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Determination of quinolones in fish by ultra-high performance liquid chromatography with fluorescence detection using QuEChERS as sample treatment
Accepted Manuscript Determination of quinolones in fish by ultra-high performance liquid chromatography with fluorescence detection using QuEChERS as sample treatment Manuel Lombardo-Agüí, Ana M. García-Campaña, Carmen Cruces-Blanco, Laura Gámiz-Gracia PII:
S0956-7135(14)00609-4
DOI:
10.1016/j.foodcont.2014.10.027
Reference:
JFCO 4127
To appear in:
Food Control
Received Date: 9 July 2014 Revised Date:
16 October 2014
Accepted Date: 17 October 2014
Please cite this article as: Lombardo-Agüí M., García-Campaña A.M., Cruces-Blanco C. & GámizGracia L., Determination of quinolones in fish by ultra-high performance liquid chromatography with fluorescence detection using QuEChERS as sample treatment, Food Control (2014), doi: 10.1016/ j.foodcont.2014.10.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Determination
quinolones
in
fish by ultra-high
performance liquid
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chromatography with fluorescence detection using QuEChERS as sample
3
treatment
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Manuel Lombardo-Agüí, Ana M. García-Campaña, Carmen Cruces-Blanco and Laura
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Gámiz-Gracia*
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Department of Analytical Chemistry, Faculty of Sciences, University of Granada.
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Campus Fuentenueva s/n, E-18071 Granada, Spain.
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(*Corresponding author: Phone: 34 958 248594; E-mail: [email protected])
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Abstract
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A simple and sensitive method is proposed for the simultaneous determination of
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quinolones (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin,
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difloxacin, oxolinic acid and flumequine) in different fish samples. Ultra-high
14
performance liquid chromatography using a partially porous C18 column coupled to
15
fluorescence detection with a wavelength excitation/emission program was used. The
16
sample treatment consisted of extraction and clean-up using the QuEChERS
17
methodology, showing a high efficiency without interferences in the chromatographic
18
determination. The method was characterized in fish tissue in terms of linearity,
19
precision, trueness and limits of detection and quantification. Limits of detection
20
between 0.1 and 4.7 µg/kg were obtained, with recoveries between 72 and 108%. The
21
proposed method has been tested in bass, trout, panga and sturgeon, showing its
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simplicity, sensitivity and suitability for routine analysis in that complex matrix.
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Keywords: Quinolones; UHPLC; fluorescence; QuEChERS; fish.
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ACCEPTED MANUSCRIPT 1
Introduction
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Quinolones are antibiotics widely used in for prophylaxis and treatment of veterinary
27
diseases in livestock farming and aquaculture. As a consequence, residues of these
28
products could be present in commercialized fish and shellfish and reach the consumers.
29
This fact has contributed to the increase of drug resistance bacteria and antibiotic-
30
resistance infections, meaning a health hazard (Hernandez-Serrano, 2005). To protect
31
consumer health, the European Union (EU) has set maximum residue limits (MRLs) for
32
different antibiotics (as quinolones) in several food matrices of animal origin, including
33
fish (European Commission, 2010).
34
In the last years, different reviews about the determination of antibiotics in fish have
35
been published (Samanidou & Evaggelopoulou, 2007; Cañada-Cañada, Espinosa-
36
Mansilla & Muñoz de la Peña, 2009). Focusing on quinolones, a recent review
37
describes the separation methods for their determination in different matrices (Saleh,
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Askal, Refaat & Abdel-aal, 2013). Although capillary electrophoresis (CE) has been
39
reported for the analysis of quinolones in aquatic products (Sun et al., 2012; Juan-
40
García et al., 2006; Juan-García et al., 2007), the most extended analytical technique for
41
the determination of quinolone residues in fish is liquid chromatography (LC) with UV
42
(Samanidou et al., 2007; Cañada-Cañada et al., 2009; Cañada-Cañada, Espinosa-
43
Mansilla, Jiménez Girón & Muñoz de la Peña, 2012), fluorescence (FL) (Cañada-
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Cañada et al., 2012; Rambla-Alegre, Peris-Vicente, Esteve-Romero & Carda-Broch,
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2010; Karbiwnyk, Carr, Turnipseed, Andersen & Miller, 2010; Paschoal, Reyes &
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Rath, 2009a; Li, Yin, Liu & Shang, 2012) or mass spectrometry (MS) detection
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(Karbiwnyk et al., 2010; Samanidou, Evaggelopoulou, Trötzmüller, Guo & Lankmayr,
48
2008; Turnipseed, Clark, Storey & Carr, 2012; Li, Hao, Ji, Xu, Chen, Shen & Ding,
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2009; Zheng, Ruan & Feng, 2009; Paschoal, Reyes & Rath, 2009b). Moreover, ultra-
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ACCEPTED MANUSCRIPT high performance liquid chromatography (UHPLC), usually coupled to MS, has
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emerged as an efficient alternative to conventional LC, offering increased throughput
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and efficiency. Although UHPLC-MS has been explored for the determination of
53
antibiotics (including quinolones) in different food matrices (Lombardo-Agüí, García-
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Campaña, Gámiz-Gracia & Cruces-Blanco, 2012; Pereira Lopes, Cazorla Reyes,
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Romero-González, Garrido-Frenich & J. L. Martínez-Vidal, 2012a; Freitas, Barbosa &
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Ramos, 2013), it has been scarcely used for the analysis of fish (Tang, Lu, Lin, Shin &
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Hwang, 2012; Pereira Lopes, Cazorla Reyes, Romero-González, Martínez Vidal &
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Garrido Frenich, 2012b). However, UHPLC coupled with a sensitive and selective
59
detection technique as FL offers a very interesting alternative for the determination of
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compounds with luminescence properties, such as quinolones. Although LC-FL has
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been extensively used to study quinolones, there are very few applications of UHPLC-
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FL for their determination in fish (Zhang, Chen, Lu, & Dai, 2010).
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An important step in the determination of antibiotics in products of animal origin is the
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extraction and clean-up procedure, as and effective sample preparation is crucial for
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achieving reliable results. As stated in a recent review about determination of drug
66
residues in food (Berendsen, Stolker & Nielen, 2013), the most frequently reported
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sample-preparation methods are solvent extraction, solid-phase extraction (SPE) and
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QuEChERS. SPE has been widely applied for the determination of quinolones in
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different food samples usually preceded by solvent extraction with organic or buffered
70
solvents (Cañada-Cañada et al., 2012; Samanidou et al., 2008; Zhang et al., 2010;
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Samanidou et al., 2007; Cañada-Cañada et al., 2009; Rambla-Alegre et al., 2010;
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Karbiwnyk et al., 2010; Turnipseed et al., 2012). On the other hand, QuEChERS
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methodology presents some advantages, such as its simplicity, minimum steps, and
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effectiveness for cleaning-up complex samples (Lehotay, Anastassiades & Majors,
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ACCEPTED MANUSCRIPT 2010). QuEChERS comprises extraction with an organic solvent –for quinolones this
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solvent is usually acidic acetonitrile (AcN), which improves their recovery (Blasco,
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Masia, Morillas & Picó, 2011)– and phase separation using a high salt content, followed
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by dispersive SPE (dSPE), when a clean-up is required. A summary of applications
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using QuEChERS for the analysis of multi-class veterinary drugs in products of animal
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origin has been recently presented (Berendsen et al., 2013). For instance, QuEChERS
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has been previously reported for the determination of quinolones in different food
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matrices, such as bee products (Lombardo-Agüí et al., 2012) or milk (Lombardo-Agüí,
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Gámiz-Gracia, Cruces-Blanco & García-Campaña, 2011), as well as for the multiclass
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determination of antibiotics in different food commodities (Pereira Lopes et al., 2012a;
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Pereira Lopes et al., 2012b; Stubbings & Bigwood, 2009; Karageorgou, Myridakis,
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Stephanou & Samanidou, 2013). However, as far as we know, only one paper has been
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reported using QuEChERS for the determination of four quinolones in fish by LC-FL
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(Li et al., 2012).
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The aim of this work was to develop a simple and sensitive method for the
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determination of eight quinolones (included in the EU regulation for foodstuff) in fish
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samples using a powerful separation technique, such as UHPLC, coupled with a
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sensitive detection system as FL. QuEChERS methodology for sample treatment has
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been proposed to improve this crucial step, increasing the sample throughput. The
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method has been evaluated in four different fishes, showing its suitability for the
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determination of these residues.
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Experimental
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ACCEPTED MANUSCRIPT 2.1
Chemicals and solutions
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Solvents were LC-MS grade and quinolones were analytical standard grade. Ultrapure
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water (Milli-Q Plus system, Millipore Bedford, MA, USA) was used to prepare buffer
101
and standard solutions. AcN and formic acid (analysis grade) were supplied by Merck
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(Darmstadt, Germany). NaOH and NaH2PO4·H2O were obtained from Panreac-Química
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(Madrid, Spain). Danofloxacin (DANO), sarafloxacin (SARA) and difloxacin (DIFLO)
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were supplied by Riedel-de Haën (Seelze, Germany); flumequine (FLUME) by Sigma
105
Aldrich (St Louis, MO, USA); and marbofloxacin (MARBO), ciprofloxacin (CIPRO),
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enrofloxacin (ENRO) and oxolinic acid (OXO) by Fluka (Steinheim, Germany).
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Individual stock standard solutions (100 mg/L) of each quinolone were prepared by
108
dissolving appropriate amounts of each analyte in AcN/0.02% formic acid aqueous
109
solution (50/50) and were stored in the dark at -20 °C. Working solutions (containing all
110
the quinolones) were prepared daily by dilution of the individual stock solutions with
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water. A 30 mM phosphate buffer solution (pH 7.0) was prepared by dissolving an
112
adequate amount of NaH2PO4·H2O in water and the pH was adjusted with 4 M NaOH
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solution.
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SampliQ EN QuEChERS extraction kits (Agilent Technologies, Waldbron, Germany)
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consisted of 4 g MgSO4, 1 g NaCl, 1 g sodium citrate and 0.5 g sodium citrate dibasic
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sesquihydrate (buffered), or 4 g MgSO4 and 1 g NaCl in (non-buffered). The dSPE kits
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(Agilent Technologies) consisted of (a) 150 mg C18 and 900 mg MgSO4; or (b) 150 mg
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C18, 150 mg PSA and 900 mg MgSO4.
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Acrodisc Nylon membrane syringe filters (0.2 µm, 13 mm, Pall Corp., MI, USA) were
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used to filter the final extract. The mobile phase was filtered before use in nylon
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membrane filters (0.2 µm, 47 mm from Supelco, Bellefonte, PA, USA).
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Instruments
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Chromatographic separation and detection was performed on an Extreme Pressure LC
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system consisting of two pumps, oven, auto sampler, mixer and degasser units (XLC
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from Jasco, Easton, MD, USA) coupled to a fluorescence detector (Jasco X-LC
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3120FP). The separation was achieved using a Poroshell 120 EC-C18 column (50×2.1
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mm, 2.7 µm) from Agilent Technologies.
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Samples were crushed before sample treatment using a kitchen blender. A pH-meter
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with a resolution of ±0.01 pH unit (Crison model pH 2000, Barcelona, Spain), a
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Universal 320R centrifuge (Hettich Zentrifugen, Tuttlingen, Germany) and a vortex-2
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Genie (Scientific Industries, Bohemia, NY, USA) were also used.
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2.3
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The chromatographic separation was achieved using a Poroshell 120 EC-C18 (50×2.1
134
mm, 2.7 µm) partially porous column. The mobile phase consisted of (A): 0.1% formic
135
acid aqueous solution (pH 4.75) and (B): AcN. A linear gradient was selected for the
136
separation with the following program: 0 min 5% B; 4.2 min 21% B; 5 min 90% B.
137
Analysis was performed at a flow rate of 500 µL/min, a column oven temperature of 35
138
°C, with an injection volume of 5 µL.
139
Detection was achieved using the following multi-wavelength excitation/emission
140
program: λex = 294 nm and λem = 514 nm from 0 to 3.2 min (detection of MARBO); λex
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= 278 nm and λem = 476 nm from 3.2 to 4.7 min (detection of CIPRO, DANO, ENRO,
142
SARA and DIFLO); and λex = 325 nm and λem = 366 nm from 4.7 min until the end of
143
the analysis (detection of FLUME and OXO).
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2.4
145
Samples of bass, trout and panga were purchased in a local market, while sturgeon was
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provided by a local fish farm. The bones were removed and the fish muscle was
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UHPLC-FL analysis
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Sample preparation
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ACCEPTED MANUSCRIPT homogenised together with the skin using a kitchen blender, divided in aliquots of 2 g
148
and kept at -20 °C until analysis.
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Samples (2 g) were placed in a 50-mL tube and spiked with a standard solution of
150
quinolones to get the required analyte concentration. Then, they were homogenized and
151
let stand for 15 min. After that, 8 mL of 30 mM NaH2PO4 pH 7.0 was added, shaking
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by hand for 10 s. Subsequently, 10 mL of 5% formic acid in AcN was added, shaking
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by hand for 30 s. Agilent SampliQ EN QuEChERS buffered extraction kit (4 g MgSO4,
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1 g NaCl, 1 g sodium citrate and 0.5 g sodium citrate dibasic sesquihydrate) was added
155
and the tube was shaken vigorously for 2 min. The tube was centrifuged at 9000 rpm for
156
5 min and 4 mL of the upper AcN layer was transferred to dSPE tube containing C18
157
(150 mg) and MgSO4 (900 mg) and shaken by vortex for 2 min. The tube was
158
centrifuged at 5000 rpm for 5 min. Then, 1 mL of supernatant was transferred to a vial,
159
dried at 35 ºC under a stream of nitrogen and re-dissolved in 1 mL of H2O/AcN/formic
160
acid 88/10/2. Before injection into UHPLC system, sample was filtered in order to
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reduce the possibility of column blockage.
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A schedule of the optimized sample treatment is shown in Figure 1.
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Results and discussion
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3.1
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Chromatographic separation and FL detection conditions were optimized with standard
167
solutions of quinolones using mobile phase as solvent.
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Separation was performed in a C18 column (Poroshell 120 EC-C18, 50×2.1 mm, 2.7
169
µm) with partially porous particles, previously reported for the analysis of quinolones
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(Cao, Mou, Gao, Geng, Zhang, Sui, Liang, Sha & Guan, 2013; Lombardo-Agüí,
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Cruces-Blanco, García-Campaña & Gámiz-Gracia, 2014). This type of column provides
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Optimization of UHPLC-FL separation
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ACCEPTED MANUSCRIPT similar or even better resolution than sub-2 µm totally porous columns, reducing the
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analyte diffusion length inside the particle while not increasing column backpressure at
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a given eluent velocity. In this way, the maximum pressure reached during the analysis
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was 210 bar.
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Attending to previous experiences in our laboratory and also from bibliography data, the
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most common solvents to achieve chromatographic separation of quinolones are water
178
and AcN with a low percentage of acid (Lombardo-Agüí et al., 2012; Lombardo-Agüí et
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al., 2011; Hermo, Barrón & Barbosa, 2006). Moreover, the acidity of the mobile phase
180
increases fluorescence emission of quinolones. Therefore, 0.1% formic acid and citric
181
acid aqueous solutions were tested together with AcN. Good separation was obtained
182
with both acids, and formic acid (pH 4.75) was finally selected as the most suitable for
183
an optimal resolution between analytes. When gradient was studied to get the best
184
separation in the shortest time, the optimum results were obtained with the conditions
185
indicated in section 2.3. Temperature was also studied in the range of 30-50 °C,
186
selecting 35 °C as a compromise between resolution and analysis time. The flow rate
187
was set at 500 µL/min, as a compromise between resolution, analysis time and
188
backpressure. The injection volume was 5 µL (full loop).
189
Concerning FL detection, the studied compounds show different excitation and
190
emission wavelengths (Cañada-Cañada et al., 2012). Therefore, λex / λem were selected
191
for each compound by means of a wavelength program as follows: 294/514 nm for
192
MARBO (0 to 3.2 min); 278/476 nm for CIPRO, DANO, ENRO, SARA and DIFLO
193
(3.2 to 4.7 min); and 325/366 nm for FLUME and OXO (4.7 min until the end of the
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analysis).
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Figure 2 shows chromatograms of a blank and a spiked bass sample. Good resolution
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without co-eluting interferents was achieved in just 5 min.
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Optimization of sample treatment
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Bass was selected as representative sample for the optimization of QuEChERS
199
procedure. A 2-g sample was placed in a 50-mL tube, and 8 mL of 30 mM NaH2PO4
200
buffer pH 7.0 was added, shaking by hand for some seconds. Quinolones under study
201
are neutral in a pH range of 5-8, thus this pH facilitates their extraction in the organic
202
phase. Afterwards, 10 mL of 5% formic acid in AcN was added to the tube, shaking by
203
hand for 30 s. Acidic AcN increases the recovery of quinolone extraction, as previously
204
reported (Berendsen et al., 2013). Then, the mixture of salts was added to achieve the
205
phase separation. Buffered and non-buffered extraction kits (whose compositions are
206
included in section 2.1) were studied at this step, obtaining slightly better results with
207
the buffered kit which was selected for the rest of the experimental work. After
208
centrifugation, 4 mL of the upper AcN phase was transferred to a second tube to carry
209
out the dSPE. Two sorbent compositions were studied: (a) C18 (150 mg) + MgSO4 (900
210
mg); and (b) C18 (150 mg) + MgSO4 (900 mg) + PSA (150 mg). When PSA was
211
included recoveries lower than 50% for CIPRO and DANO were obtained, while
212
without PSA, recoveries higher than 70% for all the compounds were achieved.
213
Therefore, sorbent composition (a) was selected for dSPE.
214
Subsequently, 1 mL of the obtained extract was dried under N2 stream at 35 °C and re-
215
dissolved in 1 mL of H2O/AcN/formic acid (88/10/2). Finally this extract was filtered
216
before analysis.
217
3.3
218
The proposed method was evaluated in terms of linearity, limits of detection (LODs)
219
and quantification (LOQs), repeatability (intraday precision) and intermediate precision
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(interday precision) using bass as representative matrix. Finally, trueness was assessed
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Characterization of the method
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by recovery experiments in four different fishes: bass, trout, panga and sturgeon. All the
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samples were previously analysed to confirm the absence of quinolone residues.
223
3.3.1
224
Calibration curves were established with spiked samples (six different concentration
225
levels in the ranges indicated in Table 1) submitted to the QuEChERS procedure
226
described in section 2.4. Each concentration level was processed in duplicate
227
(experimental replicates) and injected also in duplicate (instrumental replicates). LODs
228
and LOQs were calculated as 3×S/N and 10×S/N, respectively. As shown in Table 1,
229
LOQs lower than MRLs established by EU (European Commission, 2010) were
230
obtained.
231
The precision of the method was evaluated in terms of repeatability and intermediate
232
precision. Repeatability was assessed by application of the whole procedure in the same
233
day to five bass samples (experimental replicates) spiked at three concentration levels:
234
25, 75 and 150 µg/kg for CIPRO, ENRO, SARA and DIFLO; 2.5, 7.5 and 15 µg/kg for
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DANO; and 100, 300 and 600 µg/kg for MARBO, OXO and FLUME. Each sample was
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injected in duplicate (instrumental replicates). Intermediate precision was evaluated in a
237
similar way, but the samples were treated and analysed in five different days. The
238
results, expressed as relative standard deviation (RSD) of the peak areas, are given in
239
Table 2.
240
3.3.2
241
In order to check the trueness of the proposed methodology, recovery experiments were
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carried out in different types of fish (bass, trout, panga and sturgeon) at the same
243
concentration levels used in the precision studies. Table 3 shows the range of recoveries
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obtained for each quinolone, which were above 82% except for CIPRO (recoveries
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between 72.2 and 82.2), with RSD lower than 10.2% in all cases. It must be pointed out
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that MARBO could not be determined in sturgeon due to the presence of a co-eluting
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peak.
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4
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A rapid and simple UHPLC–FL method with sample treatment based on QuEChERS
250
has been proposed as a very attractive alternative for the determination of eight
251
quinolones in fish samples. The quinolones were separated and detected in less than 6
252
min showing high sensitivity and selectivity. The method has been applied to different
253
fish species and the LOQs are lower, in all cases, than the MRLs established by EU for
254
these compounds. The sample treatment is quick, effective and cheap, with a high
255
sample throughput, providing good recoveries and precision. Moreover, the
256
combination of QuEChERS with a high efficiency technique such as UHPLC-FL is an
257
environmentally friendly alternative for the determination of quinolones, as the
258
consumption of organic solvent is reduced in both steps of the method (sample
259
treatment and determination). To the best of our knowledge, this is the first time that
260
QuEChERS and UHPLC-FL have been combined for the determination of quinolones
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in fish.
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Conclusions
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Acknowledgements
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The authors gratefully acknowledge the financial support of the “Junta de Andalucía”
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for supporting this work (Proyecto de Excelencia Ref. P12-AGR-1647).
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(2014). Multiresidue analysis of quinolones in water by ultra-high perfomance liquid chromatography with tandem mass spectrometry using a simple and effective sample treatment. Journal Separation Science, 37, 2145-2152.
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Lombardo-Agüí, M., Gámiz-Gracia, L., Cruces-Blanco, C., & García-Campaña, A.M. (2011). Comparison of different sample treatments for the analysis of quinolones in
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milk by capillary-liquid chromatography with laser induced fluorescence detection. Journal Chromatography A, 1218, 4966-4971. Lombardo-Agüí, M., García-Campaña, A. M., Gámiz-Gracia, L., & Cruces-Blanco, C. (2012). Determination of quinolones of veterinary use in bee products by ultra-high performance liquid chromatography-tandem mass spectrometry using a QuEChERS extraction procedure. Talanta, 93, 193-199.
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ACCEPTED MANUSCRIPT Paschoal, J. A. R., Reyes, F. G. R., & Rath, S. (2009a). Determination of quinolone residues in tilapias (Orechromis niloticus) by HPLC-FLD and LC-MS/MS QToF. Food Additives Contaminants, 26, 1331-1340.
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Paschoal, J. A. R., Reyes, F. G. R., & Rath, S. (2009b). Quantitation and identity confirmation of residues of quinolones in tilapia fillets by LC-ESI-MS-MS QToF. Analytical Bioanalytical Chemistry, 394, 2213-2221.
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Pereira Lopes, R., Cazorla Reyes, R., Romero-González, R., Garrido-Frenich, A., & J. L. Martínez-Vidal (2012a). Development and validation of a multiclass method for the
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determination of veterinary drug residues in chicken by ultra-high performance liquid chromatography-tandem mass spectrometry. Talanta, 89, 201-208. Pereira Lopes, R., Cazorla Reyes, R., Romero-González, R., Martínez Vidal, J. L., &
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Garrido Frenich, A. (2012b). Multiresidue determination of veterinary drugs in aquaculture fish samples by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Journal Chromatography B, 895-896, 39-47.
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Rambla-Alegre, M., Peris-Vicente, J., Esteve-Romero, J., & Carda-Broch, S. (2010). Analysis of selected veterinary antibiotics in fish by micellar liquid chromatography
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with fluorescence detection and validation in accordance with regulation 2002/657/EC. Food Chemistry, 123, 1294-1302. Saleh, G. A., Askal, H. F., Refaat, I. H., & Abdel-aal, F. A. M. (2013). Review on recent separation methods for determination of some fluoroquinolones. Journal Liquid Chromatography and Related Technologies, 36, 1401-1420.
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ACCEPTED MANUSCRIPT Samanidou, V. F., & Evaggelopoulou, E. N. (2007). Analytical strategies to determine antibiotic residues in fish. Journal Separation Science, 30, 2549-2569. Samanidou, V. F., Evaggelopoulou, E., Trötzmüller, M., Guo, X., & Lankmayr, E.
seabream
using
liquid
chromatography-tandem
Chromatography A, 1203, 115-123.
mass
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(2008). Multi-residue determination of seven quinolones antibiotics in gilthead spectrometry.
Journal
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Stubbings, G., & Bigwood, T. (2009). The development and validation of a multiclass liquid chromatography tandem mass spectrometry (LC-MS/MS) procedure for the
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determination of veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach. Analytica Chimica Acta, 637, 6878.
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Sun, H., Zuo, Y., Qi, H., & Lu, Y. (2012). Accelerated solvent extraction combined with capillary electrophoresis as an improved methodology for simultaneous determination of residual fluoroquinolones and sulfonamides in aquatic products.
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Analytical Methods, 4, 670-675.
Tang, Y. Y., Lu, H. F., Lin, H. Y., Shin, Y. C., & Hwang D. F. (2012). Development of
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a quantitative multi-class method for 18 antibiotics in chicken, pig, and fish muscle using UPLC-MS/MS. Food Analytical Methods, 5, 1459-1468. Turnipseed, S. B., Clark, S. B., Storey, J. M., & Carr, J.R. (2012). Analysis of veterinary drug residues in frog legs and other aquacultured species using liquid chromatography quadrupole time-of-flight mass spectrometry. Journal Agricultural and Food Chemistry, 60, 4430-4439.
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ACCEPTED MANUSCRIPT Zhang, H., Chen, S., Lu, Y., & Dai, Z. (2010). Simultaneous determination of quinolones in fish by liquid chromatography coupled with fluorescence detection: comparison of sub-2 microm particles and conventional C18 columns. Journal
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Separation Science, 33, 1959-1967. Zheng, M. M., Ruan, G. D., & Feng, Y. Q. (2009). Evaluating polymer monolith in-tube solid-phase microextraction coupled to liquid chromatography/quadrupole time-of-
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flight mass spectrometry for reliable quantification and confirmation of quinolone
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antibacterials in edible animal food. Journal Chromatography A, 1216, 7510-7519.
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ACCEPTED MANUSCRIPT Figure captions:
Figure 1. Diagram of sample treatment. (a) SampliQ EN QuEChERS extraction kit: 4 g
Dispersive SPE kit: 150 mg C18; 900 mg MgSO4.
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MgSO4; 1 g NaCl; 1 g sodium citrate; 0.5 g sodium citrate dibasic sesquihydrate; (b)
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Figure 2. Chromatograms of a blank (red) and a spiked sample of bass (blue): 7.5 µg/kg for DANO (3), 75 µg/kg for CIPRO (2), ENRO (4), SARA (5) and DIFLO (6), 300
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µg/kg for MARBO (1), OXO (7) and FLUME (8).
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ACCEPTED MANUSCRIPT Table 1. Statistical and performance characteristics of the proposed method in bass samples. R2
LOD (µg/kg)
0.998 0.994 0.991 0.995 0.995 0.995 0.992 0.996
3.2 1.2 0.1 0.5 1.4 0.6 4.7 3.5
LOQ (µg/kg) MRL in fish (µg/kg)
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100 100 100 30 300 100 600
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10.5 4.1 0.3 1.7 4.5 2.0 15.6 11.7
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MARBO CIPRO DANO ENRO SARA DIFLO OXO FLUME
Calibration range (µg/kg) 20-1000 5-250 0.5-25 5-250 5-250 5-250 20-1000 20-1000
ACCEPTED MANUSCRIPT Table 2. Precision study. Repeatability and intermediate precision expressed as RSD (%) (n=10)
MARBO CIPRO DANO ENRO SARA DIFLO OXO FLUME
Level 1 1.9 3.3 2.8 3.0 4.3 5.4 2.4 3.2
Level 2 2.1 4.1 3.1 3.1 6.4 3.3 1.9 2.6
Intermediate precision Level 3 1.3 2.6 1.5 1.7 2.8 1.6 5.2 2.9
Level 1 6.7 4.2 3.0 1.8 4.1 7.9 3.7 4.4
Level 2 6.4 9.3 7.8 9.3 10.4 10.5 9.4 8.9
Level 3 3.4 5.1 2.8 3.5 5.3 4.4 5.5 3.9
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Repeatability
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Level 1: 2.5 µg/kg for DANO, 25 µg/kg for CIPRO, ENRO, SARA and DIFLO, 100µg/kg for MARBO, OXO and FLUME. Level 2: 7.5 µg/kg for DANO, 75 µg/kg for CIPRO, ENRO, SARA and DIFLO, 300 µg/kg for MARBO, OXO and FLUME. Level 3: 15 µg/kg for DANO, 150 µg/kg for CIPRO, ENRO, SARA and DIFLO, 600 µg/kg for MARBO, OXO and FLUME.
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a) Not determined due to the presence of a co-eluting peak
Panga 91.9 – 94.8 78.4 – 82.0 88.6 – 95.9 97.4 – 98.3 93.2 – 94.4 98.5 – 100.5 99.5 – 104.5 100.8 – 105.4
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Bass 86.7 – 93.7 73.8 – 82.2 86.2 – 91.8 94.6 – 98.8 93.7 – 104.6 100.4 – 106.0 100.8 – 105.6 99.1 – 107.8
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MARBO CIPRO DANO ENRO SARA DIFLO OXO FLUME
Recovery ranges % Trout Sturgeon 89.3 – 93.7 (a) 72.2 – 80.0 76.5 – 77.2 83.3 – 93.8 83.2 – 90.4 91.0 – 100.9 89.8 – 94.3 82.0 – 100.4 87.4 – 92.8 97.1 – 105.4 98.2 – 103.0 97.8 – 107.4 100.1 – 102.6 97.8 – 106.0 96.8 – 99.8
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ACCEPTED MANUSCRIPT Highlights UHPLC-FL method for the determination of quinolones regulated by EU in food QuEChERS methodology shows high extraction efficiency without matrix interferences The method was validated for bass, trout, panga and sturgeon Limits of detections were between 0.1-4.7 ppb and recoveries were between 72-108% The method is quick, effective, cheap, with a high sample throughput
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S0956-7135(14)00609-4
DOI:
10.1016/j.foodcont.2014.10.027
Reference:
JFCO 4127
To appear in:
Food Control
Received Date: 9 July 2014 Revised Date:
16 October 2014
Accepted Date: 17 October 2014
Please cite this article as: Lombardo-Agüí M., García-Campaña A.M., Cruces-Blanco C. & GámizGracia L., Determination of quinolones in fish by ultra-high performance liquid chromatography with fluorescence detection using QuEChERS as sample treatment, Food Control (2014), doi: 10.1016/ j.foodcont.2014.10.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Determination
quinolones
in
fish by ultra-high
performance liquid
2
chromatography with fluorescence detection using QuEChERS as sample
3
treatment
4
Manuel Lombardo-Agüí, Ana M. García-Campaña, Carmen Cruces-Blanco and Laura
5
Gámiz-Gracia*
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Department of Analytical Chemistry, Faculty of Sciences, University of Granada.
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Campus Fuentenueva s/n, E-18071 Granada, Spain.
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(*Corresponding author: Phone: 34 958 248594; E-mail: [email protected])
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of
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Abstract
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A simple and sensitive method is proposed for the simultaneous determination of
12
quinolones (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin,
13
difloxacin, oxolinic acid and flumequine) in different fish samples. Ultra-high
14
performance liquid chromatography using a partially porous C18 column coupled to
15
fluorescence detection with a wavelength excitation/emission program was used. The
16
sample treatment consisted of extraction and clean-up using the QuEChERS
17
methodology, showing a high efficiency without interferences in the chromatographic
18
determination. The method was characterized in fish tissue in terms of linearity,
19
precision, trueness and limits of detection and quantification. Limits of detection
20
between 0.1 and 4.7 µg/kg were obtained, with recoveries between 72 and 108%. The
21
proposed method has been tested in bass, trout, panga and sturgeon, showing its
22
simplicity, sensitivity and suitability for routine analysis in that complex matrix.
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Keywords: Quinolones; UHPLC; fluorescence; QuEChERS; fish.
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ACCEPTED MANUSCRIPT 1
Introduction
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Quinolones are antibiotics widely used in for prophylaxis and treatment of veterinary
27
diseases in livestock farming and aquaculture. As a consequence, residues of these
28
products could be present in commercialized fish and shellfish and reach the consumers.
29
This fact has contributed to the increase of drug resistance bacteria and antibiotic-
30
resistance infections, meaning a health hazard (Hernandez-Serrano, 2005). To protect
31
consumer health, the European Union (EU) has set maximum residue limits (MRLs) for
32
different antibiotics (as quinolones) in several food matrices of animal origin, including
33
fish (European Commission, 2010).
34
In the last years, different reviews about the determination of antibiotics in fish have
35
been published (Samanidou & Evaggelopoulou, 2007; Cañada-Cañada, Espinosa-
36
Mansilla & Muñoz de la Peña, 2009). Focusing on quinolones, a recent review
37
describes the separation methods for their determination in different matrices (Saleh,
38
Askal, Refaat & Abdel-aal, 2013). Although capillary electrophoresis (CE) has been
39
reported for the analysis of quinolones in aquatic products (Sun et al., 2012; Juan-
40
García et al., 2006; Juan-García et al., 2007), the most extended analytical technique for
41
the determination of quinolone residues in fish is liquid chromatography (LC) with UV
42
(Samanidou et al., 2007; Cañada-Cañada et al., 2009; Cañada-Cañada, Espinosa-
43
Mansilla, Jiménez Girón & Muñoz de la Peña, 2012), fluorescence (FL) (Cañada-
44
Cañada et al., 2012; Rambla-Alegre, Peris-Vicente, Esteve-Romero & Carda-Broch,
45
2010; Karbiwnyk, Carr, Turnipseed, Andersen & Miller, 2010; Paschoal, Reyes &
46
Rath, 2009a; Li, Yin, Liu & Shang, 2012) or mass spectrometry (MS) detection
47
(Karbiwnyk et al., 2010; Samanidou, Evaggelopoulou, Trötzmüller, Guo & Lankmayr,
48
2008; Turnipseed, Clark, Storey & Carr, 2012; Li, Hao, Ji, Xu, Chen, Shen & Ding,
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2009; Zheng, Ruan & Feng, 2009; Paschoal, Reyes & Rath, 2009b). Moreover, ultra-
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ACCEPTED MANUSCRIPT high performance liquid chromatography (UHPLC), usually coupled to MS, has
51
emerged as an efficient alternative to conventional LC, offering increased throughput
52
and efficiency. Although UHPLC-MS has been explored for the determination of
53
antibiotics (including quinolones) in different food matrices (Lombardo-Agüí, García-
54
Campaña, Gámiz-Gracia & Cruces-Blanco, 2012; Pereira Lopes, Cazorla Reyes,
55
Romero-González, Garrido-Frenich & J. L. Martínez-Vidal, 2012a; Freitas, Barbosa &
56
Ramos, 2013), it has been scarcely used for the analysis of fish (Tang, Lu, Lin, Shin &
57
Hwang, 2012; Pereira Lopes, Cazorla Reyes, Romero-González, Martínez Vidal &
58
Garrido Frenich, 2012b). However, UHPLC coupled with a sensitive and selective
59
detection technique as FL offers a very interesting alternative for the determination of
60
compounds with luminescence properties, such as quinolones. Although LC-FL has
61
been extensively used to study quinolones, there are very few applications of UHPLC-
62
FL for their determination in fish (Zhang, Chen, Lu, & Dai, 2010).
63
An important step in the determination of antibiotics in products of animal origin is the
64
extraction and clean-up procedure, as and effective sample preparation is crucial for
65
achieving reliable results. As stated in a recent review about determination of drug
66
residues in food (Berendsen, Stolker & Nielen, 2013), the most frequently reported
67
sample-preparation methods are solvent extraction, solid-phase extraction (SPE) and
68
QuEChERS. SPE has been widely applied for the determination of quinolones in
69
different food samples usually preceded by solvent extraction with organic or buffered
70
solvents (Cañada-Cañada et al., 2012; Samanidou et al., 2008; Zhang et al., 2010;
71
Samanidou et al., 2007; Cañada-Cañada et al., 2009; Rambla-Alegre et al., 2010;
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Karbiwnyk et al., 2010; Turnipseed et al., 2012). On the other hand, QuEChERS
73
methodology presents some advantages, such as its simplicity, minimum steps, and
74
effectiveness for cleaning-up complex samples (Lehotay, Anastassiades & Majors,
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ACCEPTED MANUSCRIPT 2010). QuEChERS comprises extraction with an organic solvent –for quinolones this
76
solvent is usually acidic acetonitrile (AcN), which improves their recovery (Blasco,
77
Masia, Morillas & Picó, 2011)– and phase separation using a high salt content, followed
78
by dispersive SPE (dSPE), when a clean-up is required. A summary of applications
79
using QuEChERS for the analysis of multi-class veterinary drugs in products of animal
80
origin has been recently presented (Berendsen et al., 2013). For instance, QuEChERS
81
has been previously reported for the determination of quinolones in different food
82
matrices, such as bee products (Lombardo-Agüí et al., 2012) or milk (Lombardo-Agüí,
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Gámiz-Gracia, Cruces-Blanco & García-Campaña, 2011), as well as for the multiclass
84
determination of antibiotics in different food commodities (Pereira Lopes et al., 2012a;
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Pereira Lopes et al., 2012b; Stubbings & Bigwood, 2009; Karageorgou, Myridakis,
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Stephanou & Samanidou, 2013). However, as far as we know, only one paper has been
87
reported using QuEChERS for the determination of four quinolones in fish by LC-FL
88
(Li et al., 2012).
89
The aim of this work was to develop a simple and sensitive method for the
90
determination of eight quinolones (included in the EU regulation for foodstuff) in fish
91
samples using a powerful separation technique, such as UHPLC, coupled with a
92
sensitive detection system as FL. QuEChERS methodology for sample treatment has
93
been proposed to improve this crucial step, increasing the sample throughput. The
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method has been evaluated in four different fishes, showing its suitability for the
95
determination of these residues.
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Experimental
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ACCEPTED MANUSCRIPT 2.1
Chemicals and solutions
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Solvents were LC-MS grade and quinolones were analytical standard grade. Ultrapure
100
water (Milli-Q Plus system, Millipore Bedford, MA, USA) was used to prepare buffer
101
and standard solutions. AcN and formic acid (analysis grade) were supplied by Merck
102
(Darmstadt, Germany). NaOH and NaH2PO4·H2O were obtained from Panreac-Química
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(Madrid, Spain). Danofloxacin (DANO), sarafloxacin (SARA) and difloxacin (DIFLO)
104
were supplied by Riedel-de Haën (Seelze, Germany); flumequine (FLUME) by Sigma
105
Aldrich (St Louis, MO, USA); and marbofloxacin (MARBO), ciprofloxacin (CIPRO),
106
enrofloxacin (ENRO) and oxolinic acid (OXO) by Fluka (Steinheim, Germany).
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Individual stock standard solutions (100 mg/L) of each quinolone were prepared by
108
dissolving appropriate amounts of each analyte in AcN/0.02% formic acid aqueous
109
solution (50/50) and were stored in the dark at -20 °C. Working solutions (containing all
110
the quinolones) were prepared daily by dilution of the individual stock solutions with
111
water. A 30 mM phosphate buffer solution (pH 7.0) was prepared by dissolving an
112
adequate amount of NaH2PO4·H2O in water and the pH was adjusted with 4 M NaOH
113
solution.
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SampliQ EN QuEChERS extraction kits (Agilent Technologies, Waldbron, Germany)
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consisted of 4 g MgSO4, 1 g NaCl, 1 g sodium citrate and 0.5 g sodium citrate dibasic
116
sesquihydrate (buffered), or 4 g MgSO4 and 1 g NaCl in (non-buffered). The dSPE kits
117
(Agilent Technologies) consisted of (a) 150 mg C18 and 900 mg MgSO4; or (b) 150 mg
118
C18, 150 mg PSA and 900 mg MgSO4.
119
Acrodisc Nylon membrane syringe filters (0.2 µm, 13 mm, Pall Corp., MI, USA) were
120
used to filter the final extract. The mobile phase was filtered before use in nylon
121
membrane filters (0.2 µm, 47 mm from Supelco, Bellefonte, PA, USA).
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Instruments
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Chromatographic separation and detection was performed on an Extreme Pressure LC
124
system consisting of two pumps, oven, auto sampler, mixer and degasser units (XLC
125
from Jasco, Easton, MD, USA) coupled to a fluorescence detector (Jasco X-LC
126
3120FP). The separation was achieved using a Poroshell 120 EC-C18 column (50×2.1
127
mm, 2.7 µm) from Agilent Technologies.
128
Samples were crushed before sample treatment using a kitchen blender. A pH-meter
129
with a resolution of ±0.01 pH unit (Crison model pH 2000, Barcelona, Spain), a
130
Universal 320R centrifuge (Hettich Zentrifugen, Tuttlingen, Germany) and a vortex-2
131
Genie (Scientific Industries, Bohemia, NY, USA) were also used.
132
2.3
133
The chromatographic separation was achieved using a Poroshell 120 EC-C18 (50×2.1
134
mm, 2.7 µm) partially porous column. The mobile phase consisted of (A): 0.1% formic
135
acid aqueous solution (pH 4.75) and (B): AcN. A linear gradient was selected for the
136
separation with the following program: 0 min 5% B; 4.2 min 21% B; 5 min 90% B.
137
Analysis was performed at a flow rate of 500 µL/min, a column oven temperature of 35
138
°C, with an injection volume of 5 µL.
139
Detection was achieved using the following multi-wavelength excitation/emission
140
program: λex = 294 nm and λem = 514 nm from 0 to 3.2 min (detection of MARBO); λex
141
= 278 nm and λem = 476 nm from 3.2 to 4.7 min (detection of CIPRO, DANO, ENRO,
142
SARA and DIFLO); and λex = 325 nm and λem = 366 nm from 4.7 min until the end of
143
the analysis (detection of FLUME and OXO).
144
2.4
145
Samples of bass, trout and panga were purchased in a local market, while sturgeon was
146
provided by a local fish farm. The bones were removed and the fish muscle was
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Sample preparation
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ACCEPTED MANUSCRIPT homogenised together with the skin using a kitchen blender, divided in aliquots of 2 g
148
and kept at -20 °C until analysis.
149
Samples (2 g) were placed in a 50-mL tube and spiked with a standard solution of
150
quinolones to get the required analyte concentration. Then, they were homogenized and
151
let stand for 15 min. After that, 8 mL of 30 mM NaH2PO4 pH 7.0 was added, shaking
152
by hand for 10 s. Subsequently, 10 mL of 5% formic acid in AcN was added, shaking
153
by hand for 30 s. Agilent SampliQ EN QuEChERS buffered extraction kit (4 g MgSO4,
154
1 g NaCl, 1 g sodium citrate and 0.5 g sodium citrate dibasic sesquihydrate) was added
155
and the tube was shaken vigorously for 2 min. The tube was centrifuged at 9000 rpm for
156
5 min and 4 mL of the upper AcN layer was transferred to dSPE tube containing C18
157
(150 mg) and MgSO4 (900 mg) and shaken by vortex for 2 min. The tube was
158
centrifuged at 5000 rpm for 5 min. Then, 1 mL of supernatant was transferred to a vial,
159
dried at 35 ºC under a stream of nitrogen and re-dissolved in 1 mL of H2O/AcN/formic
160
acid 88/10/2. Before injection into UHPLC system, sample was filtered in order to
161
reduce the possibility of column blockage.
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A schedule of the optimized sample treatment is shown in Figure 1.
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Results and discussion
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3.1
166
Chromatographic separation and FL detection conditions were optimized with standard
167
solutions of quinolones using mobile phase as solvent.
168
Separation was performed in a C18 column (Poroshell 120 EC-C18, 50×2.1 mm, 2.7
169
µm) with partially porous particles, previously reported for the analysis of quinolones
170
(Cao, Mou, Gao, Geng, Zhang, Sui, Liang, Sha & Guan, 2013; Lombardo-Agüí,
171
Cruces-Blanco, García-Campaña & Gámiz-Gracia, 2014). This type of column provides
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Optimization of UHPLC-FL separation
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173
analyte diffusion length inside the particle while not increasing column backpressure at
174
a given eluent velocity. In this way, the maximum pressure reached during the analysis
175
was 210 bar.
176
Attending to previous experiences in our laboratory and also from bibliography data, the
177
most common solvents to achieve chromatographic separation of quinolones are water
178
and AcN with a low percentage of acid (Lombardo-Agüí et al., 2012; Lombardo-Agüí et
179
al., 2011; Hermo, Barrón & Barbosa, 2006). Moreover, the acidity of the mobile phase
180
increases fluorescence emission of quinolones. Therefore, 0.1% formic acid and citric
181
acid aqueous solutions were tested together with AcN. Good separation was obtained
182
with both acids, and formic acid (pH 4.75) was finally selected as the most suitable for
183
an optimal resolution between analytes. When gradient was studied to get the best
184
separation in the shortest time, the optimum results were obtained with the conditions
185
indicated in section 2.3. Temperature was also studied in the range of 30-50 °C,
186
selecting 35 °C as a compromise between resolution and analysis time. The flow rate
187
was set at 500 µL/min, as a compromise between resolution, analysis time and
188
backpressure. The injection volume was 5 µL (full loop).
189
Concerning FL detection, the studied compounds show different excitation and
190
emission wavelengths (Cañada-Cañada et al., 2012). Therefore, λex / λem were selected
191
for each compound by means of a wavelength program as follows: 294/514 nm for
192
MARBO (0 to 3.2 min); 278/476 nm for CIPRO, DANO, ENRO, SARA and DIFLO
193
(3.2 to 4.7 min); and 325/366 nm for FLUME and OXO (4.7 min until the end of the
194
analysis).
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Figure 2 shows chromatograms of a blank and a spiked bass sample. Good resolution
196
without co-eluting interferents was achieved in just 5 min.
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Optimization of sample treatment
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Bass was selected as representative sample for the optimization of QuEChERS
199
procedure. A 2-g sample was placed in a 50-mL tube, and 8 mL of 30 mM NaH2PO4
200
buffer pH 7.0 was added, shaking by hand for some seconds. Quinolones under study
201
are neutral in a pH range of 5-8, thus this pH facilitates their extraction in the organic
202
phase. Afterwards, 10 mL of 5% formic acid in AcN was added to the tube, shaking by
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hand for 30 s. Acidic AcN increases the recovery of quinolone extraction, as previously
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reported (Berendsen et al., 2013). Then, the mixture of salts was added to achieve the
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phase separation. Buffered and non-buffered extraction kits (whose compositions are
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included in section 2.1) were studied at this step, obtaining slightly better results with
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the buffered kit which was selected for the rest of the experimental work. After
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centrifugation, 4 mL of the upper AcN phase was transferred to a second tube to carry
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out the dSPE. Two sorbent compositions were studied: (a) C18 (150 mg) + MgSO4 (900
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mg); and (b) C18 (150 mg) + MgSO4 (900 mg) + PSA (150 mg). When PSA was
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included recoveries lower than 50% for CIPRO and DANO were obtained, while
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without PSA, recoveries higher than 70% for all the compounds were achieved.
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Therefore, sorbent composition (a) was selected for dSPE.
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Subsequently, 1 mL of the obtained extract was dried under N2 stream at 35 °C and re-
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dissolved in 1 mL of H2O/AcN/formic acid (88/10/2). Finally this extract was filtered
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before analysis.
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3.3
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The proposed method was evaluated in terms of linearity, limits of detection (LODs)
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and quantification (LOQs), repeatability (intraday precision) and intermediate precision
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(interday precision) using bass as representative matrix. Finally, trueness was assessed
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Characterization of the method
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by recovery experiments in four different fishes: bass, trout, panga and sturgeon. All the
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samples were previously analysed to confirm the absence of quinolone residues.
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3.3.1
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Calibration curves were established with spiked samples (six different concentration
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levels in the ranges indicated in Table 1) submitted to the QuEChERS procedure
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described in section 2.4. Each concentration level was processed in duplicate
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(experimental replicates) and injected also in duplicate (instrumental replicates). LODs
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and LOQs were calculated as 3×S/N and 10×S/N, respectively. As shown in Table 1,
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LOQs lower than MRLs established by EU (European Commission, 2010) were
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obtained.
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The precision of the method was evaluated in terms of repeatability and intermediate
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precision. Repeatability was assessed by application of the whole procedure in the same
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day to five bass samples (experimental replicates) spiked at three concentration levels:
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25, 75 and 150 µg/kg for CIPRO, ENRO, SARA and DIFLO; 2.5, 7.5 and 15 µg/kg for
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DANO; and 100, 300 and 600 µg/kg for MARBO, OXO and FLUME. Each sample was
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injected in duplicate (instrumental replicates). Intermediate precision was evaluated in a
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similar way, but the samples were treated and analysed in five different days. The
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results, expressed as relative standard deviation (RSD) of the peak areas, are given in
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Table 2.
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3.3.2
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In order to check the trueness of the proposed methodology, recovery experiments were
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carried out in different types of fish (bass, trout, panga and sturgeon) at the same
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concentration levels used in the precision studies. Table 3 shows the range of recoveries
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obtained for each quinolone, which were above 82% except for CIPRO (recoveries
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between 72.2 and 82.2), with RSD lower than 10.2% in all cases. It must be pointed out
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that MARBO could not be determined in sturgeon due to the presence of a co-eluting
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peak.
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4
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A rapid and simple UHPLC–FL method with sample treatment based on QuEChERS
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has been proposed as a very attractive alternative for the determination of eight
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quinolones in fish samples. The quinolones were separated and detected in less than 6
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min showing high sensitivity and selectivity. The method has been applied to different
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fish species and the LOQs are lower, in all cases, than the MRLs established by EU for
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these compounds. The sample treatment is quick, effective and cheap, with a high
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sample throughput, providing good recoveries and precision. Moreover, the
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combination of QuEChERS with a high efficiency technique such as UHPLC-FL is an
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environmentally friendly alternative for the determination of quinolones, as the
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consumption of organic solvent is reduced in both steps of the method (sample
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treatment and determination). To the best of our knowledge, this is the first time that
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QuEChERS and UHPLC-FL have been combined for the determination of quinolones
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in fish.
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Conclusions
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Acknowledgements
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The authors gratefully acknowledge the financial support of the “Junta de Andalucía”
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for supporting this work (Proyecto de Excelencia Ref. P12-AGR-1647).
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Figure 1. Diagram of sample treatment. (a) SampliQ EN QuEChERS extraction kit: 4 g
Dispersive SPE kit: 150 mg C18; 900 mg MgSO4.
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MgSO4; 1 g NaCl; 1 g sodium citrate; 0.5 g sodium citrate dibasic sesquihydrate; (b)
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Figure 2. Chromatograms of a blank (red) and a spiked sample of bass (blue): 7.5 µg/kg for DANO (3), 75 µg/kg for CIPRO (2), ENRO (4), SARA (5) and DIFLO (6), 300
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µg/kg for MARBO (1), OXO (7) and FLUME (8).
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ACCEPTED MANUSCRIPT Table 1. Statistical and performance characteristics of the proposed method in bass samples. R2
LOD (µg/kg)
0.998 0.994 0.991 0.995 0.995 0.995 0.992 0.996
3.2 1.2 0.1 0.5 1.4 0.6 4.7 3.5
LOQ (µg/kg) MRL in fish (µg/kg)
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100 100 100 30 300 100 600
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10.5 4.1 0.3 1.7 4.5 2.0 15.6 11.7
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MARBO CIPRO DANO ENRO SARA DIFLO OXO FLUME
Calibration range (µg/kg) 20-1000 5-250 0.5-25 5-250 5-250 5-250 20-1000 20-1000
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MARBO CIPRO DANO ENRO SARA DIFLO OXO FLUME
Level 1 1.9 3.3 2.8 3.0 4.3 5.4 2.4 3.2
Level 2 2.1 4.1 3.1 3.1 6.4 3.3 1.9 2.6
Intermediate precision Level 3 1.3 2.6 1.5 1.7 2.8 1.6 5.2 2.9
Level 1 6.7 4.2 3.0 1.8 4.1 7.9 3.7 4.4
Level 2 6.4 9.3 7.8 9.3 10.4 10.5 9.4 8.9
Level 3 3.4 5.1 2.8 3.5 5.3 4.4 5.5 3.9
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Repeatability
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Level 1: 2.5 µg/kg for DANO, 25 µg/kg for CIPRO, ENRO, SARA and DIFLO, 100µg/kg for MARBO, OXO and FLUME. Level 2: 7.5 µg/kg for DANO, 75 µg/kg for CIPRO, ENRO, SARA and DIFLO, 300 µg/kg for MARBO, OXO and FLUME. Level 3: 15 µg/kg for DANO, 150 µg/kg for CIPRO, ENRO, SARA and DIFLO, 600 µg/kg for MARBO, OXO and FLUME.
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a) Not determined due to the presence of a co-eluting peak
Panga 91.9 – 94.8 78.4 – 82.0 88.6 – 95.9 97.4 – 98.3 93.2 – 94.4 98.5 – 100.5 99.5 – 104.5 100.8 – 105.4
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Bass 86.7 – 93.7 73.8 – 82.2 86.2 – 91.8 94.6 – 98.8 93.7 – 104.6 100.4 – 106.0 100.8 – 105.6 99.1 – 107.8
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MARBO CIPRO DANO ENRO SARA DIFLO OXO FLUME
Recovery ranges % Trout Sturgeon 89.3 – 93.7 (a) 72.2 – 80.0 76.5 – 77.2 83.3 – 93.8 83.2 – 90.4 91.0 – 100.9 89.8 – 94.3 82.0 – 100.4 87.4 – 92.8 97.1 – 105.4 98.2 – 103.0 97.8 – 107.4 100.1 – 102.6 97.8 – 106.0 96.8 – 99.8
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ACCEPTED MANUSCRIPT Highlights UHPLC-FL method for the determination of quinolones regulated by EU in food QuEChERS methodology shows high extraction efficiency without matrix interferences The method was validated for bass, trout, panga and sturgeon Limits of detections were between 0.1-4.7 ppb and recoveries were between 72-108% The method is quick, effective, cheap, with a high sample throughput
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