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In the present paper, a simultaneous method has been developed
and validated for estimation of gliquidone in the presence of H1-
receptor antagonists (fexofenadine hydrochloride, buclizine
hydrochloride, and levocetirizine dihydrochloride) using reversed-
phase high-performance liquid chromatographic technique. A good
chromatographic separation between these drugs was achieved
using a mobile phase containing methanol–water (80:20 v/v) at pH
3.5 with a flow rate of 1.0 mL/min; and detection was performed at
230 nm with a UV detector. Validation of the method was
performed in terms of linearity, accuracy, precision, and limit of
detection and quantification. The linearity of the calibration curves
for gliquidone, fexofenadine hydrochloride, buclizine
hydrochloride, and levocetirizine dihydrochloride were found to be
0.338–50 µg/mL (r= 0.9964), 5–50 µg/mL (r= 0.9956), 0.325–50
µg/mL (r= 0.9967), and 0.553–50 µg/mL (r= 0.9950), respectively.
There was no significant difference between the amount of drug
spiked in serum and the amount recovered, and serum did not
interfere in simultaneous estimation. Thus, the proposed method is
suitable for the simultaneous analysis of active ingredients in tablet
dosage forms and human serum.
Introduction
Diabetes mellitus is a chronic, progressive
disease characterized by deteriorating glucose
control and increased risk of micro- and
macrovascular complications (1,2). In addi-
tion, psychological troubles are considered to
be risk factors for the future development of
diabetes-related complications (3). For
patients who are diagnosed with diabetes, a
large number of medications become avail-
able for appropriate therapy (4). The second
most common oral pharmacologic strategy to
manage type 2 diabetes include the use of
agents that promote insulin release (e.g., sul-
fonylureas) (5). Gliquidone (Figure 1) belongs to the class of sul-
fonylurea derivatives and is mainly used for the treatment of
non-insulin-dependent diabetes mellitus (NIDDM). It lowers the
blood sugar level by stimulating the production and release of
insulin from the pancreas (6). Gliquidone causes a marked and
dose-dependent stimulation of acid production in gastric glands
and potentiates the stimulatory effect of both histamine and car-
bachol, which increases the rate of pepsinogen release in gastric
glands (7).
Histamine H1-receptor antagonists are the mainstays of treat-
ment for several allergic disorders, particularly rhinitis, con-
junctivitis, dermatitis, urticaria, and asthma (8). First-
generation histamine H1-receptor antagonists readily penetrate
the blood brain barrier and produce histamine blockade at his-
tamine H1-receptors in the central nervous system. In contrast,
second generation histamine H1-receptor antagonists are associ-
ated with a reduced incidence of sedation due to their poor ability
to penetrate the blood brain barrier (9). Using immunohisto-
chemical techniques, Stauber et al. (10) found that albumin
could easily enter the cerebral cortex and also that second-gen-
eration histamine H1-receptor antagonists may easily penetrate
the blood brain barrier and cause potent sedation in diabetics. It
has been recognized that patients with diabetes have a higher
prevalence of depression than the general population (4).
382
Abstract
Simultaneous Determination of Gliquidone,
Fexofenadine, Buclizine, and Levocetirizine in
Dosage Formulation and Human Serum by RP-HPLC
M. Saeed Arayne1,*, Najma Sultana2, Agha Zeeshan Mirza1,and Farhan Ahmed Siddiqui1
1Lab 9, Department of Chemistry, University of Karachi, Karachi. 75270, Pakistan and 2Research Institute of Pharmaceutical Sciences,
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi. 75270, Pakistan
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
Journal of Chromatographic Science, Vol. 48, May/June 2010
* Author to whom correspondence should be addressed:
e-mail zee_amm@hotmail.com.
Figure 1. Gliquidone (A), buclizine hydrochloride (B), levocetirizine dihydrochloride (C), and
fexofenadine hydrochloride (D).
Journal of Chromatographic Science, Vol. 48, May/June 2010
383
Antidiabetic drugs and H1-receptor antagonists can be coadmin-
istrated in a number of cases. The main objective of this study
was to develop a new method for the simultaneous determina-
tion of gliquidone with H1-receptor antagonists (fexofenadine
hydrochloride, buclizine hydrochloride, and levocetirizine dihy-
drochloride) (Figures 1).
Literature survey revealed that quantification of gliquidone
has been achieved by UV spectrophotometry (11), atmospheric
pressure chemical ionization liquid chromatographic–mass
spectrometry (APCI-LC–MS), LC–MS (12), and high-perfor-
mance liquid chromatography (HPLC) (13–15). Numerous
workers have reported determination of H1-receptor antagonists
by different techniques. There were also HPLC methods reported
for the quantitation of cetirizine dihydrochloride or fexofenadine
hydrochloride with pseudoephedrine in combined pharmaceu-
tical dosage forms (16–18), cetirizine dihydrochloride or levoce-
tirizine dihydrochloride with cefpirome (19), fexofenadine
hydrochloride (20), and buclizine hydrochloride (21,22); but the
method of all the previously mentioned coadministrated drugs
in active and dosage form are not reported simultaneously by
HPLC. Such a method is needed as the coadministration of both
drugs is possible in multiple-drug therapy. The proposed method
was successfully applied to the determination of these drugs in
commercial tablets and human serum. The established method
was validated with respect to linearity, limit of detection and
quantification, precision, accuracy, specificity, and robustness.
Experimental
Materials
The gliquidone reference standard was kindly gifted by
Pharmatec Limited Karachi (Karachi, Pakistan). The H1-
receptor antagonists (fexofenadine hydrochloride, buclizine
hydrochloride, levocetirizine dihydrochloride) of pharmaceu-
tical purity were obtained from AGP Limited Karachi (Karachi,
Pakistan). Glurenor (30 mg), Fexet (30 mg), Longifene(25 mg),
and Xyzal (45 mg) tablets were purchased from the local phar-
macy. Methanol used was HPLC-grade.
Equipments
A Shimadzu HPLC (Kyoto, Japan) system equipped with LC-
10 AT VP pump, Rheodyne manual injector fitted with a 20-µL
loop, PurospherSTAR RP18 end-capped column (25 cm ×0.46
cm, 5 µm) and SPD-10 A VP UV–Vis detector was utilized. The
chromatographic system was integrated via Shimadzu model
CBM-102 to PIV computer. Shimadzu CLASS-GC software
(Version 5.03) was used for data acquisition and mathematical
calculations.
Solution preparations
Separate stock solutions of gliquidone and H1-receptor antag-
onists (fexofenadine hydrochloride, buclizine hydrochloride, and
levocetirizine dihydrochloride) were prepared separately in a
100-mL volumetric flask by dissolving 10 mg of each drug, and
volume was made up by 100 mL of 80% (v/v) aqueous methanol
so that final concentration was 100 µg/mL. Working solutions of
5, 10, 15, 20, and 50 µg/mL were prepared by diluting with
aqueous methanol (80%, v/v) from the standard solutions.
Assay in formulations
To determine the content of all the drugs in the formulations,
20 tablets of each drug were powdered, and an equivalent to 10
mg of gliquidone and 10 mg of H1antagonists (fexofenadine
hydrochloride, buclizine hydrochloride, levocetirizine dihy-
drochloride) were weighed and transferred separately into 100-
mL calibrated flasks before 100 mL 80% aqueous methanol was
added. The content of the flask was shaken for about 60 min. This
solution was filtered through Whatman filter paper to separate
out the insoluble excipients, and further dilutions were carried
out to obtain the desired concentration. Final solutions were fil-
tered through a 0.45-µm Millipore filter (Billerica, MA) before
injection into the HPLC.
Serum drug analysis
The recovery of gliquidone, fexofenadine hydrochloride,
buclizine hydrochloride, and levocetirizine dihydrochloride in
human serum was determined by the stated chromatographic
conditions. Multiple blood samples (10 mL) of ten healthy vol-
unteers were collected in evacuated glass tubes. The blood was
then centrifuged at 3000 rpm for 10 min, and the plasma was
separated and deproteinated by acetonitrile. The supernatant
obtained was filtered through a 0.45-micron pore size mem-
brane filter. Serum thus obtained was mixed to different aliquots
of stock standard solution to produce the desired concentrations.
Figure 2. Representative chromatogram of fexofenadine, levocetirizine,
buclizine, and gliquidone in formulation (A) and representative
chromatogram of fexofenadine, levocetirizine, buclizine, and gliquidone
in human serum (B).
These were stored at –20°C, and 10 µL volume of each sample
was injected and chromatographed.
Optimization of chromatographic condition
To optimize the operating conditions for isocratic reversed-
phase (RP)-HPLC detection, parameters such as mobile phase
composition and flow rate were varied. Mobile phase composed
of methanol–water (80:20) and a flow rate of 1.0 mL/min were
chosen as the optimal settings that gave the retention times 2.71,
3.16, 5.81, and 11.05 min for fexofenadine hydrochloride, levoce-
tirizine dihydrochloride, buclizine hydrochloride, and gliq-
uidone, respectively. The pH effect showed that optimized
conditions are reached when the pH value is 3.5 because it pro-
duces well-resolved and sharp peaks for all drugs assayed. A rep-
resentative chromatogram is shown in Figure 2. In addition, the
UV spectra of individual drugs were recorded in the wavelength
range from 200 to 400 nm and compared. The choice to use
isobestic point set at 230 nm was considered satisfactory, per-
mitting the detection of all drugs with adequate sensitivity.
Result and Discussion
Method validation
The method was validated according to ICH guidelines for val-
idation of analytical procedures (23). The method was validated
for the parameters like linearity, limit of detection (LOD), limit
of quantitation (LOQ), accuracy and precision, specificity,
and robustness. The linearity of this method was proved using
linear correlation of the peak-area values and appropriate
concentrations.
Linearity, limit of detection, and quantification
Under the previously described experimental conditions,
linear correlation between the peak area and applied concentra-
tion was found in the concentration range 0.3–50 µg/mL. The
regression statistics are shown in Table I. The LOD and LOQ at
concentrations where the signal-to-noise ratios were equal to 3
and 10, respectively, were determined to be 0.10, 0.19, 0.097,
0.16 µg/mL and 0.33, 5.00, 0.32, 0.55 µg/mL for gliquidone, fex-
ofenadine hydrochloride, buclizine hydrochloride, and levoceti-
rizine dihydrochloride, respectively. The correlation coefficient
of this dependence was calculated to be 0.9964, 0.9956, 0.9967,
0.9950 for gliquidone, fexofenadine hydrochloride, buclizine
hydrochloride, and levocetirizine dihydrochloride, respectively.
Accuracy and precision
Intra-day precision and accuracy of the method were evaluated
at three different independent concentrations (i.e., 8, 10, 12
µg/mL) (n= 3) in synthetic samples using placebo mixtures. The
accuracy results (Table II) revealed that the method was accurate
for all previously mentioned purposes. %Recoveries and %rela-
tive standard deviation (RSD) values were used to express accu-
racy and precision. Standard addition and recovery experiments
were also conducted to determine the accuracy of the present
method for the quantification of gliquidone, fexofenadine
hydrochloride, buclizine hydrochloride, and levocetirizine dihy-
drochloride (Table III).
Specificity
A representative chromatogram (Figure 2) was generated to
show that other components, which could be
present in the sample matrix, are resolved from
the parent analyte. No significant changes in
retention times of the drugs in the presence and
the absence of excipients clearly indicated the
specificity of the method.
Robustness
The robustness was evaluated by minor mod-
ifications in the composition of the mobile
phase used in the proposed method. The factors
selected to examine were pH of mobile phase
and temperature (°C). A change of ± 0.1 unit of
Journal of Chromatographic Science, Vol. 48, May/June 2010
384
Table I. Regression Statistics and LOD and LOQ
Regression LOD LOQ
Drug equation R2(µg/mL) (µg/mL)
Gliquidone y = 17786x – 36988 0.9964 0.10 0.33
Fexofenadine y = 6940.8x – 46043 0.9956 0.19 5.00
Buclizine y = 9493.2x – 21326 0.9967 0.09 0.32
Levocetirizine y = 8454.4x – 1434.7 0.9950 0.16 0.55
Table II. Accuracy and Precision
Spiked Precision Accuracy
Analyte conc. (µg/mL) (% RSD) (%)
Gliquidone 8 0.49 100.0
10 2.46 100.5
12 0.93 100.7
Fexofenadine 8 2.11 99.8
hydrochloride 10 3.26 96.9
12 0.96 100.4
Buclizine 8 0.49 102.2
hydrochloride 10 0.92 99.8
12 1.03 97.8
Levocetirizine 8 0.90 99.6
dihydrochloride 10 1.00 98.4
12 1.80 99.5
Table III. Results from Recovery Studies of Gliquidone, Fexofenadine, Buclizine, and
Levocetirizine
Gliquidone Fexofenadine Buclizine Levocetirizine
Conc. Found Recovery Found Recovery Found Recovery Found Recovery
µg/mL µg/mL % µg/mL % µg/mL % µg/mL %
5 4.96 99.3 5.10 102.0 5.01 100.2 5.01 100.1
10 10.05 100.5 9.79 97.9 9.98 99.8 9.94 99.4
15 14.93 99.6 15.13 100.9 15.27 101.8 14.88 99.2
20 20.01 100.1 19.97 99.8 19.84 99.2 19.08 95.4
25 24.85 99.4 25.04 100.2 25.15 100.6 24.35 97.4
pH (pH of mobile phase) had no considerable impact on chro-
matographic performance. The effect of column temperature on
resolution was studied at 25°C and 32°C instead of room tem-
perature (28°C). It can be seen that at every employed condition,
the chromatographic parameters are in accordance with the
established value.
The results of the present study demonstrate that simulta-
neous determination of gliquidone, fexofenadine hydrochloride,
buclizine hydrochloride, and levocetirizine dihydrochloride are
very beneficial for pharmaceutical companies, clinicians, and
physicians and also can be beneficial for the studies of drug inter-
action.
Application in human serum
It was observed after spiking the analyte in the serum sample
that there was no significant difference between the amount of
drug spiked in serum and the amount recovered. Previously
developed HPLC procedures for the determination of gliquidone,
fexofenadine hydrochloride, buclizine hydrochloride, and levo-
cetirizine dihydrochloride in plasma are based on liquid–liquid
extraction from plasma samples. The method applied in our
study involved the direct injection of the plasma samples after
precipitation of protein with acetonitrile. The recovery values
(Table IV) in human serum clearly indicate the applicability of
the present method for the required purpose (Figure 2B).
Conclusion
After studying all the results obtained by HPLC studies, it was
concluded that the present method was fast and easy to perform.
The linearity range, LOD and LOQ, precision, accuracy, and
specificity were processed to determine the suitability of the
method, and the confirmed results were obtained. HPLC has sev-
eral superiorities compared with UV spectrophotometry, such as
smaller detection and quantification limits, small sample vol-
umes, and specificity. The validity of the method makes it an
acceptable for clinical studies in patients taking these medica-
tions simultaneously. Thus, the developed HPLC method is
rapid, reliable, cost-effective, and can be proposed for routine
analysis laboratories and quality control purposes.
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Journal of Chromatographic Science, Vol. 48, May/June 2010
385
Table IV. Accuracy and Precision in Human Serum
Spiked Precision Accuracy
Analyte conc. (µg/mL) (% RSD) (%)
8 1.55 102.0
Gliquidone 10 1.36 101.7
12 1.45 100.8
8 0.98 101.7
Fexofenadine 10 2.11 97.9
hydrochloride 12 1.56 101.4
8 0.41 101.5
Buclizine 10 1.92 102.0
hydrochloride 12 0.93 99.8
8 1.50 99.9
Levocetirizine 10 1.31 99.4
dihydrochloride 12 1.20 101.0