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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.
Zaazaa, Hala E.
