 1. a Representative chromatogram of HCTZ pharmaceutical suspension (30 lg mL 1);
b placebo suspension
and 50 lg mL 1) were prepared in
mobile phase. Three replicates of 20 lL
injections were made for the standard
solution to verify the repeatability of the
detector response at each concentration.
The peak areas of the chromatograms
were plotted against the concentrations
of HCTZ to obtain the calibration curve.
The five concentrations of the standard
solutions were subjected to regression
analysis by the least squares method to
calculate calibration equation and
correlation coefficient.
The LOD and LOQ were calculated
from the slope and the standard devia-
tion of the intercept of the mean of three
calibration curves. The LOQ and the
LOD were also confirmed in an experi-
mental assay.
Accuracy
The accuracy of the method was evalu-
ated by a recovery test. HCTZ samples
(10 lg mL 1) were fortified with three
known concentrations of reference stan-
dard: 5, 20 and 35 lg mL 1 to obtain
solutions of 15, 30 and 45 lg mL 1,
respectively. The recovery of the added
standard was determined in triplicate
analysis and calculated as the percentage
of the drug recovered from the formu-
lation.
Precision
The precision assay was determined by
repeatability (intra-day) and intermedi-
Chromatographia 2008, 67, April (No. 7/8)
ate precision (inter-day). The repeat-
ability was evaluated by assaying six
samples containing 30 lg mL 1 of
HCTZ, on the same day, under the same
experimental conditions. The intermedi-
ate precision was assessed by carrying
out the analysis on three different days.
The peak areas were determined and
compared. The precision was expressed
as percentage of relative standard devi-
ation (RSD %).
Robustness
The robustness of the developed method
was determined by analyzing the same
samples (30 lg mL 1) under a variety of
conditions of the method parameters,
such as: flow rate, column temperature,
and variations in the mobile phase pH
and composition.
To evaluate the stability of HCTZ in
mobile phase, a sample solution was
prepared, maintained at ambient tem-
perature, and analyzed after 48 h.
Analysis of HCTZ
in Pharmaceutical Suspension
For quantitation of HCTZ in the dosage
form, six batches of suspension contain-
ing 2.50 mg mL 1 of HCTZ were ana-
lyzed as described in preparation of
sample solution. An aliquot of 20 lL was
injected for the analysis and the amount
of drug in the suspension calculated
against the respective reference standard.
649

Fig. 2. Chromatograms obtained under stress studies. a HCTZ standard (30 lg mL 1); b CTZ
standard (10 lg mL 1); c after acid hydrolysis; d after alkaline hydrolysis; e after neutral
hydrolysis; f after oxidative degradation
Results and Discussion
Method Development
To obtain the best chromatographic
conditions, the mobile phase was opti-
mized to provide sufficient selectivity
650
and sensitivity in a short separation time.
The best peak symmetry was achieved
with a flow rate of 1.3 mL min 1 at
40 C column temperature. The acid pH
of mobile phase is to ensure HCTZ sta-
bility during the analysis [22]. In opti-
mized chromatographic conditions the
Chromatographia 2008, 67, April (No. 7/8)
HCTZ retention time was about 3.5 min,
and a typical chromatogram obtained by
the proposed method is shown in Fig. 1a.
The system suitability results showed
that the parameters are within the suit-
able range. The mean asymmetry found
was 1.17 with RSD (%) of 0.38. The
mean theoretical plates, retention time
and peak area ± RSD (%) found were
9,749 ± 1.11, 3.559 ± 0.08, and
504,966 ± 0.57, respectively.
Method Validation
Specificity
The specificity of the method was eval-
uated by analyzing a sample of suspen-
sion without HCTZ (placebo), and the
chromatograms showed that there is no
interference or overlap of the excipients
with the HCTZ peak (Fig. 1). Addi-
tionally, specificity was confirmed
through the stress studies. These studies
were performed to validate stability
indicating capability of the developed
method and to identify the key factors
which will impact the stability of the
drug product. After acid, alkaline and
neutral hydrolysis the HCTZ content
decreased and an additional peak was
observed at 3.2 min (Fig. 2c, d, e,
respectively). The identification of the
additional peak was performed through
the injection of a CTZ standard solution
(10 lg mL 1), that resulted in a peak
with the same retention time, thus con-
firming its identity (Fig. 2b). The degra-
dation of HCTZ in acid and alkaline
conditions was found to be about 17 and
18%, respectively. The neutral condition
showed more significant degradation
than other conditions, with 25% of drug
degraded after refluxing for 2 h (Fig. 2e).
Under oxidative conditions, the HCTZ
remained 91%, and two degradations
products were detected, CTZ and an-
other that has not been identified
(Fig. 2f). From the results of the pho-
tolytic studies, it was observed that the
drug in the solid state was comparatively
more photostable than in solution, with
only 0.6% of degradation. In acid,
alkaline and neutral solution 5, 8 and
6% of degradation were observed after
72 h to light exposure. Under the
Full Short Communication
thermal stress conditions there was no Table 1. Results from determination of the intra- and inter-day precision, and the accuracy of
change in the area and no additional the method
peak was detected after 72 h. % Recovered RSD (%)
The studies with the PDA detector
showed that the HCTZ peaks were free Intra-day precision
from any coeluting peak, with values of Day 1 (n = 6) 99.6 0.44
Day 2 (n = 6) 99.3 0.51
a peak purity index higher than 0.9999, Day 3 (n = 6) 98.4 0.80
thus demonstrating that the proposed Inter-day precision (n = 18) 99.4 0.98
method is specific. Accuracy (n = 3)
Fortified solution Mean concentration Recovery (%)
(lg mL 1) found (lg mL 1) (% RSD)
Linearity, and Limits of Detection (LOD) 15 14.93 98.6 (0.3)
and Quantitation (LOQ) 30 30.34 101.7 (0.6)
45 45.21 100.6 (0.5)
The linearity of detector response was
assessed for various solution standards RSD Relative standard deviation
over the range of 10–50 lg mL 1. The
value of the determination coefficient
Table 2. Chromatographic conditions and range investigated during robustness testing
calculated (r2 = 0.9998, y = 15895.67
x 69.87; where, x is concentration and Variable Range investigated Hydrochlorothiazidea %
y is the peak absolute area) indicated the
linearity of the calibration curve for the Flow rate (mL min 1) 1.2 101.80
1.3 100.12
method. The validity of the assay was 1.4 99.22
verified by analysis of variance, ANOVA Column temperature (C) 35 100.70
(Fcalculated = 8568.5 > Fcritical = 0.0001; 40 100.12
45 100.69
P = 5%).
Percent acetonitrile 25 99.89
The LOQ and LOD calculated were 30 100.12
1.23 and 0.40 lg mL 1, respectively, 35 100.90
which were confirmed experimentally, Mobile phase pH 2.7 100.80
3.0 100.12
indicating adequate sensitivity of the 3.3 100.30
method. Solution stability (48 h) – 99.80

a
Mean of three replicates
Accuracy and Precision

The accuracy was assessed from three
replicate determinations of three differ-
ent fortified solutions. No significant
differences were observed between
amounts of HCTZ added and the
amounts found (P < 0.05). The ob-
tained values were within the range of
98.0–99.0% (Table 2), satisfying the
acceptance criteria for the study.
The intra-day and inter-day precision
RSD (%) are given in Table 1. The
amounts of HCTZ found on the three
different days were equivalent
(P < 0.05), and the relative standard
deviation values were within the accep-
tance criteria of 2%.
Robustness
The results and the experimental range
of the variables evaluated in the robust-
ness assessment are given in Table 2. The
analysis performed resulted in changes in
the retention time without effect on the
determination of the drug in the phar-
maceutical formulations. No significant
changes were observed in the content of
HCTZ during solution stability experi-
ment, which confirms that the sample
solutions were stable for at least 48 h.
Analysis of HCTZ
in Pharmaceutical Suspension
The results obtained from the validation
studies proved that the method is suitable
to quantify HCTZ in the oral suspension.
The quantitative assay of six batches of
HCTZ in suspensions for pediatric use
was within 100.91–102.10%.
Conclusion
The proposed high-performance liquid
chromatographic method was validated
towards specificity, linearity, accuracy,
precision, robustness, and was proved to
be capable of separating HCTZ and its
major degradation product, CTZ,
demonstrating its suitability for use as a
stability indicating method during sta-
bility studies. The short analytical run
time of less than 4.0 min leads to a cost-
effective and rapid chromatographic
procedure. Furthermore, the developed
method showed no interference of the
formulation excipients and was success-
fully applied for the quality control of
HCTZ in pharmaceutical suspensions
for pediatric use.
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