Accepted Manuscript Title: Determination of nitroimidazole residues in aquaculture tissue using ultra high performance liquid chromatography coupled to tandem mass spectrometry Author: Anna Gadaj Valentina di Lullo Helen Cantwell Martin McCormack Ambrose Furey Martin Danaher PII: DOI: Reference:

S1570-0232(14)00262-1 http://dx.doi.org/doi:10.1016/j.jchromb.2014.04.024 CHROMB 18893

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

16-9-2013 21-3-2014 10-4-2014

Please cite this article as: A. Gadaj, V. di Lullo, H. Cantwell, M. McCormack, A. Furey, M. Danaher, Determination of nitroimidazole residues in aquaculture tissue using ultra high performance liquid chromatography coupled to tandem mass spectrometry, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.04.024 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.

Determination of nitroimidazole residues in aquaculture tissue using ultra high performance liquid chromatography coupled to tandem mass spectrometry Anna Gadaja,c, Valentina di Lullob, Helen Cantwella, Martin McCormacka, Ambrose Fureyc

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and Martin Danahera*

Food Safety Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland

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Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila,

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a

c

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Italy

Team Elucidate, Department of Chemistry, Cork Institute of Technology, Bishopstown,

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Cork, Ireland

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*Corresponding author: Food Safety Department, Teagasc Food Research Centre, Ashtown,

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Dublin 15, Ireland, Tel. +353 1 8059500; fax: +353 1 8059550

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Email address: [email protected]

Abstract

An UHPLC-MS/MS method was developed for the quantitative confirmatory analysis of residues of nitroimidazole drugs (dimetridazole, ipronidazole, metronidazole, ornidazole and ronidazole) and their corresponding hydroxy metabolites (HMMNI, ipronidazile-OH and metronidazole-OH) in aquaculture tissue. Samples were extracted by shaking in acetonitrile, water, MgSO4 and NaCl before being defatted with n-hexane pre-saturated with acetonitrile and concentrated under nitrogen. Nitroimidazole residues were determined by UHPLC-MS/MS operating in positive electrospray ionisation mode using a reversed phase BEH C18 column. The method was validated according to the EU Commission Decision 2002/657/EC guidelines.

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The following performance studies were carried out: specificity, selectivity, linearity, within laboratory repeatability (WLr)/reproducibility (WLR), accuracy, precision, decision limit (CCα), detection capability (CCβ), absolute recovery and stability. The analytical range of the

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method is 0.1 - 20 μg kg-1. Accuracy and precision of the method, under within-laboratory reproducibility conditions, ranged from 83 to 105% and 2.3 to 14.0%, respectively. CCα were

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0.07 - 1.0 μg kg-1 depending on analyte and matrix. A total of 50 samples can be analysed in a single day using the assay. The method has been extensively evaluated through application to

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real test samples.

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Keywords: nitroimidazole residues, UHPLC-MS/MS, aquaculture, prawn, shrimp, salmon,

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trout, sea bass and tilapia

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1. Introduction

The 5-nitroimidazoles are imidazole heterocycles substituted with a nitro functional

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group on the fifth position on its ring. They are rapidly metabolised and the main metabolites of DMZ, MNZ and IPZ result from the oxidation of the side chain in the C-2 position of the imidazole ring to form hydroxy metabolites, namely HMMNI, MNZ-OH and IPZ-OH, respectively [1, 2]. RNZ has a different degradation pathway but results in an identical metabolite to that of DMZ. Nitroimidazoles are known to be effective in the treatment of parasitic infections in fish and are licensed for ornamental fish [3-5]. However, these compounds are suspected to be carcinogenic and mutagenic to humans [1] and as a consequence they were banned for the use in food-producing animals or in products intended for human consumption within the European Union under Regulation 2377/90 [6], repealed by 470/2009 [7] and 37/2010 [8]. DMZ, MNZ and RNZ are classified as prohibited

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pharmacologically active substances for which no MRL can be established as they constitute a hazard to the health of the consumer at whatever limit [6]. Therefore, detection of nitroimidazole residues or their metabolites must be considered as a violation of the EU

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regulation. Consequently, individual residues of DMZ, MNZ, RNZ and their corresponding hydroxy metabolites have each been assigned a recommended level (RL) of 3 µg kg-1, for the

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performance of analytical methods, by the Community Reference Laboratory in Berlin [9]. This means that CCα values for confirmatory methods should be lower than this

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recommended value. The European Council Directive 96/23/EC requires that nitroimidazole residues are monitored in food because of the potential for illegal use of these drugs in

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aquaculture [10].

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Several analytical procedures have been developed for the determination of nitroimidazole residues and the corresponding hydroxy metabolites in a wide range of

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matrices [11], including animal tissue [12-25], eggs [13, 14, 16, 17, 21, 26], plasma [13, 16,

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27], milk[16, 29, 30], honey [16, 29, 31] and animal feed stuff [16, 32, 33] using LCMS(/MS). In recent years, LC-MS/MS has become more widely used because it allows the

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detection in the low µg kg-1 region. Published methods for the determination of nitroimidazoles have mainly employed ethyl acetate or acetonitrile as organic extraction solvent followed by a solid phase extraction (SPE) clean-up [13-17, 34] or without a further clean-up step [19, 26, 27, 29, 32].

From examining published literature, four LC-MS methods were found to be validated

to be capable of analysing nitroimidazoles in fish samples [14, 17, 28, 31]. Mottier et al. reported a LC-MS/MS method developed on a triple quadrupole instrument for nitroimidazoles in eggs, chicken muscle and fish [14]. Kaufmann et al. [31] published the multi-residue method utilizing an UHPLC-Orbitrap configuration for the analysis of DMZ, IPZ, MNZ, RNZ, TNZ and their corresponding hydroxy metabolites following SPE clean-up.

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The multi-residue screening method published by Peters et al. [17] was based on a time-offlight mass spectrometer and tested for IPZ and IPZ-OH following SPE clean-up. Finally, a the confirmatory multi-residue method published by Smith et al. [28] tested for MNZ by LC-

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ion trap mass spectrometry following a hexane wash without any further purification step. The present study reports a relatively fast, simple and sensitive method for the

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quantitative confirmatory analysis of DMZ, IPZ, MNZ, ORZ, RNZ and their corresponding hydroxy metabolies (HMMNI, MNZ-OH and IPZ-OH) in aquaculture tissue by UHPLC-

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MS/MS. The analytical method has been validated for prawns and finfish including the following fish: salmon, sea bass, trout and tilapia in accordance with the EU Commission

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Decision 2002/657/EC criteria [35]. Finally, the applicability of this analytical procedure was

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demonstrated in a survey of aquaculture samples of different origin.

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2. Experimental

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2.1 Reagents and apparatus

Ultra-pure water (18.2 MOhm) was generated in house using a Millipore water

purification system (Millipore, Cork, Ireland). Methanol (MeOH) and acetonitrile (MeCN), both LC-MS Chromasolv® grade, Chromasolv®, for HPLC, isopropyl-alcohol (i-PrOH), methanol-D, 99.5% (MeOH-D), Chromasolv®, for HPLC, ≥97.0%, n-hexane, puriss 98.0100% formic acid and puriss p.a., anhydrous powder, very fine, ≥98.0%, magnesium sulfate (MgSO4) were sourced from Sigma-Aldrich (Dublin, Ireland). n-Hexane was saturated with MeCN by adding MeCN until it was no longer miscible. Sodium chloride (NaCl) was sourced from Applichem (Darmstadt, Germany). Polypropylene tubes (50 mL and 15 mL) with screw

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caps were obtained from Sarstedt Ltd. (Wexford, Ireland). Glass dispensers Dispensette® 111 from Brand GmbH & Co. KG (Wertheim, Germany) were used for aliquoting acetonitrile, water and n-hexane pre-saturated with acetonitrile, respectively. An Ultra-Turrax probe

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blender from IKA (Staufen, Germany), a Talboys advanced multi-tube vortexer (Apex Scientific, Kildare, Ireland), a Mistral 3000i centrifuge from Davidson and Hardy (Dublin,

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Ireland), a Turbovap LV evaporator from Caliper Life Sciences (Runcorn, UK) and a Grant GLS400 water bath from Davidson & Hardy (Dublin, Ireland) were used during sample

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preparation.

Dimetridazole (DMZ, P/N 31707-250MG), metronidazole (MNZ, P/N 46461-250MG),

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ornidazole (ORZ, O5879-5G) and ronidazole (RNZ, R7635-5G) were purchased from Sigma-

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Aldrich (Dublin, Ireland). HMMNI (P/N NM002-50), ipronidazole (IPZ, P/N NM004-50), ipronidazole-OH (IPZ-OH, P/N NM006-50), metronidazole-OH (MNZ-OH, P/N NM008-50) NM001-50), HMMNI-D3

(P/N

NM003-25),

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and dimetridazole-D3 (DMZ-D3, P/N

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ipronidazole-D3 (IPZ-D3, P/N NM005-50), ipronidazole-OH-D3 (IPZ-OH-D3, P/N NM00725), metronidazole-13C2,15N2 (MNZ-13C2,15N2, P/N NM014-25), metronidazole-OH-D2

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(MNZ-OH-D2, P/N NM013-25) and ronidazole-D3 (RNZ-D3, P/N NM010-25) were purchased from Witega (Berlin, Germany). All standards and internal standards stock solutions were prepared at a concentration of 1 mg mL-1 in MeOH and MeOH-D, respectively. Intermediate mixed standard solutions were prepared at 50 and 5 µg mL-1 in MeOH. Working calibration standard solutions at concentrations of 5 (std 1), 10 (std 2), 20 (std 3), 50 (std 4), 100 (std 5), 200 (std 6) and 400 (std 7) ng mL-1 were prepared in MeOH by serial dilution. An intermediate internal standard mix solution was prepared at 6 µg mL-1 using MeOH-D as the diluent. A working internal standard mix solution was prepared at 120 ng mL-1 in MeOH-D.

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All standards and internal standards stock solutions, and intermediate mix solutions were found to be stable for at least one year when stored at -20°C. Working calibration standards and working internal standard mix solutions were found to be stable for at least

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three months when stored at -20°C.

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2.2 Preparation of extracted matrix calibrants and recovery control checks

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Extracted matrix calibrants were prepared by fortifying negative aquaculture samples (2 g ± 0.01 g) prior to extraction with 40 µL of std 1 and 100 µL of each calibration standard

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solution (std 1 - 7) to give curves in the range of 0.1 – 20 µg kg-1.

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An additional four blank samples were spiked after extraction, two with std 3 (50 µL)

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2.3 Sample Preparation

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and two with std 6 (50 µL) to monitor for loss of analytes during extraction.

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Aquaculture tissue samples were homogenised on receipt and stored at -20°C. A

portion of each sample (2 g ± 0.01 g) was weighed into a 50 mL polypropylene tube. Samples were fortified with 50 µL of a 120 ng mL-1 internal standard mix solution and left to stand for 15 min. A 12 mL volume of MeCN was added into each tube. The tube contents were homogenised using an Ultra-Turrax probe blender for 30 s and 8 mL of H2O was subsequently added. After vortexing for 60 s, approximately 1 g of NaCl and approximately 4 g of MgSO4 was added to tubes, which were then shaken vigorously by hand for a minimum of 60 s. Samples were centrifuged at 3500 rpm (2842 × g) for 12 minutes at 4oC and the supernatants were transferred into clean empty 50 mL polypropylene tubes. Following the addition of 12 mL of n-hexane pre-saturated with acetonitrile, samples were vortexed for 30 s,

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centrifuged at 3500 rpm (2842 × g) for 12 minutes at 4oC and the upper layer was aspirated to waste. A 6 mL portion of the remaining extract was transferred to clean empty 15 mL polypropylene tubes and evaporated to low volume, but not to dryness, under high flow of

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nitrogen (at least 15 Bar) at 40oC on a Turbovap LV system. This step needs to be monitored closely because over drying of extracts results in a lower recovery. Samples were reconstituted in

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H2O:MeOH (95:5, v/v; 500 µL) by vortexing (60 s) and further shaking (10 min at 40oC) in a Grant GLS400 water bath. Extracts were filtered through 0.2 µm PTFE 13 mm Millex-FG

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syringe filters (Millipore, Cork, Ireland) and 10 µL was injected onto the UHPLC-MS/MS

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system.

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2.4 UHPLC-MS/MS conditions

Separations were performed using a Waters (Milford MA, USA) Acquity™ UPLC

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system comprising of a stainless steel Acquity UPLC® BEH C18 analytical column (2.1 x 100

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mm, particle size 1.7μm) (Waters UK P/N 186002352) equipped with an Acquity UPLC®

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Column In-Line Filter Unit, containing a 0.2 µm stainless steel replacement filter (Waters UK, P/N 205000343) maintained at a temperature of 60oC and the pump was operated at a flow rate of 0.45 mL min-1. A binary gradient system was used to separate analytes comprising of mobile phase A, 0.01 % formic acid in water and mobile phase B, 0.01 % formic acid in MeOH. The gradient profile was as follows: (1) 0.0 min, 97% A, (2) 3.0 min, 85% A, (3) 5.0 min, 80% A, (4) 5.5 min, 0% A, (5) 6.5 min, 0% A, (6) 6.51 min, 97% A, (7) 8.00 min, 97% A. The injection volume was 10 µL in a full loop mode. The UHPLC autosampler was sequentially rinsed using strong and weak washes that consisted of H2O:MeOH:i-PrOH (10:80:10 v/v/v; 1000 µL) and H2O:MeOH (80:20, v/v; 2000 µL), respectively.

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Nitroimidazole residues were detected using a Waters Quattro Premier XE triple quadrupole instrument operating in positive electrospray ionisation mode (Milford, MA, USA). The UHPLC-MS/MS system was controlled by MassLynx™ software and data was

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processed using TargetLynx™ software (both from Waters). The electrospray voltage was set at 1.0 kV. The desolvation and source temperatures were set at 450 and 120oC, respectively.

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Nitrogen was employed as the desolvation and cone gases, which were set at 1000 L h-1 and 50 L h-1, respectively. Argon was employed as the collision gas, at a flow rate of 0.19 mL

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min-1, which typically gave pressures of 3.52 × 10-3 mbar. The MS conditions were optimised by teed infusion of 1 µg mL-1 standard solutions and 50% mobile phases A and B at flow rates

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of 10 µL min-1 and 0.2 mL min-1, respectively. The cone voltage was optimised for each

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precursor ion and the two most abundant product fragment ions were selected. The SRM windows were time-sectored, and dwell time and inter-channel delays were set to get

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maximum response for the instrument. These conditions are outlined in Table 1. Inter scan

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delay was set to 20 ms between successive SRM windows and inter-channel delay was set to 5 ms. Dwell times ranged from 20 ms (HMMNI) to 100 ms (MNZ-OH and ORZ).

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Stable isotope-labelled analogues of the analytes were used as internal standards. IPZ-OH-D3 was used as an internal standard for ornidazole which has no deuterated analogue available. Linear regression of the calibration data was performed with 1/x2 weighting. The calibration curves were obtained by plotting the response factor (analyte peak area / internal standard peak area) as a function of the analyte concentration.

2.5 Method validation

The method was validated according to the EU Commission Decision 2002/657/EC criteria [35]. The following performance studies were carried out: selectivity, specificity,

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linearity of calibration curve, working range of the method, within laboratory repeatability (WLr), within laboratory reproducibility (WLR), accuracy, precision, decision limit (CCα), detection capability (CCβ), matrix effects, absolute recovery and stability. Validation was

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carried out at 1.5, 3.0 and 4.5 µg kg-1. The decision limit (CCα) and the detection capability (CCβ), defined in 2002/657/EC, were calculated in accordance with ISO 11843 [35].

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Two different validations were carried out in prawn, namely within laboratory repeatability (WLr) and within laboratory reproducibility (WLR), which are described below.

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The WLr study was carried out by a single analyst, and the method was repeated on three separate days. To evaluate WLR, the method was carried out on three separate days by three

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different analysts. In total six runs were executed for prawn validation study. 18 portions

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coming from the same negative control test material were used for the establishment of WLr. Whereas, 18 portions coming from 18 different negative control test materials were used for

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the establishment of WLR. Additionally, one validation was carried out in finfish, namely

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within laboratory reproducibility (WLR). Finfish samples used in validation included the following fish: salmon, sea bass, trout and tilapia. Both in prawn and finfish validation,

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samples were fortified at 1.5 (n = 6), 3.0 (n = 6) and 4.5 (n = 6) µg kg-1. To evaluate matrix effects in both matrices, 20 blank samples from different sources

of each matrix were post-extraction spiked at a level of 3 µg kg-1 for all analytes. Matrix effects for each analyte were calculated as percentage differences between the signals obtained when matrix extracts were injected and when a standard solution of equivalent concentration was injected, divided by the signal of the latter [36].

2.6 Stability studies

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Stability studies were designed to assess the stability of residues in sample matrix and in processed sample extracts. Two studies were carried out to assess the stability of residues in sample matrix. The

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first measured the impact of frozen storage (-20°C) and the second the effect of freeze thaw cycles. Stability was assessed in both prawn and salmon muscle tissue samples fortified at a

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concentration of 3 µg kg-1 (n = 5 replicates per experimental treatment). In the frozen storage study, muscle tissue samples were analysed at day 0 and following storage at -20°C for 1, 2,

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4, 8, 12 and 20 weeks in 50 mL polypropylene tubes. In the freeze thaw cycles study, all samples were fortified on day 0 and were stored frozen at -20°C in 50 mL polypropylene

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tubes. The fortified samples were subjected to 0, 1, 2 and 3 freeze thaw cycles, which were

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staggered to allow the samples to be analysed in a single analytical run for each matrix. A further series of studies were designed to investigate the stability of nitroimidazole

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residues in processed sample extracts. The stability of nitroimidazole residues was assessed

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in the acetonitrile extracts stored at 4°C for 1 and 7 days. Additionally, the stability in the final injection solvent during storage in autosampler vials at 10°C (0, 1, 4 and 5 days), 4°C (0,

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1, 2, 3 and 4 weeks) and -20°C (0, 1, 2, 3 and 4 weeks) was determined. All extract stability studies were assessed at a concentration of 3 µg kg-1 (n = 5 replicates).

2.7 Application of the method

The method developed in this study has been applied to routine samples.

Prawns/shrimps and fish were purchased from different local markets. Altogether 244 aquaculture samples were tested including 160 prawns and shrimps and 84 fish, namely: salmon, sea bass, trout, tilapia, sea bream and catfish. The countries of origin of prawn/shrimp and finfish samples are presented in Fig. S1, Supplementary data.

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3. Results and discussion

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3.1 Method development

3.1.1 UHPLC-MS/MS conditions

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In this study, a number of different mobile phases and different additives including volatile buffer (ammonium formate) and acid (formic) were assessed. Satisfactory results

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were achieved using mobile phases composed of water and methanol containing 0.01%

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formic acid. Nitroimidazole residues were analysed by electrospray ionisation mass spectrometry (ESI-MS) using positive ionisation mode. Data was acquired in the SRM mode

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by monitoring protonated molecules [M+H]+ for each analyte. The electrospray voltage,

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desolvation and source temperatures, desolvation, cone and collision gas flow rates were optimised to get maximum response for the instrument. Positive electrospray ionisation mode

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was used for all analytes and surprisingly decreasing of electrospray voltage from 3 to 1 kV enhanced the signals for most of them. The SRM windows were time sectored and the adequate conditions were established through effective set-up of dwell times, inter-scan delay and inter-channel delay. A total of 12-15 data points were typically obtained across a peak to attain reproducible integration and thus achieve highly repeatable quantitative analysis. In order to satisfy confirmatory criteria as outlined in the EU Commission Decision 2002/657/EC, a total of four identification points are required to verify the presence of a group A substance and this criterion was fulfilled. Investigation of analytical columns included a variety of Acquity UPLC® columns, namely HSS T3, BEH C18, HSS C18, BEH Shield RP18 and CSH C18. A simple binary

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gradient was developed for the separation of the nitroimidazole residues on a BEH C18 column. Initially, a rapid chromatographic method was developed but due to the issues with separation of some analytes (namely DMZ, HMMNI and IPZ-OH) from isobaric matrix

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interferences the total run time was extended from 4 to 8 minutes. The chromatographic resolution of nitroimidazole analytes from isobaric interfering peaks is presented in Fig. 1.

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Good chromatographic resolution is critical to ensure good accuracy and precision, and to satisfy confirmatory criteria [11, 37, 38]. The impact of chromatographic resolution on

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method accuracy is shown in Fig. 2. Following implementation of the improved chromatographic separation both method accuracy and precision improved significantly.

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Isobaric matrix interferences could lead to a failure to confirm a truly positive finding or a

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reporting of a false positive finding depending on the ion affected (quantification or confirmation, respectively). As presented in Fig. 1A, in the case of HMMNI, both

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quantification (sea bass, smoked salmon and salmon samples) and confirmation ions (smoked

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salmon and salmon samples) were affected by the isobaric interfering peaks. In the case of DMZ (Fig. 1B) and IPZ-OH (Fig. 1C) only quantification ions were affected. The impact of

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isobaric interference could potentially be addressed by the selection of alternative product ions. In the case of DMZ only three suitably intense products ions at 95, 96 and 81 m/z were generated in low energy collision induced dissociation (CID) experiments. In contrast, HMMNI and IPZ-OH gave additional products ions to those selected. HMMNI gave four further product ions, namely, 67, 69, 94 and 112 m/z but were much less intense than the selected product ions (55 and 140 m/z). The selection of these ions would have resulted in a significant reduction in the sensitivity of the analytical method. Similarly, a range of product ions were observed for IPZ-OH (128, 107, 82 and 59 m/z). The product ions at 59 and 82 m/z show good potential to be used in LC-MS/MS analysis but again would likely lead to a reduction of the sensitivity of the method. It is highlighted that the product ions used in this

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paper have been used in most of the published LC-MS/MS papers but other ions should be included if possible for better qualitative analysis [11]. The impact of chromatographic resolution on the ion ratio is presented in Fig. 3.

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Results show that the short gradient resulted in two smoked salmon samples being incorrectly identified as false negative for IPZ-OH and HMMNI because MS/MS identification criteria

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was not satisfied due to a matrix interfering peak. In contrast, a matrix interfering peak present in the trace of the quantitation ion for DMZ resulted in an overestimation of

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concentrations in sample 6 (seabass) and 9 (salmon). Further overestimation was observed for HMMNI concentrations in both seabass (samples 2, 5 and 6) and salmon (25 and 28)

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samples. The improved chromatographic method presented in Fig. 4 significantly reduced the

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overestimation of concentrations. Critically assessment of ion ratio results presented for HMMNI, indicates more variation in concentration results compare to other IPZ-OH or DMZ,

3.1.2 Sample preparation

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with some results slightly overestimated.

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The aim of this study was to develop a rapid sample preparation procedure. Previous

research carried out on these compounds showed that acetonitrile is an effective extraction solvent because it extracts less fat and protein [13, 14, 17, 18, 20, 21, 26, 27, 29, 32]. It has also been shown that the addition of NaCl removes highly water-soluble polar

matrix interferences and reduces the need for frequent cleaning of the MS ionisation source [14, 21]. The addition of a combination of MgSO4 and NaCl induces phase separation of acetonitrile from aqueous phase and this salting-out principle is being commonly used in QuEChERs protocols [39]. The next step in sample preparation was to develop a clean-up step through evaluation of the effects of different sorbents for a dispersive solid phase extraction (dSPE). In this study, the

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effects of four different sorbent materials, namely C18, primary/secondary amine (PSA), ZSep/C18 and Z-Sep+ were investigated. A comprehensive review of sorbent materials has been presented elsewhere [39]. However, a combination of C18 and Z-Sep (zirconia-coated silica)

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particles is recommended for clean-up of samples containing less than 15% fat. The C18 binds fats through hydrophobic interaction, while the zirconia acts as a Lewis acid, attracting

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compounds with electron donating groups. Z-Sep+, dual bonded C18 and zirconia on silica, is recommended for clean-up of samples containing greater than 15% fat.

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The effectiveness of hexane defatting was also evaluated because this approach was successfully applied by other groups in the past [14, 18, 24, 26, 27, 32]. The aforementioned

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sorbents with and without the hexane additions were used in method optimization

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experiments. The application of PSA sorbent clean-up resulted in low recovery for DMZ and IPZ at 10 and 11%, respectively. Recovery results for Z-Sep/C18 dSPE were unsatisfactory for

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HMMNI (17%), IPZ-OH (55%) and MNZ-OH (7%). Whereas recovery improved using Z-

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Sep+ dSPE, recoveries for HMMNI, IPZ-OH and MNZ-OH were not ideal at 60%, 65% and 53%, respectively. The inclusion of C18 dSPE clean-up gave satisfactory recoveries but did

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not reduce interferences. As an alternative, low temperature fat precipitation was performed by freezing the solvent extract at -20°C. Under these conditions, the interfering compounds are frozen, whereas analytes remain in liquid phase and are subsequently separated [40]. In this application, unsatisfactory recovery results were obtained for the majority of the analytes. The outcomes from the method development show that water addition, salting-out

(MgSO4 and NaCl addition) and subsequent hexane defatting gave satisfactory results. This approach removed an adequate amount of matrix interferences and in turn allowed for quicker sample preparation times. Following extraction, the extraction solvent is evaporated at 40°C to low volume, but not to dryness, under a high stream of nitrogen. It was found that evaporation of solvent to dryness led to significant loses in analyte recovery [11]. A range of

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different reconstitution solvents were investigated, H2O:MeOH (95:5, v/v) was found to

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provide satisfactory sensitivity and precision with acceptable peak shape.

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3.2 Method validation

3.2.1 Selectivity, specificity, linearity and matrix studies

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The specificity of the method was investigated through monitoring for interferences in the UHPLC-MS/MS traces for the analytes and internal standards. The absence of cross talk

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was verified by injecting analytes and internal standards singly. The selectivity of the method

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was established through testing 160 prawn/shrimp and 86 fish samples from different sources without observed interferences. Moreover the selectivity of the method was confirmed by

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additional analysis of prawn (n = 10) and finfish (n = 20) samples fortified at 3 μg kg-1 and

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accuracy (shown in Fig. 2) and precision were calculated. Carry-over was assessed by injecting blank solvent (MeOH) following the highest calibration standard. This approach was

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applied during each analysis of the study samples and no analyte signal appeared in blank solvent (MeOH). The linearity of the method was evaluated over the range 0.1 – 20 µg kg-1 during validation studies. The linearity of the curves (measured as R2) were greater than 0.99. Matrix effects were also evaluated (Table 2) and suppression effect was observed for all analytes in both matrices. The greatest amount of suppression was observed for RNZ (66%) and IPZ (71%) in prawn and finfish matrix, respectively. However, due to the use of stable isotope-labelled internal standards, the signal loss resulting from matrix effect could be compensated to improve accuracy and precision.

3.2.2 Accuracy and precision

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The results of the study collected in Table 3 show that the method accuracy was satisfactory for all nitroimidazole residues in both WLR (prawn and finfish) and WLr (prawn only) studies. In order to satisfy accuracy criterion as outlined in the EU Commission

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Decision 2002/657/EC, an accuracy in the range of 70 – 110% is required. In prawn matrix accuracy ranged between 98 and 104% and 96 and 104% in WLr and WLR studies,

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respectively. While in finfish matrix accuracy ranged between 83 and 105% in WLR study. The EU Commission Decision 2002/657/EC states that precision calculated as CV should be

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as low as possible. The precision of the method was satisfactory for all analytes in both matrices. CVs ranged between 1.6 and 10.1% and 2.3 and 14.0% in WLr and WLR studies in

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prawn, respectively. Precision ranged between 2.5 and 12.0% in WLR study in the case of

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finfish.

3.2.3 Decision limit (CCα) and detection capability (CCβ)

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The calculated CCα values ranged between 0.07 µg kg-1 (MNZ) and 0.33 µg kg-1

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(RNZ) in prawn, and 0.08 µg kg-1 (MNZ) and 0.29 µg kg-1 (ORZ) in finfish. As required by

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the Guidelines for the Implementation of Decision 2002/657/EC [35], they were verified in both matrices by the analysis of 20 blank samples from different sources of each matrix fortified at corresponding CCα levels for all analytes. For most analytes, with the exception of HMMNI either in prawn and finfish matrix, the analyte identification was satisfactory. CCα values calculated for HMMNI ranging 0.13 µg kg-1 (prawn) and 0.22 µg kg-1 (finfish) were too low and further investigation was necessary. The identification criteria were fulfilled for HMMNI at a concentration level of 1.0 µg kg-1 in both matrices. CCα and CCβ for all analytes are listed in Table 3 and are all below the RL of 3 µg kg-1.

3.2.4 Sample survey

16 Page 16 of 34

The method developed in this study has been used to monitor for the presence of trace levels (0.1 – 20 µg kg-1) of nitroimidazole residues in aquaculture samples available on the Irish market. A total of 244 samples with different countries of origin as shown in Fig. S1,

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Supplementary data, were analysed. None of the samples tested contained detectable

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quantities of nitroimidazole residues or their corresponding hydroxy metabolites.

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3.2.5 Stability studies

3.2.5.1 Matrix stability studies

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The stability of nitroimidazole residues was assessed in fortified prawn and salmon

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muscle tissue samples stored for 20 weeks at -20°C. This study shows that nitroimidazole residues were stable in prawn and salmon muscle tissue during frozen storage. Mean accuracy

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and precision results were mostly within the acceptable levels of 70 to 110% and 0.05). Precision was

Determination of nitroimidazole residues in aquaculture tissue using ultra high performance liquid chromatography coupled to tandem mass spectrometry.

An UHPLC-MS/MS method was developed for the quantitative confirmatory analysis of residues of nitroimidazole drugs (dimetridazole, ipronidazole, metro...
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