Spectrofluorimetric determination of 3-methylflavone-8-carboxylic acid, the main active metabolite of flavoxate hydrochloride in human urine

Article abstract of DOI:10.1016/j.saa.2014.06.058

A simple, sensitive and selective spectrofluorimetric method has been developed for the determination of 3-methylflavone-8-carboxylic acid as the main active metabolite of flavoxate hydrochloride in human urine. The proposed method was based on the measurement of the native fluorescence of the metabolite in methanol at an emission wavelength 390 nm, upon excitation at 338 nm. Moreover, the urinary excretion pattern has been calculated using the proposed method. Taking the advantage that 3-methylflavone-8-carboxylic acid is also the alkaline degradate, the proposed method was applied to in vitro determination of flavoxate hydrochloride in tablets dosage form via the measurement of its corresponding degradate. The method was validated in accordance with the ICH requirements and statistically compared to the official method with no significant difference in performance.

Full text of DOI:10.1016/j.saa.2014.06.058

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 109–113
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectrofluorimetric determination of 3-methylflavone-8-carboxylic acid,
the main active metabolite of flavoxate hydrochloride in human urine
Hala E. Zaazaa a, , Afaf O. Mohamed b,1, Maha A. Hawwam , Mohamed Abdelkawy
⇑
b
a
a
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, 11562 Cairo, Egypt
National Organization for Drug Control and Research (NODCAR), 6-Abu Hazem Street, Pyramids Ave., P.O. Box 29, 35521 Giza, Egypt
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Excretion studies of flavoxate by monitoring the fluorescence character of its active metabolite (MFA) in
urine.
ꢀ
Fluorimetric determination of
flavoxate HCl via its metabolite and or
degradation product; MFA.
As MFA excreted in urine, so
excretion studies of FLV can be
performed via monitoring MFA in
urine.
ꢀ
ꢀ
ꢀ
The urinary excretion pattern has
been calculated.
The method was applied for the
analysis of FLV in pharmaceutical
formulation.
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple, sensitive and selective spectrofluorimetric method has been developed for the determination of
Received 13 March 2014
Received in revised form 28 May 2014
Accepted 8 June 2014
3-methylflavone-8-carboxylic acid as the main active metabolite of flavoxate hydrochloride in human
urine. The proposed method was based on the measurement of the native fluorescence of the metabolite
in methanol at an emission wavelength 390 nm, upon excitation at 338 nm. Moreover, the urinary
excretion pattern has been calculated using the proposed method. Taking the advantage that
Available online 20 June 2014
3
-methylflavone-8-carboxylic acid is also the alkaline degradate, the proposed method was applied to
Keywords:
Flavoxate hydrochloride
Spectrofluorimetry
in vitro determination of flavoxate hydrochloride in tablets dosage form via the measurement of its
corresponding degradate. The method was validated in accordance with the ICH requirements and statis-
tically compared to the official method with no significant difference in performance.
Ó 2014 Elsevier B.V. All rights reserved.
3
-Methylflavone-8-carboxylic acid
Pharmaceutical formulation
Urine
Introduction
series of flavone derivatives which exhibits strong smooth muscle
relaxant activity, especially on the urogenital tract [1]. It is used for
Flavoxate hydrochloride (FLV) Fig. 1, 3-methylflavone-8-
carboxylicacid b-piperidinoethyl ester hydrochloride, belongs to a
the symptomatic relief of pain, urinary frequency, and inconti-
nence associated with inflammatory disorders of the urinary tract.
It is also used for relief of vesicourethral [2]. A pharmacokinetic
study in humans has shown that FLV, when given orally, is rapidly
and completely absorbed and metabolized into 3-methylflavone-
8-carboxylic acid (MFA) which has a direct action on smooth mus-
cles of urinary tract through the inhibition of phosphodiesterase
⇑
Corresponding author. Tel.: +20 01001543567.
E-mail addresses: hazaza@hotmail.com,
H.E. Zaazaa).
hala.zaazaa@pharma.cu.edu.eg
(
1
Present Address: Umm Al-Qura University, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, P.O. Box 13174, Makkah, Saudi Arabia.
http://dx.doi.org/10.1016/j.saa.2014.06.058
1
386-1425/Ó 2014 Elsevier B.V. All rights reserved.

1
10
H.E. Zaazaa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 109–113
(
PDE) enzyme [3,4]. As MFA excreted in urine, so excretion studies
MFA working solution 50 lg/mL: prepared by transferring 2.5 mL
of FLV can be performed via monitoring MFA in urine.
of standard stock solution of MFA (1 mg/mL) into 50-mL volumet-
ric flask and the volume was completed to the mark with
methanol.
Stock standard solution of FLV 50 lg/mL: was prepared by dis-
solving 5.0 mg of FLV in 10 mL methanol, then diluting to 100 mL
The literature survey reveals that FLV was analyzed in its phar-
maceutical preparation by spectrophotometric [5,6], HPLC [7–11],
TLC [11] voltammetric [12] and potentiometric [13] methods.
Some methods are stability indicating, which analyzed FLV in the
presence of MFA as degradation product [9–11,13], others focused
on the analysis of MFA as active metabolite in biological fluids;
these include HPLC [14,15], capillary electrophoresis [16] and TLC
with methanol.
Solvents and chemicals
[
17] methods. Additionally, FLV was analyzed in British Pharmaco-
peia in pure form by non-aqueous titration using perchloric acid as
a titrant and by spectrophotometric method in tablets dosage form
Methanol used were HPLC grade (BDH Chemicals Ltd., England),
Dioxan (BDH Chemicals Ltd., England), Acetonitrile, acetone (lab.
scan-Ireland), Hydrochloric acid (Loba-Chemie Indoustranal
Co., India, 0.1 M aqueous solution), Sulphuric acid (Fischer
Scientific, UK, 0.1 M aqueous solution), Sodium hydroxide (Adwic
Co., Egypt, 0.1 M aqueous solution), Cetylpyridinium bromide
[
18].
Although chromatographic methods have a high degree of
specificity, yet, sample clean up and instrumentation limitations
preclude their use in routine clinical studies. This led us to study
fluorescence characteristics of FLV and MFA as an attempt to
develop a simple, sensitive and reliable method for their determi-
nation. The native fluorescence of MFA initiated the present study
that aimed to monitoring the excretion of FLV via its metabolite,
MFA, in human urine. Furthermore, MFA is also reported to be
the degradation product of FLV [8,9,11], thus the method was
extended to the analysis of FLV in its dosage form via MFA mea-
surement. The method is a good alternative to HPLC methods,
and the results obtained were satisfactorily accurate and precise.
(
Sigma Aldrich, St. louis, USA), Sodium lauryl sulphate (Sigma
Aldrich, St. louis, USA), Tween 40 (Merck, Munich, Germany),
Cetyltrimethyl ammonium bromide (Sigma Aldrich, St. louis, USA).
Calibration graph
Different volumes of the working standard solution of MFA
(
50
and diluted to volume with methanol to obtain concentration
range of 0.5–5.0 g/mL.
lg/mL) were transferred into a series of 10-mL volumetric flask
l
Experimental
The relative fluorescence intensity was measured at 390 nm
after excitation at 338 nm versus a blank of equivalent concentra-
tion of intact FLV. The calibration graph was obtained by plotting
the fluorescence intensities versus the final concentrations of
MFA and the regression equation was computed.
Instrumentation
ꢀ
The fluorescence intensities were measured using Shimadzu RF
1501 spectrofluorimeter equipped with 20 KW Xenon lamp,
–
excitation, emission grating monochromators and a 1 cm quartz
cell. Centrifuge (Memmert-Germany). A pH-meter, Digital pH/
MV/TEMP/ATC meter, Jenco Model-5005 (USA).
Procedure for commercial tablets
Ten tablets of GenurinÒ were weighed and finely powdered. A
portion of the powdered tablets equivalent to 200 mg of FLV were
weighed and refluxed with 1 M NaOH for 3 h till complete
degradation then neutralized with 1 M HCl, evaporated nearly to
dryness then the residue was dissolved in 100 mL methanol.
Different dilutions were prepared to suite the procedure as under
Section ‘Calibration graph’.
Materials and reagents
Pharmaceutical grade of FLV (Recordati, Milan, Italy) was used
and certified to contain 99.7% according to the official BP method,
which is non-aqueous titration using perchloric acid as titrant.
Commercial Genurin tablets were manufactured by Medical Union
Pharmaceutical, Abu-Sultan, Ismailia, Egypt, labeled to contain
2
00 mg FLV per tablet.
Laboratory prepared mixtures containing different ratios of flavoxate
HCl and its degradation product (MFA)
Standard solutions
Aliquots (0.9–0.1 mL) of MFA were accurately transferred from
its working standard solution (50
into a series of 10-mL volumetric flasks. Aliquots (0.1–0.9 mL) of
FLV stock standard solution (50 g/mL) equivalent to (5–45 g)
were added, and then the volume was completed with methanol
to prepare mixtures containing 10–90% of FLV.
lg/mL) equivalent to (45–5 lg)
Stock standard solution MFA (derived from complete alkaline
hydrolysis of 100.0 mg/mL FLV): prepared by refluxing 100 mg of
FLV with 50 mL of 1 M NaOH for 3 h then the hydrolyzed solution
was neutralized with 1 M HCl, evaporated nearly to dryness and
then the obtained residue was dissolved in methanol, filtered into
l
l
1
00 mL volumetric flask and the volume was completed with
methanol to obtain a solution of MFA derived from complete
degradation of 1 mg/mL FLV.
Procedure for spiked human urine samples
Aliquots equivalent to 5.0–40 lg of MFA were accurately trans-
ferred into 10-mL volumetric flask. After the addition of 1 mL of
blank urine, the solutions were diluted to 10 mL with methanol,
centrifuged at 3000 rpm for 15 min and then filtered through
0
.45
intensities were measured against a blank treated at the same
manner but containing 5.0–40 g of FLV instead of MFA. The
lm disposable membrane filters. The relative fluorescence
l
relative fluorescence intensity of each concentration was plotted
against the corresponding concentration to obtain the calibration
graph of MFA and the regression equation was computed.
Fig. 1. Chemical structure of flavoxate hydrochloride.

H.E. Zaazaa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 109–113
111
Table 3
Results of assay validation obtained by applying the proposed spectrofluorimetric
method for the determination of flavoxate HCl (via its degradate) in its pure
powdered form and in spiked human urine.
Parameters
Drug substance
Spiked urine
Validation of regression equation
Slope
Intercept
100.80
14.58
30.24
8.86
Correlation coefficient
0.9998
0.9996
Validation of response
Linearity range (
Accuracya
lg/mL)
0.5–5.0
0.5–4.0
Fig. 2. Excitation and emission spectra of 4
with kex 338 nm).
lg/mL MFA in methanol (at kem 390 nm
Mean ± R.S.D%
99.43 ± 0.504
99.33 ± 0.920
Precision
Repeatabilityb R.S.D%
Intermediate precision R.S.D%
0.507
0.510
0.814
0.635
b
Table 1
Effect of solvent on the fluorescence-intensity of 5
l
g/mL MFA.
Selectivity
99.85 ± 0.470
0.24
0.08
–
0.28
0.09
LOQ (
l
g/mL)
g/mL)
Solvent
Fluorescence intensity
LOD (l
Dioxan
500.0
476.0
493.0
480.0
157.0
27.6
Acetonitrile
Methanol
Acetone
Water
The LOD and LOQ were calculated using the following equations: LOD = 3.3 (SD/S)
and LOQ = 10 (SD/S) where SD is the standard deviation of response and S is the
slope of the graph.
a
n = 6.
n = 3 Â 3.
b
0
0
0
.1 M H
.1 M NaOH
.1 M HCl
2
SO
4
32.0
78.7
Results and discussion
Due to the ease and convince of spectrofluorometric method,
where analysis can be done within a short period of time by the
use of less sophisticated equipments in comparison with the
reported chromatographic methods, our work aimed to developed
simple, selective and reliable method for direct determination of
MFA. It was observed that MFA has an intense fluorescence at
390 nm emission wavelength upon excitation at a maximum of
Table 2
Effect of surfactants on the fluorescence intensity of 5
l
g/mL MFA.
Surfactant
Fluorescence intensity
Without surfactant
Cetylpyridinium bromide
Sodium lauryl sulphate
Tween 40
490.0
167.0
181.0
237.0
170.0
Cetyltrimethyl bromide
3
38 nm as shown in Fig. 2, while a similar concentration of FLV
has no emission, using the same excitation wavelength. Thus; this
idea has been used to develop a sensitive stability indicating spec-
trofluorimetric method for determination of FLV via its metabolite
and monitoring the excreted MFA in human urine for its analysis.
Furthermore, MFA is also reported to be the degradate product of
FLV [8,9,11], thus the method was extended to the analysis of
FLV in its dosage form after subjecting to complete alkaline
hydrolysis to obtain equivalent amount of MFA.
Sample preparation (in vivo procedure)
To check the ability of the proposed method for monitoring
MFA excretion in human urine at low concentrations, it was
applied to monitor the concentration of analytes after an oral
administration of a GenurinÒ formulation containing 200 mg FLV
to a normal, healthy (normal liver, kidney functions and electrocar-
diogram), female, informed adult volunteer (26 years, 80 kg,
Optimization of the method
1
69 cm height), with no past history of allergic reaction to FLV.
The volunteer was instructed to abstain from all medications for
weeks before administration and also during study. Also, the
Effect of diluting solvent
Dilution with different solvents such as: dioxan, acetonitrile,
2 4
methanol, acetone, water, 0.1 M H SO , 0.1 M NaOH and 0.1 M
2
volunteer was instructed to be sure of evacuating her bladder as
thoroughly as possible exactly before the administration of one
FLV tablet (200 mg) with about 250 mL of water. The 0-h urine
sample was collected as blank. Urine samples were collected at
0
1
–2.0, 2.0–2.5, 2.5–3.5, 3.5–5.0, 5.0–7.0, 7.0–9.0, 9.0–12.0, 12.0–
7.0, 17.0–20.0, and 20.0–24.0 h. The volume of urine specimen
was measured and recorded after each collection; 20 mL aliquots
were stored at 4 °C until determination.
Suitable volume of the urine specimen from each sampling
point was alkalinized with equal volume of 0.1 M sodium hydrox-
ide and centrifuged at 3000 rpm for 15 min. The mixture was
neutralized by addition of 0.1 M hydrochloric acid. The solution
was filtered through 0.45 lm membrane filter. Then suitable
volume of this solution was diluted to 10 mL with methanol and
its fluorescence intensity was measured versus blank urine treated
at the same manner.
The nominal content of MFA was determined from the
corresponding regression equation.
Fig. 3. Cumulative curve of amount excreted of MFA in urine after oral adminis-
tration of commercial tablet containing 200 mg FX.

1
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H.E. Zaazaa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 109–113
Table 4
Quantitative determination of flavoxate HCl (via its degradate) in pharmaceutical formulation by the proposed spectrofluorimetric method and the result of application of
standard addition technique.
Pharmaceutical formulation
Found %a of claimed amount ± R.S.D
100.3 ± 0.364%
Standard added (
lg/mL)
Recovery % a of standard added
Genurin tablets (200 mg FLV/tab) B.N. 21081
0.5
99.81
1
2
.5
.5
100.20
100.52
Mean ± R.S.D
100.2 ± 0.35%
Claimed amount (2.5
l
g/mL).
a
Average of three different determinations.
Accuracy
Table 5
Statistical comparison between results obtained by applying the proposed spectro-
fluorimetric method and the official method for determination of FLV (via its
degradate) in pure powdered form.
The accuracy was assessed from three replicate determinations
of three different solutions containing different concentrations of
MFA. The proposed method was successfully applied for the deter-
mination of the drug (via its degradate) in bulk powder and in
spiked human urine at kem 390 nm with kex 338 nm, Table 3.
aOfficial method [18]
Item
Spectrofluorimetric method
Mean
SD
R.S.D%
Variance
n
99.43
0.504
0.507
0.255
5
99.04
0.594
0.600
0.353
5
Precision
Repeatability. It was evaluated by analyzing freshly prepared solu-
tions in triplicate at concentrations of 2, 3, 4 lg/mL and 2.5, 3, 3.5
for bulk powder and spiked urine respectively using the previously
mentioned procedure under linearity. The percentage recovery and
the relative standard deviation were then calculated, Table 3.
b
t-Test (2.306)
1.112
1.384
F-test (6.400)b
a
Nonaqueous titration using perchloric acid as a titrant.
Figures in parentheses are the corresponding tabulated values at p = 0.05.
b
Intermediate precision. It was evaluated by assaying the above
mentioned concentrations in triplicate on three successive days.
The percentage recovery and the relative standard deviation were
calculated, Table 3.
HCl was studied. Although fluorescence intensity was maxima
upon using dioxan as shown in Table 1, but its usage was omitted
due to its toxicity being classified as human carcinogen [19]. The
use of methanol was the best regarding sensitivity and safety.
Selectivity
Methods selectivity was achieved by preparing several
laboratory-prepared mixtures of the studied compounds at various
concentrations within the linearity range. The laboratory-prepared
mixtures were analyzed according to the previous procedure
described under the proposed method. Satisfactory results were
obtained as indicated in Table 3.
Effect of surfactant
The influence of concentration of different surfactants on the
absorbance of the reaction product was studied hopefully they
may enhance the absorbance. It was found that by using different
concentrations of Cetylpyridinium bromide, Sodium lauryl
sulphate, Tween 40 and Cetyltrimethyl bromide decreases the
absorbance; therefore the study was performed omitting any sur-
factant Table 2.
Detection and quantitation limits
According to ICH’s guidelines method validation [22], the
detection and quantitation limits were determined, Table 3.
Choice of the excitation wavelength
Urinary excretion pattern of MFA
Several wavelengths were tested and the use of kexcitation
3
38 nm was the best regarding the sensitivity and reproducibility.
Flavoxate hydrochloride is rapidly and completely absorbed and
metabolized into MFA, which is excreted in human urine. As MFA
is present primarily in the conjugated form in human urine, so
alkaline hydrolysis of the conjugate form to the free form is needed
Validation of the method
[
15] for the precise determination of the total MFA found in urine.
Range and linearity
The proposed analytical method was applied for determination of
Aliquots equivalent to 5–50 lg (in bulk powder) and 5–40 lg
(
in spiked urine) of MFA were transferred accurately from its work-
ing standard solution (derived from complete degradation of
g/mL of FLV) into a series of 10-mL volumetric flasks and the
volume was completed to the mark with methanol. A blank was
prepared by transferring aliquots equivalent to 5–50 g of intact
FLV from standard stock solution (50 g/mL) were transferred
50 l
l
l
accurately into a series of 10-mL volumetric flasks and the volume
was completed to mark with methanol to assure that no interfer-
ence was exerted by the intact drug [20,21]. The difference in the
fluorescence intensity between the test experiment and blank
(
which has no emission upon excitation at the selected
wavelength) was measured at kem 390 nm with kex 338 nm, and
plotted versus the corresponding concentrations, and the regres-
sion equations were calculated, Table 3.
Fig. 4. Effect of the time on the stability of the fluorescence intensity of 5 lg/mL
MFA at 390 nm.

H.E. Zaazaa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134 (2015) 109–113
113
the cumulative amount of MFA excreted in urine after oral admin-
istration of 200 mg FLV formulated in Genurin tablet, which reflect
the bioavailability of FLV. The cumulative amount of MFA excreted
in urine was found to be 120.6 mg after 24 h following oral admin-
istration of one tablet containing 200 mg FLV as shown in Fig. 3.
The proposed method has been successfully applied to assay
FLV via MFA in Genurin tablets where the extracted FLV was quan-
titatively converted into MFA following the alkaline hydrolysis
process. The validity of the proposed method was further assessed
by applying the standard addition technique for the analysis of
Genurin tablet, Table 4.
[5] Y.J. Zheng, Ultra-violet spectrophotometric determination of flavoxate
hydrochloride tablets, Yaowu-Fenxi-Zazhi 13 (1993) 339–340.
[
6] M. Attimarad, Simultaneous determination of ofloxacin and flavoxate
hydrochloride by first-and ratio first-derivative UV spectrophotometry,
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The results obtained by applying the proposed method for the
analysis of the studied drug via MFA in pure form were statistically
compared with those obtained by applying the official method [18]
for FLV. The values of the calculated t and F are less than the
tabulated ones which reveals that there is no significant difference
with respect to accuracy and precision as shown in Table 5.
The stability of MFA solutions in methanol exhibited no spec-
trofluorimetric changes for 6 h when kept at ambient temperature
for 2 days when stored in refrigerator at 4 °C as shown in Fig. 4.
[
11] K.R. Nesrin, M.M. Heba, A.M. Azza, HPLC and TLC stability-indicating methods
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Conclusion
The proposed spectrofluorimetric method provides simple,
accurate, sensitive and direct quantitative analysis of MFA, the
main active metabolite of FLV in human urine without any
extraction procedure prior to determination. The urinary excretion
pattern of the metabolite was easily established. Also, the proposed
method can be used for determination of FLV in bulk powder and
dosage form via MFA measurement thus the method presents a
good alternation to other reported HPLC methods.
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