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VOLTAMMETRIC DETERMINATION OF RUTIN
IN PHARMACEUTICAL DOSAGE FORMS
SIMONA MIREL1, RADU OPREAN1, VALENTIN MIREL2,
ROBERT SĂNDULESCU1
“Iuliu Haţieganu” University of Medicine and Pharmacy Cluj-Napoca,
Faculty of Pharmacy,
2
National Institute for Research and Development of Isotopic and
Molecular Technologies, Cluj-Napoca
*corresponding author: [email protected]
1
Abstract
The electrochemical behavior of rutin was studied using cyclic (CV), liniar
sweep (LSV) and differential pulse voltammetric (DPV) techniques. Different working
electrodes: glassy carbon electrode (GCE) paste carbon electrode (CPE s) and platinum
electrode (Pt) were investigated in voltammetric assays. Optimum experimental conditions
for the determination of rutin were established. The current intensity depending linearly
with the concentration in the range of 10 -6-10-5 M/L permitted the development of
electroanalytical method to quantitative analyses. A reproducibility of oxidation potential,
height of peaks and calibration slopes were obtained. The method was applied for the
determination of rutin in pharmaceutical dosage forms.
Rezumat
A fost studiat comportamentul electrochimic al rutozidei utilizând diferite tehnici
electrochimice: voltametria ciclică (CV), voltametria cu baleiaj liniar de potenţial (LSV) şi
voltametria puls diferenţială (DPV). Experimentele au fost realizate în paralel utilizând ca
electrozi de lucru electrodul de carbon vitros (GCE), electrodul solid pastă de carbon
(CPEs) şi electrodul de platină. S-au stabilit şi optimizat parametrii de lucru. Studiul
electrooxidării a evidenţiat dependenţa liniară a curentului în funcţie de concentraţie în
domeniul 10-6-10-5 M/L, permiţând punerea la punct a unei metode voltametrice de dozare a
rutozidei prin LSV si DPV.. Metoda a fost aplicată pentru determinarea rutozidei din
comprimate.


rutin
voltammetric method
INTRODUCTION
The antioxidant activities of flavonoids are well known, due to the
properties of these natural compounds of reducing the damaging effects of
free radicals [12]. The compounds have beneficial effects on the cell
membrane, resulting in cardiovascular protection, anti-cancer activity and
anti-inflammatory effects [4, 9]. Recent studies have shown the flavonoids
immune-modulating activity and anti-viral properties [3, 7].
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Rutin is one of the most frequently studied and most active
flavonoid compound, commonly found in fruits and vegetables and often
present in human diet, having real health benefits. This phytocompound
appears in many pharmaceutical forms that are recommended in capillary
diseases, usually associated with ascorbic acid, due to their synergic effects.
Other research studies reported antianemia effect of rutosid combination
with iron and acid ascorbic [5].
That explains the interest for investigating the redox behavior of
rutin in order to develop a method for the determination of the compound in
plants, food or pharmaceutical formulation.
The methods currently used for analysis of flavonoids are:
spectrophotometric, capillary electrophoresis, chemiluminescence or high
performance liquid chromatography [6, 8, 14, 16]. In comparison with other
methods, the electroanalytical approach is simple, rapid and economic. In
the last years, many studies research developed the electrochemical methods
using different simple or modified working electrodes [1, 2, 10, 11, 17].
MATERIALS AND METHODS
Chemicals
Rutin was purchased from Biofarm Bucuresti and ascorbic acid
was purchased from Merck (Germany). All the chemicals used in buffer
preparation were of analytical grade from Merck. The solvent (ethanol) was
from Reactivul Bucuresti. Ultra pure water was used.
The 5mM stock solutions of rutin were prepared using pure
compounds which were dissolved in ethanol (70°). The solutions were
stored at dark, 4°C.
Electrochemical measurements
Electrochemical assays were performed with AUTOLAB
PGSTAT30 potentiostat with GPES software (EcoChemie, Netherlands)
using a standard 5 mL cell with three electrodes. The voltammetric behavior
was investigated with different working electrodes (glassy carbon electrode
(GCE), paste carbon electrodes (CPE) and platinum electrode (Pt). The
reference electrode was Ag/AgCl electrode and the auxiliary electrode was
platinum electrode. Cyclic voltammograms were recorded from –0.1V to
+1.0V and liniar sweep scan and pulse-differential measurements were
performed from –0.1 V to + 0.5V.
Sample solution (μL) was added to 5.0 mL of the background
electrolyte (buffer solution) in the electrochemical cell and the
voltammograms were recorded immediately after the working electrode was
introduced into solution.
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The CPEs were prepared using the solid carbon paste [13, 15],
based on solid paraffin and graphite (1: 2 ratio).
RESULTS AND DISCUSSION
The electroanalytical methods are used to study the redox
properties of the compounds. Rutin (quercetin-3-rutinoside) has the
following chemical structure (1):
OH
OH
HO
O
O
OH
O
OH
O
H2C
O
HO
O
OH
OH
OH
(1)
OH
rutin
The oxidation processes are related to the hydroxyl groups, which
all present electroactivity, and the electron transfer process could be
interpreted as a „cascade mechanism” [1]
Cyclic voltammetry (CV)
In order to establish better electrochemical parameters, the
voltammetric measurements were performed with glassy carbon electrode
(GCE), carbon paste electrodes (CPEs) and platinum electrode (Pt).
Platinum electrode had no response, but with GCE and CPEs the curves
present similar well defined oxidation peaks. The cyclic voltammetry curves
indicated an electrochemical oxidation, which is associated to the functional
OH groups of the molecule.
The voltamogramms were recorded immediately, in order to
minimize the adsorption of the compounds and their oxidation products on
the electrode surface [10, 11]. For the same reason, the electrode surface
was carefully cleaned by polishing before each series of measurements.
From this point of view, the CPE surface is easier renewed (polyshing with
paper) in comparison with GCE (polishing with alumina powder and
ultrasonified 5 minutes after each recording).
The chemical and electrochemical variables were optimized. The
dependence of the current upon pH values, the nature of buffer, and scan
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rate were investigated. The preliminary study indicated a linear dependence
of intensity (A) to the scan rate (r2 =0.994), but a lower value of the scan
rate improved the reproducibility of CV tracings. The following
measurements were recorded at 100 mV/s. The cyclic voltammograms
indicate the influence of the pH values and the reversibility of the electrode
process according to the Nicholson-Shain criteria. The reversibility of
reaction was more clearly shown in acidic medium. If the pH was
increasing, oxidation current was decreasing and oxidation potential was
less positive (Fig. 1). At pH 2.8, rutin shows one oxidation peak at +0.45V
and a reduction peak at +0.35 V. In phosphate buffer (pH 6.0) the oxidation
peak appeared at +0.3 V and the reduction peak at + 0.25 V. At pH 7.4, two
oxidation peaks appeared: the first one at + 0.24V and the second one at
+0.73V and a reduction peak at + 0.19 V. At pH 8.2 the oxidation and
reduction peaks are non-reproducibile, due to corrosion of the electrode
surface in alkaline medium.
-6
7.0x10
pH 2.8 (A)
pH 6 (B)
pH 7.7 (C)
pH 8.2 (D)
-6
6.0x10
-6
5.0x10
-6
A
4.0x10
-6
C B
I (A)
3.0x10
D
-6
2.0x10
-6
1.0x10
0.0
-6
-1.0x10
-6
-2.0x10
0.0
0.2
0.4
0.6
0.8
1.0
E vs AgCl/AgCl (V)
Figure 1
Cyclic voltamogramms of 10 –6 M/L rutin at different pH values
It can be concluded that the compound presents the higher
antioxidant properties in phosphate buffer (pH 7.4). At this pH value, the
voltammetric response is stable, depends linearly on concentration (in the
range 10-6 - 10 -5 M/L), and could be used for analytical determinations.
Voltammetric determination of rutin
Previous mentioned electrochemical method was used for the
quantification of rutin from pharmaceutical formulation. The
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voltammograms were recorded using linear sweep (LSV) and, respectively,
differential pulse voltammetry (DPV), using two different working
electrodes: GPE and CPE. The measurements were performed adding
successively different volumes (μl) of 1mM rutin alcoholic solution in 5 mL
phosphate buffer (pH 7.4)
Quantitative determination by LSP was developed for rutin, using
anodic peak, by linear voltammetric scan from +0V to +0.5V, in the same
parameters as mentioned above (pH 7.4, scan rate 100mV/s). For high
sensitivity it is also used DPV. The measurements were performed using the
mentioned parameters: initial potential -0.1V, end potential +0.5 V, step
potential 0.02V/s, modulation amplitude 0.1V, equilibration time 0.04 s,
interval time 0.5 s. In phosphate buffers (pH 7.4) rutin shows the oxidation
peak at +0.21 V (LSV) and at +0.15 V (DPV) (Fig. 2). A linear dependence
between the peak current response and the concentration was observed
(Table I). The accuracy of analyses was evaluated by comparing the
amounts of introduced reference substance (rutin) to the results of
voltammetric determinations.
30 ul
60 ul
70 ul
100 ul
150 ul
-6
3.0x10
-6
2.5x10
10 µl
20 µl
30 µl
40 µl
50 µl
-5
1.4x10
-5
1.2x10
-5
1.0x10
-6
2.0x10
-6
-6
I(A)
I (A)
8.0x10
1.5x10
-6
6.0x10
-6
1.0x10
-6
4.0x10
-6
-7
2.0x10
5.0x10
0.0
0.0
0.0
0.0
0.1
0.2
0.3
0.4
0.5
0.1
0.2
0.3
0.4
0.5
E vs Ag/AgCL (V)
E vs Ag/AgCl (V)
a
b
Figure 2
Voltamogramms at different concentrations of rutin: LSV (a) and DPV (b)
Table I
Linear correlation between peak height and concentration
Working Method
Linearity
Linear regression
r2
Accuracy
-1
electrode
(mol L )
%
.
-6
.
-5
CGE
LSV
8 10 - 9 10
y = 0.0338x - 5E-08
0.993 98.6 ± 4
DPV
2.10-6 - 2.10-5
y = 1.1347x - 5E-07
0.995 100.5 ± 3.3
CPE
LSV
2.10-6 - 9.10-5
y = 0.0246x+ 8E-08
0.994 97.2 ± 3.8
.
-6
.
-5
DPV
2 10 -1.5 10
y = 1.3308x - 2E-07
0.992 95.7 ± 5.2
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The method was used for the determination of rutin from tablets
RUTOZID Galenic Pharm Romania (40 mg rutin/tablet). The measurements
were carried out using GCE and CPEs electrodes, in both LSV and DPV
modes and the results were presented in table II.
Electrode
Sample
RUTOZID
Galenic Pharm
Romania
40 mg
rutin/tablet
RUTOZID
Galenic Pharm
Romania
40 mg
rutin/tablet
CPE
GPE
Table II
Voltammetric determination of rutin in tablets
Method Recovery *
Relative squared
mg
(%)
differences (RSD)
(%)
LSV
97.11
2.85
DPV
104.28
3.12
LSV
95.15
4.17
DPV
102.32
5.73
* average of 6 determinations
The present voltammetric method can be used for the determination
of rutin in pharmaceutical dosage forms.
CONCLUSIONS
The electrochemical behavior of rutin was studied using cyclic,
linear sweep and differential pulse voltammetric techniques. The
dependence of the current upon pH, concentration, buffer, and scan rate
were investigated with different working electrodes: glassy carbon electrode
(GCE), platinum electrode (PE) and carbon paste electrode (CPE).
Quantitative determinations were developed using anodic peak by linear
sweep and differential pulse voltammetry and the method was applied for
the determination of rutin from tablets. The electrochemical approach has
proved to be an easy, fast and reliable method for analytical determination
and could be applied with good results for determination of flavonoids in
pharmaceutical dosage forms.
Acknowledgements: The authors thank the Ministry of Education and
Research who gratefully supported this work by grants from CEEX
Matnantech 6/2005.
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