First derivative synchronous fluorescence spectroscopy for the simultaneous determination of sulpiride and mebeverine hydrochloride in their combined tablets and application to real human plasma

Article abstract of DOI:10.1007/s10895-010-0679-0

A rapid, simple and highly sensitive first derivative synchronous fluorometric method has been developed for the simultaneous analysis of binary mixture of sulpiride (SUL) and mebeverine hydrochloride (MEB). The method is based upon measurement of the synchronous fluorescence intensity of these drugs at Δλ=100 nm in water. The different experimental parameters affecting the fluorescence of the two drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.05-1 μg/mL and 0.2-3.2 μg/mL for SUL and MEB respectively with lower detection limits (LOD) of 0.006 and 0.01 μg/mL and quantification limits (LOQ) of 0.0.02 and 0.05 μg/mL for SUL and MEB, respectively. The proposed method was successfully applied for the determination of the two compounds in synthetic mixtures and in commercial tablets. The high sensitivity attained by the proposed method allowed the determination of both of SUL and MEB metabolite (veratic acid) in real human plasma samples applying second derivative synchronous fluorometric technique. The mean% recoveries (n=3) for both MEB metabolite (veratic acid) and SUL were 99.82±2.53 and 98.84±6.20 for spiked human plasma respectively, while for real human plasma, the mean% recoveries (n=3) were 91.49±4.25 and 91.36±8.46 respectively.

Full text of DOI:10.1007/s10895-010-0679-0

J Fluoresc (2010) 20:1275–1285
DOI 10.1007/s10895-010-0679-0
ORIGINAL PAPER
First Derivative Synchronous Fluorescence Spectroscopy
for the Simultaneous Determination of Sulpiride
and Mebeverine Hydrochloride in Their Combined Tablets
and Application to Real Human Plasma
M. Walash & M. Sharaf El-Din & Nahed El-Enany &
M. Eid & Sh. Shalan
Received: 10 March 2010 /Accepted: 25 May 2010 /Published online: 7 July 2010
#
Springer Science+Business Media, LLC 2010
Abstract A rapid, simple and highly sensitive first deriv-
ative synchronous fluorometric method has been developed
for the simultaneous analysis of binary mixture of sulpiride
(SUL) and mebeverine hydrochloride (MEB). The method
is based upon measurement of the synchronous fluores-
cence intensity of these drugs at Δλ=100 nm in water. The
different experimental parameters affecting the fluorescence
of the two drugs were carefully studied and optimized. The
fluorescence-concentration plots were rectilinear over the
range of 0.05–1 µg/mL and 0.2–3.2 µg/mL for SUL and
MEB respectively with lower detection limits (LOD) of
0.006 and 0.01 µg/mL and quantification limits (LOQ) of
0.0.02 and 0.05 µg/mL for SUL and MEB, respectively.
The proposed method was successfully applied for the
determination of the two compounds in synthetic mixtures
and in commercial tablets. The high sensitivity attained by
the proposed method allowed the determination of both of
SUL and MEB metabolite (veratic acid) in real human
plasma samples applying second derivative synchronous
fluorometric technique. The mean% recoveries (n=3) for
both MEB metabolite (veratic acid) and SUL were 99.82
2.53 and 98.84 6.20 for spiked human plasma respectively,
while for real human plasma, the mean% recoveries (n=3)
were 91.49 4.25 and 91.36 8.46 respectively.
Introduction
Mebeverine hydrochloride (MEB) 3,4-Dimethoxybenzoic
acid 4- [ethyl[2-(4-methoxyphenyl)-1-methylethyl]amino]-
butylester (Fig. 1) is a potent direct antispasmodic acting
mainly on the smooth muscles of the gastrointestinal tract
and particularly effective against the colonic spasm [1].
Mebeverine undergoes rapid and extensive presystemic
(first-pass) hydrolysis in the gut and/or the liver [1].
British Pharmacopoeia described a non aqueous titrimetric
method for determination of MEB in pure form [2]. Several
spectrophotometric methods [3–9], electrochemical methods
[10–12] and chromatographic methods [13–21] were reported
for its determination either per se or in pharmaceutical
preparations and biological fluids.
Sulpiride (SUL) 5-(Aminosulfonyl)-N-[(1-ethyl-2-pyrroli-
dinyl)methyl]-2-methoxy-benzamide (Fig. 1) is the most
widely prescribed anti-psychotic drug. It is a selective
dopamine D2 antagonist with antidepressant activities it
exerts a mood elevating effect and antiemetic action [1].
Regarding SUL, British Pharmacopoeia described a non
aqeous titrimetric method for determination of SUL in pure
form [2]. Several spectrophotometric methods [22–24],
electrochemical methods [25, 26], chromatographic methods
[27–34] were reported for its determination either per se or
in pharmaceutical preparations and biological fluids.
.
.
Keywords Sulpiride Mebeverine Synchronous
.
fluorimetry Pharmaceutical preparations and Mebeverine
Combination of MEB and SUL is used for treatment
gastrointestinal and colic spasms which are a consequence of
psychosomatic manifestation of nervous tention, mental stress
or anxiety. MEB and SUL were determined in their binary
mixture via derivative spectroscopy [35–37], TLC- densi-
tometry [37], HPLC [37] and chemometric techniques [38].
The aim of the present study is to establish and
develop a novel, sensitive and selective derivative
.
metabolite (veratic acid) Human plasma
:
:
:
:
M. Walash M. Sharaf El-Din N. El-Enany M. Eid (*)
S. Shalan
Department of Analytical Chemistry, Faculty of Pharmacy,
University of Mansoura,
35516 Mansoura, Egypt
e-mail: manal_eid@yahoo.com

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J Fluoresc (2010) 20:1275–1285
Fig. 1 Structural formulae
of the studied drugs
CH3
CH3
CH3
O
O
O
O
N
H3CO
OCH3
N
S
O
NH
OCH3
NH2
OCH3
Mebeverine
Sulpiride
synchronous fluorescence spectroscopic (DSFS) method
for simultaneous determination of MEB and SUL either
per se or in pharmaceutical preparations and in real
human plasma as SUL and MEB metabolite (veratic
acid).
Experimental
Material
Mebeverine and Sulpiride pure samples were purchased
from Sigma (St. Louis, Mo, USA) and used as received.
Colona® tablets labeled to contain 100 mg of MEB and
25 mg of SUL in a ratio of 4:1 (Batch # 09425) was
obtained from commercial source in the local market.
The normal synchronous fluorescence spectra of MEB
and SUL are greatly overlapped. Such problem led us to
utilize a simple first derivative synchronous fluorescence
spectroscopy (FDSFS) to solve this problem by measuring
peak intensities at 238 and 264 nm for MEB and SUL
respectively. The developed method was applied for the
simultaneous determination of MEB and SUL in their co-
formulated pharmaceutical preparation. On the other hand,
MEB is rapidly metabolized to veratic acid, The normal
synchronous fluorescence spectra of MEB metabolite and
SUL are greatly overlapped. Such problem led us to utilize
a simple second derivative synchronous fluorescence
spectroscopy (SDSFS) to solve such problem by measuring
peak intensities at 253 and 264 nm for MEB metabolite and
SUL respectively.
Synchronous fluorescence spectroscopy (SFS) has
several advantages over conventional fluorescence spec-
troscopy, including simple spectra, high selectivity and
low interference [39]. Because of its sharp, narrow
spectrum, SFS serves as a very simple, effective method
of obtaining data for quantitative determination in a single
measurement [40].
The combination of SFS and derivatives is more
advantageous than the conventional emission spectrum in
terms of sensitivity, because the amplitude of the derivative
signal is inversely proportional to the band width of the
original spectrum [41, 42].
Recently, derivative synchronous fluorometry (DSF) has
been utilized for determination of several mixtures in their
co-formulated dosage forms and biological fluids. Mixtures
of cinnarizine and domperidone [43], metoclopramide and
pyridoxine [44], aspirin with salicylic acid [45], diflunisal
and salicylic acid [46], carvedilol and ampicillin [47],
sulpirid and its alkaline degradation [48] have been
determined through this approach.
Reagents
All reagents and solvents were of Analytical Reagent
Grade, Methanol (Merck, Darmstadt, Germany). Acetate
buffer 0.2 M (pH3.6–5.6) was prepared by mixing
appropriate volume of 0.2 M acetic acid with 0.2 M sodium
acetate. Borate buffers (pH 5.5–13) were prepared by
mixing appropriate volumes of 0.02 M boric acid with
0.2 M sodium hydroxide, Dichloromethane (Aldrich, St.
Louis, MO, USA). Phosphate buffer (pH 7.4) was prepared
by mixing appropriate volume of 0.2 M KH2PO4 with
0.1 M sodium hydroxide [43] and sodium hydroxide
((BDH, UK), 0.1 M aqueous solution.
Apparatus
Fluorescence spectra and measurements were recorded
using a Perkin -Elmer UK model LS 45 luminescence
spectrometer, equipped with a 150 Watt Xenon arc lamp,
grating excitation and emission monochromators for all
measurements and a Perkin-Elmer recorder. Slit widths for
both monochromators were set at 10 nm. A 1 cm quartz cell
was used. Derivative spectra can be evaluated using
Fluorescence Data Manger (FLDM) software.
For best resolution and smoothing, number of points of 99
were used for deriving the first derivative spectra. The
fluorescence intensities of the first derivative spectra were
estimated at 238 and 264 nm for MEB and SUL, respectively.
A pH Meter (Model pHS-3C, Shanghai Leici instru-
ments Factory, China) was used for pH adjustment.
To the best of our knowledge, neither conventional nor
synchronous spectrofluorometry has been reported for the
analysis of MEB and SUL in their co-formulated tablets. As
well as, for the analysis of MEB metabolite (veratic acid)
and SUL in spiked and real plasma.
Standard solutions
Stock solutions of MEB and SUL were prepared by
dissolving 10.0 mg of the studied compounds in 100 mL

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of methanol and were further diluted with the same solvent
as appropriate. The standard solutions were stable for
10 days when kept in the refrigerator.
was added and diluted to the volume with distilled water,
and mixed well. The recommended procedure described
under calibration curve was then performed. The peak
amplitude of the first derivative technique was plotted vs
the final concentration of the drug (µg/mL) to generate the
calibration graph. Alternatively, the corresponding regres-
sion equations were derived.
Recommended procedures
Calibration curve
Preparation and separation of the hydrolytic degradation
products of MEB
Aliquots of MEB and SUL standard solutions covering the
working concentration range cited in Table 1 were
transferred into a series of 10 mL volumetric flasks. Two
mL of borate buffer (pH 6.5) was added and the solutions
were diluted to the volume with water and mixed well.
Synchronous fluorescence spectra of the solutions were
recorded by scanning both monochromators at a constant
wavelength difference Δλ=100 nm and scan rate of
600 nm min−1 using 10 nm excitation and emission
windows. The first derivative fluorescence spectra of
MEB and SUL were derived from the normal synchronous
spectra using FLDM software. The peak amplitude of the
first derivative spectra was estimated at 238 nm and 264 nm
for MEB and SUL, respectively. A blank experiment was
performed simultaneously. The peak amplitude of the first
derivative technique was plotted versus the final concen-
tration of the drug (µg/mL) to get the calibration graph.
Alternatively, the corresponding regression equations were
derived.
0.5 gm MEB powder was transferred into 100 ml
stoppered flask. 100 mL of 1 M NaOH prepared in
methanol was added and the solution was heated under
reflux at 45°C for 12 hrs till complete degradation of
MEB (investigated by the disappearance of MEB spot
upon applying qualitative TLC). The solution was
allowed to evaporate under reduced pressure to residue,
30 mL distilled water was added and the oily part was
extracted with (20×3 ml) ether in divided portions. The
ether extract was washed with 20 ml distilled water in
two fractions and then the ethereal layer was let to
evaporate at room temperature where a colorless oily
residue appeared of 4-(ethyl [2-(4-methoxyphenyl)-1-
methylethyl] amino) butan-1-ol (mebOH) [49]. The
primarily separated aqueous layer was acidified with
5 M HCl just till acidic to litmus, where a heavy white
precipitate of veratic acid was produced, which was then
filtered and washed with 30 mL distilled water added in
divided portions. The white needles of veratic acid were
dried in the oven at 90°C for 1 hr. IR and Mass spectra
were obtained to confirm the identity of the separated
degradation products of MEB.
Procedure for the synthetic mixture
Aliquot volumes of MEB and SUL standard solutions in the
pharmaceutical ratio of 4:1 were transferred into a series of
10 mL volumetric flasks. Two mL of borate buffer (pH 6.5)
Note: During the preparation of MEB hydrolytic
degradation products, heating was carried out at 45°C;
unlike what stated in the literature (reflux for three hours)
[49] to avoid the formation of methyl veratrate.
Table 1 Performance data of the proposed method
Stock solutions of MEB degradation product (veratic acid)
were prepared by dissolving 10.0 mg of the studied
compounds in 100 mL of methanol and were further diluted
with the same solvent as appropriate. The standard solutions
were stable for 10 days when kept in the refrigerator.
Parameter
Mebeverine
Sulpiride
Concentration range (µg/ml)
Limit of detection (LOD) (µg/ml)
Limit of quantification (LOQ) (µg/ml)
Correlation coefficient (r)
Slope
0.2–3.2
0.01
0.05–1.0
0.006
0.02
0.05
0.9999
39.58
16.5
0.9999
140.9
−0.20
0.421
0.262
0.472
0.646
0.228
Applications
Intercept
standard deviation of the residuals (Sy/x
standard deviation of the intercept (Sa)
standard deviation of the slope(Sb)
%RSD
)
0.352
0.227
0.129
0.318
0.112
Procedure for commercial tablets
Ten film coated tablets were bleached with methanol swab
and pulverized well. A weighed quantity of the powder
equivalent to 100 mg MEB and 25 mg of SUL (in their
pharmaceutical ratio of 4:1) was transferred into a small
conical flask and extracted with 3×30 mL of methanol. The
% Error
Sy/x, Standard deviation of the residuals; Sa, Standard deviation of the
intercept; Sb Standard deviation of the slope

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extract was filtered into a 100 mL volumetric flask. The
conical flask was washed with few milliliters of methanol.
The washings were passed into the same volumetric flask
and completed to the volume with the same solvent.
Aliquots covering the working concentration range were
transferred into 10 mL volumetric flasks. The recommen-
ded procedure under “Calibration Curve” was performed.
The nominal content of the tablets were determined either
from a previously plotted calibration graph or using the
corresponding regression equation.
Procedure for real human plasma
Three healthy volunteers (women around 35 years old)
were administered Colona® tablets after 8 h fasting.
Further, 5 mL blood samples were drawn at different time
intervals at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and
6.0 hr into tubes containing 0.5 ml of 2% EDTA solution to
prevent blood coagulation. The blood was processed to
plasma by centrifugation at 4000 rpm and 15 min. the
supernatant plasma samples transferred into test tubes and
the procedure mentioned above was followed for the
monitoring of drug concentration in plasma.
Content uniformity testing
The same procedure applied for the analysis of MEB and SUL
in tablets was followed using one tablet as a sample. Ten
tablets were analysed and the uniformity of their contents was
tested by applying the official USP [50] guidelines.
Results and discussion
Both of MEB and SUL exhibit native fluorescence with λ
maximum of 370 nm and 354 nm, after excitation at 235 nm
for MEB and SUL respectively (Fig. 2). Both the excitation
and emission spectra of MEB and SUL overlapped (Fig. 2).
This fact hindered the use of this method for the simulta-
neous determination of MEB and SUL. This problem is
aggravated if it is desired to determine these compounds in
their co-formulated preparations and biological fluids.
It was necessary to record first, the normal synchronous
spectra for MEB and SUL in order to derive the first
derivative synchronous spectra. Figure 3 shows the SF
spectra of different concentrations of MEB at 258 nm in
presence of constant concentration SUL (1.0 µg/mL),
whereas Fig. 4 illustrates the SF spectra of different
concentrations of SUL at 240 nm in presence of constant
concentration of MEB (3.2 µg/mL).
Procedure for spiked human plasma
Suitable aliquots of mixture of MEB metabolite (veratic
acid) and SUL stock solutions containing 0.5–2.0 μg/mL
and 0.05–0.10 μg/mL respectively were transferred into a
series of centrifugation tubes. 1.0 ml of human plasma was
added to each tube. Then, 0.1 mL of NaOH (1.0 M) was
added and the tubes were shaken for 1 min. The samples
were mixed well using a vortex mixer and then extracted
with 3×3 mL of ethyl acetate / dichloromethane (5:1 v/v)
by centrifugation for 5 min at 4000 rpm. The organic layer
was transferred into an evaporating dish and evaporated till
dryness. The residue was reconstituted in 3 mL methanol.
The procedure described under calibration curve was
followed. The nominal content of the drug was determined
from the previously plotted calibration graph or using the
corresponding regression equation.
Therefore the first derivative synchronous fluorescence
spectroscopy (FDSFS) technique was chosen for simulta-
neous determination of both of MEB and SUL in their
Fig. 2 Native fluorescence
spectra at pH 6.5.a, (A,A″) are
excitation and emission spectra
of SUL (1 µg/ml); b, (B,B″) are
excitation and emission spectra
of MEB (4 µg/ml) and (C,C″)
are excitation and emission
spectra of veratic acid (1 µg/ml)

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Fig. 3 Synchronous fluorescence spectra of MEB at 258 nm and
SUL. (1) a-f spectra of MEB (0.2–3.2 µg/ml); (2), spectrum of SUL
(1 µg/ml) respectively
tablets. Spectra of MEB and SUL were well separated using
FDSFS with a zero-crossing technique of measurement
(Figs. 5 and 6). Under the experimental conditions the two
peaks appeared at 238 and 264 nm for MEB and SUL
respectively. Figure 7, illustrates FDSFS for simultaneous
determination of synthetic mixture of SUL & MEB.
Fig. 5 First derivative synchronous fluorescence spectra of MEB at
238 nm and SUL, (1), a–h Spectra of MEB (0.2–3.2 µg/ml) ; (2),
Spectrum of SUL (1 µg/ml) respectively
Selection of optimum Δλ
Optimization of reaction condition
The optimum Δλ value is important for performing the
synchronous fluorescence scanning technique with regards to
its resolution, sensitivity and features. It can directly influence
Different experimental parameters affecting the performance
of the proposed method were carefully studied and optimized.
Such factors were changed individually while others were
kept constant. These factors included; Δλ selection, pH, type
of the diluting solvent, stability time and ionic strength.
Fig. 4 Synchronous fluorescence spectra of SUL at 240 nm and
MEB. (1) spectrum of MEB (3.2 µg/ml); (2), a–g spectrum of SUL
(0.05–1 µg/ml) respectively
Fig. 6 First derivative synchronous fluorescence spectra of SUL at
264 nm and MEB, (1), Spectrum of MEB (3.2 µg/ml) ; (2), a–h Spectra
of SUL (0.05–1 µg/ml) respectively

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Fig. 7 First derivative synchro-
nous fluorescence spectra of
a, SUL (0.5 µg/ml) at 264 nm.;
b, MEB (2.0 µg/ml) at 238 nm.;
c, synthetic mixture of SUL
(0.5 µg/ml) and MEB
(2.0 µg/ml)
synchronous spectral shape, band width and signal value. For
this reason a wide range of Δλ (20, 40, 60, 80, 100 and
120 nm) was examined. When Δλ was less than 100 nm, the
spectra shapes were irregular with weak fluorescence inten-
sities and noisy. On the other hand, when Δλ was more than
100 nm, overlapping of the two peaks with poor separation
was achieved. Therefore, Δλ of 100 was chosen as optimal for
separation of MEB and SUL mixtures, since it resulted in two
distinct peaks with good regular shapes and reduced the
spectral interference caused by each compound in the mixture.
(THF) and dimethyl formamide (DMF) was employed. Each
of DMSO, DMF and THF decrease the fluorescence
intensities of both compounds. This may be attributed to
heavy atom effect and inter system crossing process. Of the all
tested solvents water gave the highest synchronous fluores-
cence intensities compared with the other solvents, more over
its use adds another advantage of the method. Thus, water was
chosen as the diluting solvent through out the study.
Effect of time
Selection of optimum pH
The effect of time on the stability of the synchronous
fluorescence intensity of the drugs was also studied. It was
found that the fluorescence intensity developed instante-
ously and remained stable for more than 2 h.
The influence of pH on the synchronous fluorescence
intensities of the two drugs was studied using different
buffers covering the whole pH range, e.g. acetate buffer
over the pH range of 3.6–5.6 and borate buffer over the pH
range 5.5–13. The synchronous fluorescence intensity of
MEB and SUL is increased gradually upon increasing the
pH values up to 6.4 and remained constan until pH6.6,
further increase in pH resulted in a gradual decrease in the
synchronous fluorescence intensities, after which it ex-
tremely decreased at pH 13 (Fig. 8). Therefore, borate
buffer of pH 6.5 was used throughout the study.
Analytical performance
The first derivative synchronous fluorescence spectroscopy
(FDSFS)—concentration plots for the two drugs were linear
700
Sulpirid
b
Effect of volume of buffer
600
Mebeverine
500
Increasing volume of borate buffer (pH 6.5) resulted in a
gradual increase in the synchronous fluorescence intensities
of MEB and SUL up to 2 mL after which the fluorescence
intensities remained constant till 4 mL. Therefore, 2 mL of
borate buffer (pH 6.5) was chosen as the optimum buffer
volume throughout the study.
a
400
300
200
100
0
0.0
2.5
5.0
7.5
pH
10.0
12.5
15.0
Effect of diluting solvent
Dilution with different solvents including water, methanol,
isopropanol, dimethyl sulfoxide (DMSO), tetrahydrofurane
Fig. 8 Effect of pH on the synchronous fluorescence intensity. a,
MEB(2 µg/ml), b, SUL (1 µg/ml)

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Table 2 Application of the first
derivative synchronous fluorim-
etry to the determination of the
studied drugs in the pure form
Parameter
1-MEB
Concentration
taken(µg/ml)
Concentration
found (µg/ml)
% Found
Reference
method [37]
0.2
0.4
0.8
1.2
1.6
2.0
2.4
3.2
0.200
0.400
0.797
1.206
1.603
1.998
2.386
3.209
100.00
100.00
99.60
100.46
99.53
100.15
100.53
100.17
99.89
99.43
100.27
100.07
0.318
X’
100.04
0.473
S.D.
t-test
F-value
2-SUL
0.126 (1.833)
1.48 (4.74)
0.0499
0.099
0.05
0.1
0.2
0.4
0.5
0.6
0.8
1.0
99.37
99.23
99.37
99.49
100.36
100.79
100.25
99.58
99.76
0.645
100.9
99.0
0.199
100.3
0.398
0.502
0.605
0.802
0.996
X’
100.0
0.92
S.D.
t-test
F-value
0.470 (1.833)
2.266 (4.74)
Figures between parenthesis are
the tabulated t and F values,
respectively at p=0.05 (52)
1
over the concentration range cited in Table 1. Linear
regression analysis of the data gave the following equations:
Where D is the first derivative synchronous fluores-
cence spectroscopy, C is the concentration of the drug
(μg/mL) and r is correlation coefficient. The limits of
quantification (LOQ) was calculated according to ICH
Q2B recommendations [51]. The limits of detection (LOD)
was also calculated according to ICH Q2B recommenda-
tions [51]. The results of LOD and LOQ of MEB and SUL
respectively are abridged in Table 1.
1D ¼ 16:5 þ 39:58C ðr ¼ 0:9999Þ;
for MEB at 238 nm With Sa ¼ 0:227 and Sb ¼ 0:129
1D ¼ ꢀ 0:2 þ 140:9C ðr ¼ 0:9999Þ;
for SUL at 264 nm With Sa ¼ 0:262 and Sb ¼ 0:472
Table 3 Application of the proposed method for determination of the studied drugs in their synthetic mixtures
Sample
Concentration taken (µg/ml)
MEB SUL
Concentration found (µg/ml)
MEB SUL
0.796
Recovery %
MEB
SUL
98.54
MEB and SUL mixture
0.8
1.6
2.0
2.4
0.2
0.4
0.5
0.6
0.197
0.403
99.49
100.75
99.81
99.85
99.97
0.541
0.541
0.270
1.612
1.996
2.396
100.83
100.91
99.16
99.85
1.19
05.05
0.595
X’
S.D.
%RSD
%Error
1.19
0.595
Each result is the average of three separate determinations

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Table 4 Validation of the proposed method for determination of MEB
and SUL raw materials using FDSF mode
using Student’s t-test and variance ratio F-test, shows no
significant difference between the performance of the two
methods regarding the accuracy and precision, respectively
(Table 2). The reference method is based on HPLC
separation of the two drugs on a reversed phase, Bondapak
CN column and UV detection was done at 243 nm using
buclizine hydrochloride as internal standard [37].
Concentration added (µg/ml) % recovery
% RSD % Error
MEB
Intra-day
0.8
1.2
101.00 1.6
1.58
0.61
0.32
0.91
0.35
0.18
101.20 0.624
99.77 0.320
2.0
Analysis of synthetic mixture of MEB and SUL
Inter-day
0.8
99.10 0.9
100.06 1.10
99.96 0.35
0.90
1.09
0.35
0.52
0.63
0.20
The proposed method was applied for the simultaneous
determination of MEB with SUL in synthetic mixtures
containing different concentrations of both drugs in a ratio
of 4:1 (Fig. 7). The relative fluorescence intensities of first
derivative technique were measured for both drugs. The
first derivative signal of MEB was measured at 238 nm
which is considered as zero crossing point for SUL and the
first derivative signal for SUL was measured at 264 nm
which is the zero crossing point for MEB. The concen-
trations of both drugs in the synthetic mixture were
calculated according to their linear regression equation of
the calibration graphs. The results indicate high accuracy of
the proposed method as shown in Table 3.
1.2
2.0
SUL
Intra-day
0.2
97.58 1.62
99.98 0.38
1.66
0.38
0.45
0.95
0.21
0.26
0.3
0.5
100.72 0.461
Inter-day
0.2
97.26 1.55
98.30 1.47
102.0 0.624
1.59
1.49
0.61
0.92
0.86
0.35
0.3
0.5
LOQ and LOD were calculated according to the
following equations [51]:
Validation of the method
LOQ ¼ 10s=S
LOD ¼ 3:3s=S
The validity of the method was tested regarding; linearity,
specificity, accuracy, repeatability and precision according
to ICH Q2B recommendations [51].
Where, σ is the standard deviation of the intercept of
regression line and S is the slope of regression line of the
calibration curve. The proposed method was evaluated by
studying the accuracy as percent relative error and precision
as percent relative standard deviation. The results are
abridged in Table 1. Statistical analysis [52] of the results,
obtained by the proposed and the reference method [37]
Linearity
The regression plots showed a linear dependence of 1D
values on drug Concentration(µg/ml) over the range cited in
Table 1.
Table 5 Application of the proposed method for determination of the studied drugs in their co-formulated preparations
Preparation
Concentration
taken (µg/ml)
Concentration
found (µg/ml)
Recovery %
MEB
MEB
SUL
MEB
SUL
SUL
Colona® tabletsa (MEB 100 mg+SUL 25 mg/ tab.) Batch # 09425.
0.5
1.0
1.5
2.0
0.125
0.250
0.375
0.500
0.500
0.991
1.520
1.989
0.126
0.249
0.374
0.500
100.0
99.12
101.33
99.47
99.97
0.946
0.940
0.473
100.52
99.66
99.94
100.00
100.03
0.360
0.350
0.179
X`
S.D.
%RSD
%Error
a
Each result is the average of three separate determinations. Product of Rameda Co. for Pharmaceutical Industries and Diagnostic Reagents 6th of
October City. Egypt

J Fluoresc (2010) 20:1275–1285
1283
Table 6 Content uniformity testing of MEB and SUL in co-
formulated tablets using the proposed method
Intermediate precision
Intermediate precision was performed through replicate
analysis of MEB and SUL in pure form. The results are
shown in Table 4, for a period of 3 successive days.
Parameter
Percentage of the label claim in
Colona® tablets
MEB
SUL
Robustness of the method
98.90
100.70
100.50
101.00
102.10
99.89
99.46
100.58
100.90
100.00
98.8
99.30
102.10
101.90
100.90
100.50
102.80
102.20
100.20
100.40
101.17
1.12
The robustness of the proposed method is demonstrated by
the constancy of the synchronous fluorescence intensities
with the deliberated changes in the experimental parameters
such as pH, 6.5 0.1 for MEB and SUL respectively and
change volume of borate buffer 2 ml 1. These minor
changes that may take place during the experimental
operation didn’t greatly affect the fluorescence intensity of
the mixture.
Data
X−
100.20
1.00
%RSD
Pharmaceutical applications
%Error
0.31
0.35
Acceptance value (AV) (50)
Max. allowed AV (L1) (50)
2.42
2.71
Selectivity
15.00
The proposed method was applied for the determination of
the studied drugs in their co-formulated tablets. The
specificity of the method was investigated by observing
any interference encountered from the common tablet
excepients, such as lactose, gelatin, magnesium stearate
and starch. These excepients did not interfere with the
proposed method (Table 5).
Accuracy
The proposed methods were applied for the determination
of authentic sample of MEB and SUL over the concentra-
tion range cited in Table 2 in order to determine their
accuracy. The results obtained were in good agreement with
those obtained using reference method [37]. Using the
Student’s t-test and the variance ratio F-test, [52] revealed
no significant difference between the performance of the
two methods regarding the accuracy and precision,
respectively (Table 2). The validity of the methods was
proved by statistical evaluation of the regression lines,
using the standard deviation of the residuals (Sy/x), the
standard deviation of the intercept (Sa) and standard
deviation of the slope (Sb). The results are abridged in
Table 1. The small values of the figures point out to the
low scattering of the points around the calibration lines
and high precision.
Table 7 Application of the proposed method for determination of the
studied drugs in spiked and real human plasma
Sample
Concentration Concentration Recovery %
taken (µg/ml) found (µg/ml)
Veratic SUL Veratic SUL Veratic SUL
acid
acid
acid
1-Spiked plasma
sample
0.5
0.2
0.5
1.0
0.49
1.03
1.99
0.19
0.52
0.99
97.46
92.54
104.95
99.06
98.84
6.20
1.0
2.0
102.5
99.5
99.82
2.53
2.53
1.46
X`
S.D.
%RSD
%Error
6.27
Precision
3.5
2-Real plasma sample
1.0
Repeatability
0.22 0.870
0.22 0.920
0.215
0.180
87.00
92.00
95.47
91.49
4.25
97.9
81.8
94.4
91.36
8.46
9.26
5.34
1.0
1.0
The repeatability was evaluated by applying the proposed
method for the determination of two concentrations of
MEB and SUL in pure forms on three successive times, and
the results are abridged in Table 4. The low %Error and low
% RSD indicates high accuracy and high precision of the
proposed method respectively.
0.22 0.9547 0.207
Xˉ
S.D.
%RSD
%Error
4.64
2.68

1284
J Fluoresc (2010) 20:1275–1285
Biological applications
The high sensitivity of the proposed method allowed the
determination of MEB metabolite and SUL in biological fluids
by SDSFS method. The method was further applied to the in-
vivo determination of both drugs in real human plasma.
Sulpiride is absorbed from gastro-intestinal tract. Following
oral ingestion of a single oral dose of 50 mg of SUL, a peak
plasma concentration of 0.03–0.60 mg L−1 (mean
0.18 mg L−1) is attained in about 4–7 h [48]. This value lies
within the working concentration range of the proposed
method.
After oral administration of mebeverine HCI (270 mg) to
fasted human volunteers, measurable concentrations of the
drug were not found in plasma. By contrast, the metabolite
veratric acid achieved considerable concentrations (mean
peak plasma concentration of 13.5 µg/mL at (40–80 min)
[53]. Veratic acid (MEB metabolite) is also highly
fluorescent compound and the second metabolite is
mebeverinic alcohol which is non-fluorescent at the
studied pH value (6.5) [54]. However, after oral
administration of mebeverine HCI (2 mg), only traces
of mebeverine were found in plasma. Veratric acid again
achieved considerable concentrations (mean peak plasma
concentration of 0.90 µg/mL at (15 min-4 h). The
results show that rnebeverine undergoes rapid and
extensive first-pass metabolism involving hydrolysis of
the ester function, and that negligible circulating con-
centrations of the parent drug are found in humans [53].
Fig. 9 (a) plasma blank, (b) Second derivative synchronous fluorescence
spectrum of 1 µg/ml of (MEB metabolite) veratic acid at 253 nm and
0.022 µg/ml of SUL at 264 nm in real human plasma at 3.5 h
Content uniformity testing
Owing to the high precision of the proposed method and its
suitability for analysis of the studied drugs in their dosage
forms with sufficient accuracy, the method is ideally suited
for content uniformity testing which is time consuming
process when using conventional assay techniques. The
steps of the test were adopted according to the commer-
cially available tablets and it was found to be smaller than
the maximum allowed acceptance value (L1). The results
demonstrated good drug uniformity as shown in (Table 6).
Scheme Chemical structure
of mebeverine (1) and its
hydrolysis products veratric acid
(3,4-dimethoxybenzoic acid;(2)
and mebeverinic alcohol
(4-[ethyl-(2-[4-methoxyphenyl]—
l-methylethyl)amino] butanol;(3)
The second derivative signal of veratic acid and SUL—
concentration plot in spiked human plasma was applied
over the working concentration. Linear regression analysis
of the results gave the following equations:
2D is Second derivative synchronous fluorescence
spectroscopy (SDSFS), C is concentration in µg |mL and
r is the correlation coefficient. The (SDSFS) is specific for
the determination of veratic acid (MEB metabolite) at
254 nm and SUL at 264 nm. The proposed method was
applied to human plasma by studying the accuracy as
percent relative error and precision as relative standard
2D ¼ 18:5 þ 39:9Cðr ¼ 0:9991Þfor veratic acid:
2D ¼ 1:35 þ 110:5Cðr ¼ 0:9985Þfor SUL respectively:

J Fluoresc (2010) 20:1275–1285
1285
deviation. The results are abridged in Table 7. Figure 9
illustrates the second derivative synchronous fluorescence
spectroscopy (SDSFS) in real human plasma.
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Conclusion
A new simple and sensitive method was explored for the
simultaneous determination of MEB and SUL in their
combined tablets. The first derivative synchronous spectro-
fluorimetric method, by virtue of its high sensitivity, could be
applied to the analysis of both drugs in their co-formulated
dosage forms and biological fluids. It was possible to measure
concentrations as low as 0.01 and 0.006 µg/mL for MEB and
SUL respectively with good accuracy Moreover, first deriv-
ative spectrofluorimetric technique enables the determination
of MEB in the presence of SUL by applying the zero-crossing
technique in the spectra without prior separation steps in their
pure form. Also, the proposed method allowed therapeutic
monitoring of MEB metabolite (veratic acid) in real human
plasma without interference with SUL. Moreover, the
proposed method is simple, time saving and other advantage
of the method is the use of low cost reagent.
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