Food Chemistry 130 (2012) 11081114

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Selective determination of chloramphenicol at trace level in milk samples

by the electrode modied with molecularly imprinted polymer
Taher Alizadeh a,, Mohamad Reza Ganjali b, Mashaalah Zare b, Parviz Norouzi b
a
Department of Applied Chemistry, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran
b
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran

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

Article history: A novel voltammetric sensor for determination of chloramphenicol (CAP) in milk samples was intro-
Received 16 August 2010 duced. The CAP selective molecularly imprinted polymer (MIP) and non imprinted polymer (NIP) were
Received in revised form 19 April 2011 synthesized and then added to the carbon paste (CP) electrode composition in order to prepare MIP-CP
Accepted 4 August 2011
electrode. The MIP, embedded in the carbon paste electrode, acted as the selective recognition element
Available online 10 August 2011
and pre-concentrator agent for CAP. CAP was extracted in the electrode for a denite time and then it
was analyzed by differential pulse voltametry, found to be an effective determination method. The
Keywords:
MIP-CP showed very high CAP recognition ability, compared to NIP-CP. The electrode washing, after
Chloramphenicol
Molecularly imprinted polymer
CAP extraction, led to an enhanced selectivity. Various factors, known to affect the response behavior
Voltametric sensor of the electrode, were investigated and optimized. This sensor showed a linear response range of
Carbon paste electrode 8.0  1091.0  106 M and lower detection limit of 2.0  109 M (S/N = 3). The sensor was successfully
applied to the determination of CAP in milk samples.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Chloramphenicol [D(-)-threo-2-dichloroacetamido-1-pnitro-
phenyl-1,3-propanediol] (CAP) is a broad-spectrum antibiotic, pre-
viously used in veterinary medicine (Woodward & Watson, 2004).
The adverse effects of this compound have led to restrict its use in
both human and veterinary medicine. Among other applications,
this drug has been used for the treatment of childhood meningitis.
It is also a potent drug for typhoid fever. CAP has been used in vet-
erinary practice for the prevention and treatment of many bacterial
infections. However, toxic effects in humans such as aplastic ane-
mia have been described. It has been banned for use in foodstuffs
of animal origin in the European Union (Council, 1994; Epstein,
Henry, Holland, & Dreas, 1994).
Therefore, the detection of CAP in animal food samples is highly
important. Various methods such as liquid chromatography (Shen
& Jiang, 2005; Vinas, Balsalobre, & Hernandez-Cordoba, 2006),
liquid chromatographymass spectrometry (Bogusz, Hassan,
Al-Enazi, Ibrahim, & Al-Tufail, 2004; Gantverg, Shishani, &
Hoffman, 2003; Mottier, Parisod, Gremaud, Guy, & Stadler, 2003;
Neuhaus, Hurlbut, & Hammack, 2002; Pfenning et al., 2002; Ramos
et al., 2003; Storey et al., 2003), gas chromatography (GC), gas
chromatographymass spectrometry (GCMS) (Impens et al.,
2003; Shen & Jiang, 2005), capillary zone electrophoresis (Jin, Ye,
Corresponding author.
E-mail address: taa_55@yahoo.com (T. Alizadeh).
Yu, & Dong, 2000; Wang, Zhang, & Fang, 1999), enzyme-linked
immunosorbent assay (ELISA) (Scortichini et al., 2005), spectro-
photometry (Collado, Mantovani, Goicoechea, & Olivieri, 2000)
and chemiluminescence (Icardo, Misiewicz, Ciucu, Mateo, &
Calatayud, 2003; Lindino & Bulhoes, 2004) have been applied for
CAP determination in different samples. However, most of these
methods need expensive apparatus and reagents and they are
time-consuming. Therefore, a sensitive, rapid and cheap method
for analysis is still needed.
Electrochemical methods are widely used in the analytical
chemistry eld because, they are simple, fast, involve no more re-
agents for derivatization and low cost. Several methods such as
voltametry at electrochemically activated carbon ber microelec-
trodes (Agui, Guzman, Yanez-Sedeno, & Pingarron, 2002),
single-wall carbon nanotubegold nanoparticleionic liquid com-
posite lm modied glassy carbon electrodes (Xiao et al., 2007)
and boron-doped diamond electrode (Chuanuwatanakul,
Chailapakul, & Motomizu, 2008) have been developed for the
determination of chloramphenicol. However the sensitivity and
particularly the selectivity is still the main problem with the
developed electrochemical sensors. The interference effect of some
electroactive nitroaromatic compounds is an example of the draw-
backs for the previously developed electrochemical sensors. It
needs to apply a separation step before electrochemical determina-
tion (Xiao et al., 2007) in order to avoid the mentioned interference
effects. Application of a high selective chemical interface in the
preparation of voltametric sensors can be useful for achievement

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.08.016
 T. Alizadeh et al. / Food Chemistry 130 (2012) 11081114
the high selectivity. Moreover, it can guarantee the lower detection
limit for the resulting electrode.
Molecularly imprinted polymers (MIP) are promising biomi-
metic materials which are currently used in different elds of ana-
lytical chemistry. The preparation of electrochemical sensors by
using MIP material, as the recognition element, is an interesting to-
pic in the chemical sensors eld. Chloramphenicol has been a tar-
get for preparing of molecularly imprinted polymers (Schirmer &
Meisel, 2006; Suarez-Rodriguez & Diaz-Garcia, 2001).
Mena and his coworkers (2003) have reported the application of
chloramphenicol-MIP for the voltametric determination of chl-
oramphenocol. They extracted chloramphenicol by solid phase
extraction column. Detection of chloramphenicol was carried out
by the square-wave voltametry at an electrochemically activated
carbon ber microelectrode.
The method is interesting because of coupling of the MIP-based
solid phase extraction as a high selective pre-concentration and
separation method and the square wave voltametry as a high sen-
sitive determination technique. However, it suffers from some
drawbacks such as long analyzing time, high RSD, complexity of
the method. Furthermore, According to the method, the recogni-
tion of the target molecule is performed in an aqueous phase that
is not favorable for effectively interaction of the functional groups
of the MIP sites with the target molecules, reducing the selectivity
of the method.
Recently, we have reported several voltametric sensors based on
the carbon paste electrodes modied with MIP for various analytes
such as parathion (Alizadeh, 2009), para-nitrophenol (Alizadeh,
Ganjali, Norouzi, Zare, & Zeraatkar, 2009), 2,4,6-three nitrotoluene
(Alizadeh, Zare, Ganjali, Norouzi, & Tavana, 2010), caffeine (Ali-
zadeh, Ganjali, Zare, & Norouzi, 2010), and promethazine (Alizadeh
& Akhoundian, 2010). We showed that the MIP, used in the elec-
trode body, could act as both selective chemical interface and
pre-concentration agent. In the described method, pre-concentra-
tion of target molecule is carried out directly on the electrode sur-
face by immersion of the electrode in the continuously stirring
analyte solution. The electrode is subsequently applied as the work-
ing electrode in the electrochemical determination. In most cases, it
has been shown that an extraction time of about 10 min is enough
for completion the extraction stage which is very shorter than the
pre-concentration time of the previously described technique. Be-
sides, since the recognition of the analyte is executed on the elec-
trode surface, containing hydrophobic characteristic (because of
presence of n-eicosane as the binder), strong hydrogen bonding be-
tween the target molecule and MIP sites is effectively established.
In this work, a new, cheap and simple method was applied to de-
sign and prepare a high selective and sensitive electrochemical sen-
sor for chloramphenicol determination at very low concentrations.
The MIP, containing recognition sites for CAP, was synthesized and
then used as a recognition element in the carbon paste electrode.
This biomimetic modier functioned as a selectivity inducing agent
in the electrode. Also, it pre-concentrate CAP for improvement of
the detection limit of the sensor. The prepared electrode was used
for CAP determination in the milk samples by the three-step proce-
dure, including analyte extraction in the electrode, electrode wash-
ing and electrochemical measurement of CAP.
2. Experimental
2.1. Instruments and reagents
Electrochemical data were obtained with a three-electrode sys-
tem using a potentiostat/galvanostat model PGSTAT302, Metrohm.
Carbon paste electrode modied with the MIP or NIP was used as
the working electrodes. A platinum wire and an Ag/AgCl electrode
1109
were used as the counter and reference electrodes, respectively.
Methacrylic acid (MAA), obtained from SigmaAldrich (Munich,
Germany), was puried by passing them through a short column
of neutral alumina, followed by distillation under reduced pres-
sure. Ethylene glycol dimethacrylate (EDMA), obtained from Fluka
(Buchs, Switzerland), was distilled under reduced pressure in the
presence of hydroquinone inhibitor and stored at 4 C until use.
Metronidazole, para-nitrophenol, nitrobenzene, chloramphenicol,
n-eicosane and 2,20 -azobisisobutyronitrile (AIBN) were supplied
by SigmaAldrich (Munich, Germany) and used as received. Graph-
ite powder was purchased from Fluka (Buchs, Switzerland). Other
chemicals were of analytical grade and were purchased from
Merck (Darmstadt, Germany).
2.2. Molecularly imprinted polymers preparation
In order to produce a molecularly imprinted polymer template
molecule (1 mmol), methacrylic acid (5 mmol) and 50 mL of dry
chloroform were placed into a 100 mL round-bottomed ask and
the mixture was left in contact for 10 min. Subsequently, EDMA
(30 mmol) and AIBN (0.2 mmol) were added. The ask was sealed
and the mixture was purged with nitrogen gas for 15 min. The
polymerization of the mixture was carried out in a water bath at
60 C for 24 h. The nal polymer was powdered and then the tem-
plate was removed by soxhlet extraction with methanol for 48 h.
Complete removal of the template from the polymer was traced
by the differential pulse voltametry method. The non imprinted
polymer (NIP) was prepared similar to the MIP, except that the
template was not present in the polymerization media.
In order to obtain the ner and smaller MIP particles, the ob-
tained powder was sequentially immersed three times in acetoni-
trile and the supernatant portions were collected for nal use.
2.3. Preparation of the sensors
For the construction of the sensor (MIP-CP), 0.05 g of graphite
was homogenized in a mortar with 0.01 g of MIP for 10 min. Subse-
quently, n-eicosane (0.03 g) was melted in a dish in a water bath,
heated at 4550 C. The graphite/MIP blend was then added to the
melted n-eicosane and mixed with a stainless steel spatula. The nal
paste was used to ll a hole (2.0 mm in diameter, 3.0 mm in depth)
at the end of an electrode body, previously heated at 45 C. After
cooling at room temperature, excess of the solidied material was
removed with the aid of a paper sheet. After each experiment, the
electrode can be reused by moving the electrode surface on a paper
in order to rub out a thin layer of the electrode surface.
2.4. CAP measurement in real samples
Five 10 mL portions of milk samples were transferred to centri-
fuge tubes and spiked with 1 mL of chloramphenicol standard
solution with different concentrations. Then, 2 mL of trichloroace-
tic acid (20%) solution was added to the spiked samples for protein
precipitation. The mixture was vortexed for 1 min, and then centri-
fuged at 4000 rpm for 10 min. The supernatant was collected and
ltered through a 0.45 lm membrane lter, followed by com-
pletely evaporation in an oven (55 C). After, 10 mL of phosphate
buffer (0.05 M, pH 7) was added to the container to x pH at the
desired value. Then, the MIP-CP electrode was incubated in the
solution for 10 min. It was subsequently immersed in the washing
solution (wateracetonitrile 96:4) for 15 s. The electrode was nal-
ly immersed in the electrochemical cell containing phosphoric acid
solution (0.12 M), followed by applying the differential pulse
voltametry technique in the range of 0.4 to 1.0 V. The obtained
reduction peak of CAP was used for its determination according
to the previously established calibration curve.
1110 T. Alizadeh et al. / Food Chemistry 130 (2012) 11081114
3. Results and discussion
3.1. Cyclic voltametry behavior of CAP
Fig. 1 illustrates the obtained cyclic voltamogram for 1 mM of
CAP in 0.1 M of phosphoric acid solution at a carbon paste elec-
trode. The potential was scanned in two cycles from 1.0 to
1.0 V at a scan rate of 50 mV s1. In the rst scan, two peaks were
observed at 0.65 V and 0.47 V (vs. Ag/AgCl). In the second scan,
the cathodic peak current at 0.65 V (vs. Ag/ AgCl) decreased
slightly, whereas the anodic peak current increased slightly. More-
over, a new cathodic peak was observed at 0.02 V (vs. Ag/AgCl).
This revealed the presence of intermediates in oxidation/reduction
reactions of CAP. The rst cathodic peak (0.65 V) corresponds to
an irreversible reduction of the nitro group to the hydroxylamine
derivative. The anodic peak (0.47 V) arises from the oxidation of
the hydroxylamine derivative to a nitroso derivative during a re-
verse scan. Also, the second cathodic peak (0.02 V) results from
the reduction of the nitroso compound to the hydroxylamine
derivative (Chuanuwatanakul et al., 2008). The electrode reactions
are as follows:
R-NO2 4H 4e ! R-NHOH H2 O
R-NO 2H 2e ! R-NHOH
3.2. Electrochemical method selection
In order to achieve a high sensitive sensor, the selection of a
proper electrochemical technique is crucially important. Thus, we
tested different voltametric methods including differential pulse
voltametry (DPV) and square wave voltametry (SWV), known as
the high sensitive methods. The obtained results of these experi-
ments showed that the response of the DPV for CAP is considerably
better than that of SWV. Thus, this method was selected as the
electrochemical method for the determination of CAP by the pre-
pared sensor.
3.3. Comparison of the MIP-CP and NIP-CP electrodes and evaluation
of the washing effect
In order to study the CAP recognition ability of the MIP, the
MIP-CP, NIP-CP and CP electrodes were prepared and inserted into
Fig. 1. Electrochemical behavior of CAP on the carbon paste electrode in phosphoric
acid (0.1 M); potential scan range 1.0 to 1.0 V, potential scan rate = 0.03 V s1,
[CAP] = 1  103 M.
the CAP solutions. After 10 min, the electrodes were removed from
the CAP solution and DPV was carried out. The obtained results are
shown in Fig. 2.
As can be seen, the DPV signal of MIP-CP electrode (voltamo-
gram a) is higher than that of the NIP-CP electrodes (voltamogram
c). This indicates that the MIP-CP electrode can uptake CAP from
the aqueous solution more effectively, compared to the NIP-CP.
For further evaluation of the MIP-CP, the electrode was inserted
into the washing solution for a short time (15 s) just after its re-
moval from the CAP solution. The obtained results are shown in
Fig. 2. As it is clear, the electrodes washing after CAP extraction,
does not noticeably affect the CAP signal in the MIP-CP. However,
at the same time, the response of the NIP-CP electrode decreases
considerably by the washing. According to the voltammograms
of (e) and (f), the small signal of CP electrode disappears after
washing of the electrode. These results indicate that the MIP-CP
electrode has more afnity towards CAP compared to the NIP-CP
and CP electrodes. The washing process can remove the weakly
and nonspecically absorbed CAP molecules from the electrode
surface. This is the case with the NIP-CP and CP electrodes.
However, the CAP molecules incorporated in the selective sites of
the MIP are not removed easily by the washing process.
1 In the MIP, the cavities with incomplete or irregular shape, and
2 also the non selective binding sites cannot tightly absorb CAP mol-
ecules. Thus, the portion of CAP molecules, absorbed by such men-
tioned binding sites, can be removed from the MIP-CP electrode by
the washing process. Since the decrease in signal of the MIP-CP
electrode is not noticeable, it can be deduced that most portion
of the up-taken CAP is adsorbed strongly by the selective sites of
the MIP.
Fig. 2. Recorded CAP responses from different electrodes after immersion of them
in the CAP solution (7  107 M), (voltamogram of a, c and d); the effect of the
electrode washing on the related responses after removal of the electrodes from the
CAP solution (voltamograms of b, e and f); differential pulse voltametry conditions:
modulation time = 0.002 s, modulation amplitude = 0.40 V, scan rate = 0.2 V s1;
CAP extraction conditions: pH 7 (phosphate buffer = 0.1 M), extraction time = 7 -
min, washing time = 15 s.
 T. Alizadeh et al. / Food Chemistry 130 (2012) 11081114
3.4. Evaluation of the MIP-CP electrode response for the electroactive
nitroaromatic compounds
As described above, the main interfering species, capable to
inuence the CAP voltammetric sensors, are nitroaromatic com-
pounds. Thus, we tested the response behavior of some nitroaro-
matic compounds like metronidazole, para-nitrophenol and
nitrobenzene. The voltammograms of CAP (7  107 M), metro-
nidazol (9.0  105 M), nitrobenzene (5.0  105 M) and para-
nitrophenol (5.3  105 M) are shown in Fig. 3. It can be seen that
in the proposed electrode, the signals of the tested interfering
agents are noticeably lower than that of CAP despite the fact that
their concentrations are noticeably higher than that of CAP. This
indicates that the MIP-CP electrode can provide high selectivity
for CAP even in the presence of similar and electroactive interfering
compounds.
3.5. Optimization of the MIP-CP composition
In order to nd the best composition for the MIP-CP electrode,
the amounts of different ingredients of the electrode including
MIP, carbon and n-eicosane were changed in the xed conditions
of extraction and voltammetric determination. Then, the obtained
responses were used for conclusion. The MIP-CP electrode was pre-
pared with a xed amounts of carbon and n-eicosane and different
amounts of the MIP. The resulting electrode at each case was used
for the extraction and determination of CAP. The obtained results
are presented in Fig. 4(a). It is clear that the maximum response
Fig. 3. Evaluation of sensor selectivity; comparison of the responses of some potential interfering agents with that of CAP on the MIP-CP electrode; differential pulse
voltametry conditions: modulation time = 0.002 s, modulation amplitude = 0.40 V, scan rate = 0.3 V s1.
1111
for the prepared sensor appears in the MIP amount of 0.015 g.
Higher amounts of the MIP in the MIP-CP electrode can increase
the sensor response because of providing more recognition sites
on the electrode surface, as it is evident in the related curve. How-
ever, enhancement of the MIP amount, more than a threshold va-
lue, leads to a decrease in the prepared sensor response. This is
probably due to the decrease in the conductivity of the electrode
surface. Similar experiments were also carried out in order to
investigate the effect of carbon and n-eicosane amounts on the
prepared electrode response for CAP, by variation of these compo-
nents amounts in the MIP-CP electrode, followed by recording the
obtained results. These results are shown in Fig. 4(b) and (c). From
the corresponding curves, the optimum amounts of carbon and n-
eicosane were found to be 0.06 and 0.01 g, respectively. Initially,
the increasing of carbon content of the MIP-CP electrode leads to
an increase in the electrode response, but, after a denite point,
further increase in the carbon content results in a decrease in the
corresponding signal. The rst increase in the response can be re-
lated to the enhancement in the electron transferring capability
in the electrode. However, higher percents of carbon in the elec-
trode surface leads to the decrease in the MIP content of the elec-
trode surface, reducing the electrode recognition sites and thus its
response.
Besides, it can be seen that an optimum amount of n-eicosane is
required for the MIP-CP electrode preparation. Presence of the
higher amounts of binder (n-eicosane) in the MIP-CP electrode
leads to a decrease in the electrode response. This is because of de-
crease in the conductivity of the electrode surface.
1112 T. Alizadeh et al. / Food Chemistry 130 (2012) 11081114

Fig. 4. Optimization of composition of the MIP-CP electrode (variation of the electrode response for CAP with changing of the amounts of (a) MIP, (b) carbon and (c) n-
eicosane amounts of the and optimization of different conditions affecting the CAP extraction and determination in the MIP-CP electrode (variation of electrode response for
CAP with changing of (d) CAP solution pH, (e) electrode incubation time (f) stirring rate).

3.6. Optimization of the washing conditions
As described previously, the washing of the electrode led to re-
move the weakly adsorbed species from the electrode surface.
Since the interfering compound is usually adsorbed on the
MIP-CP surface weaker than the target molecules, the electrode
washing with a proper solution, before electrochemical determina-
tion, can be helpful for improvement the sensor selectivity and
reduction the interference effects. It was found that neutral water
containing a little amount of an organic solvent decrease the
NIP-CP signal, whereas the MIP-CP signal was not inuenced
considerably by the mentioned mixture. Different organic solvent
 T. Alizadeh et al. / Food Chemistry 130 (2012) 11081114 1113

such as ethanol, methanol, acetone and acetonitrile were added to 3.9. Analytical characterization
the water (2% in water) and tested as a washing solution. In the
case of acetonitrile, the response difference between the MIP-CP After the optimization and establishment of the determination
and NIP-CP was higher than that for other tested solvents. Thus, method for the prepared MIP-CP sensor, various ions and mole-
acetonitrile was selected as an organic solvent for adding in water. cules were examined with respect to their interference with the
The difference between the responses of the MIP-CP and NIP- determination of CAP. The tolerance limit was established as the
CP in the similar conditions of extraction and determination was maximum concentration of foreign species that caused a relative
increased by increasing the organic solvent content of the wash- error of 5% in the analytical signal. For 100 nM of CAP, the results
ing solution till 4% and after, it stayed constantly. Thus 4% aceto- showed that 650-fold molar excess of the inorganic ions such as
nitrile was chosen in this case. The time of the electrode Ca2+, Zn2+, Ni2+, Co2+, Fe2+, Cl and SO2
4 , had no signicant effect
immersion in the washing solution was also investigated. It on the CAP signal. Furthermore, Cd2+, Hg2+ and Cu2+ showed no
was found that the response difference between the MIP-CP interference effect up to 500-fold molar excess. It was found that
and NIP-CP increase till 15 s and afterwards, the aimed signal a 50-fold excess of nitrobenzene and para-nitrophenol had no
difference started to slightly decrease. Thus 15 s was chosen as inuence on the signal of 100 nM CAP. However, a 60-fold excess
optimal washing time. of nitrobenzene and a 65-fold excess of para-nitrophenol affected
signicantly the CAP signal. Metronidazol, tetracycline, sulfameth-
azine and Thiouracil inuenced signicantly the analyte signal at
3.7. Optimization of the conditions of CAP extraction
110, 400, 300 and 400-fold molar excess, respectively. These re-
sults indicate that the developed sensor have a good selectivity
The effect of pH of solution on the CAP extraction in the elec-
for CAP. The selectivity of the developed sensor in the presence
trode was studied. For this purpose, the prepared electrodes were
of nitroaromatic compound is particularly interesting, because
incubated in the CAP solutions with various pH values for 10 min
these compounds have been the essential interfering agents for
and at the constantly stirring state. Then, the electrodes were re-
the voltammetric sensors, introduced previously for the determi-
moved from the solution and immersed into the solution of the
nation of CAP.
electrochemical cell. The results of these experiments are shown
The calibration graph of the developed MIP-CP sensor is shown
in Fig. 4(d). As can bee seen, the neutral pH is favorable for the
in Fig. 5. As can be seen, the prepared sensor shows a linear rela-
extraction of CAP in the MIP modied electrode. It is clear that at
tionship over CAP concentration in the range of 8  109 to
the below and above pH 7, the electrode signal is decreased due
1.0  106 M with a detection limit of 2  109 M (S/N = 3). Each
to the decrease in the extraction amount of CAP in the electrode.
point of the calibration graph is the average of three replications.
At higher pH values, the carboxylic acid groups of the MIP sites
can be deprotonated whereas, at the lower pH values, the target
molecule (CAP) may be protonated. Both of these phenomenons 3.10. Determination of CAP in milk samples
are not good for properly interaction of the target molecules with
the selective sites of the MIP. According to these results, pH 7, xed The analytical usefulness of the prepared electrochemical sen-
by phosphate buffer (0.05 M), was chosen as an optimum pH for sor was demonstrated by applying it to the determination of CAP
CAP extraction in the electrode. in milk samples. The absence of CAP was rst veried in the
In order to optimize the stirring rate in the extraction period, non-spiked samples by applying the proposed method to the
CAP was extracted in the prepared MIP-CP electrodes at various non-spiked samples and observing no CAP related response. Table
stirring rates, whereas the other extraction parameters such as 1 summarizes the results obtained for the seven samples analyzed.
time, pH and CAP extraction were xed at constant values. The As can be seen, the recoveries achieved are acceptable for all tested
obtained results as the differential pulse voltametry signals of samples.
CAP versus the stirring rates are shown in Fig. 4(e). It can be seen In order to statistically verify the performance of the proposed
that as the stirring rate is enhanced the electrode response for method, a gas chromatographic method (Pfenning et al., 2002)
CAP increases, indicating the great effect of the stirring rate on was applied as the reference method for the determination of
the CAP extraction in the MIP-CP electrode. The growth in CAP
voltametric response as the result of increase in the stirring rate
continues up to 400 rpm. However, further enhancement in the
stirring rate does not considerably affect the CAP extraction.
Therefore, the stirring rate of 500 rpm was chosen as optimal
for this variation.
The effect of extraction time on the electrode signal was also
checked. The obtained results are shown in Fig. 4(f). According to
this gure, the increase in the extraction time leads to an intensive
growth in the CAP extraction amount till about 10 min and after-
wards, the response of the electrode do not grow further by
increasing of the extraction time. In order to decrease the analyz-
ing time of CAP, as much as possible, 10 min was selected for the
extraction stage.

3.8. Selection of the proper supporting electrolyte

It was found that acidic media is proper for electrochemical

determination of CAP. Thus, different electrolyte solutions such
as HCl, HNO3 and H3PO4 were tested as the supporting electrolyte
and phosphoric acid (0.12 M) was found to be the best option Fig. 5. Calibration curve obtained for the developed sensors at the optimized
among the tested electrolytes. conditions; (inset: linear range of the calibration curve).
1114 T. Alizadeh et al. / Food Chemistry 130 (2012) 11081114

Table 1 Chuanuwatanakul, S., Chailapakul, O., & Motomizu, S. (2008). Electrochemical

Determination of CAP in milk samples (p-value = 0.05). analysis of chloramphenicol using boron-doped diamond electrode applied to a
ow-injection system. Analytical Sciences, 24(4), 493498.
Sample Spiked Average concentration Average RSD Collado, M. S., Mantovani, V. E., Goicoechea, H. C., & Olivieri, A. C. (2000).
concentration found (lM)a recovery (%) (%) Simultaneous spectrophotometric-multivariate calibration determination of
(lM) several components of ophthalmic solutions: Phenylephrine, chloramphenicol,
antipyrine, methylparaben and thimerosal. Talanta, 52(5), 909920.
1 50.00b 50.40 100.8 3.8 Council, R. (1994). J. Eur. Commun. L, 6.
2 0.50 0.47 94.0 3.4 Epstein, R. L., Henry, C., Holland, K. P., & Dreas, J. (1994). International validation-
3 0.70 0.72 102.9 2.5 study for the determination of chloramphenicol in bovine muscle. Journal of
4 1.00 1.03 103.0 1.9 AOAC International, 77(3), 570576.
5 0.50 0.51 102.0 2.6 Gantverg, A., Shishani, I., & Hoffman, M. (2003). Determination of chloramphenicol
6 0.25 0.23 92.0 4.1 in animal tissues and urine Liquid chromatographytandem mass
7 10.55b 10.32 97.8 3.7 spectrometry versus gas chromatographymass spectrometry. Analytica
Chimica Acta, 483(12), 125135.
a
Number of sample assayed = 5. Icardo, M. C., Misiewicz, M., Ciucu, A., Mateo, J. V. G., & Calatayud, J. M. (2003). FI-on
b
nM. line photochemical reaction for direct chemiluminescence determination of
photodegradated chloramphenicol. Talanta, 60(23), 405414.
Impens, S., Reybroeck, W., Vercammen, J., Courtheyn, D., Ooghe, S., DeWasch, K.,
et al. (2003). Screening and conrmation of chloramphenicol in shrimp tissue
CAP in a milk sample spiked with 100 nM of CAP. The result of GC using ELISA in combination with GCMS2 and LCMS2. Analytica Chimica Acta,
method (100.5 5.7) was compared with that of the developed 483(12), 153163.
method (101.1 3.8). The results obtained by the MIP-CP and the Jin, W. R., Ye, X. Y., Yu, D. Q., & Dong, Q. (2000). Measurement of chloramphenicol by
capillary zone electrophoresis following end-column amperometric detection
reference GC method were statistically compared by the paired t- at a carbon ber micro-disk array electrode. Journal of Chromatography B,
test. This test revealed the absence of signicant differences be- 741(2), 155162.
tween the obtained results (at 95% condence level). There was Lindino, C. A., & Bulhoes, L. O. S. (2004). Determination of chloramphenicol in
tablets by electrogenerated chemiluminescence. Journal of the Brazilian
thus no evidence of the presence of systematic error in the results.
Chemical Society, 15(2), 178182.
Mena, M. L., Agui, L., Martinez-Ruiz, P., Yanez-Sedeno, P., Reviejo, A. J., & Pingarron, J.
M. (2003). Molecularly imprinted polymers for on-line clean up and
4. Conclusion preconcentration of chloramphenicol prior to its voltammetric determination.
Analytical and Bioanalytical Chemistry, 376(1), 1825.
Mottier, P., Parisod, V., Gremaud, E., Guy, P. A., & Stadler, R. H. (2003). Determination
A differential pulse voltammetric sensor with a high selectivity
of the antibiotic chloramphenicol in meat and seafood products by liquid
for chloramphenicol determination at trace concentrations was chromatography Electrospray ionization tandem mass spectrometry. Journal
proposed. The MIP functioned as both pre-concentrator and high of Chromatography A, 994(12), 7584.
selective recognition element in the carbon paste structure. Wash- Neuhaus, B. K., Hurlbut, J. A., & Hammack, W. (2002). LC/MS/MS analysis of
chloramphenicol in shrimp. US FDALaboratory Information Bulletin, 18(9).
ing of the MIP-CP electrode, after CAP extraction, led to an en- Pfenning, A., Turnipseed, S., Roybal, J., Burns, C., Madson, M., Storey, J., et al. (2002).
hanced selectivity without considerable loss in the sensitivity Conrmation of multiple phenicol residues in shrimp by electrospray LCMS.
and detection limit of the sensor. The proposed sensor showed US FDALaboratory Information Bulletin, 18(5).
Ramos, M., Munoz, P., Aranda, A., Rodriguez, I., Diaz, R., & Blanca, J. (2003).
interesting resistance against the interference effects of the elec- Determination of chloramphenicol residues in shrimps by liquid
troactive nitroaromatic compounds. This method can be used as chromatographymass spectrometry. Journal of Chromatography B Analytical
an alternative method for reliable CAP determination in the com- Technologies in the Biomedical and Life Sciences, 791(12), 3138.
Schirmer, C., & Meisel, H. (2006). Synthesis of a molecularly imprinted polymer for
plex real samples like milk. the selective solid-phase extraction of chloramphenicol from honey. Journal of
Chromatography A, 1132(12), 325328.
Scortichini, G., Annunziata, L., Haouet, M. N., Benedetti, F., Krusteva, I., & Galarini, R.
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