Talanta 55 (2001) 685– 692

www.elsevier.com/locate/talanta

Chronoamperometric determination of paracetamol using an

avocado tissue (Persea americana) biosensor
Orlando Fatibello-Filho a,*, Karina Omuro Lupetti a, Iolanda Cruz Vieira b
a
Departamento de Quı́mica, Centro de Ciências Exatas e de Tecnologia, Uni6ersidade Federal de São Carlos,
Via Washington Luiz, km 235, Caixa Postal 676, 13.560 -970 São Carlos SP, Brazil
b
Faculdade de Farmácia e Bioquı́mica, Uni6ersidade de Cuiabá (UNIC), A6. Beira Rio, 3100, CEP 78.015 -480 Cuiaba MT, Brazil

Received 31 January 2001; received in revised form 30 May 2001; accepted 31 May 2001

Abstract

A biosensor based on vaseline/graphite modified with avocado tissue (Persea americana) as the source of polyphenol
oxidase was developed and used for the chronoamperometric determination of paracetamol in pharmaceutical
formulations. This enzyme catalyses the oxidation of paracetamol to N-acetyl-p-benzoquinoneimine whose electro-
chemical reduction back to paracetamol was obtained at a potential of − 0.12 V. After addition of paracetamol
reference solutions in glass cell and stirring for 60 s for the accumulation of N-acetyl-p-benzoquinoneimine at the
electrode surface under open-circuit conditions, the current response was monitored by 120 s without stirring. The
currents obtained at 70 s were proportional to the paracetamol concentration from 1.2 × 10 − 4 to 5.8×10 − 3 mol l − 1
(r =0.9927) with a detection limit of 8.8 × 10 − 5 mol l − 1. The recovery of paracetamol from two samples ranged from
97.9 to 100.7% and a relative standard deviation lower than 0.5% for a solution containing 5.0 ×10 − 3 mol l − 1
paracetamol in 0.10 mol l − 1 phosphate buffer solution (pH 7.0; n =10) was obtained. The results obtained for
paracetamol in pharmaceutical formulations using the proposed biosensor and those obtained using a pharmaco-
poeial procedure are in agreement at the 95% confidence level. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Biosensor; Chronoamperometry; Paracetamol; Persea americana

1. Introduction in many countries as an alternative to aspirin and

Paracetamol (N-acetyl-p-aminophenol or acet-
aminophen) is an acylated aromatic amide, which
was firstly introduced into medicine as an an-
tipyretic/analgesic by Von Mering in 1893 [1,2]. It
is the most used medicine after acetilsalicylic acid
* Corresponding author. Fax: + 55-16-2608-350.
E-mail address: bello@dq.ufscar.br (O. Fatibello-Filho).
phenacetin and is an effective and safe analgesic
agent used worldwide for the relief of mild to
moderate pain associated with headache, back-
ache, for arthritis pain and postoperative pain [3].
Nevertheless, when consumed in overdose quanti-
ties, may cause severe hepatic toxicity or death
[1–3], thus, the development of a simple, precise
and accurate procedure for the determination of
this drug in pharmaceutical products is very
useful.

0039-9140/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 3 9 - 9 1 4 0 ( 0 1 ) 0 0 4 8 2 - 9
686 O. Fatibello-Filho et al. / Talanta 55 (2001) 685–692
Both American Pharmacopoeia [4] and
A.O.A.C. [5] recommend HPLC and spectropho-
tometric techniques. Paracetamol can be readily
oxidized at a carbon paste or glass carbon elec-
trodes [2], but these amperometric procedures are
non-selective, since the potential involved in this
process ranges from 0.6 to 0.8 V and various
substances are electroactive in this potential inter-
val. However, the application of biosensors for
this task can be operated at potentials much lower
than those normally used, thus decreasing the
interference.
Only an amperometric biosensor for the deter-
mination of paracetamol in whole blood was pro-
posed by Vaughan et al. [6]. The assay uses aryl
acylamidase (E.C. 3.5.1.13) to convert paraceta-
mol to p-aminophenol, which was monitored am-
perometrically by oxidation at +250 mV vs
Ag/AgCl and the oxidation current was related to
the concentration of paracetamol from 3.0× 10 − 4
to 2.8×10 − 3 mol l − 1.
Amperometry and/or voltammetry are electro-
analytical techniques which can be used in con-
junction with disposable sensors and/or
biosensors showing inherent advantages as sim-
plicity, low cost and rapidity [7,8]. Biosensors are
devices that combine higher selectivity and sensi-
tivity of a biological component with a suitable
transducer. The biological sensing element, usu-
ally an enzyme or an antibody, recognizes the
complementary molecule and the resulting bio-
chemical changes are transduced into a propor-
tional concentration signal [9,10].
We have developed several biosensors and en-
zymatic batch and flow injection procedures for
determining phenolic compounds [11– 13], L-dopa
and carbidopa [14], methyldopa and dopamine
[15], sulfite [16], L-ascorbic acid [17], epinephrine
and dopamine [18] and hydroquinone [19–21]
using crude extracts or tissues of various vegeta-
bles instead of isolated enzymes. The use of such
biological materials is very attractive because of
their high stability, high enzyme activity concen-
tration, very low cost and fewer cofactor require-
ments in comparison with the pure enzymes.
This paper describes the construction, perfor-
mance and application of a novel biosensor based
on vaseline/graphite modified with avocado tissue
powder. The influence of different experimental
parameters such as vaseline/graphite and tissue
composition was investigated to optimize this en-
zyme electrode. After a cyclic voltammetric study,
the biosensor was used for the chronoamperomet-
ric determination of paracetamol in pharmaceuti-
cal formulations.
2. Experimental
2.1. Apparatus
All electrochemical experiments were carried
out in a 15 ml thermostated glass cell at 25 °C. A
three-electrode assembly incorporating vaseline/
graphite powder modified with avocado tissue
(vaseline carbon paste tissue electrode, VCPTE)
as working electrode, an Ag/AgCl (3.0 mol l − 1
KCl) reference and a platinum auxiliary elec-
trodes were used in all measurements. Cyclic
voltammetric and chronoamperometric measure-
ments were performed with an EG&G PAR,
Model 273 A Potentiostat/Galvanostat with an
interface GPIB-IIA/IIA and a PC equipped with a
data acquisition and treatment software was used
to record the signal generated in the electrochemi-
cal cell.
2.2. Reagents and solutions
All reagents were of analytical-reagent grade
and all solutions were prepared with water from a
Millipore (Bedford, MA) Milli-Q system (Model
UV Plus Ultra-Low Organics Water).
Vaseline/graphite electrode was prepared using
vaseline (Fluka chemicals) and graphite powder
(grade no. 38) from Fisher. The supporting elec-
trolyte used for all experiments was a 0.1 mol l − 1
phosphate buffer solution (pH 7.0).
Paracetamol (acetaminophen) was purchased
from Sigma (St. Louis, MO) and a 2.5× 10 − 2
mol l − 1 stock solution was prepared daily in 0.10
mol l − 1 phosphate buffer solution (pH 7.0) and
reference solutions were obtained by appropriate
stock solution dilution with phosphate buffer so-
lution at pH 7.0.
 O. Fatibello-Filho et al. / Talanta 55 (2001) 685–692
Solutions of citric acid, sodium benzoate, mag-
nesium stearate and saccharin were prepared by
dissolving the respective compound in phosphate
buffer solution (pH 7.0) and used in the interfer-
ence studies.
Four Brazilian pharmaceutical products such as
Resprin (Johnson & Johnson, São José dos Cam-
pos, SP), Resfenol (Galenogal, Porto Alegre, RS),
Tylenol (Cilag Farmacêutica Ltda, São José dos
Campos, SP) and Vick Pyrena (Procter & Gam-
ble, Louveira, SP) were obtained from a local
drugstore and analyzed using the proposed
biosensor.
2.3. Preparation of the a6ocado tissue powder
Healthy avocado (Persea americana), pur-
chased from a local producer was selected,
washed, hand-peeled, chopped, dried in vacuum-
oven (Sheldon Manufacturing, Inc.) for 3 h at
30 °C. A domestic blender was used to obtain
fine powder and the particle size was selected by
passing them in known mesh sieves lower than
200 mm. Then, this dry powder tissue was stored
in a desiccator at 25 °C and used as the enzy-
matic source of polyphenol oxidase (PPO;
E.C.1.14.18.1) in the construction of the biosen-
sors [20,21]. PPO activity present in the dry av-
ocado tissue was determined in triplicate as
described elsewhere [11– 16].
2.4. VCPE and VCPTE electrodes construction
For the vaseline carbon paste electrode (VCPE)
preparation, the carbon paste electrode was pre-
pared by adding in a mortar 0.350 g of graphite
powder (70% w/w) and a mass 0.075 g of solid
bovine serum albumin (15% w/w) and homogeniz-
ing for 20 min. Then, 0.075 g of vaseline oil (15%
w/w) was mixed for 20 min.
The VCPTE (biosensor) was prepared mixing
initially 0.350 g of graphite powder (70% w/w)
with 0.075 g of avocado powder (15% w/w) in a
mortar for 20 min. Thus, this mixture was added
to 0.075 g of vaseline oil (15% w/w) and mixed for
at least 20 min to produce the final paste. A
portion of each mixture (about 0.167 g) was
packed into the tip of a 1 ml insulin plastic
687
syringe and a silver wire was inserted to obtain
the external electric contact [18–21]. In this work,
the VCPE was used for comparison and blank
experiments.
2.5. Procedure and determination of paracetamol
in pharmaceutical products
As mentioned, all measurements were made in
a 15 ml thermostated glass cell at 25.09 0.2 °C in
0.10 mol l − 1 phosphate buffer solution (pH 7.0).
An accurate volume of 1.0 ml of each pharma-
ceutical product (Resprin, Resfenol, Tylenol) was
stirring until complete dissolution and then di-
luted to 10, 20 and 50 ml with 0.1 mol l − 1
phosphate buffer solution (pH 7.0), respectively.
A mass of 0.05 g of Vick Pyrena was dissolved
with the help of ultrasonic bath and diluted to 10
ml with the same buffer solution. Finally, an
aliquot of 400 ml of each medicine solution was
added to the glass cell containing 10 ml of 0.10
mol l − 1 phosphate buffer solution. The measure-
ments were performed after successive additions
of paracetamol or sample solutions. After each
addition, cyclic voltammograms were recorded by
cycling the potential between + 0.6 and −0.5 V
at a scan rate of 100 mV s − 1. The chronoampero-
metric measurements were performed at −0.12
V. After each addition of paracetamol reference
solution in the glass cell, the solution was stirred
for 60 s for the accumulation of N-acetyl-p-ben-
zoquinoneimine produced at the electrode surface
under open-circuit conditions. After that, the
biosensor was immediately polarized at a fixed
potential of − 0.12 V and the current response
curve was monitored for 120 s without stirring.
The analytical curve was obtained using the cur-
rent at 70 s for each paracetamol reference
solution.
3. Results and discussion
3.1. Principle of the measurements and cyclic
6oltammetry study
The PPO catalyses the oxidation of paraceta-
mol to N-acetyl-p-benzoquinoneimine, at the elec-
688 O. Fatibello-Filho et al. / Talanta 55 (2001) 685–692
trode surface and this product was electrochemi-
cally reduced to paracetamol at a potential of
− 0.12 V, as illustrated in Fig. 1.
Fig. 2 shows the cyclic voltammograms ob-
tained using a VCPTE containing 15% w/w of
avocado powder tissue (biosensor) in an unstirred
0.10 mol l − 1 phosphate buffer solution (pH 7.0)
for paracetamol at the following concentrations:
(1) 9.8× 10 − 4 mol l − 1; (2) 1.9× 10 − 3 mol l − 1;
(3) 4.1× 10 − 3 mol l − 1; (4) 6.1× 10 − 3 mol l − 1;
(5) 8.3× 10 − 3 mol l − 1; (6) 1.0 × 10 − 2 mol l − 1
and (7) 1.2× 10 − 2 mol l − 1. In this study, cyclic
voltammetric measurements were performed by
scanning the potential between + 0.6 and − 0.5 V
vs Ag/AgCl at a scan rate of 100 mV s − 1.
3.2. Effect of 6aseline/graphite and tissue
composition
Using a fixed amount of 0.075 g of avocado
tissue powder (15% w/w) in each electrode, the
effect of vaseline weight varying from 0.050 to
0.125 g (10–25% w/w) and graphite powder rang-
ing from 0.300 to 0.375 g (60– 75% w/w) on the
biosensor response for 0.05 mol l − 1 paracetamol
in 0.10 mol l − 1 phosphate buffer solution (pH
7.0) was investigated. The best vaseline/graphite
composition was found using 10% (w/w) vaseline
and 75% (w/w) graphite powder. This composi-
tion was then selected in further experiments.
Fig. 1. Schematic representation of the enzymatic process
between paracetamol and PPO catalyzed by the avocado tissue
powder incorporated into the vaseline/graphite electrode.
The effect of the avocado tissue powder compo-
sition varying from 5 to 20% w/w (0.025–0.100 g)
in a fixed amount of graphite powder of 75% w/w
on the VCPET response was also investigated.
VCPET containing 0.075 g (15% w/w) of tissue
powder, 0.050 g (10% w/w) of vaseline and 0.375
g (75% w/w) of graphite powder showed the best
response (best signal/noise), so this composition
was used in the construction of further VCPET.
In another study carried out in our laboratory,
avocado tissue was entrapped on the surface of a
carbon-working electrode with a dialysis mem-
brane [22]. The analytical response of this biosen-
sor was very similar to that developed in this
work. Nevertheless, the lifetime of that biosensor
was shorter than the proposed in this work
(VCPET). Table 1 summarizes the range over
which each variable was investigated and the opti-
mal value found in the optimization of this
biosensor (VCPET).
3.3. Chronoamperometric determination of
paracetamol
As pointed out by Wang and Chen [23], the
accumulation of a substance produced in an enzy-
matic electrode near the transducer surface can
greatly enhance the sensitivity of the analytical
procedure.
In order to obtain a signal amplification, in the
present work time periods ranging from 15 to 120
s, under open-circuit conditions, were studied in
the accumulation of the N-acetyl-p-benzo-
quinoneimine near the biosensor surface. The sub-
sequent chronoamperometric response was
performed after an instantaneous change of the
potential from 0 to − 0.12 V where the quinone is
reduced. The best accumulation time found was
60 s. Therefore, this time was selected for the
further experiments.
Thus, after addition of paracetamol reference
solutions in glass cell and stirring for 60 s for the
accumulation of N-acetyl-p-benzoquinoneimine,
the current response was monitored for 120 s
without stirring. Fig. 3(A) and (B) show a sche-
matic diagram of the potential step applied at the
biosensor and the current responses obtained as a
function of time, respectively. The magnitude of
 O. Fatibello-Filho et al. / Talanta 55 (2001) 685–692 689

Fig. 2. Cyclic voltammograms obtained using an avocado tissue modified vaseline/graphite electrode for: (A) phosphate buffer
solution (pH 7.0) and (B) (1) 9.8 × 10 − 4 mol l − 1; (2) 1.9 × 10 − 3 mol l − 1; (3) 4.1 ×10 − 3 mol l − 1; (4) 6.1 ×10 − 3 mol l − 1; (5)
8.3 × 10 − 3 mol l − 1; (6) 1.0× 10 − 2 mol l − 1 and (7) 1.2 ×10 − 2 mol l − 1 paracetamol in 0.10 M phosphate buffer solution (pH 7.0).
Scan rate of 100 mV s − 1 and Ecp of − 0.12 V at 25 °C.

currents obtained at 70 s were proportional to the (100%), catechol (98%), dopamine (66.7%), hy-
paracetamol concentration from 1.2× 10 − 4 to droquinone (45.2%), L-dopa (27.9%), protocate-
5.8×10 − 3 mol l − 1 (r = 0.9927) with a detection chuic aldehyde (17.8%), fenoterol (0%);
limit of 8.8× 10 − 5 mol l − 1 (three times the blank isoprenaline (0%), and terbutaline (0%). As these
standard deviation/slope) (Fig. 3(B)).
The proposed chronoamperometric procedure Table 1
described in this paper is more sensitive, present- Optimization of biosensor (VCPET) parameters
ing a better detection limit and a wider linearity
than that biosensor described by Vaughan et al. Biosensor parameter Range studied Optimal value
[6].
Graphite powder 60–75 75
(% w/w)
3.4. Effect of excipient substances Vaseline oil (% w/w) 10–25 10
Avocado tissue 5–20 15
(% w/w)
Table 2 presents an initial study of the relative pH 4.0–10.0 7.0
responses (%) of the biosensor (VCPET) toward Scan rate 20–200 mV s−1 100 mV s−1
various phenolic compounds such as paracetamol
690 O. Fatibello-Filho et al. / Talanta 55 (2001) 685–692

Table 3
Recoveries of paracetamol standard solution

Sample Paracetamol (g l−1) Recovery (%)

Added Found

Resprin 0.190 0.190 100.0

0.376 0.371 98.7
0.552 0.556 100.7
Resfenol 0.136 0.135 99.1
0.340 0.333 97.9
0.552 0.550 99.6
Tylenol 0.190 0.190 100.0
0.376 0.371 98.7
0.552 0.550 99.6
Vick Pyrena 0.190 0.190 100.0
0.376 0.372 98.9
0.552 0.556 100.7

Fig. 3. Schematic diagram of (A) the potential step applied at with paracetamol in pharmaceutical formulations
the biosensor: E1 = 0 V; E2 = − 0.12 V; t1 = 0 s; t2 =60 s and such as citric acid, sodium benzoate, magnesium
t3 = 180 s; and (B) the current responses of (a) plain buffer, (b) stearate and saccharin were evaluated using the
1.2 ×10 − 4 mol l − 1, (c) 3.5 ×10 − 4 mol l − 1, (d) 9.8 ×10 − 4
proposed biosensor. The concentration ratios be-
mol l − 1, (e) 1.5 × 10 − 3 mol l − 1, (f) 2.5 × 10 − 3 mol l − 1, (g)
3.3× 10 − 3 mol l − 1, (h) 4.6 ×10 − 3 mol l − 1, (i) 5.8 ×10 − 3
tween paracetamol and those excipient substances
mol l − 1, (j) 6.9× 10 − 3 mol l − 1, (k) 8.3 ×10 − 3 mol l − 1 and were fixed at 0.1, 1 and 10. None of these sub-
(l) 9.6 × 10 − 3 mol l − 1 paracetamol at − 0.12 V as a function stances interfered in the proposed procedure.
of time (chronoamperograms).
3.5. Study of reco6ery, repeatability,
phenolic compounds are not presented in the reproducibility and lifetime
pharmaceutical preparations that contain parac-
etamol, this biosensor was able to perform the Recoveries from 97.9 to 100.7% of paracetamol
analytical determination of this analyte. Thus, the (Table 3) from four commercial products as Re-
effect of excipient substances frequently found sprin, Resfenol, Tylenol and Vick Pyrena (n=5)
were obtained using the VCPET. In this study,
0.190, 0.376 and 0.552 g l − 1 of paracetamol solu-
Table 2
Relative responses (%) of the biosensor (VCPET) toward
tions were added to each sample and current
various phenolic compounds responses were obtained chronoamperometrically.
The recovery results obtained suggested an ab-
Phenolic compounds Relative response sence of matrix effect on those determinations.
The relative standard deviation was lower than
Paracetamol 100
Catechol 98.0
0.5% for a solution containing 5.0× 10 − 3 mol l − 1
Dopamine 66.7 paracetamol in 0.10 mol l − 1 phosphate buffer
Hydroquinone 45.2 solution (pH 7.0; n= 10) and the reproducibility
L-Dopa 27.9 of four biosensors showed only a slight variation
Protocatechuic aldehyde 17.8 ( 3%) of the analytical curve slope.
Fenoterol 0.0
Isoprenaline 0.0
The lifetime of the proposed biosensors was at
Terbutaline 0.0 least 3 months (over 350 samples were determined
for each VCPET amount used in the syringe),
 O. Fatibello-Filho et al. / Talanta 55 (2001) 685–692 691

Table 4
Determination of paracetamol in pharmaceutical products using the biosensor

Sample Label value Pharmacopoeia Biosensor Relative error (%)

E1 E2

Resprin 40 40.3 90.8 41.0 9 0.1 +2.5 +1.7

Resfenol 100 100.1 90.5 99.1 9 0.2 −0.9 −1.0
Tylenol 200 199.7 90.1 200.8 9 0.2 +0.4 +0.6
Vick Pyrena 100 100.0 90.3 99.0 90.1 −1.0 −1.0

n =6, Confidence level of 95%. E1 = biosensor vs label value and E2 =biosensor vs pharmacopoeia value.

confirming, as expected, the high stability of the
avocado tissue electrode.
3.6. Analytical characteristics and application
Under the optimum conditions above estab-
lished (Table 1), i.e., biosensor containing
75:10:15% (w/w) graphite:vaseline:avocado tissue
in 0.10 mol l − 1 phosphate buffer solution (pH
7.0), chronoamperometric measurements were
performed at − 0.12 V potential for 120 s, with
previous product accumulation at open-circuit
condition for 60 s.
This biosensor presented a response time of 40
s (Fig. 3(B)). This small response time can be
attributed to the intimate contact between the
graphite powder and the tissue (with particle sizes
lower than 200 mm) used and/or potential pulse
applied at the work enzyme electrode.
The proposed procedure was applied to deter-
mine chronoamperometrically paracetamol in
pharmaceutical formulations. Table 4 presents the
results obtained for four commercial samples us-
ing the proposed biosensor and those obtained
using a pharmacopoeial [4] procedure. Applying
t-test for paired data, it was found that all results
are in agreement at the 95% confidence level and
within an acceptable range of error, showing thus
the usefulness of the proposed tissue biosensor.
4. Conclusions
The biosensor based on vaseline/graphite
modified with avocado tissue presented in this
article is reliable, simple, rapid to prepare, of low
cost and precise. The chronoamperometric deter-
mination of paracetamol in pharmaceutical prod-
ucts directly in phosphate buffer solution (pH 7)
without time-consuming sample treatment pro-
vides results comparable to those obtained using a
pharmacopoeial procedure.
Acknowledgements
Financial support from FAPESP (Processes
1991/2637-5 and 1994/4822-2) PADCT/CNPq
(Process 62.0060/91-3), CNPq (Process 50.1638/
91-1), scholarship granted by FAPESP (Process
98/01252-1 and 00/11521-2) to K.O.L. are grate-
fully acknowledged.
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