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Pak. J. Pharm. Sci., 2006, Vol.19(4), 326-329
326
ORIGINAL ARTICLE
QUANTITATION OF BUCLIZINE HYDROCHLORIDE IN PHARMACEUTICAL
FORMULATIONS AND HUMAN SERUM BY RP-HPLC
M. SAEED ARAYNE, NAJMA SULTANA* AND FARHAN AHMED SIDDIQUI
Department of Chemistry, University of Karachi, Karachi-75270, Pakistan
*Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi
ABSTRACT
An isocratic reversed phase high-performance liquid chromatographic (HPLC) method with ultraviolet detection at
230 nm has been developed for the determination of buclizine hydrochloride in human serum and dosage
formulation. Methylparaben was successfully used as an internal standard. Good chromatographic separation
between buclizine and internal standard peaks was achieved by using a stainless steel analytical column Nucleosil,
C18 (10µm, 25 cm x 0.46 cm). The system was operated at room temperature using a mobile phase consisting of
acetonitrile–water (1:1) (pH 2.6) with phosphoric acid 85% at a flow rate of 2 ml/min. The calibration curve for
buclizine hydrochloride in human serum was linear over the tested concentration range of 10, 3, 1.5, 0.5, 0.15, 0.05,
and 0.025 µg/ml with a correlation coefficient of 0.9999. The intra- and inter-run precision and accuracy results
were 98.07 to 100.34. The proposed method was validated for selectivity, linearity, accuracy, and precision. The
method was found to be suitable for the quality control of buclizine hydrochloride in bulk drug as well as in human
serum.
Keywords: Buclizine, methylparaben, UV detection, RP-HPLC, dosage formulation, method validation.
INTRODUCTION
Buclizine hydrochloride or (RS)-1-(4-tert-butylbenzyl)-4-(4-
chlorobenzhydryl)piperazine dihydrochloride, is white or
slightly yellowish, crystalline powder. It is piperazine
derivative having antihistaminic, antimuscarinic, antiemetic,
sedative properties and is used in motion sickness [The
Merck Index (2001), Martindale, The Extra Pharmacopoeia.
1996, British Pharmacopoeia 2003].
N
N
Cl
C(CH
3
)
3
Buclizine
In literature any single liquid chromatography method for
the analysis of buclizine has not been reported, only one
method of LC was reported (El Walily et al., 1999) in which
buclizine was used as internal standard, but quantification
was not done. The main purpose of this study was to
develop a simple and reliable method to quantitate buclizine
hydrochloride in a relatively short time with high linearity
using a commercially available internal standard. Therefore,
this study focused on the development of simple and rapid
isocratic RP-HPLC method which can be employed for the
routine analysis of buclizine hydrochloride in bulk drug
formulations and in human serum. The established method
was validated with respect to specificity, linearity, precision,
accuracy and ruggedness.
EXPERIMENTAL
Materials and reagents
All chemicals and reagents were of analytical grade quality.
Buclizine hydrochloride was supplied from AGP (Private)
Limited. A stock solution of 100 ppm buclizine
hydrochloride was prepared in acetonitrile and water (1:1)
and stored in the dark at 4 °C. More dilute solutions were
prepared daily with same solvent just before use.
Pharmaceutical dosage form
Longifene tablet, AGP (Private) Limited labeled to contain
25 mg buclizine hydrochloride drug.
Chemicals
HPLC grade acetonitrile and phosphoric acid were
purchased from Merck Germany. Methylparaben was
purchased from market. Other chemicals used were
analytical grade. LongifeneTM tablets were purchase from
market.
Instrumentation and chromatographic conditions
An HPLC system consisted of an LC-10 AT VP Shimadzu
pump, SPD-10AV VP Shimadzu UV visible detector, a
Nucleosil, C18 (10µm, 25 cm x 0.46 cm) column was used
for separation. The chromatographic and integrated data
were recorded using a CBM-102 communication Bus
Module Shimadzu. The separation was carried out under
isocratic elution with mobile phase was acetonitrile and
Corresponding author: e-mail: arayne@gawab.com
M. Saeed Arayne et al.
Pak. J. Pharm. Sci., 2006, Vol.19(4), 326-329 327
water (80:20). The pH of this mobile phase was adjusted to
2.6 with phosphoric acid (85 %). The flow rate was
2.0 ml min−1, the wavelength was monitored at 230 nm, and
the injection volume was 10 µl. The assay procedure was
performed using internal standard method with
methylparaben as internal standard.
Preparation of standard and sample solutions
Standard preparation
Standard stock solutions of 100 ppm of buclizine
hydrochloride and methylparaben in acetonitrile and water
(1:1) were prepared in separate volumetric flasks. Working
solutions were prepared by diluting the stock solutions with
the same solvent to contain 10, 3, 1.5, 0.5, 0.15, 0.05, and
0.025 µg/ml. The resolution should not be less than 1.5
according to the USP 2005.
Procedure for longifene tablets
The drug content of twenty tablets was weighed, finely
powdered and mixed. The average mass per tablet was
determined. A quantity of the powder equivalent to 10 mg
of buclizine hydrochloride was transferred accurately into a
100-ml calibrated dark flask containing acetonitrile and
water mixture (1:1). The content of the flask was shaken for
about 60 min and diluted to volume with same solvent. The
solution was then filtrated through a 0.45-µm milli-pore
filter (Gelman, Germany), that to separate out the insoluble
excipients, rejecting the first portion of the filtrate. The
desired concentration for the drug was obtained by accurate
dilution and the analysis was followed up as in the general
analytical procedure.
Procedure for human serum
Plasma samples, obtained from healthy volunteers, were
collected and stored frozen to 1.0 ml of plasma, 10.0 ml of
acetonitrile was added, the mixture was vortexed for one
minute and than centrifuged for 10 minutes at 10,000 rpm.
Obtained supernatant was filtered by 0.45-micron pore size
membrane filter. An aliquot serum sample was fortified with
buclizine hydrochloride to achieve final concentration 10, 3,
1.5, 0.5, 0.15, 0.05, and 0.025 µg/ml. These solutions were
stored at –20°C and analyzed regularly at interval of two
days up to eight days for serum drug analysis. 10 µl of this
solution was injected and chromatographed. Calibrated
glassware’s from Pyrex were used for the solution and
mobile phase preparation.
RESULTS AND DISCUSSION
Development and optimization of isocratic HPLC
conditions
A reversed-phase assay was deemed most appropriate for
initial testing. A UV scan of buclizine hydrochloride
showed a maximal absorbance at or near 230 nm. Initial
method development was conducted on a Nucleosil, C18
(10µm, 25 cm x 0.46 cm) column was used for separation at
room temperature. This column provides efficient and
reproducible separations of nonpolar compounds while
minimizing solvent usage. Conse-quently, it was selected
for method development and remains the column utilized in
the validated assay.
Preliminary method development of suitable isocratic
conditions to resolve buclizine hydrochloride on the C18
column was conducted with acetonitrile-water as the mobile
phase. A mobile phase of acetonitrile-water (80:20 v/v)
adjusted pH with phosphoric acid to 2.6 was found to
provide a reproducible, baseline resolved peak. These
conditions allowed for separation of buclizine hydro-
chloride from internal standard.
The chromatographic conditions were optimized with
respect to specificity, resolution and time of analysis. The
specificity of the method was established through the study
of resolution factor of buclizine hydrochloride peak from
the internal standard peak. Peaks were identified using
retention times compared with those of standards.
For validation of analytical methods, the guidelines of the
International Conference on the Harmonization of Technical
Requirements for the Registration of Pharmaceuticals for
Human Use [ICH 1996] and [USP 2002] have
recommended the accomplishment of accuracy tests,
precision, specificity, linearity, work strip and robustness of
the method. The type of method and its respective use
determine the parameters to be evaluated, especially, when
the samples are complex biologic matrices, the calibration
curve of buclizine was 10, 3, 1.5, 0.5, 0.15, 0.05, and 0.025
µg/ml were linear in the range mentioned as above. The
representative linear equation was y = 1x + 0.006 (n = 6; r =
0.9999) where y represents injected concentration and x the
recovered concentration. The detection limit, taken as the
lowest absolute concentration of analyte in a sample, which
can be detected but not necessary quantified under the stated
experimental condition, was, 0.005 µg/ml. The limit of
quantitation, taken as the lowest concentration of analyte in
a sample, which can be determined with acceptable
precision and accuracy, was 0.025.
System suitability
The HPLC system was equilibrated with the initial mobile
phase composition, followed by 10 injections of the same
standard. These 10 consecutive injections were used to
evaluate the system suitability on each day of method
validation.
The system suitability parameters including capacity factor
>2, resolution > 3 and asymmetric factor < 2. All parameters
were satisfactory with good specificity for the stability
assessment of buclizine hydrochloride. Theoretical plates of
the column were > 3000.
Quantitation of buclizine hydrochloride in pharmaceutical formulations
Pak. J. Pharm. Sci., 2006, Vol.19(4), 326-329
328
Linearity
Linearity was tested by assaying serum spiked with known
volumes of buclizine hydrochloride stock solution at
concentrations 10, 3, 1.5, 0.5, 0.15, 0.05, and 0.025 µg/ml.
Injected concentration versus recovered concentration were
plotted and the correlation coefficients were calculated.
Precision and accuracy
Assay precision was determined by spiking human serum
with varying concentrations of buclizine hydrochloride and
fixed concentration of internal standard. Buclizine
hydrochloride concentrations were determined in batches of
three on the same run and on different runs. Intra and inter-
run assay precision was determined by calculating
coefficient of variation (CV) of the obtained data shown as
table 1.
Intraday and inter-day accuracy and precision
The precision of the method was investigated with respect to
repeatability. For intra-day precision, ten samples of seven
concentrations were analyzed on the same day. Table 1
summarizes the correlation coefficient, standard error,
standard error of estimate, and slope. Generally acceptable
repeatability of the results with in one day and day-to-day
was observed.
The statistical summary includes coefficient of variance
(C.V) and recovery (%). Inter and intraday accuracy of the
method ranged from (98.07% to 100.34%), (CV%, 0.9 to
3.5±) as shown in table 1.
The recovery of buclizine hydrochloride in dosage
formulation was performed at three concentration levels (60,
90, and 120%) in dosage formulation, accuracy of the
method in dosage formulation ranged from 98.66 % to
101.6% as shown in table 2.
Relative accuracy was determined by calculating the percent
accuracy using the equation:
% Recovery = (observed concentration/nominal
concentration) potency of standard
Table 2: Recovery characteristics of buclizine in dosage
formulation
Buclizine
% label claim % Recovery % Error (±)
60 102 2.00
80 98.66 1.34
100 101.6 1.6
120 99 1.0
Label claim: Buclizine HCl 25 mg
Limit of quantitaition and detection
The limit of detection (LOD) and limit of quantitation
(LOQ) of this method were determined from the coefficient
of variation of a known concentration of buclizine
hydrochloride, as per (ICH Q2B guidelines 1997) and
(FDA, Guidance for Industry 2000).
The LOD for this assay, calculated from three times the
noise level of the response, is 0.005 µg/ml. The LOQ for
this assay calculated from ten times the noise level of the
response, is 0.025 µg/ml.
Specificity
The specificity of the chromatographic method was
determined to ensure separation of internal standard and
buclizine hydrochloride as shown in figure 1. Specificity
was also determined by screening four different samples of
controlled human serum, which were free from interfering
endogenous plasma components. This is evidenced by the
Table 1: Recovery and regression characteristics of buclizine, in human serum
Concentration <–––––––––––––– CV % (n=6) –––––––––––––>
ug/ml Day 1 Day 2 Day 3 Day 4 Recovery (%)
0.025 1.7 1.4 3.5 2.3 98.07
0.05 1.5 1.3 1.1 2.4 98.48
0.15 1 1.4 1.1 1.6 99.51
0.5 0.9 1.4 1 1.6 100.25
1.5 1.7 1.6 1 1.6 100.34
3 0.9 1.4 1 1.5 100
10 1 2 0.9 0.8 99.94
Correlation coefficient (r) 0.9999
Standard error of estimate 0.0025
Standard error 0.0013
Intercept 0.006
Slope 1
M. Saeed Arayne et al.
Pak. J. Pharm. Sci., 2006, Vol.19(4), 326-329 329
lack of interfering peaks in the chromatograms of serum
samples. Figure 2 represents a typical chromatogram of
blank serum and serum containing drug respectively.
Solutions of buclizine hydrochloride containing aspirin,
acetaminophen, caffeine and nicotine were prepared and
then injected to check for interference from these commonly
used drugs. The method demonstrated good resolution
between the peaks and found to be free of interferences.
Figure 1: Representative chromatogram of buclizine
reference standard with internal standard, Peak 1- Internal
Standard, Peak 2- Buclizine
Figure 2: Representative chromatogram of and buclizine in
human serum with internal standard.
Peak 1- Internal Standard, Peak 2- Buclizine
Ruggedness
The ruggedness was established by determining buclizine
hydrochloride in dosage formulation and in human serum
using the same chromatographic system and the same
column by two analysts on a different day. The assay result
indicated that the method was capable with high precision
(table 1). Additionally, good separations were always
achieved which suggested that the method was selective for
all components under the test.
Robustness
Stability of standard solution and sample solutions were
determined by assay after 24 and 48 hours at –20°C
temperature against fresh standard solutions. It shows that
all the drugs are stable and does not show much variation in
the time span of 48 hours.
CONCLUSION
A simple and reliable HPLC method for measuring
buclizine hydrochloride in human serum and pharmaceutical
dosage formulation has been developed. A fully validated
RP-HPLC procedure for the assay of buclizine
hydrochloride drug in bulk, tablets and human serum is
described for the first time. Hence, it can be recommended
for the routine quality control of this antibiotic drug.
Methylparaben could be successfully used as an internal
standard. The low volume of blood or plasma needed, the
availability of the internal standard, the simplicity of the
separation procedure, the short run time and the low volume
of injection make this method suitable for quick and routine
analysis. The intra-run and inter-run variability and
accuracy results were in acceptable limit.
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Received: 09-10-06 – Accepted: 31-10-2006