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Accurate Quantitation and Analysis of Nitrofuran Metabolites, Chloramphenicol, and Florfenicol in Seafood by UHPLCMS/MS: Method Validation and Regulatory Samples Fadi Aldeek, Kevin Hsieh, Obiadada Ugochukwu, Ghislain Gerard, and Walter Hammack J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04360 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017
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Journal of Agricultural and Food Chemistry
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Accurate Quantitation and Analysis of Nitrofuran Metabolites, Chloramphenicol, and
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Florfenicol in Seafood by UHPLC-MS/MS: Method Validation and Regulatory Samples
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Fadi Aldeek*#, Kevin C. Hsieh, Obiadada N. Ugochukwu, Ghislain Gerard, Walter Hammack
4
Florida Department of Agriculture and Consumer Services, Division of Food Safety, 3125 Conner
5
Boulevard, Tallahassee, Florida 32399-1650
6
ABSTRACT
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We developed and validated a method for the extraction, identification, and quantitation of
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four nitrofuran metabolites: (3-amino-2-oxazolidinone (AOZ); 3-amino-5-morpholinomethyl-2-
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oxazolidinone (AMOZ); semicarbazide (SC); and 1-aminohydantoin (AHD)), as well as
10
chloramphenicol and florfenicol, in a variety of seafood commodities. Samples were extracted
11
by liquid-liquid extraction techniques, analyzed by UHPLC-MS/MS, and quantitated using
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commercially sourced, derivatized nitrofuran metabolites, with their isotopically-labelled
13
internal standards in-solvent. We obtained recoveries of 90-100% at various fortification levels.
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The limit of detection (LOD) was set at 0.25 ng/g for AMOZ and AOZ, 1 ng/g for AHD and SC,
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and 0.1 ng/g for the phenicols. Various extraction methods, standard stability, derivatization
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efficiency, and improvements to conventional quantitation techniques were also investigated.
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We successfully applied this method to the identification and quantitation of nitrofuran
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metabolites and phenicols in 102 imported seafood products. Our results revealed that 4 of the
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samples contained residues from banned veterinary drugs.
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KEYWORDS:
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Liquid/Liquid Extraction, Method Development, Validation
Nitrofurans,
Metabolites,
Chloramphenicol,
Florfenicol,
UHPLC/MS-MS,
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#
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Richmond, VA 23219.
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Current address: Eurofins Lancaster Laboratories (PSS) Insourcing Solutions, 601 E Jackson St,
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INTRODUCTION
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Nitrofurans and phenicols are antibiotic drugs that have been widely used to combat
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bacterial diseases in animal production because of their low cost, ready availability, and
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effectiveness against resistant infections.1-4 In past decades, the use of these two classes of
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antibiotics in aquatic products has drastically increased due to the high mortality rates of
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bacterial diseases and significant financial losses thus incurred by the aquaculture industry
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worldwide.5-8
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However, nitrofurans and their metabolites have been banned in Europe and other
34
countries due to their carcinogenic, mutagenic, and genotoxic effects in humans.8-10
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Chloramphenicol has also been restricted by many organizations, such as the Food and Drug
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Administration (FDA), for use in animals due to its potential side effects in humans, e. g.
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hematological abnormalities, grey baby syndrome, and, most fatally, aplastic anemia – all of
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which it can cause in humans who may be exposed to it during application or upon ingestion of
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food products that contain residues of the drug.11-13
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The most commonly tested nitofurans are furazolidone, furaltadone, nitrofurazone and
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nitrofurantoin (Figure 1).
In vivo, these nitrofurans are quickly metabolized, so the
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concentration of the parent compound rapidly drops below the detection and quantitation
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limits of modern analytical methods.14 The metabolites formed from the above nitrofurans are: 2
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3-amino-2-oxazolidinone
(AOZ);
3-amino-5-morpholinomethyl-2-oxazolidinone
(AMOZ);
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semicarbazide (SC); and 1-aminohydantoin (AHD), respectively (Figure 1).15 These toxic
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metabolites then bind to protein tissues, forming metabolite-protein adducts that are stable for
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long periods of time (Figure 2).16 An acidic hydrolysis step has been the most commonly used
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method to liberate the covalently bound metabolites.17 Due to the low molecular weight (75-
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to-201 amu), high polarity, poor retention on reversed phase columns, poor ionization, and
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strong covalent bindings of these metabolites with protein tissues, derivatization with 2-nitro-
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benzaldehyde (NBA) (Figure 3) is also a crucial step to allowing their detection using the
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selective and sensitive LC-MS/MS technique.18 Currently, nitrofurans are regulated at a target
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level of 1 ng/g – and chloramphenicol at 0.3 ng/g – in the United States, EU, and Canada.
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Various methods for the extraction and detection of nitrofurans and/or phenicols in seafood
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using LC-MS/MS have been described in the literature – although none have analyzed
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florfenicol in addition to the nitrofurans and chloramphenicol.19-26
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Three selected reports, which were also experimentally explored, are detailed herein. It is
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important to note that the recoveries in the reports are those calculated by the authors, which
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were not “absolute”. For example, Ye et. al described a sensitive method for the detection of
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nitrofuran metabolites using LC-MS/MS.27 They used a liquid-liquid extraction with ethyl
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acetate and aqueous phosphate buffer. Analyte recoveries in that method were found to be
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between 62-to-91% depending on the extracted analyte. The limits of detection (LOD) and
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quantitation (LOQ) were found to be 0.15 ng/g and 0.3 ng/g, respectively. Recently, An et al.
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developed a water-acetonitrile liquid-liquid extraction method of nitrofuran metabolites and 3
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chloramphenicol in shrimp, followed by LC-MS/MS detection.28 In that report, analyte
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recoveries were studied at 0.15, 0.3, and 0.6 ng/g for chloramphenicol and 0.5, 1.0, and 2.0
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ng/g for the nitrofuran metabolites. They reported an average recovery of 98-109% with intra-
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day and inter-day relative standard deviations (RSD) of 12% and 17.7%, respectively. More
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recently, Veach et. al reported on the quantitation of chloramphenicol and nitrofuran
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metabolites in various aquaculture matrices including shrimp, catfish, and crawfish.
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Derivatization of nitrofuran metabolites was performed using microwave assistance, followed
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by automated solid phase extraction (SPE).29 They fortified their samples with 5 spike levels for
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each analyte. Recoveries were found to be between 89 and 107% with RSD ≤ 8.3%. They
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determined an LOD of 0.06 ng/g and LOQ of 0.2 ng/g for nitrofuran metabolites and an LOD of
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0.01 ng/g and LOQ of ≤0.03 ng/g for chloramphenicol.
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Despite the method development effort made on the extraction and LC-MS/MS detection of
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phenicols and nitrofuran metabolites in different seafood matrices, recovery and quantitation
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of these analytes was sub-optimal for a critical reason: in previous reports, recoveries were
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often calculated using a fortified matrix-extracted calibration curve (“calibrating samples”). This
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was done due to the unavailability of commercially-sourced, derivatized nitrofuran metabolites.
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Calibrating samples were prepared by adding the non-derivatized metabolites at different
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concentrations of the curve to matrix blanks, and then derivatizing and extracting these
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metabolites from the fortified matrix to form a multi-point, calibrating samples curve. This
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prevented optimal recovery and quantitation of nitrofuran metabolites, since the fortified
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spikes and the curve built from calibrating samples were identical in terms of extraction 4
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procedure. It was therefore impossible to detect physicochemical loss of analyte during
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extraction, which led to the reporting of erroneously high, non-absolute recoveries.
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Absolute recovery is a measure of the quantity of analyte lost during an extraction
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procedure, by physical or chemical processes. It describes the efficiency of an extraction
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method. To accurately calculate the absolute recovery of spikes – which methods that employ
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calibrating samples curves cannot do – our method used underivatized nitrofuran metabolites
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for spikes only. These spikes were then quantitated with calibration curves made using
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commercially-sourced, derivatized nitrofuran metabolites spiked into extracted-matrix blanks
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post-extraction. This allowed us to compare the absolute recovery of our method to the
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absolute recoveries of the other methods after replicating them (An and Ye) or approximating
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them (Veach; a conventional oven was substituted in place of a microwave oven) to
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accommodate our equipment.
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After establishing absolute recoveries for this comparison, we began to correct the
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recoveries by placing isotopically-labelled underivatized internal standards in spikes, and
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isotopically-labelled derivatized internal standards in solvent curves. This provided better
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quantitation by correcting for analyte loss through internal standards. The process also
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demonstrated whether the derivatization was complete.
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Finally, we evaluated the applicability of the validated method by taking part in a FAPAS-
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administered proficiency test, alongside 98 other laboratories. The test results support the
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applicability of the method. Furthermore, we analyzed 102 composite samples of a variety of
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imported seafood – including shrimp, tilapia, frog legs, salmon, and swai fish – and discovered
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several violations above the FDA action level. 5
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MATERIALS AND METHODS
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Reagents. AOZ, AMOZ, AHD-HCl, SC-HCl, AOZ-d4, AMOZ-d5, AHD-13C3, SC-HCl-13C,
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AOZ, 2-NP-AMOZ, 2-NP-AHD, 2-NP-SC, 2-NP-AOZ-d4, 2-NP-AMOZ-d5, 2-NP-AHD-13C3, 2-NP-SC-
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HCl-13C, 15N2, 2-NP-AOZ, 2-nitrobenzaldehyde, dibasic potassium phosphate, hydrochloric acid,
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and ammonium acetate were obtained from Sigma-Aldrich (St. Louis, MO). Chloramphenicol
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and florfenicol in methanol (500 ppm) were provided by Restek Corporation (Bellefonte, PA).
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Chloramphenicol-d5 (100 ppm) was provided by Cambridge Isotopes (Tewksbury, MA). HPLC
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grade acetonitrile (with and without 1% formic acid), methanol, and water (with and without
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1% formic acid), and ethyl acetate were obtained from Fisher Scientific (Pittsburgh, PA).
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Solutions Preparation. The ammonium acetate in methanol (20:80 NH4OAc/MeOH, v/v)
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solution was prepared by dissolving ammonium acetate (655 mg) in 1 L of DI water. This
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solution was then combined with methanol to form the final 20:80 (v/v) solution. The 0.125 M
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hydrochloric acid solution was prepared by diluting 6 M hydrochloric acid with DI water. The
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phosphate buffer solution (PBS) (0.1 M) was prepared by dissolving anhydrous dipotassium
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hydrogen phosphate (174 g) in 1000 mL of DI water. Lastly, 0.1 M 2-nitrobenzaldehyde (2-NBA)
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solution was prepared by dissolving 148 mg in 10 mL of methanol.
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Preparation of Standard Stock Solutions. Individual stock solutions of AOZ, AMOZ, AHD-HCl,
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SC-HCl, AOZ-d4, AMOZ-d5, AHD-13C3, SC-HCl-13C, 15N2, 2-NP-AOZ, 2-NP-AMOZ, 2-NP-AHD, 2-NP-
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SC, 2-NP-AOZ-d4, 2-NP-AMOZ-d5, 2-NP-AHD-13C3, 2-NP-SC-HCl-13C,
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prepared by dissolving each standard after adjusting for salt content, purity, and 2-NBA in
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methanol. The contribution factors are 46.37%, 36.06%, 43.41%, and 60.19% for AHD, SEM,
15
15
N2, 2-NP-
N2 at 100 µg/mL were
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AOZ, and AMOZ. Isotopically labelled internal standards were checked upon receipt for the
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absence of native standards. This is a procedure that need only be done once, prior to method
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validation.
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Preparation of Mixed Intermediate Standard Solutions (INT-A, -B, -C, -D, -E, and -F) (1 µg/mL =
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1 µg/mg). Intermediate standard solutions of the non-isotopically-labelled underivatized (INT-
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A), isotopically-labeled underivatized (INT-B), non-isotopically-labelled derivatized (INT-C), and
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isotopically-labeled derivatized (INT-D) nitrofuran metabolites were prepared by adding 100 µL
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of each stock solution in 10 mL of NH4OAc/MeOH (20:80, v/v). Intermediate standard solution
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of the phenicols (INT-E) was prepared by adding 100 µL of each standard (500 µg/mL) to a 50
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mL volumetric flask and diluting with methanol, resulting in a 1 µg/mL concentration of each
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analyte. The intermediate internal standard solution for the phenicols (INT-F) was prepared
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similarly by adding 100 µL of chloramphenicol-d5 standard (100 µg/mL) into a 10 mL volumetric
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flask and diluting with methanol, resulting in a 1 µg/mL concentration of the analyte.
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Preparation of Underivatized Nitrofuran Metabolites Intermediate Solutions for Spikes (INT-
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S-A and INT-IS-B). The series of working dilutions, INT-S-A1 to INT-S-A6, were prepared from
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INT-A to give a concentration of 2, 5, 20, 100, 200, and 300 ng/mL of the calibration standards.
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The internal, INT-IS-B, was prepared from 1 mL of INT-B and diluted with NH4OAc:MeOH (20:80,
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v/v) to 25 mL to give a concentration of 40 ng/mL.
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Preparation of Derivatized Nitrofuran Metabolites Intermediate Solutions for Calibration
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Curves (INT-S-C and INT-IS-D). The series of working dilutions, INT-S-C1 to INT-S-C6, were
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prepared from INT-C to give a concentration of 2, 5, 20, 100, 200, and 300 ng/mL of the 7
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calibration standards. The internal, INT-IS-D, was prepared from 1 mL of INT-D and diluted with
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NH4OAc:MeOH (20:80, v/v) to 25 mL to give a concentration of 40 ng/mL.
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Preparation of Phenicols Intermediate Solutions (INT-S-E and INT-IS-F). The series of working
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dilutions, INT-S-E1 to INT-S-E6, was prepared from INT-E to give a concentration of 0.5, 1, 2, 5,
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10, and 20 ng/mL of the calibration standards. The internal, INT-IS-F, was prepared by diluting
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125 µL of INT-F with MeOH to a volume of 25 mL to give a concentration 5 ng/mL.
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Sample Fortification. The fortified samples were made by spiking 2 g of the different seafood
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matrices (tilapia and shrimp) with 100 µL of four intermediate mixed nitrofuran standard
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solutions – INT-S-A2, INT-S-A3, INT-S-A4, and INT-S-A5, 100 µL of the internal standard
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intermediate solution, INT-IS-B (40 ng/mL), 100 µL of 4 intermediate mixed phenicol standard
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solutions – INT-S-E3, INT-S-E4, INT-S-E5, and INT-S-E6, and 100 µL of the internal standard
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intermediate solution for phenicols, INT-F (5 ng/mL). Overall, spikes were made for each matrix
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to reach final concentrations of 0.25, 1, 5, and 10 ng/g of AMOZ, AOZ, AHD, and SC and 0.1,
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0.25, 0.5, and 1 ng/g of chloramphenicol and florfenicol in each spike. It is worth mentioning
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that reagent (2 g of water) and matrix blanks were not fortified with internal standard and were
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used as negative controls Fortifying blanks with standard would not allow them to serve their
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purpose of identifying the presence of contamination somewhere in the chain of analysis.
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Calibration Curve. To prepare 800 µL of the individual calibration standards, 100 µL of INT-S-C1
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to INT-S-C6 and 100 µL of INT-S-E1 to INT-S-E6 were added to create six calibration standards,
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SC1 to SC6, respectively. The remaining 600 µL was comprised of 100 µL of INT-IS-D, INT-IS-F,
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and 400 µL of NH4OAc:MeOH (20:80, v/v) solution. The final concentration of nitrofurans and
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phenicols were 0.1, 0.25, 1, 5, 10, and 15 ng/g and 0.025, 0.05, 0.1, 0.25, 0.5 and 1 ng/g. The
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final concentration of the internal nitrofurans and phenicols were 2 and 0.25 ng.
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Extraction of Nitrofuran Metabolites and Phenicols from Fortified Seafood Matrices. Seafood
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samples were purchased and homogenized to a powder-like (when frozen) or paste consistency
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using a Robot Coupe (Ridgeland, MS) with a 7 mm thick stainless steel blade and immediately
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stored at -80 ̊C. A 2 g (±0.05 g) portion was weighed into a 50 mL polypropylene centrifuge
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tube for each sample a day before the extraction. A reagent blank was prepared by measuring 2
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g (±0.05 g) of DI water into a 50 mL disposable centrifuge tube. A matrix blank was prepared by
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measuring 2 g (±0.05 g) of a blank seafood matrix into a 50 mL disposable centrifuge tube.
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Spikes were prepared by measuring 2 g (± 0.05 g) of blank seafood matrix into a 50 mL
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disposable centrifuge tube. Spikes were fortified with internal standard solutions (100 µL of
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INT-IS-B and INT-IS-F) as well as four spike levels of intermediate mixed standard solutions, in
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triplicate. Then, 10 mL of 0.125 M HCl, followed by 200 uL of 100 mM 2-NBA (prepared daily),
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was added to all blanks and spikes and shaken for 10 min at 1100 rpm using a Geno Grinder
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(Metuchen, NJ). The samples were placed in the oven at 37°C overnight to allow the nitrofurans
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to derivatize.
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The following day, the samples were cooled for 15 minutes at room temperature. Then,
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5 mL of 1.0 M dibasic potassium phosphate buffer solution was added to each tube to adjust
189
the pH to 7.3. If further adjustment is needed, 0.1 M NaOH or HCl is used. After the pH
190
adjustment, the contents were vortexed for 20 s and centrifuged at 4000 rpm for 15 min.
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Following centrifugation, the supernatant is transferred from each tube into a clean 50 mL
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centrifuge tube. In the remaining centrifugate, 6 mL of water was added, vortexed for 15 s, and 9
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centrifuged for 5 mins at 4000 rpm. The supernatant was then decanted into the same 50 mL
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centrifuge tube that contained the previous supernatant. Sodium chloride (2 g), followed by 6
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mL of ethyl acetate, were added to the supernatant, shaken for 30 s, and centrifuged at 4000
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rpm for 5 min. The ethyl acetate layer was then transferred to a clean 50 mL centrifuge tube.
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The aqueous layer was extracted again using the same amount of ethyl acetate, shaken, and
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centrifuged at 4000 rpm for 5 min. After collecting both ethyl acetate portions into the same
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tube, the liquid was evaporated to complete dryness with flowing nitrogen gas at 15 psi, 40°C
200
for 30 min. The dried extracts were reconstituted with 0.8 mL of NH4OAc:MeOH (20:80, v:v)
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and sonicated for 1 min. The contents were then transferred using a pipette and filtered
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through a 0.2 µm PDVF filter.
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Derivatization Efficiency and Standard Stability Tests. The curves used for quantitation were
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prepared as in-vial combinations of: 100 uL, sequentially, of the intermediate standard mixed
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solutions of the derivatized nitrofurans (INT-S-C1 through INT-S-C6) and phenicols (INT-S-E1
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through INT-S-E6); 100 uL each of the intermediate internal standard mixed solutions of the
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derivatized nitrofurans (INT-IS-D) and chloramphenicol (INT-IS-F); and 400 uL each of
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NH4OAc:MeOH (20:80, v:v) for a final volume of 800 uL. To test their stability over time, curves
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were made, run, and compared on a weekly basis against those previously prepared. Data
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showed that curves were stable for up to three weeks when stored in the LC auto-sampler at 10
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ᵒC. To test the efficiency of the in-situ derivatization, in-vial combinations were made of: 100
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uL, sequentially, of the intermediate standard mixed solutions of the underivatized nitrofurans
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(INT-S-A1 through INT-S-A6); 100 uL each of 2-NBA (0.1 M); 50 uL each of HCl (0.125 M); and
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550 uL each of NH4OAc:MeOH (20:80, v:v) for a final volume of 800 uL. They were derivatized
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overnight using the oven at 37 ᵒC.
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LC-MS/MS Method for the Analysis of Nitrofurans and Phenicols. The LC-MS/MS apparatus
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included Waters Acquity UHPLC systems (Waters, Milford, MA) coupled to AB Sciex 5500 or
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6500 Q-Trap (AB Sciex, Toronto, Canada) triple quadrupole mass spectrometers. Separation of
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compounds was achieved on UHPLC systems that were equipped with Acquity C18 columns (1.7
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um particle size, 150 mm length, 2.1 mm internal diameter) maintained at 40 (± 1) deg. C. The
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mobile phase was composed of: (A) 0.1% formic acid in water and (B) 0.1% formic acid in
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acetonitrile. The gradient began at 95:5 of A:B with a flow of 0.2 mL per min. The gradient was
223
changed to 5:95 of A:B over a course of 10 min followed by a 5 min hold at 5:95 of A:B. Over 0.1
224
min, the gradient was changed to 95:5 of A:B with a concomitant change in flow rate to 0.4 mL
225
per min. After 4 min, the flow was reduced to 0.2 mL per min over the course of 0.1 min. This
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was held for 0.8 min to complete the LC gradient (20 min total run time).
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An alternate, 10-minute method was also developed as a proof-of-concept using the
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same mobile and stationary phases. The gradient began at 95:5 of A:B with a flow of 0.2 mL per
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min. The gradient was changed to 5:95 of A:B over a course of 5 min followed by a 2.5 min hold
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at 5:95 of A:B. Over 0.05 min, the gradient was changed to 95:5 of A:B with a concomitant
231
change in flow rate to 0.4 mL per min. After 2 min, the flow was reduced to 0.2 mL per min over
232
the course of 0.05 min. This was held for 0.4 min to complete the LC gradient.
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All analyses of samples were carried out using electrospray ionization (ESI) using
234
multiple-reaction monitoring (MRM). Sample analyses were done in duplicate, with one each in
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positive-ion mode (nitrofurans) and negative-ion mode (phenicols). MS source parameters for 11
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the positive-ion mode experiments (nitrofurans) were: ion source voltage (4500 V), curtain gas
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(30 psi), ion source gas (GS1 = 40 psi and GS2 = 45 psi), ion source temperature (400°C), and
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entrance potential (10 V) and for the negative-ion mode experiments (phenicols) were: ion
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source voltage (-4500 V), curtain gas (30 psi), ion source gas (GS1 = 40 psi and GS2 = 45 psi), ion
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source temperature (400°C), and entrance potential (-10 V). By infusing 1 ng/uL solutions of NP-
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NF metabolites, chloramphenicol, and florfenicol in acidified methanol at 10 uL/min and using
242
the automatic and manual tuning modes of the Analyst software (Version 1.6.1), optimal values
243
for the formation of stable product ions were found for the following MS parameters:
244
declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit
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potential (CXP) (Table 1).
246 247
RESULTS AND DISCUSSION
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Background. The nitrofurans and phenicols (chloramphenicol and florfenicol) are broad-
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spectrum, synthetic antibiotics. Their efficacy and low cost make them attractive options in
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controlling the bacterial diseases prevalent in populations of terrestrial and aquatic animals
251
raised for human consumption. Despite these economic incentives for their application, they
252
are banned in the US and EU for such veterinary uses due to their recognized dangers to human
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health. It is for this reason that developing methods for their detection and quantitation are
254
crucial from a public health standpoint.
255
Of the two classes of antibiotics analyzed in this paper, the chemistry of nitrofurans
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presented the greatest obstacle to their analysis. Due to their rapid conversion in vivo to highly
257
polar, low mass metabolites (listed in Figure 1), nitrofurans are difficult to resolve on reversed12
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phase UHPLC columns and analyze in MS/MS systems. These metabolites also have high
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affinities for binding with the amino acids found in animal muscle tissue (depicted in Figure 2)
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and forming very stable metabolite-protein adducts. These facts require samples to be
261
subjected to chemical methods of analyte liberation and derivatization before analysis. The
262
phenicols, however, need no such consideration. Additionally, quantitation of the phenicols is
263
not affected by the steps taken to prepare the nitrofurans, so they can be extracted
264
simultaneously. Liberation of the nitrofuran metabolites was achieved by acid hydrolysis of the
265
imine bonds formed between the metabolites and amino acids (Figure 2); derivatization of the
266
nitrofuran metabolites was achieved by reaction of the newly-liberated amine moiety of the
267
nitrofuran metabolites with the aldehyde group of 2-nitrobenzaldehyde (Figure 3) via a
268
nucleophilic addition, followed by the elimination of water to form the imine. Conveniently,
269
these steps can all be carried out simultaneously in the same reaction vessel.
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Extraction Method Optimization. To optimize the extraction method of the nitrofuran
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metabolites, chloramphenicol, and florfenicol from various seafood matrices, we explored
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three extraction protocols that have been reported in the literature.27-29 In our laboratory, we
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use two important criteria to qualify a method of extraction: the signal-to-noise ratio of the
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analyte peak of interest, and the absolute recovery before correction with internal standards.
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We used matrix-matched calibration curves (in-matrix curves) to determine the “absolute
276
recovery” by controlling for the effect of the matrix on ionization, thereby exposing
277
physicochemical losses of analyte during extraction.
278
We calculated absolute recoveries of the analytes from fortified (spiked) seafood
279
samples using matrix-matched calibration curves, with correlation coefficient values (R2) that 13
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were consistently greater than 0.99. To generate these curves, commercially available
281
derivatized nitrofuran metabolites and phenicols standards were dissolved in a solution
282
(containing extracted matrix components) at concentrations varying from 0.1 to 15 ng/g for
283
nitrofuran metabolites and 0.025to 1 ng/g for phenicols. LC-MS/MS data points were fitted to
284
the curve using a quadratic, 1/x-weighted regression. Matrix effects on the area counts for the
285
analytes in the three matrices were also investigated, and any cases of interference,
286
suppression, etc. are reported here.
287
The three FDA extraction methods we explored started with the same steps: weighing
288
the sample, fortification with standards, addition of HCl and 2-NBA, oven incubation for 16 h,
289
and addition of K2HPO4 buffer. After these steps, the extractions began to vary. For example,
290
the first method investigated, by An et al., used acetonitrile to extract the analytes via a
291
liquid/liquid extraction.28 In this method, the absolute recoveries we obtained ranged from 50-
292
to-65% depending on the extracted analyte. These absolute recoveries are satisfying and within
293
our acceptable criteria. However, a matrix interference was found to co-elute with the AHD
294
metabolite, which made it very difficult to quantify at the LOQ level for this analyte. We
295
hypothesize that due to its relative non-polarity, the acetonitrile used in this method was
296
efficient at solubilizing oily, low-polarity components of the seafood matrices that interfered
297
with analysis, such as the AHD interference in question.
298
The second method explored, by Veach et al., used a solid phase extraction (SPE)
299
column to extract the analyte and remove interferences.29 We replicated the method, with the
300
exception of our substitution of a conventional oven for the microwave oven used by Veach, et
301
al. Given the 99% derivatization efficiency achieved by our oven method (Figure 6), we 14
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conclude that any differences arising from this substitution were minimal. The absolute
303
recoveries we obtained by this method ranged from 33-53%, depending on the analyte in
304
question.
305
Finally, we tried a third method, published by Ye et al., and successfully used it as the
306
basis for the rest of the method development and validation work.27 In this method, samples
307
were extracted by liquid-liquid extraction using two 6 mL aliquots of water, followed by
308
extraction of the combined 12 mL of water with two 6 mL aliquots of ethyl acetate. The
309
subsequent addition of NaCl to the water increased the polarity of the aqueous phase, “salting
310
out” the desired analytes into the less polar, organic ethyl acetate. Reconstituted extracts were
311
also filtered through 0.2 um PVDF filters to remove macroscopic pieces of matrix material that
312
could clog or dirty the LC-MS/MS system and interfere with analysis.
313
With this procedure, the absolute recovery for the nitrofuran metabolites was found to
314
be between 60-70%, with a much cleaner background. Ion suppression and enhancement
315
effects were found to be negligible on extracts produced from this method. It is worth
316
mentioning that in the above methods, the authors calculated their recoveries by comparing
317
spikes with calibration curves created from spiked samples (“calibrating samples”). Herein, we
318
describe the absolute recoveries calculated by our lab upon replication of their methods, and
319
compare them to the absolute recovery of our method. Absolute recoveries are calculated by
320
using extracted-matrix calibration curves as described in the first part of this section.
321
LC-MS/MS Analysis Optimization. 1 ng/uL solutions of the NP-NFs in acidified methanol,
322
chloramphenicol, and florfenicol were infused directly into the AB Sciex 6500 and 5500 QTrap
323
triple quadrupole mass spectrometers to determine precursor ion masses, product ion masses, 15
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324
and the values of the parameters necessary to produce the most stable and responsive product
325
ions.
326
First, Q1 of the MS/MS system was scanned to determine the mass of the precursor ion.
327
When found, this mass was monitored while values for the declustering potential (DP) were
328
optimized. Following this, Q1 was fixed at the selected m/z ratio of the precursor ion while Q3
329
was scanned to determine potential product ion masses. After this, values for the DP, collision
330
energy (CE), entrance potential (EP), and collision cell exit potential (CXP) that gave the most
331
stable and responsive product ions were found and documented (Table 1). The data thus
332
obtained was placed into an acquisition method, and single standards of each compound were
333
run on the instrument to determine retention times (Table 1) and ion ratios between precursor
334
and product ions, both of which were used for analyte identity confirmation. With the latest-
335
eluting analytes emerging from the column at about 6 min, the necessity of derivatization in
336
reducing the polarity of the nitrofuran metabolites can be seen.
337
Figures 4A and 5A depict the extracted ion chromatograms for each analyte of interest,
338
obtained from LC injection at 0.25 ng/mL for AMOZ and AOZ, 1 ng/mL for AHD and SC, and 0.1
339
ng/mL for chloramphenicol and florfenicol in solvent. These levels represent the LOD levels of
340
this method. The obtained retention times reflect expectations based on the molecular shapes
341
and polarity of these compounds (Figure 1). For example, the retention time of 2-NP-AMOZ was
342
found to be 4.15 min whereas 2-NP-AOZ exhibited a retention time of 6.03 min. The short
343
retention time of 2-NP-AMOZ in comparison to the three other derivatized nitrofuran
344
metabolites was attributed the presence of a morpholine ring in its structure. Figures 4B and 5B
345
show the same chromatograms at the LOD level in tilapia matrix. Matrix interferences were not 16
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observed, demonstrating the performance of the extraction method. For example, the area
347
count of the 335 → 291 transiVon for 2-NP-AMOZ is 2.22x105 in solvent and 2.26x105 in matrix.
348
As mentioned in the Materials and Methods, the LC method can be shortened to 10
349
minutes, for those desiring higher throughput. Retention times changed as such: 2-NP-AMOZ
350
to 3.36 min; 2-NP-AOZ to 4.46 min; 2-NP-AHD to 4.17 min; 2-NP-SC at 4.05 min;
351
chloramphenicol at 4.29 min; and florfenicol at 4.19 min. We, however, chose not to implement
352
this shortened run, due to our desire to expand this method to include other antibiotic and
353
drug residues in the future. A shortened LC run would increase elution overlap not only with
354
analytes, but with interferences.
355
Checking Standards and Derivatization. It is our policy in the laboratory to check our standards
356
before performing the analysis of any batch. This was done by running a calibration curve,
357
made up as previously described in Materials and Methods. In comparison to the derivatized
358
nitrofuran metabolites (2-NP-AMOZ, 2-NP-AOZ, 2-NP-AHD, 2-NP-SC), it was impossible to
359
directly analyze a calibration curve built to verify the quality of the commercially-sourced
360
underivatized nitrofurans – used for spiking – due to their low masses and high polarities. To
361
check the integrity of the commercially-sourced underivatized nitrofurans standards, a multi-
362
point calibration curve was prepared, followed by in-vial derivatization in acidic conditions
363
using an excess of 2-NBA. This curve was left in the oven at 37 ᵒC overnight and run against
364
another multi-point calibration curve made using the commercially-sourced derivatized
365
nitrofuran metabolites. Figure 6 shows the high degree of agreement between these in-house
366
made calibration curves prepared from derivatized vs underivatized standards. This experiment
367
allowed us not only to check the quality and stability of our standards, but also to verify the 17
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368
yield of our derivatization step in the extraction. The efficiency of the derivatization process
369
was >99%.
370
Fortification and Recovery. Tables 2-5 present the validation results. The method was tested in
371
triplicate to determine its accuracy and precision. All the nitrofuran metabolites and phenicols
372
were examined in two different seafood commodities (shrimp and tilapia) at four different
373
concentrations (0.25, 1, 5 and 10 ng/g for Nitrofurans and 0.1, 0.25, 0.5, 1 ng/g for phenicols).
374
Preliminary experiments indicated that the UHPLC-MS/MS instrumental response varied
375
with the absence or presence of seafood matrix. On one hand, to compensate for the effects of
376
the matrix suppression, matrix-matched calibration standard curves (in-matrix curves) were
377
used to quantitate the analytes and obtain the absolute recovery, which is a measure of the
378
mass of analyte lost during the extraction procedure (Tables 4 and 5). On the other hand, the
379
availability of derivatized and underivatized isotopically-labeled internal standards made it
380
possible to correct the recoveries, accounting for physical loss and chemical degradation of
381
analytes during the extraction process, as well as ion suppression in the mass spectrometer
382
(Tables 2 and 3).
383
Interestingly, the effect of matrix on the area counts for all analytes in the two studied
384
matrices was found to be negligible due to the clean extraction method used. In other words,
385
the area counts of analytes in-solvent or in-extracted-matrix-blanks were very similar. For all
386
four nitrofuran residues, chloramphenicol and florfenicol, the reproducibility errors (CV) and
387
standard deviations (SD) were lower than 10% and the corrected recoveries with internal
388
standards were between 92 and 102% for nitrofuran metabolites and 85 and 110% for
389
chloramphenicol in tilapia (Table 2 and 3). An internal standard for florfenicol was not available. 18
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390
The average absolute recoveries using matrix-matched calibration curves without internal
391
standards were 60 % for AMOZ, 68% for AHD, 63% for AOZ, 51% for SC, 79% for
392
chloramphenicol, and 87% for florfenicol in tilapia (Table 4 and 5).
393
Matrix-matched calibration curves were prepared by spiking the final extracts of
394
seafood blanks with solutions of the commercially-sourced derivatized nitrofurans and
395
phenicols. Two product-ion transitions per analyte, along with the ion ratios, were used for data
396
acquisition and analyte confirmation, resulting in a highly selective method. No interference
397
was observed in the negative controls at the retention time of the analytes. The limit of
398
detection (LOD) for each analyte was defined to be 0.25 ng/g for AMOZ and AOZ, 1 ng/g for
399
AHD and SC, and 0.1 ng/g for chloramphenicol and florfenicol. The limit of quantitation (LOQ)
400
was defined to be 1 ng/g for AMOZ, AOZ, AHD and SC, and 0.25 ng/g for chloramphenicol and
401
florfenicol. The lower LODs for AMOZ and AOZ were due to their much greater response in the
402
LC-MS/MS analysis than AHD and SC.
403
Method Validation and Analysis. The analytical method was validated by independently
404
analyzing two different matrices of commercially available seafood: tilapia and shrimp. Each
405
matrix was spiked in triplicate at four spike levels with underivatized nitrofuran metabolites and
406
phenicols. A multi-point, in-solvent calibration curve with isotopically-labelled internal
407
standards was used to calculate the concentration in the spikes of the in-situ derivatized
408
nitrofuran metabolites as well as chloramphenicol and florfenicol. Calibrating standards ranged
409
in concentration from 0.1 ng/g to 15 ng/g for the nitrofurans and 0.025 ng/g to 1 ng/g for the
410
phenicols. At least 5 data points were necessary for quantitation. Chromatographic peaks at
411
each of these thresholds are depicted for the nitrofurans and phenicols in Figures 4 and 5. 19
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412
The primary source of improvement in quantitation came from the fortification of each
413
sample with an internal standard containing isotopically-labelled forms of the underivatized
414
nitrofuran metabolites and chloramphenicol. The internal standard allowed the calculation of
415
corrected recoveries by comparing the ratio of the analyte/internal standard responses in the
416
sample to the ratio of the analyte/internal standard responses in the solvent curve, which
417
afforded the ability to correct for analyte lost during the extraction procedure.
418
It is the policy of the laboratory to set LOD and LOQ values at levels greater than the
419
minimum 3x and 10x general requirements such that daily instrument variance, matrix effects,
420
procedural errors, etc. do not result in failures to meet established and validated LODs and
421
LOQs for studies taking place over extended periods of time. In our laboratory, we may set the
422
LOD and LOQ values to levels greater than the minimum detected signal at 3x or 10x on the
423
instrument based on the needs of the method and the desired ruggedness of the method.
424
Instrument blanks were prepared by using ammonium acetate with no sample preparation.
425
Acceptance criteria for method spikes were evaluated using control charts. Recoveries should
426
be within +/- 3 process sigma. For initial control charting purposes, the use of method
427
validation data had to establish a suitable recovery range. Once a suitable population of spike
428
recoveries were generated at the spike levels, these results were utilized to determine control
429
limits and for evaluation of any trending in the data.
430
Analyte identification required at least 2 product-ions. The ion ratio must be within +/-
431
20% of the average of the reference standards in each analytical run. Standards which fail to
432
meet method performance criteria such as consistent instrument response (i.e. drift no greater
433
than 30% relative difference between bracketed standards) are not included in this average. In 20
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434
other words, we assessed the ratios of the highest responding ion to an additional confirming
435
ion, then determined the difference (if any) between the ratios for in solvent and spiked
436
analytes. Differences in ratios above 20% were deemed failing. The retention time of the
437
sample peak must match the average reference standard peaks within +/- 0.25 minutes.
438
Method performance characteristics are often influenced by time and fiscal constraints
439
of the customer and the laboratory. Therefore, we do not try to set any criteria prior to
440
completion of the validation for these types of projects. We do, however, have performance
441
minimums that we consider when reviewing these types of data packages. In general, we
442
consider any recoveries inside of 50-150% for low and sub-ppb level analysis acceptable, and
443
we view any coefficient of variation less than 30% to be acceptable for our typical multi-level
444
replicate analysis at these levels. These values represent our minimums and methods often
445
exceed these. For this method, these parameters were exceeded and the data are considered
446
fit for the intended purpose by our laboratory, and the Florida Department of Agriculture and
447
Consumer Services currently uses this method to test imported seafood samples.
448
Quantitation of Nitrofuran Metabolites. Most reports on the quantitation of nitrofuran
449
metabolites in the literature utilized the “calibrating samples” method for their calibration
450
curves. This was done due to the unavailability of commercially-sourced, derivatized nitrofuran
451
metabolites. Researchers would fortify five or six blank matrix samples with varying
452
concentrations of standard solutions containing mixtures of underivatized nitrofuran
453
metabolites. Then, these samples would be derivatized in-situ using 2-NBA and HCl in an oven
454
overnight and extracted using different liquid-liquid extraction or SPE methods.
21
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455
In this scenario, absolute recoveries cannot be accurately quantitated since the
456
calibrating samples and spikes are made identically. Thus, a 100% recovery is misleading when
457
the recovery of a spike is calculated using a sample that has been fortified and extracted
458
identically. To overcome this problem and calculate the recovery of spikes with higher accuracy
459
while maintaining precision, we purchased the underivatized nitrofuran metabolites and their
460
isotopically labelled internal standards and used them for fortifications (spikes) only. These
461
spikes were then quantitated with calibration curves which were made using commercially-
462
sourced, derivatized nitrofuran metabolites and their isotopically labelled internal standards –
463
in-solvent. The commercially-sourced, derivatized standards provide better quantitation and
464
demonstrate whether the derivatization was complete or not. If the calculation, preparation,
465
and derivatization of standards are performed accurately, the multi-point calibration curve of
466
the in-situ derivatized nitrofuran metabolites should match that made using the commercially-
467
sourced derivatized metabolites. This correlation was observed using our method (Figure 6).
468
Proficiency Test. To independently evaluate the ability of the method to extract, analyze and
469
quantitate analytes, we engaged in a FAPAS-administered (ISO 17043-accredited) proficiency
470
test with 98 other laboratories.30 AHD and AOZ were the two nitrofurans analyzed. Their
471
assigned values were set at 2.06 ng/g (AHD) and 2.21 ng/g (AOZ). Lab performance was
472
described by FAPAS through the calculation of z-scores (Figures 7 & 8). Z-scores within a range
473
of -2 ≤ z ≤ 2 indicate that the method is considered fit-for-purpose by our laboratory, in
474
accordance with IUPAC guidelines.31 We obtained values of 2.5 ng/g (AHD) and 2.8 ng/g (AOZ),
475
for z-scores of 1.0 (AHD) and 1.2 (AOZ), demonstrating the ability of the method.
22
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476
Regulatory Samples. To evaluate the applicability of the validated method, 102 composite
477
samples of a variety of imported seafood, including shrimp, tilapia, frog legs, salmon, and swai
478
fish, were analyzed. Composites were made from different lots of the same commodity to
479
provide a more representative sample. The analyzed samples were imported, frozen, from
480
non-EU countries. They were collected from different grocery stores and warehouses
481
throughout Florida.
482
reagent blank, a matrix blank, and several spiked blank samples were evaluated with the aim of
483
checking the quality of the results. The reagent and matrix blanks were obtained by weighing 2
484
g of water and seafood samples with the objective of eliminating possible false positives
485
because of contamination in the instrument or solvent used. Spiked samples at two levels of
486
fortification (1 and 10 ng/g for nitrofurans and 0.25 and 1 ng/g for phenicols) were used to
487
monitor the extraction efficiency.
Internal quality controls, including an in-solvent calibration curve, a
488
Among the 102 analyzed composite samples, 4 samples tested were positive for
489
nitrofuran metabolites and chloramphenicols. A swai fish sample tested positive for the SC
490
nitrofuran metabolite at 2.5 ng/g; a shrimp sample was contaminated with chloramphenicol at
491
0.44 ng/g; a sample of frog legs tested positive for AOZ nitrofuran metabolite at 2.7 ng/g;
492
another sample of frog legs was found contaminated with AOZ at 3 ng/g and chloramphenicol
493
at 17 ng/g. This last sample was heavily contaminated with chloramphenicol, requiring a 20-fold
494
dilution followed by a standard addition for accurate quantitation. Two product-ion transitions
495
per analyte, along with ion ratios, were used for data acquisition and analyte confirmation of
496
these violations. The ion ratios were calculated by dividing the area counts of two product ions
497
of the analyte, in-solvent (standard) and in-sample, and multiplying this quotient by 100 to 23
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498
obtain the ion ratio as a percentage. The percentage difference between ion ratios in-standard
499
and in-sample is then calculated, and must be lower than 20% for a sample to confirm.
500
The contaminated samples were also re-extracted to confirm the presence of a
501
violation. For example, the swai fish sample was extracted 4 different times in 4 separate
502
batches. These 4 batches were analyzed, one per week, over 4 weeks. The SC concentration
503
was found to be 2.56, 2.58, 2.31 and 2.42 ng/g for the 4 analyzed swai fish samples. These 4
504
samples were weighed from 4 different homogenized sub-samples (multiple cups of the same
505
sample), scheduled in 4 batches and extracted by multiple chemists. These findings show the
506
precision and accuracy of the homogenization process, validated extraction method, and
507
quantitation. Our laboratory conducted further investigation into one of the violative samples
508
of frog legs. This composite sample was initially made from 20 lots of frog legs collected from a
509
warehouse. Our analysis revealed that 17 of the 20 samples had been heavily contaminated
510
with (0.1-to-19 ng/g) AOZ and chloramphenicol (0.8-to-140 ng/g).
511 512
Conclusion
513
A UHPLC-MS/MS method for the detection, quantitation, and confirmation of
514
nitrofurans and phenicols in several seafood matrices was developed and validated for use in
515
analyzing regulatory samples. This method produces improvements in accuracy on those
516
previously reported in the literature, with a derivatization yield of >99%. While underivatized
517
nitrofuran metabolites and their isotopically labelled internal standards were used for
518
fortifications (spikes), commercially-sourced derivatized nitrofuran metabolites and their
519
isotopically labelled internal standards were used for building in-solvent calibration curve, 24
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520
which is the key improvement that led to the improved accuracy that we report. For the
521
nitrofurans, chloramphenicol, and florfenicol, the reproducibility errors (CV) and standard
522
deviations (SD) were lower than 10%, and the recoveries corrected with internal standards
523
were between 90 and 100% for nitrofuran metabolites and 85 and 110% for chloramphenicol.
524
The absolute recovery, calculated using in matrix matched calibration curve, was found to be
525
between 60-70% for the nitrofuran metabolites and 70-80% for the phenicols. Finally, this
526
validated method was successfully used to analyze and identify violations in imported seafood
527
samples, which unequivocally demonstrates the ability of the method to extract and quantify
528
varying levels of incurred residues.
529 530
AUTHOR INFORMATION
531
Corresponding Author
532
*Corresponding author (Tel: +1-804-335-3119 and E-mail:
[email protected])
533
Notes
534
The authors declare no competing financial interest.
535 536
ACKNOWLEDGMENTS
537
We thank the Food Emergency Response Network for financial support. We would like to thank
538
Rebecca Kitlica, Carion Haynes, and Amy Brown from the Department of Agriculture and
539
Consumer Services (FDACS), Tallahassee, for their assistance with standards and samples
540
preparation and homogenization. We also thank Ghislain Gerard, Walter Hammack, Jo-Marie
541
Cook, Matthew Standland and Mark Crosswhite for many helpful discussions. 25
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and
gas
599
chromatography-tandem mass spectrometry (GC/MS/MS). Marine Pollution Bulletin 2016, 113, 579-
600
584.
601
21. Venable, R.; Haynes, C.; Cook, J. M., Reported prevalence and quantitative LC-MS methods for the
602
analysis of veterinary drug residues in honey: a review. Food Additives & Contaminants: Part A 2014,
603
31, 621-640.
604
22. Wang, K.; Lin, K.; Huang, X.; Chen, M., A Simple and Fast Extraction Method for the Determination of
605
Multiclass Antibiotics in Eggs Using LC-MS/MS. Journal of Agricultural and Food Chemistry 2017, 65,
606
5064-5073.
607
23. Barbosa, J.; Freitas, A.; Mourão, J. L.; Noronha da Silveira, M. I.; Ramos, F., Determination of
608
Furaltadone and Nifursol Residues in Poultry Eggs by Liquid Chromatography–Electrospray
609
Ionization Tandem Mass Spectrometry. Journal of Agricultural and Food Chemistry 2012, 60, 4227-
610
4234.
28
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Journal of Agricultural and Food Chemistry
611
24. Van Poucke, C.; Detavernier, C. l.; Wille, M.; Kwakman, J.; Sorgeloos, P.; Van Peteghem, C.,
612
Investigation into the Possible Natural Occurence of Semicarbazide in Macrobrachium rosenbergii
613
Prawns. Journal of Agricultural and Food Chemistry 2011, 59, 2107-2112.
614
25. Zhao, H.; Zulkoski, J.; Mastovska, K., Development and Validation of a Multiclass, Multiresidue
615
Method for Veterinary Drug Analysis in Infant Formula and Related Ingredients Using UHPLC-
616
MS/MS. Journal of Agricultural and Food Chemistry 2017, 65, 7268-7287.
617
26. Wittenberg, J. B.; Simon, K. A.; Wong, J. W., Targeted Multiresidue Analysis of Veterinary Drugs in
618
Milk-Based Powders Using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS).
619
Journal of Agricultural and Food Chemistry 2017, 65, 7288-7293.
620 621
27. Ye, L.; Chamkasem, N.; Williams, A.; Dimandja, J., Determination of Nitrofuran Metabolites in Shrimp by LC/MS/MS. Laboratory Information Bulletin 2011, 4466, 1-20.
622
28. An, H.; Parrales, L.; Wang, K.; Cain, T.; Hollins, R.; Forrest, D.; Liao, B.; Paek, H. C.; Sram, J.,
623
Quantitative analysis of nitrofuran metabolites and chloramphenicol in shrimp using acetonitrile
624
extraction and liquid chromatograph-tandem mass spectrometric detection: a single laboratory
625
validation. Journal of AOAC International 2015, 98, 602-608.
626
29. Veach, B. T.; Baker, C. A.; Kibbey, J. H.; Fong, A.; Broadaway, B. J.; Drake, C. P., Quantitation of
627
chloramphenicol and nitrofuran metabolites in aquaculture products using microwave-assisted
628
derivatization, automated SPE, and LC-MS/MS. Journal of AOAC International 2015, 98, 588-594.
629
30. Fapas Report 02334, 2017, Fera Science Ltd, www.fapas.com (published to participants 01/12/2017)
630
31. Thompson, M.; Ellison, S. L. R.; Wood, R. The International Harmonized Protocol for the Proficiency
631
Testing of Analytical Chemistry Laboratories. Pure and Applied Chemistry 2006, 78 (1), 145–196.
632 633
29
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634
CAPTIONS FOR FIGURES
635
Figure 1. Column-by-column, from left to right, the structures of: the parent nitrofuran drugs as
636
administered; the nitrofuran metabolites after in vivo transformation; the nitrofuran
637
metabolites after derivatization with 2-NBA; and the phenicols.
638
Figure 2. Depictions of: the in vivo transformation of the parent nitrofuran into its metabolite
639
(top, left and center); the formation of the metabolite-protein adduct (top, right); the acid
640
hydrolysis of the metabolite-protein adduct to liberate the metabolite (bottom).
641
Figure 3. Schematic representation of the chemical reaction between the AOZ, nitrofuran
642
metabolite, and 2-nitrobenzaldhyde in acidic condition.
643
Figure 4. Extracted ions chromatograms of two product ions of nitrofuran metabolites
644
standards in solvent (A), in matrix (B) and of fortified (spiked) samples in tilapia (C), each at the
645
LOD levels.
646
Figure 5. Extracted ions chromatograms of two product ions of chloramphenicol and florfenicol
647
standards in solvent (A), in matrix (B) and of fortified (spiked) samples in tilapia (C), each at the
648
LOD levels.
649
Figure 6. Multi-points calibration curve of derivatized nitrofuran metabolites standards (red
650
squares) and in-laboratory derivatized nitrofuran metabolites (blue circle).
651
Figure 7. A bar graph of z-scores for quantitation of total AHD; our laboratory is “Lab 4”.
652
Reproduced from Fapas Report 02334, Fera Science Ltd, 2017.30
653
Figure 8. A bar graph of z-scores for quantitation of total AOZ; our laboratory is “Lab 4”.
654
Reproduced from Fapas Report 02334, Fera Science Ltd, 2017.30
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Journal of Agricultural and Food Chemistry
Nitrofuran
Furazolidone
Metabolite
AOZ
2-NBA Derivatized Metabolite
Phenicols
2-NP-AOZ
Chloramphenicol
Furaltadone
AMOZ
Nitrofurantoin
AHD
2-NP-AMOZ
2-NP-AHD Florfenicol
656 657
Nitrofurazone
SC
2-NP-SC
Figure 1.
658 659 660 661
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N N
O
O
Metabolizes
+ Cleavage
Furazolidone
Protein
AOZ Bound Protein
H Cl O
N NH
Acidic Hydrolysis
O O H
H3O, OH OH
+ AOZ Metabolite
662 663
Figure 2.
664 665 666 667 668 669 670 671
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Journal of Agricultural and Food Chemistry
H3O
672 673
Figure 3.
674
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A Intensity (cps)
8.0x104
Area = 2.224 x 105 m/z = 335 → 291
3.0x104 4.15 min
Area = 7.810 x 104 2.5x104 m/z = 236 → 134
4.0x104 6.11 min
Area = 7.7033 x 104 m/z = 249 → 134
1.5x104
5.63 min
Area = 2.896 x 104 m/z = 209 → 192
5.46 min
3.0x104
4
6.0x10
2.0x104
1.0x104
2.0x104
1.5x104
4.0x104
4
2.0x104
Page 34 of 44
1.0x10
AMOZ
AOZ
1.0x104
5.0x103
AHD
SC
3
5.0x10
0.0 0.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 Time (min)
Intensity (cps)
2.0x104
Area = 5.970 x 104 m/z = 335 → 128
4.16 min
Area = 4.214 x 104 m/z = 236 → 104
0.0 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4
Time (min)
Time (min) 2.0x104
1.5x104
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
2.0x104 6.09 min
4
1.5x10
1.5x10
Area = 3.800 x 104 m/z = 249 → 104
Time (min) 1.5x104
5.60 min
Area = 3.153 x 104 m/z = 209 → 166
5.45 min
4
1.0x104 1.0x104
1.0x104
1.0x104
5.0x103
5.0x103
5.0x103
5.0x103
0.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
0.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0
Time (min)
B Intensity (cps)
8.0x10
4
Area = 2.262 x 105 m/z = 335 → 291
3.0x10 4.15 min
6.0x104
Area = 8.27 x 104 2.5x104 m/z = 236 → 134
2.0x104
0.0 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4
Time (min) 4
4
3.0x10 6.11 min
Area = 7.449 x 104 m/z = 249 → 134
Time (min)
1.5x10
4
5.62 min
2.0x104
2.0x104 4.0x104
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
Time (min)
Area = 3.316 x 104 m/z = 209 → 192
5.45 min
1.0x104
1.5x104
AMOZ
1.0x104
AOZ
1.0x104
AHD
5.0x103
SC
5.0x103 0.0 0.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 Time (min) Time (min)
Intensity (cps)
2.5x104 2.0x10
4
Area = 6.022 x 104 m/z = 335 → 128
2.0x104 4.16 min 1.5x10
Area = 4.099 x 104 m/z = 236 → 104
1.5x10
1.5x104 5.62 min
Area = 3.156 x 104 m/z = 209 → 166
5.45 min
4
1.0x104
1.0x104
5.0x103
5.0x103
5.0x103
0.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0
6.0x10
4
Area = 1.428 x 105 m/z = 335 → 291
2.5x10
4.16 min
4.0x104 1.5x10
5.0x10
2.5x10 6.10 min
2.0x104
1.0x104
AOZ
2.0x104
1.5x10
Area = 4.120 x 104 m/z = 335 → 128
1.5x104
1.0x104
AHD
Area = 2.180 x 104 m/z = 236 → 104
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
1.5x104
Area = 2.509 x 104 m/z = 249 → 104
SC
Time (min)
2.5x104 5.62 min
4
1.0x104
5.47 min
0.0 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4
Time (min)
6.10 min
Area = 4.816 x 104 m/z = 209 → 192
5.0x103
5.0x10
Time (min)
4.15 min
2.0x104 1.5x104
3
0.0 0.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2
Time (min) 2.5x104
5.63 min
1.5x10
3
Time (min)
Area = 5.036 x 104 m/z = 249 → 134
4
4
1.0x104
AMOZ
Area = 5.418 x 104 m/z = 236 → 134
0.0 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4
Time (min) 4
4
2.0x104
2.0x104
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
Time (min)
Time (min)
2.0x104
Area = 5.058 x 104 m/z = 209 → 166
5.46 min
1.0x104
1.5x104 1.0x104 5.0x103
1.0x104
5.0x103
5.0x103
5.0x103
0.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 Time (min)
675 676
Time (min)
1.0x104
3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Intensity (cps)
Area = 4.048 x 104 m/z = 249 → 104
1.5x104
0.0
Intensity (cps)
2.0x104 6.11 min
4
0.0 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4
Time (min)
1.0x104
5.0x103
C
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
0.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 Time (min)
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
Time (min)
0.0 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 Time (min)
Figure 4. 34
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Journal of Agricultural and Food Chemistry
Intensity (cps)
A
8.0x102
Area = 2.307 x 103 m/z = 321 → 152
2.5x103 5.90 min
6.0x102
Area = 7.342 x 103 m/z = 356 → 336
5.66 min
1.5x103 4.0x102
1.0x103
CP 2
2.0x10
1.0x103 8.0x102
FF
5.0x102
0.0 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8
Intensity (cps)
2.0x103
Time (min) Area = 2.058 x 103 m/z = 321 → 257 5.91 min
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
1.5x103
Time (min) Area = 4.498 x 103 m/z = 356 → 185
5.65 min
1.0x103
6.0x102 4.0x102
5.0x102
2
2.0x10
0.0
Intensity (cps)
B
5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 Time (min)
1.6x103
Area = 4.561 x 103 m/z = 321 → 152
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 Time (min)
2.5x103 5.90 min
2.0x103
1.2x103
Area = 9.136 x 103 m/z = 356 → 336
5.66 min
1.5x103 8.0x102
4.0x10
2
1.0x103
CP
FF
5.0x102 0.0 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8
Intensity (cps)
1.5x103
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
Time (min)
Area = 4.073 x 103 m/z = 321 → 257
Time (min) 1.8x103
5.91 min
1.0x103
1.5x103 1.2x10
Area = 6.157 x 103 m/z = 356 → 185
5.66 min
3
9.0x102
5.0x102
6.0x102 3.0x102
0.0
Intensity (cps)
C
3.2x103
5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 Time (min) Area = 9.498 x 103 m/z = 321 → 152
2.4x103
1.6x103
2.0x103
5.67 min
3.2x103
FF
CP
2.5x103
Time (min) Area = 1.950 x 104 m/z = 356 → 336
4.8x103
1.6x103
0.0 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8
Intensity (cps)
6.4x103
5.92 min
8.0x102
Time (min) Area = 7.080 x 103 m/z = 321 → 257
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
4.0x103
5.91 min
Time (min)
Area = 1.137 x 103 m/z = 356 → 185
5.66 min
3.0x103
1.5x103
2.0x103 1.0x103
1.0x103
5.0x102 0.0 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 Time (min)
677 678
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
0.0 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 Time (min)
Figure 5. 35
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AMOZ
1.4x107
Page 36 of 44
AOZ
4.0x106
Intensity (cps)
1.2x107 3.0x106
1.0x107 8.0x106
Standard R2 = 0.9977
In-situ R2 = 0.9928
2.0x106
6
6.0x10
In-situ R2 = 0.9916
Standard R2 = 0.9995
6
4.0x10
1.0x106
2.0x106 0.0
0.0 0
2
4
6
8
1.4x106
10 12 14 16 18
0
2
4
6
8
1.5x106
AHD
10 12 14 16 18
SC
1.2x106 Intensity (cps)
1.2x106 1.0x106 9.0x105
In-situ R2 = 0.9987
8.0x105 6.0x105
Standard R2 = 0.9998
5
6.0x10 Standard R2 = 0.9985
5
4.0x10
In-situ R2 = 0.9979
3.0x105 2.0x105 0.0
0.0 0
2
4
6
8
10 12 14 16 18
Concentration (ppb)
0
2
4
6
8
10 12 14 16 18
Concentration (ppb)
679 680
Figure 6.
681 682 683 684 685 686 687 688 689 690 36
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691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718
Journal of Agricultural and Food Chemistry
Figure 7.
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719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746
Page 38 of 44
Figure 8.
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747 748 749
Journal of Agricultural and Food Chemistry
Table 1. Table of optimal parameters for each analyte obtained from the infusion of 1ng uL-1 stock solutions into an AB Sciex 6500 QTrap Mass Spectrometer. Precursor ion (amu)
Analyte fraction
R.T (min)
DP (V)
EP (V)
CE (V)
CXP (V)
209>192
2-NP-SC-1
5.39
50
10
16
20
209>166
2-NP-SC-2
5.39
50
10
15
20
249>134
2-NP-AHD-1
5.54
51
10
17
14
249>104
2-NP-AHD-2
5.54
51
10
27
12
235>134
2-NP-AOZ-1
6.03
60
10
17
12
235>104
2-NP-AOZ-2
6.03
60
10
29
16
335>291
2-NP-AMOZ-1
4.15
70
10
17
16
335>128
2-NP-AMOZ-2
4.15
70
10
29
16
212>195
2-NP-SC- C3 N-1
5.39
60
10
18
25
212>168
2-NP-SC- C3 N-2
5.39
60
10
18
25
252>134
2-NP-AHD- C3-1
5.54
81
10
45
12
252>104
2-NP-AHD- C3-2
5.54
81
10
67
14
240>134
2-NP-AOZ-D4-1
6.01
80
10
17
16
240>104
2-NP-AOZ-D4-2
6.01
80
10
29
12
340>296
2-NP-AMOZ-D5-1
4.13
60
10
17
16
340>133
2-NP-AMOZ-D5-2
4.13
60
10
31
16
321>152
Chrloramphenicol-1
5.83
-55
-10
-24
-15
321>257
Chrloramphenicol-2
5.83
-45
-10
-16
-25
321>194
Chrloramphenicol-3
5.83
-60
-10
-18
-17
326>157
Chrloramphenicol-D5
5.82
-50
-10
-24
-13
356>336
Florfenicol-1
5.56
-60
-10
-14
-31
356>185
Florfenicol-2
5.56
-65
-10
-26
-17
13
15
13
15
13
13
750
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751 752
Page 40 of 44
Table 2. Table of internal standard-corrected recoveries of AMOZ, AOZ, AHD, and SC in tilapia and shrimp. Tilapia Fortification (ng/g)
AMOZ 335→291 Da Recovery SD (%)
CV
AHD 249→134 Da Recovery SD
(%)
(%)
CV
AOZ 236→134 Da Recovery SD
(%)
(%)
CV
SC 209→192 Da Recovery SD
(%)
(%)
CV (%)
0.25
92
1
1.1
89
3
3.6
97
1
1.6
123
9
8.9
1
90
2
3.9
91
1
1.7
97
3
3.9
98
4
5.2
5
89
5
7.2
93
12
15.5
107
9
10.8
97
7
9.7
10
96
1
1.2
90
3
3.9
103
0
0.6
96
4
4.5
Overall
92
4
4.9
91
6
8.4
102
6
7.2
97
13
5.8
0.25
86
2
3.1
58
7
15.4
90
4
4.8
98
4
4.4
1
91
1
1.9
73
2
2.7
90
1
1.3
93
1
1.9
5
94
3
3.8
87
8
11.8
93
2
2.2
94
3
4.4
10
93
1
1.9
93
3
3.9
93
2
3.3
99
2
3.0
Overall
93
4
2.8
84
15
12.4
92
3
2.6
95
4
4.0
Shrimp
753 754 755 756 757 758 759 760 761 762 763 764 765 40
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Journal of Agricultural and Food Chemistry
Table 3. Table of internal standard-corrected recoveries of chloramphenicol in tilapia and shrimp.
Tilapia
Chloramphenicol 321→152 Da Fortification (ng/g) Recovery (%) SD CV (%) 0.1
110
15
16.2
0.25
110
1
1.1
0.5
106
3
3.4
1
113
3
2.9
Overall
110
8
3.4
0.1
89
0
0.6
0.25
86
2
2.4
0.5
81
4
6.1
1
85
3
3.8
Overall
84
4
4.4
Shrimp
767 768 769 770 771 772 773 774 775 776 777 778 779 41
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Page 42 of 44
Table 4. Table of absolute recoveries of AMOZ, AOZ, AHD, and SC in tilapia and shrimp. Tilapia Fortification (ng/g)
AMOZ 335→291 Da Recovery CV SD (%) (%)
AHD 249→134 Da Recovery CV SD (%) (%)
AOZ 236→134 Da Recovery CV SD (%) (%)
SC 209→192 Da Recovery CV SD (%) (%)
0.25
61
1
2.5
65
4
8.4
61
2
4.1
67
2
3.5
1
58
5
9.6
66
2
4.7
61
2
4.1
58
1
3
5
60
4
8.6
68
4
6.7
62
0
0
45
1
3.8
10
63
1
1.8
70
1
1.4
65
2
3.1
48
3
7.8
Overall
60
4
7.5
68
4
5.0
63
2
4.2
51
9
12.4
0.25
55
0
1.0
77
3
5.3
62
1
2.8
64
8
17.7
1
58
2
3.6
70
7
11.4
68
1
1.5
53
1
1.9
5
57
1
2.0
69
7
11.8
70
0
0.8
49
2
5
10
57
1
2.0
73
3
5.6
70
1
1.7
50
4
8.7
Overall
57
1
2.3
71
6
8.9
69
3
1.9
51
6
6.2
Shrimp
781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 42
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Table 5. Table of absolute recoveries of chloramphenicol and florfenicol tilapia and shrimp.
Tilapia
Chloramphenicol Florfenicol 321→152 Da 356→ 185 Da Fortification (ng/g) Recovery (%) SD CV (%) Recovery (%) SD CV (%) 0.1
77
10
16.2
83
5
7.0
0.25
76
3
5.3
86
2
3.4
0.5
80
0
0
85
1
1.7
1
80
1
1.4
90
4
5.7
Overall
79
6
4.5
87
4
4.5
0.1
73
4
7.6
83
4
6.7
0.25
70
2
3.0
76
3
4.6
0.5
69
1
2.2
76
1
2.3
1
72
1
2.4
78
1
2.2
Overall
70
3
2.8
77
4
3.0
Shrimp
797 798 799 800 801 802 803 804 805 806 807 808 809 43
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Journal of Agricultural and Food Chemistry
810
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TOC Graphic
811
812 813
44
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