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Quim. Nova, Vol. 35, No. 1, 207-212, 2012
Nota Técnica
*e-mail: giskuminek@gmail.com
DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING HPLC METHOD FOR THE
DETERMINATION OF BUCLIZINE HYDROCHLORIDE IN TABLETS AND ORAL SUSPENSION AND ITS
APPLICATION TO DISSOLUTION STUDIES
Gislaine Kuminek*, Hellen K. Stulzer, Monika P. Tagliari, Paulo R. Oliveira, Larissa S. Bernardi, Gabriela Rauber e
Simone G. Cardoso
Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, Campus Universitário Reitor João David
Ferreira Lima, 88040-900 Florianópolis - SC, Brasil
Recebido em 10/3/11; aceito em 24/5/11; publicado na web em 22/7/11
A method using liquid chromatography has been developed and validated for determination of buclizine in pharmaceutical
formulations and in release studies. Isocratic chromatography was performed on a C18 column with methanol:water (80:20 v/v, pH
2.6) as mobile phase, at a flow rate of 1.0 mL/min, and UV detection at 230 nm. The method was linear, accurate, precise, sensible
and robust. The dissolution test was optimized and validated in terms of dissolution medium, apparatus agitation and rotation speed.
The presented analytical and dissolution procedures can be conveniently adopted in the quality and stability control of buclizine in
tablets and oral suspension.
Keywords: buclizine; dissolution test; liquid chromatography.
INTRODUCTION
Buclizine Hydrochloride (BCZ), (RS)-1-(4-tert-butylbenzyl)-4-
(4-chlorobenzhydryl)piperazine dihydrochloride (Figure 1),1 is a pi-
perazine derivative having antihistaminic, antimuscarinic, antiemetic
and moderate sedative properties. BCZ, as its analogue meclizine, is
mainly used in the prevention of motion sickness, nausea, vomiting,
with analgesics in the treatment of migraine and as an appetite stimu-
lant due to its orexigenic effect. This drug has also been used in the
treatment of vertigo associated with disorders of the vestibular system,
although its value in these conditions remains to be established.1-3
Analytical methods have been published for the analysis of BCZ
in tablet and human serum by LC4,5 and by spectrophotometry.6
However, these methods lack stability indicating nature. Stability
indicating methods can detect changes with time of drug substances
and drug products. Information of degradation products over time is
important for safety of drugs.7 Moreover, the published methods were
not applied for oral suspension or for in vitro dissolution studies. This
is important because the quality of oral solid pharmaceutical dosage
forms depends on their ability to release the active ingredients in
aqueous media in a consistent and reproducible manner, making the
active substances available for gastrointestinal absorption. Therefore,
the performance of oral solid dosage forms should be verified prefe-
rably with an in vitro dissolution test before the product is released
to the market.8 The dissolution test has emerged as a valuable quality
control tool to assess batch-to-batch product release performance and
detection of manufacturing deviations. Besides, it may be relevant to
the prediction of in vivo performance of the drug. In addition, dissolu-
tion is a requirement for regulatory approval for product marketing.9-12
At the moment, no dissolution test has been described in literature
for BCZ. The present work has two main objectives. The first is to
develop and validate a stability-indicating method for determination
of BCZ in tablets and oral suspension. The second objective is to
apply the assay to the optimization and validation of dissolution test.
EXPERIMENTAL
Reagents and chemicals
BCZ standard was kindly donated by Medley (Campinas, SP,
Brazil). Tablets and oral suspension where purchased at the local
market. The tablets (Profol®) labeled to contain 25 mg of the BCZ,
20 mg of tryptophan, 20 mg of pyridoxine hydrochloride, 50 µg of
cyanocobalamin and the excipients: talc, dibasic calcium phosphate
dehydrate, povidone, magnesium stearate and starch. The oral sus-
pension (Profol®) labeled to contain 1 mg/mL of BCZ, 2 mg/mL of
tryptophan, 2 mg/mL of pyridoxine hydrochloride and 5 µg/mL of
cyanocobalamin and the excipients: hydrochloric acid, sodium cy-
clamate, methylparaben, propylparaben, polysorbate 80, propylene
glycol, simethicone, sorbitol, sodium tartatre, cysteine hydrochloride
monohydrate and deionized water. Sodium lauryl sulfate (SLS) was
from Vetec (Rio de Janeiro, RJ, Brazil). HPLC-grade methanol was
obtained from Tedia (Fairfield, OH, USA). Phosphate and acetate buf-
fer solutions were prepared according to USP.13 For all the analyses,
ultrapure water was obtained from Milli-Q® apparatus (Millipore,
Bedford, MA, USA). All chemicals used were of pharmaceutical or
special analytical grade.
HPLC method
Chromatographic analyses were performed with a Shimadzu
LC 10A vp system (Shimadzu, Kyoto, Japan). The UV detector
was set at 230 nm and peak areas were integrated automatically by
computer using a Shimadzu Class VP V 6.14 software program. The
Cl
N
N
C(CH3)3
Figure 1. Chemical structure of buclizine
Kuminek et al.
208 Quim. Nova
experiments were carried out on a reversed-phase Waters Corpora-
tion (Ireland) X-Bridge C18 column (250 mm x 4.6 mm I.D., 5 µm),
maintained at 25 ± 1 ºC. The mobile phase consisted of a mixture of
methanol and water (80:20, v/v) at pH 2.6 adjusted with phosphoric
acid, was eluted isocratically at a 1.0 mL/min flow rate. The injection
volume was 20 µL and the retention time was about 6 min.
Preparation of standard solution
A standard stock solution containing 1 mg/mL of BCZ chemical
reference standard was prepared in mobile phase. This solution was
kept refrigerated and protected from light. Working solutions were
prepared by diluting the stock solution as appropriate in mobile phase
or dissolution medium depending on the analysis.
Preparation of sample solutions
Tablets
Twenty tablets were weighted and finely powdered. An amount
of tablet equivalent to 50 mg of BCZ was accurately weighted,
transferred into a 50 mL volumetric flask and 30 mL of the mobile
phase was added. The solution was subjected to sonication for 45
min, brought to volume with mobile phase, and filtered through a
0.45 µm nylon membrane. Finally, 700 µL of the filtered solution
was directly transferred to a 50 mL volumetric flask and diluted
to volume with mobile phase obtaining the final concentration of
14 µg/mL.
Oral suspension
The suspension vial was stirred on a magnetic stir plate during
2 min to assure the homogeneity at the time to take the aliquot. An
aliquot of 700 µL of the suspension was quantitatively transferred
to a 50 mL volumetric flask containing 30 mL of mobile phase, and
stirred in an ultrasonic bath for 45 min. The volume was completed
with the mobile phase to obtain the final concentration of 14 µg/
mL, and the resulted solution was filtered through a 0.45 µm nylon
membrane before use.
Validation of the HPLC method
Specificity
In order to determine the specificity of the method, the absence
of interference was checked by the placebo solution (tablets and
oral suspension only with excipients and other combined drugs).
Moreover, the specificity was determined according to ICH14 by
the accelerated degradation. The stability-indicating capability of
the method was determined by subjecting a standard stock solution
(1000 µg/mL) to accelerated degradation by acidic, basic, oxidative,
and photolytic conditions. It was taken 2 mL from this solution and
transfered to 10 mL volumetric flask and brought to volume with
2 M hydrochloric acid, 2 M sodium hydroxide and 30% hydrogen
peroxide to achieve a concentration of 200 µg/mL. The studies in
acid conditions were carried out under reflux at 80 °C for 4 h, and
the solution was cooled at room temperature and neutralized with
base. The alkaline degradation was induced by storing the samples
solutions under reflux at 80 °C for 4 h, cooled at room temperature
and neutralized with acid. Oxidative degradation was obtained by
treating the drug with 30% hydrogen peroxide, maintaining it pro-
tected from light at ambient temperature for 48 h. Photodegradation
was achieved by exposing the standard solution in a photostability
chamber to 200 W h/m2 of near ultraviolet light for 48 h. After the
described treatments, all the samples were diluted to 14 µg/mL with
mobile phase, and analyzed.
Linearity
Linearity was determined by constructing three independent
calibration curves. For the construction of each calibration curve
5 standard concentrations of BCZ in the range of 0.5-24 µg/mL
were prepared using stock solution and mobile phase. The solutions
were injected in triplicate and the linearity was estimated by linear
regression.
Limits of quantitation and detection
The quantification limit (LOQ) and detection limit (LOD) were
based on the standard deviation of the response and the slope of the
mean of three calibration curves, as described in ICH.14
Accuracy
Accuracy of the LC method was evaluated by the recovery test of
known amounts of the standard added to a sample solution (containing
the excipients and 7 µg/mL of BCZ) to obtain solutions with final
concentrations corresponding to 80, 100 and 120% of the nominal
analytical concentration. The accuracy was calculated as the percent-
age of drug recovered from the formulation matrix.
Precision
The precision assay was investigated with respect to repeatability
(intra-day) and intermediate precision (inter-day). The repeatability
was evaluated by assaying 6 tablets and oral suspension samples
(14 µg/mL), during the same day and under the same experimental
conditions. The intermediate precision was assessed by carrying out
the same analysis on 3 different days. The precisions were expressed
as % RSD.
Robustness
The robustness of the developed method was examined against
small and deliberate variations of critical parameters such as pH,
composition of the mobile phase, flow rate and column temperature.
System suitability
The system suitability was evaluated by six replicate analyses
of a BCZ solution at a concentration of 14 µg/mL. The calculated
parameters were: peak area %, retention time, number of theoretical
plates and tailing factor.
Dissolution testing
Dissolution study
The development and validation of the dissolution test was
performed using a Varian dissolution test system, model VK 7000
multi-bath (n = 8) in accordance to USP Pharmacopoeia13 general
method. All the dissolution samples were analyzed by LC assay as
described above.
Solubility determination and sink conditions
The sink conditions were determined in different media. 0.01, 0.1
M HCl, aqueous solution with 0.5% of sodium lauryl sulfate (SLS),
aqueous solution with 1.0% of SLS, aqueous solution with 1.5% of
SLS, phosphate buffer pH 6.8 and acetate buffer pH 4.1 were tested.
Vessels (n = 3) containing 240 mL of medium were pre-heated to 37
± 0.5 °C before adding an excess of BCZ (20 mg). The samples were
gently rotated. Aliquots (5 mL) were withdrawn from each vessel
after 1 and 2 h, filtered, neutralized, and analyzed.
Dissolution test conditions
The influence of different apparatus (USP basket and paddle) and
different rotation speeds were evaluated. The medium volume used
Development and validation of a stability-indicating HPLC method 209
Vol. 35, No. 1
was 900 mL pre-heated to 37 ± 0.5 °C. The dissolution medium pH
was monitored before and after the performance of the tests. Sample
aliquots of 10 mL were withdrawn at 5, 10, 15, 20, 30, 60, 90 and 120
min and replace with an equal volume of fresh medium to maintain
a constant total volume. After the end of each test time, samples
aliquots were filtered, diluted in methanol (1:1, v/v) and quantified.
The assay of the tablets and oral suspension was performed using the
validated LC method and the contents results were used to calculate
the percentage of drug released on each time of dissolution profile.
The cumulative percentage of drug released was plotted against time,
in order to obtain the release profile and to calculate the in vitro disso-
lution data. BCZ stability in dissolution medium was evaluated using
the same test conditions. Aliquots of the samples were tested after
2 h. The responses for the solutions were evaluated comparing with
a freshly prepared standard. The assay was performed in triplicate.
The filtration of BCZ standard and samples (tablets and oral suspen-
sion dissolved in dissolution medium) was evaluated using 0.45 µm
cellulose acetate membrane filter (Vertical, Thailand), quantitative
filter (J. Prolab, Brazil) and cellulose membrane filter together with
quantitative filter. Unfiltered (centrifuged) solution was not evaluated
because it could damage the column.
Validation of the dissolution method
The developed dissolution test was validated for specificity,
linearity, accuracy, and precision according to ICH guidelines and
US Pharmacopoeia.13,14
Specificity
Specificity was evaluated by preparing samples of placebo which
consisted of all excipients present in formulations. Their concentra-
tions were determined based in Handbook of Pharmaceutical Exci-
pients15 and calculated for medium weight content. The samples of
placebo were transferred to separate vessels (n = 3), filled with 900
mL of dissolution medium at 37 ± 0.5 °C and stirred for 120 min
at 100 rpm using basket. Samples aliquots were filtered, diluted in
methanol (1:1, v/v) and analyzed.
Linearity
The nominal concentration of BCZ after the dissolution experi-
ments – assuming quantitative dissolution – is 27.8 µg/mL (25 mg
BCZ per tablet or 25 mL of a suspension (1 mg/mL) in 900 mL of
dissolution medium). Before the injection, these solutions were
diluted in methanol (1:1, v/v), to obtain good peak resolution, to a
final concentration of 13.9 µg/mL. In order to bracket effectively the
above mentioned concentration, linearity was validated in the range
of 0.5-24 µg/mL, in dissolution medium, corresponding to approxi-
mately 4-180% of the target value.
Accuracy
The accuracy of the dissolution method was evaluated by the
recovery of known amounts of BCZ added to placebo. Aliquots of
5, 25 and 30 mL of a 1 mg/mL BCZ standard solution dissolved in
mobile phase was added to vessels containing dissolution medium
for a final volume of 900 mL (final concentrations were 5.5, 27.8
and 33.3 µg/mL, respectively), and subjected to the dissolution test
conditions described above. Placebo samples were prepared in the
same way previously described in the specificity test.
Precision
The precision was determined by measuring the repeatability
(intra-day) and the intermediate precision (inter-day), both expressed
as % RSD. Tablets and oral suspension were subjected to dissolution
test conditions (900 mL of dissolution medium pre-heated at 37 °C ±
0.5, basket with stirring rate of 100 rpm for tablets and paddle with
stirring rate of 25 rpm for oral suspension, both for 120 min) in the
same day and in three different days.
RESULTS AND DISCUSSION
HPLC method
To obtain the best chromatographic condition, the mobile phase
was optimized to provide adequate peak symmetry and sensitivity.
Methanol, acetonitrile and water in different proportions were tested.
The method proposed by Arayne et al. was also considered. The
optimal results were achieved with a mobile phase of methanol in
combination with water (80:20, v/v) pH adjusted to 2.6 with phospho-
ric acid, at a flow rate of 1 mL/min and 25 °C of column temperature.
In optimized chromatographic conditions the BCZ retention time was
about 5.8 min, and a typical chromatogram obtained by the proposed
method is shown in Figure 2a.
Figure 2. Chromatograms obtained under stress studies. (A) BCZ standard
(14 μg/mL); (B) after photolytic degradation (UV 254nm); (C) after acid
hydrolysis (2M HCl); (D) after oxidative (30% H2O2) degradation
Kuminek et al.
210 Quim. Nova
Dissolution testing
Solubility data were used as the basis for the selection of a
dissolution medium for BCZ. Drug solubility was determined in
different media, using an amount of the drug equivalent a three
times of the dose in the pharmaceutical formulations. According to
Table 3, aqueous solution with 1.5% of SLS was the best medium
for the BCZ dissolution test and also ensured sink conditions. In
this medium, BCZ was stable for 2 h (variation less than 2%). The
filter evaluation is necessary to determine if it could be used in the
dissolution test without adsorption of the drug. Besides, it removes
insoluble excipients that may otherwise cause high background or
turbity.16 The quantitative and 0.45 µm cellulose acetate filters sho-
wed recoveries between 98-102%. Nevertheless, due to low cost of
quantitative filter when compared to 0.45 µm cellulose acetate filter,
the first one was chosen.
Dissolution profile of tablets
The selection of the dissolution medium was based on the so-
lubility data. Figure 3 shows the dissolution profiles of BCZ tablets
obtained in 900 mL of aqueous solution with 1.5% of SLS medium
in rotation speeds of 50, 75 and 100 rpm using basket and 50 and
75 rpm using paddle (n = 6). It was observed that more than 80% of
drug was dissolved at 120 min in basket 75 and 100 rpm. In the other
conditions the % drug dissolved was < 75 %. The analysis of variance
of the dissolution efficiency (DE%) showed significant difference (p
< 0.05) between the results obtained at 75 and 100 rpm (67.2 and
75.8%, respectively). It was observed that drug release percent and
the DE were higher at 100 rpm. Basket apparatus is recommended,
as a general rule, for dissolution test of capsules. However, by using
Table 1. Intra-day and inter-day precision data of the LC method fo r BCZ
Sample Intra-day Inter-day
RSDb
Day Recoverya (%) ± RSDb
Tablet 1 100.5 ± 0.30 0.75
2 99.3 ± 0.68
3 100.6 ± 0.64
Oral Suspension 1 101.5 ± 0.26 0.59
2 100.3 ± 0.42
3 101.0 ± 0.09
aMean of six replicates; brelative standard deviation
Table 2. Chromatographic conditions and range investigated during robustness testing for BCZ
Variable Range investigated Tablets assaya % ± RSDbOral suspension assaya % ± RSDb
Flow rate (mL/min) 0.8 100.2 ± 1.05 101.4 ± 1.20
1.0 100.3 ± 0.49 100.9 ± 0.65
1.2 98.0 ± 0.77 98.7 ± 0.56
Column temperature (ºC) 25 100.1 ± 0.34 100.2 ± 0.44
30 100.7 ± 0.22 100.8 ± 0.37
40 100.5 ± 0.12 100.9 ± 0.28
Mobile phase pH 2.4 98.1 ± 0.96 99.0 ± 0.98
2.6 99.7 ± 0.63 100.3 ± 0.39
2.8 97.8 ± 0.75 98.7 ± 0.47
aMean of three replicates; brelative standard deviation
The chromatograms obtained with specificity test (not shown)
showed that there is no interference or overlap of the excipients
with the BCZ peak. Furthermore, specificity was confirmed through
the stress study. 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. The
alkaline condition was excluded from the study, due to the fact that
BCZ is insoluble in this condition of pH and precipitates, which makes
impossible to quantify it. In this way, the stress study was performed
only with photolytic, acid and oxidative conditions. Under photolytic
conditions 91% of BCZ remained, and four well-separated degrada-
tions products were detected (Figure 2b). After acid hydrolysis the
BCZ content decreased and two additional peaks were observed at
5 and 9 min (Figure 2c). Peaks at approximately 3 min correspond
to HCl. The degradation of BCZ in acid conditions was found to be
about 27% after 4 h of study. From the results of the oxidative studies
(Figure 2d), it was observed only 0.7% of degradation without any
additional peak. Peak at 2.8 min is due to H2O2. All additional peaks
were resolved from BCZ peak.
The calibration curves constructed for BCZ were obtained by
plotting peak area ratio against the concentration of the drug. Good
linearity was observed in the 0.5 to 24 µg/mL range. The response
for the drug was linear and the calibration equation was y = 37657x
– 9081 (where, x is concentration and y is the peak absolute area),
which showed good correlation coefficient (r = 0.9995) and intercept
not different from 0 (p > 0.05). The detection and quantification
limits were 0.06 µg/mL and 0.21 µg/mL respectively, showing the
sensibility of the method.
Mean recovery of BCZ standard for low, medium and high level
were, respectively, 98.5, 98.6 and 99.8% for tablets and 100.5, 100.2
and 101.2% for oral suspension, satisfying the acceptance criteria
for the study.
A satisfactory intra-day and inter-day variability was obtained,
as can be observed in Table 1. The amounts of BCZ found on 3 di-
fferent days were equivalent (p > 0.05), and RSD values were found
to be below 2.0%.
The degree of reproducibility of the results obtained as a result
of small deliberate variations in the method parameters has proven
that the method is robust and the data are summarized in Table 2.
The results obtained with system suitability tests confirmed that
chromatographic system was adequate for the analyses. The RSD of
peak area (0.46%) and retention time (0.04%) are within 2%. The
efficiency of the column as expressed by number of theoretical plates
was 5520 and the tailing factor was 1.26.
Development and validation of a stability-indicating HPLC method 211
Vol. 35, No. 1
paddle apparatus the BCZ tablets have adhered on the bottom of the
vessel, making it unable to use. In this way, aqueous solution with
1.5% of SLS medium and basket in rotation speed of 100 rpm were
the conditions selected for the dissolution test of tablets.
Dissolution profile of oral suspension
Figure 4 shows the dissolution profiles of BCZ oral suspension
in 900 mL of aqueous solution with 1.5% of SLS medium in rotation
speeds of 25 and 50 rpm, using paddle apparatus. The statistical analy-
sis at 0.05 significance level showed significant difference between
the DE of 25 and 50 rpm (84.3 and 91.7, respectively).Using 50 rpm
the dissolution proceeds too quickly, almost 100% of the drug was
dissolved in 30 min. On the other hand, rotational speed of 25 rpm
produced a better discriminating dissolution profile. Based in these
results, the selected rotation for dissolution test validation was 25 rpm.
Validation of dissolution test
The chromatogram obtained through the injection of placebo
solution did not present any other peak in the same retention time
of BCZ. According to the USP Forum16 the lack of chromatographic
peaks from the placebo formulation demonstrates the specificity of
the method.
The representative linear equation was y = 34083x – 5074 (where,
x is concentration and y is the peak absolute area) and the correlation
coefficient was r = 0.9990, indicating good linearity. The validity of
the assay was verified by means of ANOVA, which demonstrated
significant linear regression and non-significant linearity deviation (p
> 0.05). In the accuracy test, the mean recovery percentages for low,
medium and high level were, respectively, 96.9, 101.8 and 100.4% for
tablets and 104.5, 101.3 and 103.9% for oral suspension, confirming
the accuracy of the method.
All the data for the repeatability and intermediate precision (Table
4) are within the acceptance criteria of 5%.16
CONCLUSIONS
A stability-indicating LC method was developed and validated to
the routine quality control of BCZ in pharmaceutical formulations and
was satisfactory in the quantitation of BCZ tablets and oral suspension
from the dissolution tests. The method showed to be specific, linear,
accurate, precise, sensible and robust. In dissolution test, the use of
900 mL of aqueous solution with 1.5% of SLS medium, basket at 100
rpm stirring speed and paddle at 25 rpm stirring speed as apparatus for
tablets and oral suspension, respectively, provided satisfactory results
for both products. In these conditions, the BCZ stability was guarantee.
The dissolution test was validated and showed to be specific, linear,
precise and accurate. The % dissolved for all products was > 75% in
30 min, and the suggested acceptance criteria could be 80% in 45 min.
REFERENCES
1. Martindale; The Extra Pharmacopoeia, 31th ed., Pharmaceutical Press:
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Table 3. Percent of buclizine dissolved in different media
Medium % Dissolved
1 h 2 h
0.01 M HCl 7.6 14.4
0.1 M HCl 55.5 68.3
Phosphate buffer (pH 6.8) 40.7 43.9
Acetate buffer (pH 4.1) 1.8 2.0
Aqueous solution with 0.5% of SLS 4.2 7.5
Aqueous solution with 1.0% of SLS 28.1 43.2
Aqueous solution with 1.5% of SLS 67.3 99.0
Figure 3. Dissolution profiles of buclizine tablets in aqueous solution with
1.5% of sodium lauryl sulfate medium using basket (50, 75 and 100 rpm) and
paddle (25 and 50 rpm)
Figure 4. Dissolution profiles of buclizine oral suspension in 1.5% sodium
lauryl sulfate medium using paddle at 25 and 50 rpm
Table 4. Intra-day and inter-day precision data of the dissolution method
for BCZ
Sample Inter-day Intra-day
RSDb (%)
Day Recoverya (%) ± RSDb
Tablet 1 99.6 ± 1.71 1.13
2 99.5 ± 1.50
3 101.5 ± 0.92
Oral Suspension 1 101.2 ± 0.31 0.17
2 101.2 ± 0.23
3 101.5 ± 0.41
aMean of 6 replicates; brelative standard deviation
Kuminek et al.
212 Quim. Nova
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