ANALELE UNIVERSITII DUNREA DE JOS GALAI

MEDICIN
FASCICULA XVII, no 1, 2012

ORIGINAL STUDY
ELECTROCHEMICAL DETERMINATION OF CATECHIN IN
PHARMACEUTICAL DOSAGE FORMS
I.M. Apetrei1,*, D. Tutunaru1, M.L. Rodriguez-Mendez2
1

Dunarea de Jos University of Galati, Faculty of Medicine and Pharmacy,

Department of Inorganic Chemistry, E. T. Ingenieros Industriales, University of Valladolid, Spain.

irina_apetrei@yahoo.com

ABSTRACT
A novel biosensor based on screen-printed technology was constructed, characterized and used to
evaluate catechin in pharmaceutical dosage forms. The biosensor performances were analyzed by
chronoamperometry in model solution of catechin. The amperometric studies show an intense cathodic peak
depending catechin concentration. The cathodic peak was attributed to the reduction of enzymatically produced
o-quinone at the biosensor surface. The optimal conditions for biosensor were founded. In optimal conditions,
the kinetics of the enzymatic reaction fitted into a MichaelisMenten type kinetics, as confirmed by the h
performances of the biosensor. Pharmaceutical samples were analyzed with biosensor to evaluate its real
feasibility in pharmaceutical analysis.
KEYWORDS: biosensor, tyrosinase, pharmaceutical analysis

The electrochemical behavior of several organic and

1. Introduction

inorganic compounds has been studied at the surface

of screen printed electrodes [1, 5-10]. Numerous

In the last years, screen-printing technology

researches have been dedicated to polyphenolic

has been widely used for the production of

compounds. The interest in polyphenolic compounds

microelectronic devices [1]. This novel technology is

stems from their protective role against oxidative

an alternative for the production of simple, sensitive,

damage diseases (i.e. coronary heart disease, stroke,

rapid, cheap and reproducible electrochemical sensors

and cancers) [11-13].

[2-4]. Numerous materials were used for the

From this class of compounds, flavonoids

fabrication of screen printed electrodes such as

have been increasingly becoming the subject of

graphite, gold, platinum and other. These materials

medical research. Catechin has been reported to be

used for the construction of the screen-printed

present in a diversity of foods such as red wine, green

electrode influence the electrochemical behavior of

tea, fruits etc. [14]. Catechin is a hepatoprotective

the sensor.

agent, known for its antioxidant and membrane

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FASCICULA XVII

stabilizing properties [10]. Catechin could be used as

solutions of catechin were freshly prepared daily.

antioxidant in oils and a health functional component

Ultrapure water was used for the preparation of

in various foods and dietary supplements [11].

solutions. A 60gL1 solution of tyrosinase in

Different analytical methods were used for the

buffer phosphate 0.01 M (pH=7) was used for the

determination of catechin such as spectrophotometry

enzyme immobilization on screen-printed electrode

and

surface.

high-performance

liquid

chromatography

(HPLC) [15-16]. However, most of these methods are

Screen-printed based biosensor

difficult and expensive as they necessitate several

The

enzyme,

tyrosinase

(Tyr),

was

operations including pre-treatment of the sample and

immobilized on the carbon screen-printed electrodes

costly equipments [16].

by casting technique followed by cross-linking. 5L

In comparison with other methods, the

of 0.01 M phosphate buffer (pH 7.0) containing

electroanalytical methods are simple, rapid and

60gL1 of enzyme, was added onto carbon

economic.

screen-printed electrode surface.

Numerous

amperometric

biosensors

employing polyphenol oxidase (tyrosinase)

evaluation

of

polyphenolic

compounds

for

After drying, the biosensor was immersed in a

were

glutaraldehyde solution (5%) for 20 minutes followed

developed [17-22].

by drying in air at room temperature for cross-linking

In this work, screen-printed biosensors based

process.

on tyrosinase as biocatalyst were prepared and their

The

enzyme-immobilized

biosensor

was

capability to detect catechin has been evaluated. For

washed with phosphate buffer solution thrice in order

this purpose, catechin detection has been carried out

to remove any unbound enzyme. The biosensor was

in aqueous solutions.

additionally dried at 10C and stored at 4C.

Optimum operational conditions for this

Electrochemical measurements

biosensor, such as, pH, temperature and applied

Electrochemical

potential were evaluated.

Also,

analytical

preformed
characteristics

of

the

using

measurements
an

EG&G,

Model

were
263

potentiostat/galvanostat driven by Echem software.

biosensors such as coefficient of variation, detection

Electrochemical cell with three electrodes was

limit, signal to noise ratio, linear dynamic range, as

used. The Ag/AgCl was the reference electrode. A

well as enzyme kinetics were also investigated.

platinum wire was used as counter electrode. A

The capacity of biosensor to detect and

tyrosinase modified screen printed carbon electrode

quantify the catechin in pharmaceutical dosage forms

was used as working electrode. These sensors were

was studied.

fabricated at the Dropsens, Spain, using screenprinting technology. They have graphite working
electrodes with an area of about 4 mm2 [23]. All

2. Material and methods

measurements were carried out in 0.01M phosphate

buffer adjusted to pH 7.0.

Reagents and solutions

All chemicals used were of analytical grade

Pharmaceutical analysis

used

purification.

Applicability of screen-printed biosensor was

Catechin acid was purchased from Sigma-Aldrich.

studied by analyzing the catechin in green tea

Phosphate buffer solution was prepared by mixing

commercial pharmaceutical dosage forms.

and

without

any additional

suitable amounts of Na2HPO4 and NaH2PO4. Stock

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ANALELE UNIVERSITII DUNREA DE JOS GALAI

FASCICULA XVII

with successive addition of catechin (in 50M steps)

3. Results and discussions

are given in Figure 3. Further, the response of

Amperometric response of biosensor

unmodified carbon screen printed electrodes (without

Catechin has two different pharmacophores,

enzyme) recorded over the same range of catechin

the catechol group in ring B and resorcinol group in

concentrations

was

found

to

be

insignificant

the ring A, as well as the OH group at position 3 in

compared with those of the tyrosinase modified

ring C (Figure 1).

electrodes.
As a result of enzymatic activity, catechin is
oxidized to catechin-orthoquinone which is reduced
amperometrically at -0.080 V (vs. Ag/AgCl).
Figure 3 shows the amperometric response for
the biosensor at -0.080 V after the addition of
successive aliquots of catechin to the 0.01 M PBS
(pH 7.0) under constant stirring. The resulting
reduction current is directly proportional to the
concentration of catechin present in the solution,
which results from the electrochemical reduction of

Figure 1. Chemical structure of catechin

o-quinone species enzymatically formed.

Tyrosinase (polyphenol oxidase - PPO) is a
copper-containing enzyme that is widely distributed
in plants and other organisms. These enzyme
catalyses two reactions in the presence of molecular
oxygen:

hydroxylation

of

monophenols

to

o-

diphenols and oxidation of o-diphenols to o-quinones.

The

scheme

of

the

enzymatic

and

electrochemical reactions at biosensor surface is

presented in Figure 2.
Figure 3. Amperometric response of biosensor to
catechin in 0.01 M PBS solution (pH=7).
Optimization of the experimental variables
The modification of biosensor response on the
applied potential was investigated over a potential
range of -0.25 to +0.2 V, using 10-4 M catechin in
Figure 2. Scheme of processes taking place at
biosensor surface

0.01 M phosphate buffer (pH 7.0).

The effect of applied potential for the

The tyrosinase modified carbon screen printed

biosensor on the amperometric signal is illustrated in

sensors produced a stable baseline response towards

Figure 4. The maximum of the response versus

background electrolyte solution. The results of the

background current is obtained at -0.080 V. This

steady state amperometric response of the biosensor

potential is favorable, since only some chemical

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ANALELE UNIVERSITII DUNREA DE JOS GALAI

species expected to be present in samples are reduced

FASCICULA XVII

According to the optimization process, the

at such a low potential [24, 25].

optimum working potential with respect to applied

potential of the biosensor was found to be -0.080V.
Optimum pH of the biosensor was found to be 7.
Effect of catechin concentration
Figure 6 showed the relationship between the
cathodic current of the biosensor and the catechin
concentration in PBS (pH 7.0) at -0.080V (calibration
curve).

Figure 4. Biosensor response at different values of

applied potentials
The performance of the biosensor is affected
by the pH value of solution due to the participation of
protons in the electrochemical reaction. The effect of
pH for the biosensor in 0.01 M PBS containing 10-4
M catechin is shown in Figure 5.
Figure 6. Calibration curve between the cathodic
current and the concentration of catechin in 0.01 M
PBS solution (pH 7.0).
It can be seen in Figure 6 that the response
current is linear with catechin concentration in the
range from 10 to 160M. The sensitivity of the
biosensors is 0.064AM-1. The corresponding
detection limits were calculated according to the
3sb/m criterion, where m is the slope of the
Figure 5. Biosensor response at different pH values

calibration graph, and sb was estimated as the

standard deviation (n = 7) of the amperometric

The reduction current increases somewhat as

signals from different solutions of the substrate at the

the pH changing from 5.0 to 7.0, and then decreases

concentration level corresponding to the lowest

progressively from 7.0 to 9.0. The current achieves

concentration of the calibration plot. The detection

the maximum value at a pH of 7. This pH value is in

limits calculated were 1.66 M.

accordance with the pH at which the enzymatic

From the calibration data, the Hill coefficient

activity is maximum in solution [26]. In order to

(h) can be calculated by representing the log[I/(Imax-

obtain the maximum response, a pH of 7.0 for the

I)] vs. log [catechin]. A Hill coefficient of 0.96 was

PBS was selected for the following studies.

calculated for the reduction process of o-quinone

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ANALELE UNIVERSITII DUNREA DE JOS GALAI

formed from the enzymatic reaction on the electrode

FASCICULA XVII

Table I. The found catechin concentration values in

pharmaceutical formulations by chronoamperometry.

surface (R2=0.985). The value obtained for the h

parameter, calculated from the corresponding Hills

Pharmaceutical product

plot, was close to unity demonstrated that the kinetics

of the enzymatic reaction fitted into a Michaelis

Green Tea

Mega Green

Extract

Tea

70

338

Labeled claim (mg

Menten type kinetics.

epigallocatechin gallate)

The parameters for MichaelisMenten kinetics

were calculated from the steady-state currents and the

Amount found (mg)

64

330

Standard deviation (mg)

2.14

5.56

electrochemical version of the LineweaverBurk

equation:

4. Conclusions

K Mapp
1
1

I I max I max S

A novel screen-printed based biosensor were

constructed and characterized for detection of
catechin in aqueous medium with desirable analytical

where: I is the steady-state current after the addition

characteristics such as sensitivity, response time,

of analyte, [S] is the concentration of catechin, Imax

signal/noise ratio, detection limit and lifetime

is the maximum rate of the enzymatic reaction, and

stability. The biosensor exhibits high sensitivity for

is the apparent MichaelisMenten constant.

the amperometric detection of catechin because of the

high loading of tyrosinase and the rapid electron

The Imax can be calculated from the intercept

transfer

and slope. The Imax is 15.34A, and is 78.12M.

between

the

enzymatically-produced

From the above results it could be concluded

quinones and the biosensor surface. Potential

that biosensor has good quality performances,

applications of biosensor include quantification of

comparable with those of previously reported

catechin from pharmaceutical dosage forms.

biosensors [19,21, 27-29].

Acknowledgments

Pharmaceutical studies
This work has benefited from financial support through the 2010

Different pharmaceutical formulations were

analyzed

with biosensor

to

evaluate

POSDRU/89/1.5/S/52432 project, ORGANIZING THE NATIONAL

its real

INTEREST

feasibility.

POSTDOCTORAL

BIOTECHNOLOGIES

WITH

SCHOOL
IMPACT

OF
ON

APPLIED
ROMANIAN

BIOECONOMY, project co-financed by the European Social Fund

A simple pretreatment of the pharmaceutical

through the Sectoral Operational Programme Human Resources

sample was made.

Development 2007-2013.

The samples were dissolved in water and then

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ANALELE UNIVERSITII DUNREA DE JOS GALAI

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