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Research article
Received: 25 June 2014,
Revised: 7 November 2014,
Accepted: 16 November 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2828
Micelle-enhanced spectroﬂuorimetric
determination of amlexanox in bioadhesive
buccal tablets: application to content
uniformity testing
M. I. Walash, F. Belal, M. M. Tolba and M. I. Halawa*
ABSTRACT: A highly sensitive, simple and rapid spectroﬂuorimetric method was developed for the determination of
Amlexanox (AMX) in its bioadhesive buccal tablets. The proposed method is based on measuring the native ﬂuorescence
of the methanolic solution of AMX at 400 nm after excitation at 242 nm in 0.2 M borate buffer (pH 10) and 0.5% w/v sodium
dodecyl sulfate (SDS) solution. The interaction of AMX with SDS was studied, and the enhanced ﬂuorescence intensity was
exploited to develop an assay method for the determination of AMX. The relative ﬂuorescence intensity–concentration plot
was rectilinear over the range 5.0–80.0 ng/mL, with a lower detection limit of 0.57 ng/mL and a lower quantiﬁcation limit of
1.74 ng/mL. The proposed method was successfully applied to the analysis of AMX in its commercial tablets. Moreover, content uniformity testing was conducted by applying ofﬁcial USP guidelines. Statistical evaluation and comparison of the data
obtained using the proposed and comparison methods revealed good accuracy and precision for the proposed method.
Copyright © 2015 John Wiley & Sons, Ltd.
Keywords: Amlexanox; spectroﬂuorimetric; bioadhesive buccal tablets; content uniformity testing
Introduction
Amlexanox is a novel anti-inﬂammatory and anti-allergic agent.
Clinically, it is the only drug approved by Food and Drug Administration (FDA) for the treatment of aphthous ulcers (1). AMX
effectively treats aphthous ulcers by accelerating healing and
complete pain resolution. It potently inhibits the release of histamine and leukotrienes from mast cells, basophils and neutrophils
under in vitro settings, possibly by increasing the intracellular
cyclic AMP content of inﬂammatory cells, a membrane-stabilizing
effect or inhibition of calcium inﬂux (1,2).
Chemically, AMX is 2-amino-7-isopropyl-5-oxo-5H-[1]benzopyrano
[2,3-b]pyridine-3-carboxylic acid (Figure 1). It is commercially available as a 5% oral paste, bioadhesive buccal tablets and as a biodegradable muco-adhesive disc (1).
A review of the literature revealed that few HPLC methods
have been developed for the analysis of AMX in bulk drugs (3)
or human serum (4) or in its paste in the presence of related substances (5). Also, AMX was determined simultaneously with
cromolyn and tranilast using the HPLC method (6). Nothing
has been reported in the literature regarding the determination
of AMX in its bioadhesive buccal tablets. In addition, no
spectroﬂuorimetric method has been described for the determination of AMX in its tablets and this initiated the present study.
Based on micellar enhancement of the native ﬂuorescence intensity of AMX, a simple and highly sensitive spectroﬂuorimetric
method has been developed for determination of AMX in its tablets. The use of surfactants in various areas of analytical chemistry has attracted much interest in the last three decades. It has
been used in separation science as a modiﬁer of mobile and stationary phases (7). Micellar catalysis has been used to enhance
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several chromogenic derivatization reactions by the simultaneous alteration of various physical properties of the solution
and physicochemical properties of the reagents, intermediates
and products (8).
The use of micellar media is the most commonly applied
method for increasing the ﬂuorescence intensity of target
ﬂuorophores and consequently, for lowing their limit of detection (LOD) (9). When a solute passes from an aqueous medium
to a micellar medium, several properties (e.g. reactivity, solubility
or spectroscopic characteristics) undergo important changes, increasing ﬂuorescence intensity being the most outstanding and
advantageous (10). In general, micellar media provide a very
rigid microenvironment capable of restricting the freedom of
ﬂuorophores, and so diminish the probabilities of non-radiative
processes and provide relatively high viscous microenvironments that can inhibit quenching by molecular oxygen. These
factors might increase the ﬂuorescence quantum yield and enhance the ﬂuorescence signals of guest molecules (9).
Sodium dodecyl sulfate (SDS), also known as sodium lauryl
sulfate (C12H25SO4Na), is an anionic surfactant that has recently
found many applications in different areas of analytical chemistry. The critical micelle concentration (CMC) of SDS in pure water
at 25 °C is 8.2 × 10-3 M and the aggregation number at this concentration is usually considered to be 62 (11). SDS has been used
* Correspondence to: M. I. Halawa, Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516 Mansoura, Egypt. E-mail:
[email protected]
Department of Analytical Chemistry, University of Mansoura, Mansoura,
Egypt
Copyright © 2015 John Wiley & Sons, Ltd.
M. I. Walash et al.
Procedures
Figure 1. Chemical formula of AMX.
as a ﬂuorescence enhancer for the determination of some
pharmaceutical compounds viz., verapamil hydrochloride (12),
citalopram (13), labetalol (14), gatiﬂoxacin (15), levoﬂoxacin
(16), histamine (17) and epinephrine (18).
The method developed here is simple, timesaving and does
not require elaborate treatment compared with the reported
chromatographic method (5). In addition, content uniformity
testing was conducted according to USP guidelines (19).
Experimental
Instruments
All ﬂuorescence measurements were made using an RF-1501
Shimadzu spectroﬂuorometer, equipped with a 150 W xenon
arc lamp (Kyoto, Japan). The excitation and emission wavelengths were 242 and 400 nm, respectively. The slit widths were
5 nm for both excitation and emission wavelengths, and the
photomultiplier voltage was set to auto. A 1 cm quartz cuvette
was used. A Consort NV P-901 pH meter (Belgium, Europe) was
used for pH measurements.
Construction of the calibration graph. Aliquots of methanolic
AMX standard solution were transferred to a series of 10 ml volumetric ﬂasks to give ﬁnal concentrations of 5.0–80.0 ng/mL.
One milliliter of 0.2 M borate buffer (pH 10) was added to each
ﬂask followed by 0.6 ml of 0.5% w/v SDS solution. The volume
was completed with methanol, the contents of the ﬂasks were
mixed well and the ﬂuorescence intensity was measured at
400 nm after excitation at 242 nm. Relative ﬂuorescence intensity (RFI) was plotted against ﬁnal drug concentration (ng/mL)
to give a calibration graph. Alternatively, the corresponding regression equation was derived.
Twenty Aphthtab® tablets
were crushed and a weight equivalent to 10.0 mg AMX was
transferred quantitively into a 100 ml volumetric ﬂask, ~ 80 ml
of methanol was added and the ﬂasks were sonicated for
30 min. Dissolution of the tablets was enhanced with the aid of
a magnetic stirrer. The solutions were then diluted to volume
with methanol, mixed and ﬁltered. Aliquots covering a working
concentration range of 5.0–80.0 ng/mL were transferred into
10 ml volumetric ﬂasks. The procedure described under ‘Construction of the calibration graph’ was performed. The nominal
contents of the tablets were calculated using the calibration
graph or the corresponding regression equation.
Analysis of AMX in aphthtab® tablets.
Content uniformity testing of aphthtab tablets. Ten different tablets were analyzed using the procedure applied to the analysis of
the studied compound in tablets. The uniformity of the tablet
contents was tested by applying the ofﬁcial USP guideline [see
Chapter 905 (19)].
Reagents and materials
Results and discussion
All chemicals used were of analytical reagents (AR) grade, and
the solvents were HPLC grade. AMX lot # 080112G (certiﬁed to
have a purity of 98%) was purchased from AK Scientiﬁc, Inc.
(Union City, CA, USA) and was used as received without further
puriﬁcation. Aphthtab® tablets labeled as containing 2 mg AMX
per tablet (batch # 203369) were from EVA Pharma for Pharmaceuticals and Medical Appliances (Giza, Egypt).
The following surfactants were used as 0.5% (w/v) aqueous
solutions: SDS (95%) and cetyl trimethylammonium bromide
(CTAB; 99%) (London, England); β-cyclodextrin (β-CD) and hydroxy propyl-β-cyclodextrin (HP-β-CD) (Darmstadt, Germany);
carboxymethylcellulose (El-Nasr Pharmaceutical Chemicals Co.,
Cairo, Egypt); Tween-80 (El-Nasr Pharmaceutical Chemicals Co.);
methanol (Paris, France), n-propanol and acetonitrile (SigmaAldrich Chemie GmbH, Germany); dimethyl sulfoxide (RiedeldeHäen, Sleeze, Germany); glacial acetic acid, sodium acetate
trihydrate, boric acid, sodium hydroxide and dimethyl formamide (El-Nasr Pharmaceutical Chemical Co.). Acetate buffer
(0.2 M, pH 3.5–5.5) and borate buffer (0.2 M, pH 6.5–10.5) solutions were freshly prepared.
AMX is formulated in a very minute amount in tablets
(2 mg/tablet); therefore, there is an urgent need to explore a sufﬁciently sensitive and speciﬁc method for its determination in
tablets. Fluorimetric analysis, by virtue of its high inherent sensitivity, might successfully overcome this problem. A methanolic
solution of AMX was found to exhibit three emission bands of
different intensities at 400 nm, after excitation at 242, 281 or
350 nm. The highest sensitivity was achieved upon using
242 nm as the excitation wavelength, and so this value was selected. It is well known that, in many cases, the addition of a surfactant at a concentration above its CMC to a given ﬂuorophore
solution increases its molar absorbtivity and/or the ﬂuorescence
quantum yield (20,21). This has been used to improve the performance of spectroﬂuorimetric methods for various analytes. The
ﬂuorescence properties of AMX in various micellar media were
studied; there was signiﬁcant enhancement of the ﬂuorescence
intensity in the presence of SDS compared with a methanolic
solution, therefore SDS was used as a ﬂuorescence enhancer to
develop a new sensitive spectroﬂuorimetric method for the determination of AMX.
Standard solutions
Fluorescence spectra of AMX in methanolic solution and in
the SDS system
Stock solution equivalent to μg/mL of AMX was prepared by
dissolving 10.0 mg of the drug in 100 ml of methanol. This solution was further diluted using the same solvent as appropriate.
Standard solutions were stable for 10 days when kept in the
refrigerator.
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The ﬂuorescence spectra of AMX in both the methanolic and SDS
systems were studied. Figure 2 illustrates the ﬂuorescence spectra of AMX in the two systems. The ﬁrst system was methanolic
borate buffer (pH 10) and the second was the same solution
Copyright © 2015 John Wiley & Sons, Ltd.
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Spectroﬂuorimetric determination of amlexanox
a
b
Figure 2. (a) Fluorescence spectra of: (A, B) AMX (60 ng/mL) in methanolic borate buffer (pH 10). Where: A = excitation band; B1 = emission band at λex = 242 nm;
B2 = emission band at λex = 281 nm; B3 = emission band at λex = 350 nm. (b) Fluorescence spectra of: (A, B) AMX (60 ng/mL) in methanolic borate buffer (pH 10) (SDS system); (A′, B′) blank. Where: A, A′ = excitation spectra; B, B′ = emission spectra.
but in the presence of SDS as a ﬂuorescence enhancer. The percentage ﬂuorescence enhancement in the presence of SDS was
signiﬁcant when compared with the native ﬂuorescence intensity of the drug in a methanolic medium.
Optimization of the experimental conditions
Effect of organized media. The effect of various organized
media on the ﬂuorescence intensity of AMX was studied by adding
1.0 ml of a 0.5% (w/v) aqueous solution of each to the drug solution. Different surfactants such as SDS (anionic surfactant), CTAB
(cationic surfactant) and Tween-80 (nonionic surfactant) and also
macromolecules like β-CD and HP-β-CD were studied.
All the organized media studied caused a slight decrease in
the RFI of the drug (Fig. 3). Only SDS gave a considerable increase in the RFI, so it was used throughout the present study.
Effect of the volume of SDS. The inﬂuence of SDS on the RFI
was studied using increasing volumes of 0.5% w/v SDS aqueous
solution. It was found that increasing volumes of SDS solution
resulted in a corresponding increase in RFI up to 0.4 ml, after
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Figure 3. Effect of different organized media (1 ml of a 0.5% w/v solution of each)
on the RFI of AMX (60 ng/mL).
Copyright © 2015 John Wiley & Sons, Ltd.
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M. I. Walash et al.
which no further increase in RFI was obtained. Therefore, 0.6 ml
of a 0.5% w/v SDS solution was chosen as the optimum volume
for AMX (Fig. 4). An SDS volume > 1 ml was not used because of
a high blank reading.
Effect of pH. The inﬂuence of pH on the micelle-enhanced
ﬂuorescence of AMX was studied using different types of buffers
covering the whole pH range, for example, 0.2 M acetate buffer
over the pH range 3.5–5.5 and 0.2 M borate buffer over the pH
range 6.5–10.5. It was found that maximum and constant RFI
was achieved over a pH range of 9.5–10.5; therefore, pH 10
was chosen as the optimum value for the study. Use of 0.2 M acetate buffer caused a gradual decrease in the RFI of AMX (Fig. 5).
Effect of diluting solvent. The effect of dilution with different
solvents such as distilled water, methanol, acetonitrile, n-propanol,
dimethyl sulfoxide and dimethyl formamide was studied. It was
found that methanol was the best solvent for dilution, as it gave
the highest RFI and lowest blank reading Table 1. A distinct and
sharp decrease in RFI was observed on using acetonitrile or
n-propanol. Addition of these solvents may result in a reduction
in the size of the micelles and a progressive breakdown of the
surfactant aggregate at very high concentrations (22). Both
dimethyl sulfoxide and dimethyl formamide gave high blank readings and greatly quenched the ﬂuorescence intensities of AMX
because they exhibited an intersystem-crossing process (similar
to the heavy atom effect) (23).
Table 1. Effect of diluting solvents on the ﬂuorescence intensity (FI) of AMX (60 ng/mL)
Solvent
FI
Water
Methanol
n-propanol
Acetonitrile
Dimethyl sulfoxide
Dimethyl formamide
420
660
365
340
135
50
Effect of time. The effect of time on the RFI of the drug was
also studied. It was found that the ﬂuorescence intensity developed immediately and remained stable for more than 2 h.
Effect of temperature. Another factor that may affect the
ﬂuorescence intensity is temperature. The effect of temperature
was studied in the range 40–100 °C in a thermostatically controlled water bath. It was found that increasing the temperature
resulted in a decrease in the RFI. This can be explained by higher
internal conversion as the temperature increases, facilitating
nonradiative deactivation of the excited singlet state and an increase in the loss of energy via collision with solvent molecules
(24). Therefore, all experiments were carried out at room
temperature.
Method validation
The validity of proposed method was evaluated regarding linearity, limit of detection (LOD), limit of quantiﬁcation (LOQ), accuracy, precision, robustness and speciﬁcity.
Linearity and range. The calibration graph for the determination of AMX using the proposed method was constructed
by plotting the RFI against the concentration of the drug
(ng/mL). The calibration graph showed a linear dependence
of RFI on drug concentration over a range of 5.0–80.0 ng/mL
for AMX. Linear regression analysis of the data gave the
following equation:
RFI ¼ 44:73 þ 9:82C
Figure 4. Effect of the volume of SDS (0.5% w/v) on the RFI of AMX (60 ng/mL).
Figure 5. Effect of pH on the RFI of AMX (60 ng/mL).
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Where RFI is the relative ﬂuorescence intensity and C is the
concentration of the drug in ng/mL for AMX.
Statistical analysis (25) of the data gave high values for the
correlation coefﬁcients (r) of the regression equations, small for
the standard deviation of residuals (Sy/x), of intercept (Sa) and
of slope (Sb), and small value for the percentage relative standard deviation (%RSD) and the percentage relative error Table 2.
These data proved the linearity of the calibration graph of the
studied drug.
LOQ and LOD. LOQ and LOD were calculated according to the
ICH Q2 (R1) recommendations (26). LOQ values were determined by establishing the measurable concentrations below
which the calibration graph is nonlinear. LOD values were determined by evaluating the lowest analyte concentrations that can
be readily detected. The results are also summarized in Table 2.
The values of LOQ and LOD were calculated according to the following equations (26):
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015
Spectroﬂuorimetric determination of amlexanox
signiﬁcant difference between the performance of the methods
regarding accuracy and precision Tables 3 and 4. The comparison
HPLC method involved the use of a mobile phase consisting of
methanol : 0.01 M phosphate buffer (70:30, v/v; pH 4.8) and an
Octadecylsilane (ODS) column with UV detection at 350 nm (5).
Table 2. Analytical performance data for the
spectroﬂuorimetric determination of AMX
Parameter
AMX
Wavelength [λex/λem] (nm)
Concentration (ng/mL)
Intercept (a)
Slope (b)
Correlation coefﬁcient (r)
SD of residuals (Sy/x)
SD of intercept (Sa)
SD of slope (Sb)
% RSD
LOD (ng/mL)
LOQ (ng/mL)
242/400
5.0–80.0
44.73
9.82
0.9999
2.64
1.70
0.04
0.80
0.57
1.74
Precision. Intraday precision was evaluated by determining
three concentrations of AMX in its pure form on three successive
occasions over the course of one day. Interday precision was
also evaluated by replicate analysis of three concentrations over
a period of three successive days. The results of intraday and
interday precision are summarized in Table 4.
Robustness of the method. The robustness of the adopted
method was demonstrated by the constancy of the RFI with minor changes in the experimental conditions, such as a change in
the pH (10 ± 0.2) and SDS volume (0.6 ± 0.2 ml). These minor
changes that may occur during the experimental operation did
not affect the RFI.
LOQ ¼ 10Sa =b
LOD ¼ 3:3Sa =b
Where Sa is the standard deviation of the intercept of the regression line and b is the slope of the calibration graph.
Accuracy. To prove the accuracy of the proposed method, the
results of the drug assay were compared with those obtained
using a comparison HPLC method (5). Statistical analysis (25) of
the results obtained from the proposed and comparison methods
using Student’s t-test and variance ratio F-test showed no
Speciﬁcity. The speciﬁcity of the method was investigated by
observing any interference encountered from common tablet
excipients. It was shown that these compounds did not interfere
with the results of the proposed method (Table 3).
Pharmaceutical applications
Application of the proposed method to the analysis of AMX
in bioadhesive buccal tablets. The proposed method was
successfully applied to the assay of AMX in its conventional tablets. Percent recoveries of different concentrations were based
Table 3. Assay results for the determination of AMX in raw material and its bioadhesive buccal tablets using the proposed and
comparison methods
Compound
Amount taken
(ng/mL)
AMX
Mean ± SD
% RSD
t
F
Aphthtab tabletsb
(AMX 2 mg/
tablet)
Mean ± SD
t
F
Comparison method[5]
Proposed method
5.0
10.0
20.0
30.0
40.0
50.0
60.0
80.0
20.00
40.00
60.00
Amount found
(ng/mL)
4.915
10.006
19.883
29.963
40.247
50.430
59.594
79.958
19.776
39.792
60.120
% Found
Amount taken
(μg/mL)
98.31
100.07
99.42
99.88
100.62
100.86
99.32
99.95
99.80 ± 0.80
0.80
0.89 (2.26)
1.17 (4.74)
98.88
99.48
100.20
99.52 ± 0.66
1.05 (2.78)
4.53 (19.0)
6.0
8.0
10.0
Amount found
(μg/mL)
5.9670
8.0200
10.118
% Found
99.45
100.25
101.18
100.29 ± 0.87
0.86
6.0
8.0
10.0
5.949
8.156
10.030
99.15
101.95
100.30
100.47 ± 1.41
a
Average of three separate determinations.
Labeled to contain 2 mg AMX/tablet (batch # 203369), product of EVA Pharma for Pharmaceuticals and Medical Appliances, Giza,
Egypt.
c
Values in parentheses are the tabulated t and F values at P = 0.05 (25).
b
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M. I. Walash et al.
Table 4. Precision data for the determination of AMX using the proposed method
Parameter
AMX (ng/mL)
10.0
Intra-day
% Found
Inter-day
Mean (SD)
% RSD
% Found
Mean (SD)
% RSD
100.35
101.20
99.45
100.33 (0.88)
0.87
100.48
98.78
101.82
100.36 (1.53)
1.52
Table 5. Results of content uniformity testing of AMX tablets using the proposed method
Parameter
Data
Mean (SD)
% RSD
% Error
Acceptance value (AV) (19)
Max. allowed AV (L1) (19)
Tablet no.a
1
2
3
4
5
6
7
8
9
10
Percentage of the
label claim
98.85
100.72
101.45
101.35
99.64
100.18
99.28
97.92
102.55
101.98
100.39 (1.48)
1.47
0.47
3.55
15.0
a
Aphthtab tablets: labeled to contain 2 mg AMX per tablet
(batch # 203369), product of EVA Pharma for Pharmaceuticals and Medical Appliances, Giza, Egypt.
on the average of three replicate determinations. The results
were in good agreement with those obtained using the comparison HPLC method (5).
Content uniformity testing for tablets. Because of the high
sensitivity of the proposed method and its ability to rapidly measure the ﬂuorescence intensity of a single tablet extract with sufﬁcient accuracy, it is ideally suited for content uniformity testing,
which is a time-consuming process when using conventional assay techniques. The steps of the test were adopted according to
the USP (19) procedure. The acceptance value (AV) was calculated and found to be smaller than the maximum allowed AV
(L1). The results demonstrated excellent drug uniformity, as
shown in Table 5.
Conclusion
A simple and sensitive spectroﬂuorimetric method was developed for the determination of AMX through enhancement of
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20.0
99.58
101.45
101.10
100.71 (0.99)
0.99
99.30
101.55
99.76
100.20 (1.19)
1.19
40.0
99.82
100.45
99.30
99.86 (0.58)
0.58
98.60
99.95
101.20
99.92 (1.30)
1.30
its native ﬂuorescence upon using SDS. The proposed method
is rapid, time-saving and does not require elaborate treatment
compared with the reported chromatographic methods. By virtue of its simplicity and sensitivity, the proposed method can
be applied to the analysis of Amlexanox for the ﬁrst time in its
bioadhesive buccal tablets, and moreover, in quality control laboratories. The proposed method is suitable for use in content
uniformity testing.
Acknowledgements
The authors extend their appreciation to Analytical Chemistry
Department Mansoura University for providing instruments and
chemicals. Also Ak Scientiﬁc company and EVA Pharma company
for kindly providing pure powder and tablets of amlexanox.
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