Accurate Quantitation and Analysis of Nitrofuran Metabolites

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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

2

Florfenicol in Seafood by UHPLC-MS/MS: Method Validation and Regulatory Samples

3

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

8

four nitrofuran metabolites: (3-amino-2-oxazolidinone (AOZ); 3-amino-5-morpholinomethyl-2-

9

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

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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,

1

ACS Paragon Plus Environment


23

#

24

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

28

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

36

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

6


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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,

146

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

8


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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

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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)

201

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

211

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

10


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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

226

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

228

same mobile and stationary phases. The gradient began at 95:5 of A:B with a flow of 0.2 mL per

229

min. The gradient was changed to 5:95 of A:B over a course of 5 min followed by a 2.5 min hold

230

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

235

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

237

(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:

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declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit

245

potential (CXP) (Table 1).

246 247

RESULTS AND DISCUSSION

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Background. The nitrofurans and phenicols (chloramphenicol and florfenicol) are broad-

249

spectrum, synthetic antibiotics. Their efficacy and low cost make them attractive options in

250

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

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are banned in the US and EU for such veterinary uses due to their recognized dangers to human

253

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

256

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

259

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

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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

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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.

270

Extraction Method Optimization. To optimize the extraction method of the nitrofuran

271

metabolites, chloramphenicol, and florfenicol from various seafood matrices, we explored

272

three extraction protocols that have been reported in the literature.27-29 In our laboratory, we

273

use two important criteria to qualify a method of extraction: the signal-to-noise ratio of the

274

analyte peak of interest, and the absolute recovery before correction with internal standards.

275

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


Page 15 of 44


302

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



Page 16 of 44

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


Page 17 of 44


346

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



Page 18 of 44

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


Page 19 of 44


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



Page 20 of 44

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


Page 21 of 44


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



Page 22 of 44

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


Page 23 of 44


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



Page 24 of 44

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


Page 25 of 44


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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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



Page 30 of 44

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

655 30


Page 31 of 44


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

31



Page 32 of 44

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

32


Page 33 of 44


H3O

672 673

Figure 3.

674

33



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


Page 35 of 44


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



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


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


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


Page 37 of 44

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


Figure 7.

37



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.

38


Page 39 of 44

747 748 749


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

39



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


Page 41 of 44

766


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



780

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


Page 43 of 44

796


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



810

Page 44 of 44

TOC Graphic

811

812 813

44


Accurate Quantitation and Analysis of Nitrofuran Metabolites

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