Journal of Food Composition and Analysis 56 (2017) 140–146

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Journal of Food Composition and Analysis

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

Original research article

Determination of fluoroquinolones in fishes using microwave-assisted

extraction combined with ultra-high performance liquid
chromatography and fluorescence detection
J. Aufartováa,b , I. Brabcováa,b , M.E. Torres-Padróna , P. Solichb , Z. Sosa-Ferreraa ,
J.J. Santana-Rodrígueza,*
a
Instituto Universitario de Estudios Ambientales y Recursos Naturales (i-UNAT), Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran
Canaria, Spain
b
Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 50005 Hradec Králové, Czechia

A R T I C L E I N F O A B S T R A C T

Article history:
Received 7 July 2016 A new analytical methodology based on ultra-high performance liquid chromatography with
Received in revised form 5 December 2016 fluorescence detection (UHPLC-FD) has been developed for the determination of five fluoroquinolone
Accepted 12 December 2016 antibiotics (FQs) used in aquaculture (norfloxacin, ciprofloxacin, enrofloxacin, danofloxacin and
Available online 12 December 2016 sarafloxacin) in fish muscles (e.g. gilthead seabream (Sparus aurata)). The extraction and determination
process was carried out by combining microwave-assisted extraction (MAE) along with a clean-up and
Chemical compounds studied in this article: pre-concentration step with solid phase extraction (SPE) followed by UHPLC-FD detection. The effects of
Norfloxacin (PubChem CID:4539) different variables on MAE-SPE were studied and optimized using an experimental design. Validation
Ciporfloxacin (PubChem CID:2764)
was made in accordance with Decision 2002/657/EC. CCa and CCb were determined according to the
Enrofloxacin (PubChem CID:71188)
maximum residue limit (MRL). The method detection limit (MDL) of the entire process ranged between
Danofloxacin (PubChem CID:71335)
Sarafloxacin (PubChem CID:56208) 0.1 and 6.0 ng g 1. The recoveries obtained at two spiked concentrations levels (25 and 250 ng g 1) were
greater than 90%, and the relative standard deviation was less than 8.7%. The developed methodology was
Keywords: successfully applied for the evaluation of the presence of FQs in fish muscle samples from aquaculture
Food analysis farms bought in various commercial shops in Gran Canaria Island (Spain).
Food composition © 2016 Elsevier Inc. All rights reserved.
Microwave-assisted extraction
Fluoroquinolones
Solid-phase extraction
Ultra-high performance liquid
chromatography
UHPLC
MAE
SPE
Gilthead seabream
Fishes

1. Introduction Aquaculture fish species are susceptible to diseases. The factors

Before aquaculture began, fish species were traditionally reared
in lagoons and coastal areas. Over the last few decades, global
aquaculture production has grown dramatically (Rabasso and
Hernández, 2015). This fact has caused undesirable effects, like
ingestion of contaminants or escaped fishes, on ecosystems for
feed waste, faeces, medicines and pesticides emissions (Read and
Fernandes, 2003).
* Corresponding author.
E-mail address: josejuan.santana@ulpgc.es (J.J. Santana-Rodríguez).
that contribute to the morbidity and mortality of farmed fish
include stress and the poor water quality as well as disease-causing
organism, i.e., bacteria, fungi, parasites and viruses (Banerjee et al.,
2014; Cañada-Cañada et al., 2009; Quesada et al., 2013; Shao,
2001).
Antimicrobial compounds are used in aquaculture production
with the objectives of inhibiting the growth of microorganisms as
well as the treatment and prevention of diseases (Banerjee et al.,
2014; Quesada et al., 2013). Their residues may affect the microbial
community and also accumulate in the tissue of fishes, causing
potential health risks for consumers (Brooks et al., 2005; Mimeault
et al., 2005; Schwaiger et al., 2004). Of the drugs approved for

http://dx.doi.org/10.1016/j.jfca.2016.12.012
0889-1575/© 2016 Elsevier Inc. All rights reserved.
 J. Aufartová et al. / Journal of Food Composition and Analysis 56 (2017) 140–146
agriculture, antibiotics are among the most widely administered
for animal health and management, especially for aquaculture.
Administration methods include water treatment, incorporation in
feed and direct injection (Shao, 2001).
Fluoroquinolones (FQs) are a group of synthetic antimicrobials
developed to prevent and treat infections. Some of them were
developed directly for veterinary treatment such enrofloxacin
(ENRO) or sarafloxacin (SARA). They are also used for the
promotion of growth in fish farms because they are approved
for aquaculture treatment. These antibiotics are added directly into
the water in fish farms leading to high concentrations in the water
and sediment (Rusu et al., 2015). Ciprofloxacin (CIPRO) is one of the
most prescribed antibiotics, and it is a major and active metabolite
of enrofloxacin in organisms (Martínez-Carballo et al., 2007).
Different Annexes to Council Regulation No 2377/90 include
different pharmacologically active substances to protect public
health, on the basis of the scientific assessment of the safety of
those substances (EEC, 1990). Maximum residue limits (MRLs) of
veterinary medicinal products in foodstuffs of animal origin were
established for these compounds. As specified in this regulation,
MRLs indicate the maximum concentration of a residue resulting
from the use of a veterinary medicinal product (expressed in
mg kg 1 or pg kg 1 on a fresh weight basis) that may be accepted
by the Community to be legally permitted or recognized as
acceptable in or on a food. The Commission approved administra-
tive MRLs for some FQ traces in animal tissues to 10–100 mg kg 1 in
Europe.
This regulation has been repealed and replaced by the Council
Regulation No 470/2009 (EEC, 2009) and, recently, for the
regulation No 37/2010 that includes these pharmacologically
active substances and their classification regarding their MRLs in
foodstuffs of animal origin (EEC, 2010). In this document, ENRO
and CIPRO present MRL values of 100 mg kg 1 in muscle, while
SARA has a MRL value of 30 mg kg 1 in salmon muscle because
Table 1
Selected fluoroquinolones in this work.
Compound Abbreviation Identification number Retention time (min)
Norfloxacin NOR 1 2.93
Ciprofloxacin CIPRO 2 3.40
Enrofloxacin ENRO 3 3.97
Danofloxacin DANO 4 4.28
Sarafloxacin SARA 5 6.23
141
some of these are making their way into food chains and reaching
the consumer (Tittlemier et al., 2007). These compounds together
with norfloxacin (NOR) and danofloxacin (DANO) have been
selected as representative members of the FQ family that were
chosen for this study. Their structures and characteristics are
shown in Table 1.
Different methods for the extraction, preconcentration and
determination of FQs have been developed to determine the
presence of these compounds in various matrices. In this sense,
different FQs have been determined in different environmental
samples such as waters (Montesdeoca-Esponda et al., 2012a;
Seifrtova et al., 2010; Speltini et al., 2016) and wastewater
(Lindberg et al., 2004), soil and sediment (Montesdeoca-Esponda
et al., 2012b; Vázquez-Roig et al., 2010) or compost from sewage
sludge (Dorival-García et al., 2015) between them.
However, the MRLs established in Europe require sensitive and
specific methods to monitor and determine the FQ residues in
aquaculture products because these data play an important role in
guaranteeing the safety of food. Food-based matrices are very
complex, and chromatography combined with mass spectrometry
has been one of the most sensitive and selective analytical
methodologies used (Dufresne et al., 2007; Hernando et al., 2006;
Rezk et al., 2015; Romero-González et al., 2007; Samanidou et al.,
2008; Storey et al., 2014). However, LC combined with fluorescence
(Kirbis et al., 2005; Zhang et al., 2010) or photodiode array
detection (Evaggelopoulou et al., 2014) has also been used to
obtain similar results.
In all cases, solvent extraction combined with centrifugation
and solid phase extraction (SPE) has been used as a preparation
method in liquid samples. The extraction of analytes from solid
samples is complicated, especially in environmental applications,
because the solute biological matrix interactions are very difficult
to predict and overcome (Camel, 2001; Montesdeoca-Esponda
et al., 2012b). Conventional extraction methods required a high
Structure pKa log Kow CAS
6.3 1.09 70458 96-7
5.9 0.4 85721 33-1
6.3 1.1 93106 60-6
6.04 0.3 112398 08-0
6.22 0.3 98105 99-8
142 J. Aufartová et al. / Journal of Food Composition and Analysis 56 (2017) 140–146
volume of organic solvent or dilute the analytes, and additionally,
they are time consuming.
Microwave-assisted extraction (MAE) is an interesting sample
extraction technique for solid samples because it reduces the
volume of solvents and extraction time and it improves the
analytical parameters (precision and recovery) while increasing
sample throughput (Sánchez-Prado et al., 2015). Due to these
reasons, the method decreases the cost and represents a viable
alternative to other methodologies. Experimental design is a
powerful tool to use in the MAE optimization because it allows for
fast sample preparation development and simplifies the method
optimization of parameters that have to be evaluated (Morales-
Muñoz et al., 2004; Montesdeoca-Esponda et al., 2012b).
The aim of the present study was to develop and validate an
analytical method that permits the determination of FQs in
gilthead seabream (Sparus aurata) because this species has a high
consumption, and it is regularly present in the markets of Europe.
We have selected microwave-assisted extraction (MAE) coupled to
solid phase extraction (SPE) for the preconcentration and clean-up
step previous to determination using ultra-high performance
liquid chromatography coupled to a fluorescence detector (UHPLC-
FD). This sample preparation methodology speeds up the process
and allows the use of lower volumes of sample and organic
solvents.
We have optimized the parameters that affect the MAE process
(i.e., extraction time, power, volume of extractant) using an
experimental design to obtain the best results. The proposed
method was successfully applied to extract and determine the
selected FQs in aquaculture gilthead seabream bought in different
markets of Gran Canaria Island (Spain). Some of the selected
compounds (NOR, CIPRO, ENRO and DANO) were determined in a
previous work in a multi-residue and multi-class determination of
antibiotics in this species (Freitas et al., 2014). In our work, we have
selected, these compounds and SARA (see Table 1), used as
veterinary medicines in aquaculture. This proposed method could
be a viable alternative for the determination of these analytes in
this type of sample.
2. Experimental
2.1. Chemical and reagents
Standards of the fluoroquinolones, norfloxacin (NOR), cipro-
floxacin (CIPRO), enrofloxacin (ENRO), danofloxacin (DANO) and
sarafloxacin (SARA) were acquired from Sigma-Aldrich (Madrid,
Spain). Stock solutions with a concentration of 1000 mg mL 1 were
prepared by dissolving appropriate amounts of the commercial
products in methanol and then stored in glass-stopper bottles at
18  C.
LC-grade methanol used to dissolve the standards and prepare
the mobile phase was obtained from Panreac Química (Barcelona,
Spain). HPLC-grade glacial acetic acid was used to adjust the pH of
the mobile phase to 2.5 and was obtained from Scharlau Chemie S.
A. (Barcelona, Spain).
Ultra-high quality water, obtained by a Milli-Q (Millipore,
Bedford, MA, USA) water purification system, was used in the solid
phase extraction protocol, mobile phase as well as to dilute
samples. The 0.45-mm syringe-driven filter employed for the
purification of the extract was provided by Scharlau Chemie S.A.
(Barcelona, Spain).
2.2. Instrumentation and chromatographic conditions
The microwave oven used for the extraction of the selected
compounds was a multiwave equipped with a 6 EVAP rotor and 6
MF100 vessels (Anton Paar, Graz, Austria). The cartridges (6 mL)
employed in this study were Oasis HLB cartridges (200 mg) from
Waters (Milford, MA, USA). A Varian Vac Elut 20 SPE manifold
(Varian Inc., CA, USA) coupled to a Sartorius vacuum pump was
used for the extractions.
The chromatographic system was an Acquity UHPLC system
from Waters (Milford, MA, USA). The instrument consists of the
following different parts: (a) a quaternary solvent manager pump;
(b) an autosampler manager-FTN and (c) a fluorescence detector
(FD). Separation of the FQs was achieved on a Waters Acquity BEH
C18 column (5  2.1 mm, 1.7 mm particle size; Waters Cromatog-
rafía, Barcelona, Spain) with a gradient mobile phase consisting of
MeOH:H2O (adjusted to a pH of 2.5) at a flow rate of 0.3 mL min 1
(0–5 min 16:84 v/v, 5–9 min 30:70 v/v). The injected sample
volume was 5 mL, and the fluorescence detector was operated at
an excitation wavelength of 280 nm and an emission wavelength of
450 nm. The retention time for each compound is listed in Table 1.
2.3. Preparation of spiked samples
Fishes for the method optimization were dissected, and the
muscle without skin was carefully extracted and dried in an oven at
70  C until a constant weight was achieved. Finally, the muscles
were homogenized using a mortar. All fish samples used for
optimization of the method were provided from sea cultures and
bought in a local market of Las Palmas de Gran Canaria. These
samples were previously analysed to verify that they did not
contain any quantity of the analytes in the study (Montesdeoca-
Esponda et al., 2012b). Muscle samples were spiked with a mix of
the selected compounds in methanol to obtain a final concentra-
tion of 1 mg g 1 to determine the best extraction conditions. Then,
the samples were stirred until homogenized and air-dried
overnight in the dark at room temperature to obtain a dry
homogeneous sample. For the analysis of real samples, we selected
three commercially available gilthead seabream from aquaculture
bought in different local markets.
2.4. MAE-SPE procedure
For the MAE process, one gram of the spiked matrix or sample
was allocated to the polytetrafluorethylene (PTFE) vessel. Then,
10 mL of methanol was added to the samples, and the vessels were
closed to avoid the loss of extract during the extraction process.
The microwave program consisted of the application of 150 W for
8 min. These conditions were enough to reach a temperature near
the extractant boiling point and obtain the best extraction
efficiencies for the selected compounds. Once the program
concluded, the vessels were first cooled for 5 min with the
microwave fan and then allowed to stand for 10 min at room
temperature before the microwave was opened. The entire
extraction solution (approximately, 9 mL) was removed from the
vessels, filtered with a 0.45 mm syringe-driven filter and diluted
with Milli-Q water until a final volume of 100 mL. Afterwards, a SPE
process was applied as a clean-up step (Montesdeoca-Esponda
et al., 2012b). An Oasis HLB cartridge (6 mL, 200 mg) was activated
with 5 mL of methanol and 10 mL of Milli-Q water. The extracted
sample was passed through the cartridges under vacuum at a flow
rate slower than 10 mL min 1. The cartridge was washed with 5 mL
of Milli-Q water and dried under a vacuum. Finally, the retained
analytes were eluted with 2  1 mL of methanol, and then they
were transferred to glass vials for analysis by UHPLC-FD.
2.5. Software and statistical analysis
The experimental designs for the optimization of the MAE
conditions were performed using the Statgraphics Plus software,
version 5.1 (Manugistic Rockville, MD, USA). Statistical analyses of
 J. Aufartová et al. / Journal of Food Composition and Analysis 56 (2017) 140–146
the partial and bivariate correlations were performed using the
SPSS 17.0 program.
3. Results and discussion
3.1. Optimization of MAE and SPE conditions
Prior to beginning the optimization of the parameters that
affect the microwave extraction (extractant volume, irradiation
time and power), it was necessary to refine the conditions of the
SPE clean-up and pre-concentration step developed by Montes-
deoca-Esponda et al. (2012b). According to the literature, the
extraction solvents used in MAE are polar liquid or mixtures of
polar liquids because only polar solvents are able to absorb
microwave energy. Methanol is characterised by a high dielectric
constant and dissipation factor (Eskilsson and Björklund, 2000).
For that, selection of the extraction solvent were made evaluating
methanol and acetonitrile. Although results were similar, emet-
hanol was used as the extraction solvent for MAE because this
solvent was compatible with mobile phase. After MAE extraction,
approximately 9 mL of extractant was obtained. We checked the
effects of the dilution of the extracts used in the SPE process, which
follows the MAE in the sample preparation process. For that, we
added different water volumes to obtain final solutions of 200, 100
and 50 mL corresponding to 4.5%, 9% and 18%, v/v, respectively, for
SPE elution. Recoveries obtained for 200 and 100 mL (4.5% and 9%,
v/v) did not show any significant differences (range from 93% to
109%) as shown as Table 2. However, low retention efficiencies
were observed in the SPE cartridge using higher percentages of
methanol in the extract. FQs were probably eluted together with
the organic sample phase and not retained on the sorbent. Based
on these results, the MAE extract (9 mL) was mixed with Milli-Q
water to reach a final volume of 100 mL (that is, 9% (v/v)) in the
MAE extracts, which was chosen to obtain the best results.
The optimization of the MAE parameters was performed using a
factorial design strategy because it permits to evaluate a large
number of parameters and optimize the experimental conditions,
minimizing the number of experiments and allowing all of the
possible interactions to be considered (Bazhdanzadeh et al., 2011;
Bingol and Kulcu, 2011). The factorial design was conducted in two
different stages: a first screening phase to determine the influence
of each variable on the analyte recovery and a second phase to
select the optimum values of the variables that have a greater
influence on the extraction efficiency based on a response surface
design (Vega-Morales et al., 2011).
The screening phase was performed on 1 g samples (containing
1000 ng g 1 of each analyte) that were duplicated and randomized.
In this stage of MAE optimization, a 23 factorial design (three
variables at two levels, consisting of 8 combinations) was
employed. The experimental parameters studied were the power
(100 and 300 W), irradiation time (5 and 10 min) and extractant
volume (10 and 15 mL). By applying this factorial design, it was
possible to investigate different correlations among the variables
and their influence on the extraction efficiency.
As observed in Table 3, the partial and bivariate correlations
between the dependent parameters were calculated. Irradiation
Table 2
Recoveries (with%RSD) obtained for the selected FQs in the SPE elution step (n = 3).
Compound 4.5% (%RSD) 9% (%RSD)
NOR 103.2 (4.1) 103.48 (8.2)
CIPRO 96.0 (3.5) 103.17 (8.0)
ENRO 100.63 (5.4) 95.24 (4.9)
DANO 106.06 (4.3) 87.67 (0.4)
SARA 103.84 (1.0) 94.15 (0.4)
143
Table 3
Results of the first experimental design. Relevant variables and correlation between
them under design 23.
NOR CIPRO ENRO DANO SARA
Time 0.686 0.085 0.931 0.146 0.809
Power 0.121 0.577 0.227 0.813 0.423
Volume 0.262 0.383 0.251 0.192 0.001
Bold values signify a = 0.05.
time and extraction power exerted the greatest influence on the
analyte recoveries. Although the extractant volume have little
influence on the extraction, lower methanol volumes were not
used because we had some problems in removing it from the
PTFE vessels. Therefore, we have selected 10 mL like solvent
volume.
Irradiation time and power were studied using a 32 factorial
design in the second phase. The matrix was constructed with these
variables at three levels (4, 6 and 8 min and 100, 150 and 200 W,
respectively). This design consisted of 12 randomly distributed
runs spread into three blocks with a central point for each block. All
analyses were carried out in triplicate.
Using the polynomial fits of the results, the response surface for
each analyte was composed. In Fig. 1, it can observed that the
fluorescence signal improves with high values of the power and
irradiation time for NOR (fig. 1(A)),SARA (Fig. 1(B)) and CIPRO.
However, ENRO and DANO presented the best results for
irradiation times less than 8 min and power of 150 W which could
be explained due to the similarity of their structures.
To achieve a compromise between the optimal conditions for all
of the target analytes, we have selected parameters with a higher
response for NOR, CIPRO and SARA. The responses of ENRO and
DANO still provide sufficient sensitivity for their determination in
real samples under the selected conditions.
Summarizing, higher recoveries of the MAE method were
obtained with 1 g of fish muscle using 8 min of irradiation time,
150 W of extraction power and 10 mL of methanol as the
extractant volume. The 9 mL of obtained extract was diluted
until a final volume of 100 mL prior to the SPE step, and the
analytes were eluted with 2 mL of methanol prior to analysis by
UHPLC-FD. Under these conditions, we obtained a preconcentra-
tion factor of 5.
3.2. Analytical parameters
The proposed method using MAE-SPE-UHPLC-FD was validated
using with the requirements IUPAC, ISO and AOAC International,
between them (Taverniers et al., 2004). In this sense, method
validation is done by evaluating linearity, precision, recovery,
method detection (MDL) and method quantification limit (MQL).
CCa and CCb have been included following the requirements of
European Union regulation 2002/657 . . . Calibration curves were
obtained using spiked samples at different concentration levels
(between 0.5 and 1000 ng g 1) of each compound. Each point on
the calibration curves corresponds to the mean value obtained
from three area measurements. The correlation coefficients were
higher than 0.997 in all cases.
The MDLs and MQLs were calculated from the signal-to-noise
ratios of the individual peaks, assuming minimum detectable
signal-to-noise levels of 3 and 10 (Taverniers et al., 2004),
18% (%RSD)
respectively (Table 4). We have obtained MDLs between 0.1 ng g 1
for DANO and 6.0 ng g 1 for CIPRO.
38.9 (4.5)
42.2 (6.3)
Our results are better than those obtained by other authors with
80.3 (9.3) fluorescence (Zhang et al., 2010) and DAD detection (Evaggelo-
20.0 (4.0) poulou et al., 2014), and they were similar to those obtained in
46.9 (9.4) other published works as shown in Table 5. In this sense, Hernando
et al. (2006) obtained a MDL for ENRO of 2.5 ng g 1 using solid-
144 J. Aufartová et al. / Journal of Food Composition and Analysis 56 (2017) 140–146

1
Fig. 1. Response surface for the effect of power and extraction time of NOR (A) and SARA (B) using MAE-SPE-UHPLC-FD (FQs concentration 1000 ng g ).

Table 4
Linearity range, method limit detection (MDL) and quantification (MQL), precision and recoveries for the proposed method.

Compound Linearity range MDL MQL CCa CCb Inter-day precision (% Intra-day precision (% % Recovery (n = 3)
(ng g 1) (ng g 1
) (ng g 1
) (ng g 1
) (ng g 1
) RSD) (n = 9) RSD) (n = 9)
1 1
25 250 25 250 25 (ng g ) 250 (ng g )
1 1 1 1
(ng g ) (ng g ) (ng g ) (ng g )
NOR 5–1000 1.0 3.5 0.8 2.4 7.1 3.1 6.6 3.1 90.2  2.3 93.8  4.0
CIPRO 25–1000 6.0 20.2 100.3 101.1 6.7 4.2 6.3 4.2 103.7  4.8 102.7  7.1
ENRO 1–1000 0.2 0.5 100.2 100.6 8.2 4.4 8.7 4.4 107.1  4.1 92.3  2.6
DANO 0.5–1000 0.1 0.2 100.2 100.6 5.1 6.7 0.7 6.7 94.7  6.5 106.3  5.2
SARA 5–1000 1.2 3.9 102.0 106.6 6.1 5.0 4.1 5.0 98.3  3.5 99.5  4.2

Table 5
Analytical parameters of similar methods for determination of FQs in fishes.

Fluoroquinoles studied Sample preparation Analytical Recovery MDL Reference

1
method (%) (ng g )
SARA SPE LC-SFD-DAD 82 1 Tyrpenou et al. (2002)
CIPRO, DANO, ENRO, SARA Solvent extraction, centrifugation LC–MS/MS 36–82 1–3 Johnston et al. (2002)
ENRO, CIPRO Solvent extraction, centrifugation LC-FD 51–81 1 Kirbis et al. (2005)
ENRO Solid-liquid extraction LC–MS 40–100 2.5 Hernando et al. (2006)
NOR, DANO, CIPRO, ENRO, Solvent extraction, centrifugation LC–MS/MS 57–96 0.1–1 Dufresne et al. (2007)
SARA
ENRO, NOR SPE LC-FD 75–83 5–23 Zhang et al. (2010)
CIPRO, DANO, ENRO, SARA LLE-SPE LC-DAD 94–105 2–10 Evaggelopoulou et al.
(2014)
NOR, CIPRO, ENRO Dummy molecular imprinted polymer (DMIP)-matrix solid phase LC-FD 64.4–103 0.06–0.22 Sun et al. (2014)
dispersion (MSPD)
ENRO, NOR, CIPRO Solvent extraction, centrifugation-SPE LC–MS/MS 91.1–109 3.3–3.6 Wagil et al. (2014)
NOR, CIPRO, ENRO Solvent extraction, centrifugation LC–MS/MS 88–105 0.2–0.3 Rezk et al. (2015)

liquid extraction and LC–MS detection. Dufresne et al. (2007) and
Johnston et al. (2002) obtained MDLs between 0.1 and 3.6 ng g 1
for selected compounds using different solvent extractions and
LC–MS/MS detection. Using the same detection and ultrasonic
extraction, Samanidou et al. (2008) obtained MDLs between 6 and
8 ng g 1 for CIPRO, DANO, ENRO y SARA. Tyrpenou et al. (2002) and
Kirbis et al. (2005) obtained similar results for ENRO, CIPRO and
SARA. However, the best results were obtained by Sun et al. (2014),
with MDLs between 0.06–0.22 ng g 1 for NOR, CIPRO and ENRO
obtained using dummy molecular imprinted polymer
(DMIP)-matrix solid phase dispersion (MSPD) coupled with LC-
FD. Wagil et al. (2014) determined ENRO, NOR and CIPRO in fish
tissues obtaining MDLs approximately 3.5 ng g 1 using solvent
extraction and LC–MS/MS.
CCa and CCb were determined according to the maximum
residue limit (MRL) for some selected compounds. We have
calculated them following the European Commision Decision
(2002) and the equations published by Freitas et al. (2014) (see
Table 4). Results indicated that CCa and CCb could be near to MDL
and MQL, respectively. Precision, including inter- and intra-day
precisions (relative standard deviations, % RSD) and recoveries
were calculated at two different levels (25 and 250 ng g 1 of each
analyte) from six spiked muscle samples. As can be observed in
Table 4, recoveries were in the range between 90% and 107%,
depending on the compound followed the guidelines marked by
European commission. Relative standard deviations (% RSD) were
lower than 8.7% in all cases, values below the limits defined in
European Commision Decision (2002).
 J. Aufartová et al. / Journal of Food Composition and Analysis 56 (2017) 140–146
Fig. 2. Chromatogram of a spiked fish muscle matrix (DANO 10 ng g 1; NOR, ENRO and SARA 50 ng g 1 and CIPRO 100 ng g 1) and different fish mucle samples from
aquaculture using MAE-SPE-UHPLC-FD procedure. (1) Norfloxacin (2) Ciprofloxacin (3) Enrofloxacin (4) Danofloxacin (5) Sarafloxacin.
3.3. Determination of FQs in gilthead seabream (Sparus aurata)
samples
Regulation No 37/2010 (EEC, 2010) includes the pharmacologi-
cally active substances and their classification regarding their
MRLs in foodstuffs of animal origin. ENRO and CIPRO present MRL
values of 100 mg kg 1 in muscle, while SARA has a MRL value of
30 mg kg 1 in salmon muscle.
Once the effectiveness of the proposed method was verified, it
was applied for the determination of the target analytes in various
real samples obtained from commercial supermarket chains in Las
Palmas de Gran Canaria (Spain). Different fish muscle samples of
gilthead seabream were provided from aquacultural breeding in
this study. Samples were extracted and analysed by the optimized
MAE-SPE-UHPLC-FD method in triplicate. In this work, no matrix
effect was observed. Chromatograms of spiked matrix, blank and
real samples under the final conditions are shown in Fig. 2. Studied
FQs were not detected in the investigated real fish samples.
4. Conclusions
In this work, a trace analytical method based on microwave-
assisted extraction and solid phase extraction clean-up (MAE-SPE)
combined with UHPLC-FD was developed for the determination of
five fluoroquinolones in fishsamples, especifically in gilthead
seabream muscles.
We have optimized different parameters that affect the
extraction process. The best extraction conditions of MAE were
obtained with 10 mL of methanol as the extraction solvent, an
extraction power of 150 W and an extraction time of 8 min. MDLs
and MQLs similar to those of other works were reached due to the
use of a previously optimized SPE clean-up and preconcentration
step. This procedure was a key step for the quantitative
determination of the target compounds because it eliminated
the interferences present in this type of sample. This method also
reduces the well-known matrix effect and provides comparable
results to the other methods with high initial investments.
Although Freitas et al. (2014) tested 14 different extractions
including SPE, by shaking, MAE-SPE procedure with methanol like
extractant demonstrated that it could provide a viable alternative
to other extraction methodologies because it could reduce the time
145
and amount of extractant required, thus considerably lowering the
cost.
The developed method was applied in real samples obtained
from local supermarkets. The results obtained demonstrate that
the optimized methodology could represent a powerful tool in the
evaluation of the occurrence of the target compounds in gilthead
seabream muscle. The proposed methodology combines selectivi-
ty, a high resolution capacity and fast analysis as well as a lower
cost than other conventional sample preparation procedures.
Acknowledgements
The publication is co-financed by the European Social Fund and
the state budget of the Czech Republic, Project no. CZ.1.07/2.3.00/
30.0061. This work was parcially supported by funds provided by
the Spanish Ministry of Economy and Competitiveness, Research
Project CTM2015-66095-C2-1-R.
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