Sensors and Actuators B 133 (2008) 398–403

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Voltammetric sensor for amoxicillin determination in human

urine using polyglutamic acid/glutaraldehyde film
Daniela P. Santos, Márcio F. Bergamini, Maria Valnice B. Zanoni ∗
Institute of Chemistry – University of São Paulo State, UNESP, C.P. 355, 14801-970 Araraquara, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: A voltammetric sensor for determination of amoxicillin (AMX) was developed based on a glutaralde-
Received 23 October 2007 hyde cross-linked polyglutamic acid modified glassy carbon electrode. The proposed method is based on
Received in revised form 15 February 2008 pre-concentration of AMX by cathodic accumulation as its oxidative product before being determined
Accepted 28 February 2008
indirectly at potential as low as +0.23 V by square wave voltammetry. Linear response range, sensitivity
Available online 6 March 2008
and limit of detection were 2.0–25.0, 1.06 and 0.9.2 ␮mol L−1 , respectively, for AMX in 0.1 mol L−1 acetate
buffer pH 5.2, pre-accumulation time of 60 s and accumulation potential of +1.0 V. It was demonstrated
Keywords:
that the glassy carbon electrode modified with PGA/GLU could be used for AMX determination in human
Sensor voltammetric
Films of polyglutamic acid
urine without any separation step.
Amoxicillin © 2008 Elsevier B.V. All rights reserved.
Human urine

1. Introduction ␤-lactam antibiotics in samples of bovine milk using capillary elec-

Amoxicillin (AMX), d-␣-amino-p-hydroxybenzylpenicillin tri-
hydrate, Fig. 1), is one of the most frequently used ␤-lactam
antibiotics in the world and its employed to treat humans and ani-
mals [1,2]. As others ␤-lactam antibiotics present a structure based
on a ␤-lactam ring responsible for the antibacterial activity and
variable side chains that account for the major differences in their
chemical and pharmacological properties. After a single oral dose
of 500 mg, 60–86% of the drug is excreted unchanged in the urine
during the first 6 h. Despite a high level of clinical success, a seri-
ous mechanism of resistance had emerged demanding high dose
regimen and new pharmacokinetic combination. AMX is one of
the more important antibiotics used in the treatment of bacterial
infections and its determination.
Various analytical methods have been reported for the separa-
tion and determination of amoxicillin based on spectrophotometric
[3–9] and chromatographic [10–14] techniques. HPLC has been the
most widely used technique for determination of this group of
drugs, but presents some disadvantages, such as it requires large
amount of high purity organic solvents, long equilibration and
derivatizing treatment. In addition, its low solubility in organic sol-
vent makes it difficult to develop procedures based on extraction
and pre-concentration steps. The iodometry has been the method
officially requested by the American Pharmacopoeia 1990 [15].
It is possible to find methods for simultaneous determination of
∗ Corresponding author. Tel.: +55 16 33016619; fax: +55 16 33227932.
E-mail address: boldrinv@iq.unesp.br (M.V.B. Zanoni).
trophoresis [16,17] and liquid chromatography coupled to mass
spectrometry [18–20] and also in animal tissues [21] and in human
plasma [22].
Although numerous methods are available for the determi-
nation of penicillins and cephalosporins [23–28], the most part
of them shows lack of selectivity since they are based on pro-
duction of electroactive intermediates generated after hydrolysis
reaction [26]. Although, the electrochemical behavior of penicillins
has been extensively studied, papers about electrochemical oxida-
tion of amoxicillin describe very ill defined waves and high positive
potential. For this, Lyle and Yassin [29] describe its polarographic
reduction only after complexes with copper, nickel, cobalt and zinc.
The oxidation of AMX on gold and platinum electrodes has been
reported by Koprowski et al. [30], presents very ill defined waves.
The use of modified electrode to improve the voltammetric tech-
nique is also investigated. Carbon paste electrode modified with
poly-4-vinyl pyridine [31], poly-N-vinyl imidazole [32] and [N,N-
ethylene-bis(salicylideneaminato)] oxovanadium(IV), [VO (Salen)]
[33] have being reported as indirect method for its determination
in pharmaceutical formulations. But, all the methods require very
positive potential and presents low sensitivity.
The use of chemically modified electrodes by polyglutamic acid
(PGA) (Fig. 2) has been described in the literature with easy applica-
tion to construct voltammetric sensor for hydrazine [34], ascorbic
acid [35], uric acid [36] and caffeic acid [37]. Besides the ver-
satile application of the polyamino acid in the construction of
electrochemical sensor polyglutamic acid has been investigated as
promise candidate to form polymer–drug conjugate or use as drug
delivery [38,39].

0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2008.02.045
 D.P. Santos et al. / Sensors and Actuators B 133 (2008) 398–403
Fig. 1. Chemical structure of amoxicillin (AMX).
Fig. 2. Structure of polyglutamic acid (PGA).
In this study, we report the application of a polyglutamic acid
chemically modified electrode to investigate the interaction of the
polyamino acid with amoxicillin as mimetic model of complex
molecules. A simple and sensitive method was developed based
on its pre-accumulation, during 60 s at applied potential of +1.0 V,
which product is incorporated on the PGA film and oxidized at
+0.23 V. The method is successful applied for determination of
amoxicillin in human urine without any pretreatment of the
sample.
2. Experimental
2.1. Apparatus and reagents
Voltammetric measurements were carried out using an
AUTOLAB PGSTAT 30 potentiostat and software GPES 4.9. A three-
electrode system (EG&G PARC) consisting of an SCE as reference
electrode, a platinum wire as auxiliary electrode and a glassy carbon
electrode as working electrode were used. The glassy carbon elec-
trode (GCE) (3 mm diameter) was polished with alumina (0.3 mm,
BUEHLER), washed, and dried at room temperature before use. The
pH measurements were carried out using a Micronal pH meter
B222 model with a Micronal combined pH reference electrode. All
experiments were done at temperature of 25 ◦ C.
Suprapure grade reagents supplied by Merck and purified water
from a Millipore Milli-Q system (conductivity of 18.2 m cm−1 ,
pH 6.9) were used in the preparation of all solutions. Acetate
buffer 0.1 mol L−1 , used as supporting electrolyte was prepared by
mixing appropriate amounts of acetic acid and sodium acetate solu-
tion. Solutions of AMX (DEG-Chemical Products) were prepared
using analytical grade of 99.8%. Polyglutamic acid (MM = 50,000;
Sigma–Aldrich) solution 1% (w/v) in water was utilized in all exper-
iments.
2.2. Preparation of modified glassy carbon electrode
Searching for a better adherence of the films of PGA on the
electrode surface, it was used glutaraldehyde, which has been
extensively used for immobilization of biological materials [40].
Its bi-functional nature facilitates the formation of Schiff base,
involving the direct reaction between aldehyde groups from GLU
399
with the free amino group present in the polyglutamic acid
[41].
For this, an aliquot of 4 ␮L of the glutaraldehyde (GLU) (0.05%,
v/v) solution was placed on the polished GCE surface, followed by
addition of 12 ␮L of the polyglutamic acid (PGA) (1%, w/v) solu-
tion. The in situ prepared PGA/GLU modified GCE was then set
aside and allowed to dry in a sterile atmosphere at room temper-
ature. The voltammetric experiments with the PGA film modified
GCE was obtained after submitted it to a conditioning procedure
in phosphate buffer 0.1 mol L−1 pH 7.0, where successive voltam-
metric scan were recorded between −0.8 and +2.0 V (15 cycles, at
100 mV s−1 ). Then the electrodes were washed and transferred into
the solutions.
2.3. Analysis in the commercial sample
The human urine samples were spiked with 1.0 × 10−3 mol L−1
of AMX treated in 0.2 mL of methanol for subsequent removal of the
proteins. These samples were agitated and placed in a micro cen-
trifuge during 3 min at 6000 rpm. The superior liquid was removed
and transferred to a solution of 20 mL of acetate buffer pH 5.2. All
the measurements were carried out using the same PGA/GLU film
modified electrode.
3. Results and discussion
3.1. Oxidation of amoxicillin on PGA film modified electrode
Fig. 3 shows a typical cyclic voltammograms, obtained for
electrochemical oxidation of 5.0 × 10−4 mol L−1 AMX in 0.1 mol L−1
acetate buffer, pH 5.2 on bare and PGA/GLU modified electrode.
In agreement with literature [32], the oxidation of amoxicillin on
bare glassy carbon electrode (Fig. 3, curve 1) characterizes by a ill
defined wave around +0.7 V (peak Ia) attributed to oxidation of the
phenolic ( ∅ OH) substituent to respective carbonyl group ( ∅ O)
on the side chain of the molecule. On the modified electrode by
PGA/GLU film (Fig. 3, curve 2), the oxidation occurs around +0.9 V
and the product generated after the electron transfer is reduced
at +0.18 V (peak Ic). The peak current is much higher than bare
electrode, clearly indicating that AMX is accumulated on the film
coating. Taking into consideration that the free carboxylic groups
on the film presents pKa = 4.07 and AMX at pH 5.0 occurs pre-
dominantly in the zwitterion form [33], with both non-protonated
carboxylic group and the protonated amino group, probably AMX
is pre-concentrated on the electrode surface due electrostatic
interaction and/or cross-linked reaction between the amino group
of the drug and carboxylic group of the film. The generated
product of AMX oxidation is strongly retained into the film, which
Fig. 3. Oxidation of 5.0 × 10−4 mol L−1 AMX in 0.1 mol L−1 acetate buffer pH 5.2 on
bare glassy carbon electrode before (1) and after (2) modified with film of PGA:GLU.
Scan rate = 50 mV s−1 .
400 D.P. Santos et al. / Sensors and Actuators B 133 (2008) 398–403

Fig. 5. Linear scan voltammograms obtained to 1.0 × 10−4 mol L−1 AMX in
0.1 mol L−1 acetate buffer pH 5.2 on glassy carbon electrode modified by PGA:GLU
Fig. 4. Successive cyclic voltammograms obtained for glassy carbon electrode modi- film submitted to previous oxidation at +1.0 V after accumulation time of (1) 0 s and
fied by PGA:GLU film immersed in a solution of 1.0 × 10−4 mol L−1 AMX in 0.1 mol L−1 (2) 60 s. Scan rate = 100 mV s−1 .
acetate buffer solution pH 5.2. (1) first potential scan (2) second potential scan. Scan
rate = 100 mV s−1 .
The influence of the accumulation time on the peak current was
investigated oxidizing 1.0 × 10−4 mol L−1 of AMX in acetate buffer
product can be identified by its reduction at +0.18 V in the reverse
pH 5.2 at +1.0 V under controlled time from 0 to 120 s, which results
scan.
are shown in Fig. 6. The increase in the peak current was observed
An interesting behavior is obtained when cyclic voltammgrams
up to 60 s with a slight decrease at longer accumulation times. For
recorded for the first and second potential scan are compared, as
further measure it was chosen accumulation time of 60 s.
shown Fig. 4. Despite the first potential scan indicates only one
The scan rate dependence on the peak currents values was
peak for oxidation of AMX at EpIa = +0.9 V (peak Ia, Fig. 4), this
investigated using a PGA/GLU film modified electrode immersed in
peak is decreased and an extra pair of redox peaks (peak Ic/IIa)
1.0 × 10−4 mol L−1 solution of AMX in 0.1 mol L−1 acetate buffer pH
are seen on the second potential scan at less positive potential
5.2, using pre-accumulation time of 60 s and accumulation poten-
(+0.23 V/+0.17 V). The voltammetric parameters involving this pair
tial of +1.0 V. For scan rates from10 to 100 mV s−1 it was obtained
of peaks were investigated at different scan rate and present val-
a straight line for the plot of the peak current of the anodic peak
ues of Epa − Epc = 60 mV and values of Ipa /Ipc close to 1 at scan rate
(IIa) vs. the square root of scan rate (), following the equation:
≥200 mV s−1 . These results indicate that after first oxidation of AMX
Ip (␮A) = 2.44 + 1.11 1/2 ( = mV s−1 ; R = 0.999, n = 7). Previous stud-
at more positive potential can be forming an stable intermediate,
ies [43] have indicated that PGA/GLU films modified GCE presented
which could be stabilized on the electrode surface coated by film of
a very uniform and smooth morphology, with very well defined
PGA and reducing/oxidizing at less positive potential (peaks Ic/IIa)
large pores, from 80 up to 180 nm width and 70–150 nm deep
involving a reversible electrodic process of one electron transfer
formed by a polymeric network chains and identified by AFM spec-
[42]. This extra peak could be a good alternative to follow AMX
troscopy. This indicates that the electrode process is controlled
by measuring the electrochemical reduction of the drug previously
by diffusion through the polyelectrolyte film. The oxidation of the
oxidized at potential higher than +0.9 V and immobilized on the
intermediate through the PGA film immobilized on the GCE can
modified electrode by films of PGA.
be facilitated due to diffusion of the reactant trapped into the film
In order to confirm this possibility linear scan voltammograms
domains of the PGA [44].
were recorded from 0 to +0.5 V for modified electrode immersed
The effect of glutaraldehyde in the composition of the resulting
in 1.0 × 10−4 mol L−1 AMX in 0.1 mol L−1 acetate buffer pH 5.2 and
film of PGA and GLU could interfere in the efficiency of the modified
submitted to previous oxidation at +1.0 V after accumulation time
electrode to pre accumulated of the drug. For this, it was investi-
of 60 s. Fig. 5 compared the respective voltammograms obtained in
gated the effect of the ratio of PGA/GLU in the composition of the
relation to one obtained without accumulation time (curve 1). At
resulting film testing oxidation of 1.0 × 10−4 mol L−1 amoxicillin in
this condition it is possible to see the occurrence of a well defined
peak at +0.23 V (curve 2) that increase with amoxicillin concen-
tration and can be the basis of its voltammetric determination at
less positive potential than described in the literature for its usual
oxidation.
The influence of pH on the incorporation of AMX on the modified
electrode with PGA was investigated from pH 2.0 to 12.0. Although,
direct oxidation of AMX presents voltammetric signal on all pH
range, maximum peak value are obtained at pH < 6.0 and the occur-
rence of the intermediate oxidized at +0.23 V is observed only at
pH ≤ 6.0. When the pH is increased it is observed that the peak
potential shifts to less positive values up to a break in the slope at
pH 7.0. Taking into account that AMX presents three pKa values at
2.4, 7.1 and 9.4, probably at pH value higher than 7.0 there is no
interaction with PGA film, since both drug and polyamino acids are
in non-protonated form and the interaction is minimized. For this,
Fig. 6. Influence of the accumulation time on the peak current of AMX on glassy
pH 5.0 was chosen as optimum condition to pre-concentrate the carbon electrode modified by PGA:GLU films submitted to previous oxidation at
drug and its derivative after oxidation on the film of PGA. +1.0 V. Scan rate = 100 mV s−1 .
 D.P. Santos et al. / Sensors and Actuators B 133 (2008) 398–403
Fig. 7. Effect of glutaraldehyde in the composition of the resulting films to oxidation
de 1.0 × 10−4 mol L−1 of AMX in 0.1 mol L−1 acetate buffer pH 5.2 with accumulation
time of 60 s at +1.0 V. Scan rate = 100 mV s−1 .
acetate buffer pH 5.2, accumulation of 60 s and potential of +1.0 V.
The respective graph obtained for peak current and different com-
position of glutaraldehyde/polyglutamic acid is shown in Fig. 7. The
results showed that decreasing the amount of GLU in the film com-
position increases the peak current due to the largest number active
sites of PGA for the interaction with the AMX, but better adherence
is obtained in relation to the film constructed without glutaralde-
hyde. Optimum film composition was observed to 87.5%:12.5% of
PGA/GLU, and so this ratio was used for further studies.
In order to increase the detection limit, the analytical properties
of the PGA/GLU film modified GCE were evaluated for determining
AMX in 0.1 mol L−1 acetate buffer pH 5.2 using square wave voltam-
metry (SWV). The voltammograms in Fig. 8 shows a characteristic
square wave voltammogram obtained for 1.0 × 10−4 mol L−1 of AMX
in acetate buffer pH 5.2 on modified electrode using accumulation
time of 60 s at +1.0 V. Square wave voltammograms clearly show
that the resultant current is due to the contribution of the forward
current and reverse current. This is the most suitable situation for
SWV analysis since the resulting current is the sum of forward and
reverse contributions, the latter only being present in reversible
processes [45].
The experimental parameters evaluated by SWV technique were
frequency, f, (from 10 to 75 Hz), scan increment, Es , (from 2 to
10 mV) and pulse amplitude, Esw , (from 10 to 100 mV). The optimum
Fig. 8. Square wave voltammograms obtained for 1.0 × 10−4 mol L−1 AMX in
0.1 mol L−1 acetate buffer solution pH 5.2 on glassy carbon electrode modified by
PGA:GLU films using accumulation time of 60 s at +1.0 V. f = 20 Hz, E = 50 mV and
Es = 2.0 mV. (a) Resulting current, (b) forward current and (c) reverse current.
401
Fig. 9. (A) Square wave voltammograms obtained for AMX in 0.1 mol L−1 acetate
buffer solution pH 5.2 in different concentrations: (a) = 4.0; (b) = 7.0; (c) = 11.0;
(d) = 17.0; (e) = 25.0; (f) = 37.0 ␮mol L−1 . (B) Analytical curve. f = 20 Hz, E = 50 mV
and Es = 2.0 mV.
voltammetric signal was obtained for frequency of 20 Hz, pulse
amplitude of 50 mV and Es of 2.0 mV, where a combination of bet-
ter voltammogram definition associated with higher peak currents
is seen.
In order to develop a new method to monitored AMX by the
proposed method square wave voltammograms were recorded
at concentration from 2.0 to 25.0 ␮mol L−1 in 0.1 mol L−1 acetate
buffer pH 5.2, as shown Fig. 9A. The peak current increases linearly
at all investigated concentration interval, as shown Fig. 9B, follow-
ing the equation: IpIIa (␮A) = −0.89 + 1.06 C (C = ␮mol L−1 ) R = 0.996
and n = 6. The repeatability of the proposed sensor, evaluated in
term of relative standard deviation, was measured as 3.7% for 10
experiments in 10.0 ␮mol L−1 AMX using the same electrode. The
detection limits (LD) was evaluated by statistical treatment 3 S.D./b,
where (S.D.) is equivalent to the standard deviation of the average
of the signal of five measurements of the blank in the peak potential
of AMX and (b) is the slope of the analytical curve. The value of LD
was 9.2 × 10−7 mol L−1 .
3.2. Analysis of AMX in human urine
The method proposed was applied to the determination of
amoxicillin in human urine samples using GCE coated by PGA/GLU
monitoring the response by SWV procedure. The determination of
the amount of AMX was carried out in triplicates using the method-
ology described in the experimental section submitting the sample
to standard addition method of AMX 1.0 × 10−3 mol L−1 solution.
A mean recovery of 106.7 ± 2.52% was obtained for 3 repetitions.
The results were compared with a spectrophotometric method
402 D.P. Santos et al. / Sensors and Actuators B 133 (2008) 398–403
Table 1
Results (3 determinations) obtained for the determination of AMX in human urine by
square wave voltammetric procedure in comparison with the spectrophotometric
method
Method AMX add AMX found Recovery (%)
Proposed 6.0 ␮mol L−1 6.4 ␮mol L−1 106.7 ± 2.5
Spectrophotometer 10.0 ␮mol L−1 9.5 ␮mol L−1 96.2 ± 1.4
proposed in literature [9], as shown in Table 1. The results show
good agreement between the two methods indicating absent of
the matrix effect in the spiked urine sample, although the spec-
trophotometric requires higher concentration of AMX. This result
demonstrating that the PGA/GLU film modified glassy carbon elec-
trode can be successfully employed for the reliable determination
of AMX in real urine samples.
4. Conclusions
The electrochemical behavior of AMX on a PGA/GLU modified
glassy electrode shows that such electrodes can be successfully
used for determination of AMX in human urine. The method has
been developed here in which AMX is pre-concentrated by cathodic
accumulation as its oxidative product before being determined
indirectly at potential as low as +0.23 V. The proposed voltam-
metric method has the interesting advantage in the determination
AMX in less positive potential than obtained through direct oxi-
dation, once that in the literature is made at high potential values
and present very ill defined response. It was demonstrated that
the glassy carbon electrode modified with PGA/GLU could be used
for AMX determination in human urine without any separation
step.
Acknowledgements
The authors thank financial support from FAPESP [process no.
03/06598-3 (DPS) and 04/00111-8 (MFB)], CAPES and CNPq.
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