Accurate Quantitation and Analysis of Nitrofuran Metabolites

Article Cite This: J. Agric. Food Chem. 2018, 66, 5018−5030

pubs.acs.org/JAFC

Accurate Quantitation and Analysis of Nitrofuran Metabolites, Chloramphenicol, and Florfenicol in Seafood by UltrahighPerformance Liquid Chromatography−Tandem Mass Spectrometry: Method Validation and Regulatory Samples Fadi Aldeek,*,† Kevin C. Hsieh, Obiadada N. Ugochukwu, Ghislain Gerard, and Walter Hammack Division of Food Safety, Florida Department of Agriculture and Consumer Services, 3125 Conner Boulevard, Tallahassee, Florida 32399-1650, United States ABSTRACT: We developed and validated a method for the extraction, identification, and quantitation of four nitrofuran metabolites, 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), semicarbazide (SC), and 1-aminohydantoin (AHD), as well as chloramphenicol and florfenicol in a variety of seafood commodities. Samples were extracted by liquid−liquid extraction techniques, analyzed by ultrahigh-performance liquid chromatography−tandem mass spectrometry (UHPLC−MS/MS), and quantitated using commercially sourced, derivatized nitrofuran metabolites, with their isotopically labeled internal standards in-solvent. We obtained recoveries of 90−100% at various fortification levels. The limit of detection (LOD) was set at 0.25 ng/g for AMOZ and AOZ, 1 ng/g for AHD and SC, and 0.1 ng/g for the phenicols. Various extraction methods, standard stability, derivatization efficiency, and improvements to conventional quantitation techniques were also investigated. We successfully applied this method to the identification and quantitation of nitrofuran metabolites and phenicols in 102 imported seafood products. Our results revealed that four of the samples contained residues from banned veterinary drugs. KEYWORDS: nitrofurans, metabolites, chloramphenicol, florfenicol, UHPLC−MS/MS, liquid/liquid extraction, method development, validation

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INTRODUCTION Nitrofurans and phenicols are antibiotic drugs that have been widely used to combat bacterial diseases in animal production because of their low cost, ready availability, and effectiveness against resistant infections.1−4 In the past few decades, the use of these two classes of antibiotics in aquatic products has drastically increased as a result of the high mortality rates of bacterial diseases and significant financial losses thus incurred by the aquaculture industry worldwide.5−8 However, nitrofurans and their metabolites have been banned in Europe and other countries as a result of their carcinogenic, mutagenic, and genotoxic effects in humans.8−10 Chloramphenicol has also been restricted by many organizations, such as the U.S. Food and Drug Administration (FDA), for use in animals as a result of its potential side effects in humans, e.g., hematological abnormalities, gray baby syndrome, and, most fatally, aplastic anemia, all of which can be caused in humans who may be exposed to it during application or upon ingestion of food products that contain residues of the drug.11−13 The most commonly tested nitofurans are furazolidone, furaltadone, nitrofurazone, and nitrofurantoin (Figure 1). In vivo, these nitrofurans are quickly metabolized; therefore, the concentration of the parent compound rapidly drops below the detection and quantitation limits of modern analytical methods.14 The metabolites formed from the above nitrofurans are 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), semicarbazide (SC), and 1aminohydantoin (AHD) (Figure 1).15 These toxic metabolites © 2017 American Chemical Society

then bind to protein tissues, forming metabolite−protein adducts that are stable for long periods of time (Figure 2).16 An acidic hydrolysis step has been the most commonly used method to liberate the covalently bound metabolites.17 As a result of the low molecular weight (75−201 amu), high polarity, poor retention on reversed-phase columns, poor ionization, and strong covalent bindings of these metabolites with protein tissues, derivatization with 2-nitrobenzaldehyde (2-NBA) (Figure 3) is also a crucial step to allow for their detection using the selective and sensitive liquid chromatography−tandem mass spectrometry (LC−MS/MS) technique.18 Currently, nitrofurans are regulated at a target level of 1 ng/g, with chloramphenicol at 0.3 ng/g, in the United States, European Union (EU), and Canada. Various methods for the extraction and detection of nitrofurans and/or phenicols in seafood using LC−MS/MS have been described in the literature, although none have analyzed florfenicol in addition to nitrofurans and chloramphenicol.19−26 Three selected reports, which were also experimentally explored, are detailed herein. It is important to note that the recoveries in the reports are those calculated by the authors, which were not “absolute”. For example, Ye et al. described a Special Issue: 54th North American Chemical Residue Workshop Received: Revised: Accepted: Published: 5018

September 20, 2017 December 6, 2017 December 28, 2017 December 28, 2017 DOI: 10.1021/acs.jafc.7b04360 J. Agric. Food Chem. 2018, 66, 5018−5030

Article

Journal of Agricultural and Food Chemistry

Figure 1. Column-by-column, from left to right, the structures of the parent nitrofuran drugs as administered, nitrofuran metabolites after in vivo transformation, nitrofuran metabolites after derivatization with 2-NBA, and phenicols.

Figure 2. Depictions of the in vivo transformation of parent nitrofuran into its metabolite (top, left, and center), formation of the metabolite−protein adduct (top and right), acid hydrolysis of the metabolite−protein adduct to liberate the metabolite (bottom).

Figure 3. Schematic representation of the chemical reaction between AOZ, nitrofuran metabolite, and 2-nitrobenzaldhyde in acidic conditions.

sensitive method for the detection of nitrofuran metabolites using LC−MS/MS.27 They used a liquid−liquid extraction with ethyl acetate and aqueous phosphate buffer. Analyte recoveries in that method were found to be between 62 and 91% depending upon the extracted analyte. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.15 and 0.3 ng/g, respectively. Recently, An et al. developed a

water−acetonitrile liquid−liquid extraction method of nitrofuran metabolites and chloramphenicol in shrimp, followed by LC−MS/MS detection.28 In that report, analyte recoveries were studied at 0.15, 0.3, and 0.6 ng/g for chloramphenicol and 0.5, 1.0, and 2.0 ng/g for the nitrofuran metabolites. They reported an average recovery of 98−109% with intra- and interday relative standard deviations (RSDs) of 12 and 17.7%, 5019

DOI: 10.1021/acs.jafc.7b04360 J. Agric. Food Chem. 2018, 66, 5018−5030

Article


AHD−13C3, 2-NP−SC−HCl−13C, 15N2, 2-NP−AOZ, 2-NBA, dibasic potassium phosphate, hydrochloric acid, and ammonium acetate were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Chloramphenicol and florfenicol in methanol (500 ppm) were provided by Restek Corporation (Bellefonte, PA, U.S.A.). Chloramphenicol-d5 (100 ppm) was provided by Cambridge Isotopes (Tewksbury, MA, U.S.A.). Highperformance liquid chromatography (HPLC)-grade acetonitrile (with and without 1% formic acid), methanol, water (with and without 1% formic acid), and ethyl acetate were obtained from Fisher Scientific (Pittsburgh, PA, U.S.A.). Solution Preparation. The ammonium acetate in methanol (20:80, v/v, NH4OAc/MeOH) solution was prepared by dissolving ammonium acetate (655 mg) in 1 L of deionized (DI) water. This solution was then combined with methanol to form the final 20:80 (v/ v) solution. The 0.125 M hydrochloric acid solution was prepared by diluting 6 M hydrochloric acid with DI water. The phosphate buffer solution (PBS, 0.1 M) was prepared by dissolving anhydrous dipotassium hydrogen phosphate (174 g) in 1000 mL of DI water. Lastly, 0.1 M 2-NBA solution was prepared by dissolving 148 mg in 10 mL of methanol. Preparation of Standard Stock Solutions. Individual stock solutions of AOZ, AMOZ, AHD−HCl, SC−HCl, AOZ-d4, AMOZ-d5, AHD−13C3, SC−HCl−13C, 15N2, 2-NP−AOZ, 2-NP−AMOZ, 2-NP− AHD, 2-NP−SC, 2-NP−AOZ-d 4 , 2-NP−AMOZ-d 5 , 2-NP− AHD−13C3, 2-NP−SC−HCl−13C, and 15N2 at 100 μg/mL were prepared by dissolving each standard after adjusting for the salt content, purity, and 2-NBA in methanol. The contribution factors are 46.37, 36.06, 43.41, and 60.19% for AHD, SEM, AOZ, and AMOZ. Isotopically labeled internal standards were checked upon receipt for the absence of native standards. This is a procedure that only needs to be performed once, prior to method validation. Preparation of Mixed Intermediate Standard Solutions (INTA, INT-B, INT-C, INT-D, INT-E, and INT-F) (1 μg/mL = 1 μg/mg). Intermediate standard solutions of the non-isotopically labeled underivatized (INT-A), isotopically labeled underivatized (INT-B), non-isotopically labeled derivatized (INT-C), and isotopically labeled derivatized (INT-D) nitrofuran metabolites were prepared by adding 100 μL of each stock solution in 10 mL of NH4OAc/MeOH (20:80, v/v). An intermediate standard solution of the phenicols (INT-E) was prepared by adding 100 μL of each standard (500 μg/mL) to a 50 mL volumetric flask and diluting with methanol, resulting in a 1 μg/mL concentration of each analyte. The intermediate internal standard solution for the phenicols (INT-F) was prepared similarly by adding 100 μL of chloramphenicol-d5 standard (100 μg/mL) into a 10 mL volumetric flask and diluting with methanol, resulting in a 1 μg/mL concentration of the analyte. Preparation of Underivatized Nitrofuran Metabolite Intermediate Solutions for Spikes (INT-S-A and INT-IS-B). The series of working dilutions, from INT-S-A1 to INT-S-A6, were prepared from INT-A to give a concentration of 2, 5, 20, 100, 200, and 300 ng/ mL of the calibration standards. The internal, INT-IS-B, was prepared from 1 mL of INT-B and diluted with NH4OAc/MeOH (20:80, v/v) to 25 mL to give a concentration of 40 ng/mL. Preparation of Derivatized Nitrofuran Metabolite Intermediate Solutions for Calibration Curves (INT-S-C and INT-ISD). The series of working dilutions, from INT-S-C1 to INT-S-C6, were prepared from INT-C to give a concentration of 2, 5, 20, 100, 200, and 300 ng/mL of the calibration standards. The internal, INTIS-D, was prepared from 1 mL of INT-D and diluted with NH4OAc/ MeOH (20:80, v/v) to 25 mL to give a concentration of 40 ng/mL. Preparation of Phenicol Intermediate Solutions (INT-S-E and INT-IS-F). The series of working dilutions, from INT-S-E1 to INT-SE6, was prepared from INT-E to give a concentration of 0.5, 1, 2, 5, 10, and 20 ng/mL of the calibration standards. The internal, INT-IS-F, was prepared by diluting 125 μL of INT-F with MeOH to a volume of 25 mL to give a concentration of 5 ng/mL. Sample Fortification. The fortified samples were made by spiking 2 g of the different seafood matrices (tilapia and shrimp) with 100 μL of four intermediate mixed nitrofuran standard solutions, INT-S-A2, INT-S-A3, INT-S-A4, and INT-S-A5, 100 μL of the internal standard

respectively. More recently, Veach et. al reported on the quantitation of chloramphenicol and nitrofuran metabolites in various aquaculture matrices, including shrimp, catfish, and crawfish. Derivatization of nitrofuran metabolites was performed using microwave assistance, followed by automated solid-phase extraction (SPE).29 They fortified their samples with five spike levels for each analyte. Recoveries were found to be between 89 and 107% with RSD of ≤8.3%. They determined a LOD of 0.06 ng/g and LOQ of 0.2 ng/g for nitrofuran metabolites and a LOD of 0.01 ng/g and LOQ of ≤0.03 ng/g for chloramphenicol. Despite the method development effort made on the extraction and LC−MS/MS detection of phenicols and nitrofuran metabolites in different seafood matrices, recovery and quantitation of these analytes were sub-optimal for a critical reason: in previous reports, recoveries were often calculated using a fortified matrix-extracted calibration curve (“calibrating samples”). This was performed as a result of the unavailability of commercially sourced, derivatized nitrofuran metabolites. Calibrating samples were prepared by adding the nonderivatized metabolites at different concentrations of the curve to matrix blanks and then derivatizing and extracting these metabolites from the fortified matrix to form a multipoint, calibrating sample curve. This prevented optimal recovery and quantitation of nitrofuran metabolites, because the fortified spikes and the curve built from calibrating samples were identical in terms of the extraction procedure. It was therefore impossible to detect physicochemical loss of analyte during extraction, which led to the reporting of erroneously high, non-absolute recoveries. Absolute recovery is a measure of the quantity of analyte lost during an extraction procedure, by physical or chemical processes. It describes the efficiency of an extraction method. To accurately calculate the absolute recovery of spikes, in which methods that employ calibrating sample curves cannot do, our method used underivatized nitrofuran metabolites for spikes only. These spikes were then quantitated with calibration curves made using commercially sourced, derivatized nitrofuran metabolites spiked into extracted-matrix blanks post-extraction. This allowed us to compare the absolute recovery of our method to the absolute recoveries of the other methods after replicating them (An and Ye) or approximating them (Veach; a conventional oven was substituted in place of a microwave oven) to accommodate our equipment. After establishing absolute recoveries for this comparison, we began to correct the recoveries by placing isotopically labeled underivatized internal standards in spikes and isotopically labeled derivatized internal standards in-solvent curves. This provided better quantitation by correcting for analyte loss through internal standards. The process also demonstrated whether the derivatization was complete. Finally, we evaluated the applicability of the validated method by taking part in a Fapas-administered proficiency test, alongside 98 other laboratories. The test results support the applicability of the method. Furthermore, we analyzed 102 composite samples of a variety of imported seafood, including shrimp, tilapia, frog legs, salmon, and swai fish, and discovered several violations above the U.S. FDA action level.

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MATERIALS AND METHODS

Reagents. AOZ, AMOZ, AHD−HCl, SC−HCl, AOZ-d4, AMOZd5, AHD−13C3, SC−HCl−13C, 15N2, 2-NP−AOZ, 2-NP−AMOZ, 2NP−AHD, 2-NP−SC, 2-NP−AOZ-d4, 2-NP−AMOZ-d5, 2-NP− 5020


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Table 1. Table of Optimal Parameters for Each Analyte Obtained from the Infusion of 1 ng μL−1 Stock Solutions into an AB Sciex 6500 QTrap Mass Spectrometer precursor ion (amu) 209 209 249 249 235 235 335 335 212 212 252 252 240 240 340 340 321 321 321 326 356 356

> > > > > > > > > > > > > > > > > > > > > >

192 166 134 104 134 104 291 128 195 168 134 104 134 104 296 133 152 257 194 157 336 185

analyte fraction

RT (min)

DP (V)

EP (V)

CE (V)

CXP (V)

2-NP−SC-1 2-NP−SC-2 2-NP−AHD-1 2-NP−AHD-2 2-NP−AOZ-1 2-NP−AOZ-2 2-NP−AMOZ-1 2-NP−AMOZ-2 2-NP−SC−13C315N-1 2-NP−SC−13C315N-2 2-NP−AHD−13C3-1 2-NP−AHD−13C3-2 2-NP−AOZ-d4-1 2-NP−AOZ-d4-2 2-NP−AMOZ-d5-1 2-NP−AMOZ-d5-2 chrloramphenicol-1 chrloramphenicol-2 chrloramphenicol-3 chrloramphenicol-d5 florfenicol-1 florfenicol-2

5.39 5.39 5.54 5.54 6.03 6.03 4.15 4.15 5.39 5.39 5.54 5.54 6.01 6.01 4.13 4.13 5.83 5.83 5.83 5.82 5.56 5.56

50 50 51 51 60 60 70 70 60 60 81 81 80 80 60 60 −55 −45 −60 −50 −60 −65

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 −10 −10 −10 −10 −10 −10

16 15 17 27 17 29 17 29 18 18 45 67 17 29 17 31 −24 −16 −18 −24 −14 −26

20 20 14 12 12 16 16 16 25 25 12 14 16 12 16 16 −15 −25 −17 −13 −31 −17

intermediate solution, INT-IS-B (40 ng/mL), 100 μL of four intermediate mixed phenicol standard solutions, INT-S-E3, INT-SE4, INT-S-E5, and INT-S-E6, and 100 μL of the internal standard intermediate solution for phenicols, INT-F (5 ng/mL). Overall, spikes were made for each matrix to reach final concentrations of 0.25, 1, 5, and 10 ng/g of AMOZ, AOZ, AHD, and SC and 0.1, 0.25, 0.5, and 1 ng/g of chloramphenicol and florfenicol in each spike. It is worth mentioning that reagent (2 g of water) and matrix blanks were not fortified with internal standard and were used as negative controls. Fortifying blanks with standard would not allow them to serve their purpose of identifying the presence of contamination somewhere in the chain of analysis. Calibration Curve. To prepare 800 μL of the individual calibration standards, 100 μL of INT-S-C1−INT-S-C6 and 100 μL of INT-S-E1− INT-S-E6 were added to create six calibration standards, from SC1 to SC6, respectively. The remaining 600 μL was comprised of 100 μL of INT-IS-D and INT-IS-F and 400 μL of NH4OAc/MeOH (20:80, v/v) solution. The final concentrations of nitrofurans and 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, respectively. The final concentrations of the internal nitrofurans and phenicols were 2 and 0.25 ng, respectively. Extraction of Nitrofuran Metabolites and Phenicols from Fortified Seafood Matrices. Seafood samples were purchased and homogenized to a powder-like (when frozen) or paste consistency using a Robot Coupe (Ridgeland, MS, U.S.A.) with a 7 mm thick stainless-steel blade and immediately stored at −80 °C. A 2 g (±0.05 g) portion was weighed into a 50 mL polypropylene centrifuge tube for each sample a day before the extraction. A reagent blank was prepared by measuring 2 g (±0.05 g) of DI water into a 50 mL disposable centrifuge tube. A matrix blank was prepared by measuring 2 g (±0.05 g) of a blank seafood matrix into a 50 mL disposable centrifuge tube. Spikes were prepared by measuring 2 g (±0.05 g) of blank seafood matrix into a 50 mL disposable centrifuge tube. Spikes were fortified with internal standard solutions (100 μL of INT-IS-B and INT-IS-F) as well as four spike levels of intermediate mixed standard solutions in triplicate. Then, 10 mL of 0.125 M HCl, followed by 200 μL of 100 mM 2-NBA (prepared daily), was added to all blanks and spikes and shaken for 10 min at 1100 rpm using a Geno Grinder (Metuchen, NJ, U.S.A.). The samples were placed in the oven at 37 °C overnight to allow nitrofurans to derivatize.

The following day, the samples were cooled for 15 min at room temperature. Then, 5 mL of 1.0 M dibasic potassium phosphate buffer solution was added to each tube to adjust the pH to 7.3. If further adjustment is needed, 0.1 M NaOH or HCl is used. After the pH adjustment, the contents were vortexed for 20 s and centrifuged at 4000 rpm for 15 min. Following centrifugation, the supernatant is transferred from each tube into a clean 50 mL centrifuge tube. In the remaining centrifugate, 6 mL of water was added, vortexed for 15 s, and centrifuged for 5 min at 4000 rpm. The supernatant was then decanted into the same 50 mL centrifuge tube that contained the previous supernatant. Sodium chloride (2 g), followed by 6 mL of ethyl acetate, were added to the supernatant, shaken for 30 s, and centrifuged at 4000 rpm for 5 min. The ethyl acetate layer was then transferred to a clean 50 mL centrifuge tube. The aqueous layer was extracted again using the same amount of ethyl acetate, shaken, and centrifuged at 4000 rpm for 5 min. After collection of both ethyl acetate portions into the same tube, the liquid was evaporated to complete dryness with flowing nitrogen gas at 15 psi and 40 °C for 30 min. The dried extracts were reconstituted with 0.8 mL of NH4OAc/ MeOH (20:80, v/v) and sonicated for 1 min. The contents were then transferred using a pipet and filtered through a 0.2 μm polyvinylidene fluoride (PDVF) filter. Derivatization Efficiency and Standard Stability Tests. The curves used for quantitation were prepared as in-vial combinations of 100 μL, sequentially, of the intermediate standard mixed solutions of the derivatized nitrofurans (from INT-S-C1 to INT-S-C6) and phenicols (from INT-S-E1 to INT-S-E6), 100 μL each of the intermediate internal standard mixed solutions of the derivatized nitrofurans (INT-IS-D) and chloramphenicol (INT-IS-F), and 400 μL each of NH4OAc/MeOH (20:80, v/v) for a final volume of 800 μL. To test their stability over time, curves were made, run, and compared on a weekly basis against those previously prepared. Data showed that curves were stable for up to 3 weeks when stored in the LC autosampler at 10 °C. To test the efficiency of the in situ derivatization, in-vial combinations were made of 100 μL, sequentially, of the intermediate standard mixed solutions of the underivatized nitrofurans (from INT-S-A1 to INT-S-A6), 100 μL each of 2-NBA (0.1 M), 50 μL each of HCl (0.125 M), and 550 μL each of NH4OAc/MeOH (20:80, v/v) for a final volume of 800 μL. They were derivatized overnight using the oven at 37 °C. 5021


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Journal of Agricultural and Food Chemistry LC−MS/MS Method for the Analysis of Nitrofurans and Phenicols. The LC−MS/MS apparatus included Waters Acquity ultrahigh-performance liquid chromatography (UHPLC) systems (Waters, Milford, MA, U.S.A.) coupled to AB Sciex 5500 or 6500 Q-Trap (AB Sciex, Toronto, Ontario, Canada) triple quadrupole mass spectrometers. Separation of compounds was achieved on UHPLC systems that were equipped with Acquity C18 columns (1.7 μm particle size, 150 mm length, and 2.1 mm internal diameter) maintained at 40 (±1) °C. The mobile phase was composed of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. The gradient began at 95:5 of A/B with a flow of 0.2 mL/min. The gradient was 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 min, the gradient was changed to 95:5 of A/B with a concomitant change in the flow rate to 0.4 mL/ min. After 4 min, the flow was reduced to 0.2 mL/min over the course of 0.1 min. This was held for 0.8 min to complete the LC gradient (20 min total run time). An alternate, 10 min method was also developed as a proof of concept using the same mobile and stationary phases. The gradient began at 95:5 of A/B with a flow of 0.2 mL/min. The gradient was changed to 5:95 of A/B over a course of 5 min, followed by a 2.5 min hold at 5:95 of A/B. Over 0.05 min, the gradient was changed to 95:5 of A/B with a concomitant change in the flow rate to 0.4 mL/min. After 2 min, the flow was reduced to 0.2 mL/min over the course of 0.05 min. This was held for 0.4 min to complete the LC gradient. All analyses of samples were carried out using electrospray ionization (ESI) using multiple reaction monitoring (MRM). Sample analyses were performed in duplicate, with one each in positive-ion mode (nitrofurans) and negative-ion mode (phenicols). Mass spectrometry (MS) source parameters for the positive-ion mode experiments (nitrofurans) were as follows: ion source voltage, 4500 V; curtain gas, 30 psi; ion source gas, GS1 of 40 psi and GS2 of 45 psi; ion source temperature, 400 °C; and entrance potential, 10 V. MS source parameters for the negative-ion mode experiments (phenicols) were as follows: ion source voltage, −4500 V; curtain gas, 30 psi; ion source gas, GS1 of 40 psi and GS2 of 45 psi; ion source temperature, 400 °C; and entrance potential, −10 V. By infusion of 1 ng/μL solutions of NP−NF metabolites, chloramphenicol, and florfenicol in acidified methanol at 10 μL/min and using the automatic and manual tuning modes of the Analyst software (version 1.6.1), optimal values for the formation of stable product ions were found for the following MS parameters: declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) (Table 1).

no such consideration. Additionally, quantitation of phenicols is not affected by the steps taken to prepare nitrofurans; therefore, they can be extracted simultaneously. Liberation of the nitrofuran metabolites was achieved by acid hydrolysis of the imine bonds formed between the metabolites and amino acids (Figure 2); derivatization of the nitrofuran metabolites was achieved by reaction of the newly liberated amine moiety of the nitrofuran metabolites with the aldehyde group of 2-NBA (Figure 3) via a nucleophilic addition, followed by the elimination of water to form the imine. Conveniently, these steps can all be carried out simultaneously in the same reaction vessel. Extraction Method Optimization. To optimize the extraction method of the nitrofuran metabolites, chloramphenicol, and florfenicol from various seafood matrices, we explored three extraction protocols that have been reported in the literature.27−29 In our laboratory, we use two important criteria to qualify a method of extraction: the signal-to-noise ratio of the analyte peak of interest and the absolute recovery before correction with internal standards. We used matrixmatched calibration curves (in-matrix curves) to determine the “absolute recovery” by controlling for the effect of the matrix on ionization, thereby exposing physicochemical losses of analyte during extraction. We calculated absolute recoveries of the analytes from fortified (spiked) seafood samples using matrix-matched calibration curves, with correlation coefficient values (R2) that were consistently greater than 0.99. To generate these curves, commercially available derivatized nitrofuran metabolites and phenicol standards were dissolved in a solution (containing extracted matrix components) at concentrations varying from 0.1 to 15 ng/g for nitrofuran metabolites and from 0.025 to 1 ng/g for phenicols. LC−MS/MS data points were fitted to the curve using a quadratic, 1/x-weighted regression. Matrix effects on the area counts for the analytes in the three matrices were also investigated, and any cases of interference, suppression, etc. are reported here. The three U.S. FDA extraction methods that we explored started with the same steps: weighing the sample, fortification with standards, addition of HCl and 2-NBA, oven incubation for 16 h, and addition of K2HPO4 buffer. After these steps, the extractions began to vary. For example, the first method investigated, by An et al., used acetonitrile to extract the analytes via a liquid/liquid extraction.28 In this method, the absolute recoveries that we obtained ranged from 50 to 65% depending upon the extracted analyte. These absolute recoveries are satisfying and within our acceptable criteria. However, a matrix interference was found to co-elute with the AHD metabolite, which made it very difficult to quantify at the LOQ level for this analyte. We hypothesize that, as a result of its relative nonpolarity, acetonitrile used in this method was efficient at solubilizing oily, low-polarity components of the seafood matrices that interfered with analysis, such as the AHD interference in question. The second method explored, by Veach et al., used a SPE column to extract the analyte and remove interferences.29 We replicated the method, with the exception of our substitution of a conventional oven for the microwave oven used by Veach et al. Given the 99% derivatization efficiency achieved by our oven method (Figure 6), we conclude that any differences arising from this substitution were minimal. The absolute recoveries that we obtained by this method ranged from 33 to 53% depending upon the analyte in question.

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RESULTS AND DISCUSSION Background. Nitrofurans and phenicols (chloramphenicol and florfenicol) are broad-spectrum, synthetic antibiotics. Their efficacy and low cost make them attractive options in controlling the bacterial diseases prevalent in populations of terrestrial and aquatic animals raised for human consumption. Despite these economic incentives for their application, they are banned in the U.S. and EU for such veterinary uses as a result of their recognized dangers to human health. It is for this reason that developing methods for their detection and quantitation are crucial from a public health standpoint. Of the two classes of antibiotics analyzed in this paper, the chemistry of nitrofurans presented the greatest obstacle to their analysis. As a result of their rapid conversion in vivo to highly polar, low mass metabolites (listed in Figure 1), nitrofurans are difficult to resolve on reversed-phase UHPLC columns and analyze in MS/MS systems. These metabolites also have high affinities for binding with the amino acids found in animal muscle tissue (depicted in Figure 2) and forming very stable metabolite−protein adducts. These facts require samples to be subjected to chemical methods of analyte liberation and derivatization before analysis. The phenicols, however, need 5022


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Figure 4. Extracted ions chromatograms of two product ions of nitrofuran metabolite standards in solvent (A), in matrix (B), and of fortified (spiked) samples in tilapia (C), each at the LOD levels.

Finally, we tried a third method, published by Ye et al., and successfully used it as the basis for the rest of the method development and validation work.27 In this method, samples were extracted by liquid−liquid extraction using two 6 mL aliquots of water, followed by extraction of the combined 12 mL of water with two 6 mL aliquots of ethyl acetate. The

subsequent addition of NaCl to water increased the polarity of the aqueous phase, “salting out” the desired analytes into less polar, organic ethyl acetate. Reconstituted extracts were also filtered through 0.2 μm PVDF filters to remove macroscopic pieces of matrix material that could clog or dirty the LC−MS/ MS system and interfere with analysis. 5023


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Journal of Agricultural and Food Chemistry With this procedure, the absolute recovery for the nitrofuran metabolites was found to be between 60 and 70%, with a much cleaner background. Ion suppression and enhancement effects were found to be negligible on extracts produced from this method. It is worth mentioning that, in the above methods, the authors calculated their recoveries by comparing spikes to calibration curves created from spiked samples (“calibrating samples”). Herein, we describe the absolute recoveries calculated by our lab upon replication of their methods and compare them to the absolute recovery of our method. Absolute recoveries are calculated using extracted-matrix calibration curves, as described in the first part of this section. LC−MS/MS Analysis Optimization. Solutions (1 ng/μL) of the NP−NFs in acidified methanol, chloramphenicol, and florfenicol were infused directly into the AB Sciex 6500 and 5500 QTrap triple quadrupole mass spectrometers to determine precursor ion masses, product ion masses, and values of the parameters necessary to produce the most stable and responsive product ions. First, Q1 of the MS/MS system was scanned to determine the mass of the precursor ion. When found, this mass was monitored, while values for DP were optimized. Following this, Q1 was fixed at the selected m/z ratio of the precursor ion, while Q3 was scanned to determine potential product ion masses. After this, values for DP, CE, EP, and CXP that gave the most stable and responsive product ions were found and documented (Table 1). The data thus obtained was placed into an acquisition method, and single standards of each compound were run on the instrument to determine retention times (Table 1) and ion ratios between precursor and product ions, both of which were used for analyte identity confirmation. With the latest eluting analytes emerging from the column at about 6 min, the necessity of derivatization in reducing the polarity of the nitrofuran metabolites can be seen. Figures 4A and 5A depict the extracted ion chromatograms for each analyte of interest, obtained from liquid chromatography (LC) injection at 0.25 ng/mL for AMOZ and AOZ, 1 ng/mL for AHD and SC, and 0.1 ng/mL for chloramphenicol and florfenicol in solvent. These levels represent the LOD levels of this method. The obtained retention times reflect expectations based on the molecular shapes and polarity of these compounds (Figure 1). For example, the retention time of 2-NP−AMOZ was found to be 4.15 min, whereas 2-NP− AOZ exhibited a retention time of 6.03 min. The short retention time of 2-NP−AMOZ in comparison to the three other derivatized nitrofuran metabolites was attributed the presence of a morpholine ring in its structure. Figures 4B and 5B show the same chromatograms at the LOD level in the tilapia matrix. Matrix interferences were not observed, demonstrating the performance of the extraction method. For example, the area count of the 335 → 291 transition for 2-NP− AMOZ is 2.22 × 105 in the solvent and 2.26 × 105 in the matrix. As mentioned in the Materials and Methods, the LC method can be shortened to 10 min, for those desiring higher throughput. Retention times changed as such: 2-NP−AMOZ 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, chloramphenicol at 4.29 min, and florfenicol at 4.19 min. We, however, chose not to implement this shortened run as a result of our desire to expand this method to include other antibiotic and drug residues in the future. A shortened LC run would increase elution overlap with not only analytes but also interferences.

Figure 5. Extracted ions chromatograms of two product ions of chloramphenicol and florfenicol standards in solvent (A), in matrix (B), and of fortified (spiked) samples in tilapia (C), each at the LOD levels.

Checking Standards and Derivatization. It is our policy in the laboratory to check our standards before performing the analysis of any batch. This was performed by running a calibration curve, made up as previously described in the Materials and Methods. In comparison to the derivatized nitrofuran metabolites (2-NP-AMOZ, 2-NP-AOZ, 2-NP-AHD, and 2-NP-SC), it was impossible to directly analyze a calibration curve built to verify the quality of the commercially sourced underivatized nitrofurans, used for spiking, as a result of their low masses and high polarities. To check the integrity of the commercially sourced underivatized nitrofuran standards, a multi-point calibration curve was prepared, followed by in-vial derivatization in acidic conditions using an excess of 2-NBA. This curve was left in the oven at 37 °C overnight and run against another multi-point calibration curve made using the commercially sourced derivatized nitrofuran metabolites. Figure 6 shows the high degree of agreement between these in-house 5024


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Figure 6. Multi-point calibration curve of derivatized nitrofuran metabolite standards (red squares) and in-laboratory derivatized nitrofuran metabolites (blue circles).

Table 2. Table of Internal-Standard-Corrected Recoveries of AMOZ, AOZ, AHD, and SC in Tilapia and Shrimp AMOZ

AHD

335 → 291 Da fortification (ng/g)

recovery (%)

SD

AOZ

249 → 134 Da CV (%)

recovery (%)

0.25 1 5 10 overall

92 90 89 96 92

1 2 5 1 4

1.1 3.9 7.2 1.2 4.9

89 91 93 90 91

0.25 1 5 10 overall

86 91 94 93 93

2 1 3 1 4

3.1 1.9 3.8 1.9 2.8

58 73 87 93 84

SD

SC

236 → 134 Da CV (%)

Tilapia 3 3.6 1 1.7 12 15.5 3 3.9 6 8.4 Shrimp 7 15.4 2 2.7 8 11.8 3 3.9 15 12.4

209 → 192 Da

recovery (%)

SD

CV (%)

recovery (%)

SD

CV (%)

97 97 107 103 102

1 3 9 0 6

1.6 3.9 10.8 0.6 7.2

123 98 97 96 97

9 4 7 4 13

8.9 5.2 9.7 4.5 5.8

90 90 93 93 92

4 1 2 2 3

4.8 1.3 2.2 3.3 2.6

98 93 94 99 95

4 1 3 2 4

4.4 1.9 4.4 3.0 4.0

of the seafood matrix. On one hand, to compensate for the effects of the matrix suppression, matrix-matched calibration standard curves (in-matrix curves) were used to quantitate the analytes and obtain the absolute recovery, which is a measure of the mass of analyte lost during the extraction procedure (Tables 4 and 5). On the other hand, the availability of derivatized and underivatized isotopically labeled internal standards made it possible to correct the recoveries, accounting for physical loss and chemical degradation of analytes during the extraction process as well as ion suppression in the mass spectrometer (Tables 2 and 3). Interestingly, the effect of the matrix on the area counts for all analytes in the two studied matrices was found to be negligible as a result of the clean extraction method used. In

made calibration curves prepared from derivatized versus underivatized standards. This experiment allowed us not only to check the quality and stability of our standards but also to verify the yield of our derivatization step in the extraction. The efficiency of the derivatization process was >99%. Fortification and Recovery. Tables 2−5 present the validation results. The method was tested in triplicate to determine its accuracy and precision. All of the nitrofuran metabolites and phenicols were examined in two different seafood commodities (shrimp and tilapia) at four different concentrations (0.25, 1, 5, and 10 ng/g for nitrofurans and 0.1, 0.25, 0.5, and 1 ng/g for phenicols). Preliminary experiments indicated that the UHPLC−MS/ MS instrumental response varied with the absence or presence 5025


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Journal of Agricultural and Food Chemistry Table 3. Table of Internal-Standard-Corrected Recoveries of Chloramphenicol in Tilapia and Shrimp

fortification (ng/g)

chloramphenicol

chloramphenicol

florfenicol

321 → 152 Da

321 → 152 Da

356 → 185 Da

recovery (%)

SD

Tilapia 110 110 106 113 110 Shrimp 89 86 81 85 84

0.1 0.25 0.5 1 overall 0.1 0.25 0.5 1 overall

Table 5. Table of Absolute Recoveries of Chloramphenicol and Florfenicol Tilapia and Shrimp

CV (%)

15 1 3 3 8

16.2 1.1 3.4 2.9 3.4

0 2 4 3 4

0.6 2.4 6.1 3.8 4.4

other words, the area counts of analytes in-solvent or inextracted-matrix blanks were very similar. For all four nitrofuran residues, chloramphenicol, and florfenicol, the reproducibility errors [coefficient of variation (CV)] and standard deviations (SDs) were lower than 10% and the corrected recoveries with internal standards were between 92 and 102% for nitrofuran metabolites and between 85 and 110% for chloramphenicol in tilapia (Tables 2 and 3). An internal standard for florfenicol was not available. The average absolute recoveries using matrixmatched calibration curves without internal standards were 60% for AMOZ, 68% for AHD, 63% for AOZ, 51% for SC, 79% for chloramphenicol, and 87% for florfenicol in tilapia (Tables 4 and 5). Matrix-matched calibration curves were prepared by spiking the final extracts of seafood blanks with solutions of the commercially sourced derivatized nitrofurans and phenicols. Two product-ion transitions per analyte, along with the ion ratios, were used for data acquisition and analyte confirmation, resulting in a highly selective method. No interference was observed in the negative controls at the retention time of the analytes. The LOD for each analyte was defined to be 0.25 ng/g for AMOZ and AOZ, 1 ng/g for AHD and SC, and 0.1 ng/g for chloramphenicol and florfenicol. The LOQ was defined to be 1 ng/g for AMOZ, AOZ, AHD, and SC and 0.25 ng/g for chloramphenicol and florfenicol. The lower LODs for AMOZ


recovery (%)

0.1 0.25 0.5 1 overall

77 76 80 80 79

0.1 0.25 0.5 1 overall

73 70 69 72 70

SD

CV (%)

recovery (%)

SD

CV (%)

83 86 85 90 87

5 2 1 4 4

7.0 3.4 1.7 5.7 4.5

83 76 76 78 77

4 3 1 1 4

6.7 4.6 2.3 2.2 3.0

Tilapia 10 16.2 3 5.3 0 0 1 1.4 6 4.5 Shrimp 4 7.6 2 3.0 1 2.2 1 2.4 3 2.8

and AOZ were due to their much greater response in the LC− MS/MS analysis than AHD and SC. Method Validation and Analysis. The analytical method was validated by independently analyzing two different matrices of commercially available seafood: tilapia and shrimp. Each matrix was spiked in triplicate at four spike levels with underivatized nitrofuran metabolites and phenicols. A multipoint, in-solvent calibration curve with isotopically labeled internal standards was used to calculate the concentration in the spikes of the in situ derivatized nitrofuran metabolites as well as chloramphenicol and florfenicol. Calibrating standards ranged in concentration from 0.1 to 15 ng/g for the nitrofurans and from 0.025 to 1 ng/g for the phenicols. At least 5 data points were necessary for quantitation. Chromatographic peaks at each of these thresholds are depicted for nitrofurans and phenicols in Figures 4 and 5. The primary source of improvement in quantitation came from the fortification of each sample with an internal standard containing isotopically labeled forms of the underivatized nitrofuran metabolites and chloramphenicol. The internal standard allowed for the calculation of corrected recoveries by comparing the ratio of the analyte/internal standard responses in the sample to the ratio of the analyte/internal standard responses in the solvent curve, which afforded the

Table 4. Table of Absolute Recoveries of AMOZ, AOZ, AHD, and SC in Tilapia and Shrimp


AMOZ

AHD

AOZ

SC

335 → 291 Da

249 → 134 Da

236 → 134 Da

209 → 192 Da

recovery (%)

SD

CV (%)

recovery (%)

0.25 1 5 10 overall

61 58 60 63 60

1 5 4 1 4

2.5 9.6 8.6 1.8 7.5

65 66 68 70 68

0.25 1 5 10 overall

55 58 57 57 57

0 2 1 1 1

1.0 3.6 2.0 2.0 2.3

77 70 69 73 71

SD

CV (%)

Tilapia 4 8.4 2 4.7 4 6.7 1 1.4 4 5.0 Shrimp 3 5.3 7 11.4 7 11.8 3 5.6 6 8.9 5026

recovery (%)

SD

CV (%)

recovery (%)

SD

CV (%)

61 61 62 65 63

2 2 0 2 2

4.1 4.1 0 3.1 4.2

67 58 45 48 51

2 1 1 3 9

3.5 3 3.8 7.8 12.4

62 68 70 70 69

1 1 0 1 3

2.8 1.5 0.8 1.7 1.9

64 53 49 50 51

8 1 2 4 6

17.7 1.9 5 8.7 6.2


Article


Figure 7. Bar graph of z scores for quantitation of total AHD. Our laboratory is “lab 4”. This figure was reproduced with permission from Fapas Report 02334, Fera Science, Ltd. Copyright 2017.30

ability to correct for analyte lost during the extraction procedure. It is the policy of the laboratory to set LOD and LOQ values at levels greater than the minimum 3× and 10× general requirements, such that daily instrument variance, matrix effects, procedural errors, etc. do not result in failures to meet established and validated LODs and LOQs for studies taking place over extended periods of time. In our laboratory, we may set the LOD and LOQ values to levels greater than the minimum detected signal at 3× or 10× on the instrument based on the needs of the method and the desired ruggedness of the method. Instrument blanks were prepared using ammonium acetate with no sample preparation. Acceptance criteria for method spikes were evaluated using control charts. Recoveries should be within ±3 process sigma. For initial control charting purposes, the use of method validation data had to establish a suitable recovery range. Once a suitable population of spike recoveries was generated at the spike levels, these results were used to determine control limits and for evaluation of any trending in the data. Analyte identification required at least 2 product ions. The ion ratio must be within ±20% of the average of the reference standards in each analytical run. Standards that fail to meet method performance criteria, such as consistent instrument response (i.e., drift no greater than 30% relative difference between bracketed standards), are not included in this average. In other words, we assessed the ratios of the highest responding ion to an additional confirming ion and then determined the difference (if any) between the ratios for in-solvent and spiked analytes. Differences in ratios above 20% were deemed failing. The retention time of the sample peak must match the average reference standard peaks within ±0.25 min. Method performance characteristics are often influenced by time and fiscal constraints of the customer and the laboratory. Therefore, we do not try to set any criteria prior to completion of the validation for these types of projects. We do, however, have performance minimums that we consider when reviewing

these types of data packages. In general, we consider any recoveries inside of 50−150% for low and sub-parts per billion (ppb) level analysis acceptable, and we view any coefficient of variation less than 30% to be acceptable for our typical multilevel replicate analysis at these levels. These values represent our minimums, and methods often exceed these. For this method, these parameters were exceeded and the data are considered fit for the intended purpose by our laboratory, and the Florida Department of Agriculture and Consumer Services currently uses this method to test imported seafood samples. Quantitation of Nitrofuran Metabolites. Most reports on the quantitation of nitrofuran metabolites in the literature used the “calibrating samples” method for their calibration curves. This was performed as a result of the unavailability of commercially sourced, derivatized nitrofuran metabolites. Researchers would fortify five or six blank matrix samples with varying concentrations of standard solutions containing mixtures of underivatized nitrofuran metabolites. Then, these samples would be derivatized in situ using 2-NBA and HCl in an oven overnight and extracted using different liquid−liquid extraction or SPE methods. In this scenario, absolute recoveries cannot be accurately quantitated because the calibrating samples and spikes are made identically. Thus, a 100% recovery is misleading when the recovery of a spike is calculated using a sample that has been fortified and extracted identically. To overcome this problem and calculate the recovery of spikes with higher accuracy while maintaining precision, we purchased the underivatized nitrofuran metabolites and their isotopically labeled internal standards and used them for fortifications (spikes) only. These spikes were then quantitated with calibration curves, which were made using commercially sourced, derivatized nitrofuran metabolites and their isotopically labeled internal standards in-solvent. The commercially sourced, derivatized standards provide better quantitation and demonstrate whether the derivatization was complete or not. If the calculation, preparation, and derivatization of standards are performed 5027


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Figure 8. Bar graph of z scores for quantitation of total AOZ. Our laboratory is “lab 4”. This figure was reproduced with permission from Fapas Report 02334, Fera Science, Ltd. Copyright 2017.30

accurately, the multi-point calibration curve of the in situ derivatized nitrofuran metabolites should match that made using the commercially sourced derivatized metabolites. This correlation was observed using our method (Figure 6). Proficiency Test. To independently evaluate the ability of the method to extract, analyze, and quantitate analytes, we engaged in a Fapas-administered (ISO 17043 accreditation) proficiency test with 98 other laboratories.30 AHD and AOZ were the two nitrofurans analyzed. Their assigned values were set at 2.06 ng/g (AHD) and 2.21 ng/g (AOZ). Lab performance was described by Fapas through the calculation of z scores (Figures 7 and 8). z scores within a range of −2 ≤ z ≤ 2 indicate that the method is considered fit for purpose by our laboratory, in accordance with International Union of Pure and Applied Chemistry (IUPAC) guidelines.31 We obtained values of 2.5 ng/g (AHD) and 2.8 ng/g (AOZ) for z scores of 1.0 (AHD) and 1.2 (AOZ), demonstrating the ability of the method. As expected, our reported values were higher than those obtained by most other laboratories. This could be attributed to the accuracy of our quantitation method using the derivatized standards in-solvent curve and the fact that we did not use the conventional calibrating samples curve. As mentioned earlier in the manuscript, the calibrating samples curve used by other laboratories does not account for physicochemical loss of the analytes during the extraction. This may lead to an under-estimation of the analytes concentration that should be found. Regulatory Samples. To evaluate the applicability of the validated method, 102 composite samples of a variety of imported seafood, including shrimp, tilapia, frog legs, salmon, and swai fish, were analyzed. Composites were made from different lots of the same commodity to provide a more representative sample. The analyzed samples were imported, frozen, from non-EU countries. They were collected from different grocery stores and warehouses throughout Florida. Internal quality controls, including an in-solvent calibration curve, a reagent blank, a matrix blank, and several spiked blank

samples, were evaluated with the aim of checking the quality of the results. The reagent and matrix blanks were obtained by weighing 2 g of water and seafood samples, with the objective of eliminating possible false positives as a result of contamination in the instrument or solvent used. Spiked samples at two levels of fortification (1 and 10 ng/g for nitrofurans and 0.25 and 1 ng/g for phenicols) were used to monitor the extraction efficiency. Among the 102 analyzed composite samples, 4 samples tested were positive for nitrofuran metabolites and chloramphenicols. A swai fish sample tested positive for the SC nitrofuran metabolite at 2.5 ng/g; a shrimp sample was contaminated with chloramphenicol at 0.44 ng/g; a sample of frog legs tested positive for AOZ nitrofuran metabolite at 2.7 ng/g; and another sample of frog legs was found contaminated with AOZ at 3 ng/g and chloramphenicol at 17 ng/g. This last sample was heavily contaminated with chloramphenicol, requiring a 20-fold dilution followed by a standard addition for accurate quantitation. Two product-ion transitions per analyte, along with ion ratios, were used for data acquisition and analyte confirmation of these violations. The ion ratios were calculated by dividing the area counts of two product ions of the analyte, in-solvent (standard) and in-sample, and multiplying this quotient by 100 to obtain the ion ratio as a percentage. The percentage difference between ion ratios instandard and in-sample is then calculated and must be lower than 20% for a sample to be confirmed. The contaminated samples were also re-extracted to confirm the presence of a violation. For example, the swai fish sample was extracted 4 different times in 4 separate batches. These 4 batches were analyzed, one per week, over 4 weeks. The SC concentration was found to be 2.56, 2.58, 2.31, and 2.42 ng/g for the 4 analyzed swai fish samples. These 4 samples were weighed from 4 different homogenized sub-samples (multiple cups of the same sample), scheduled in 4 batches and extracted by multiple chemists. These findings show the precision and accuracy of the homogenization process, validated extraction 5028


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randomized controlled trials. J. Antimicrob. Chemother. 2015, 70, 979− 996. (3) Murugasu-Oei, B.; Dick, T. Bactericidal activity of nitrofurans against growing and dormant Mycobacterium bovis BCG. J. Antimicrob. Chemother. 2000, 46, 917−919. (4) Tangallapally, R. P.; Yendapally, R.; Daniels, A. J.; Lee, R. E.; Lee, R. E. Nitrofurans as novel anti-tuberculosis agents: Identification, development and evaluation. Curr. Top. Med. Chem. 2007, 7, 509−526. (5) Defoirdt, T.; Boon, N.; Sorgeloos, P.; Verstraete, W.; Bossier, P. Alternatives to antibiotics to control bacterial infections: Luminescent vibriosis in aquaculture as an example. Trends Biotechnol. 2007, 25, 472−479. (6) Aitken, S. L.; Dilworth, T. J.; Heil, E. L.; Nailor, M. D. Agricultural Applications for Antimicrobials. A Danger to Human Health: An Official Position Statement of the Society of Infectious Diseases Pharmacists. Pharmacotherapy 2016, 36, 422−432. (7) Kemper, N. Veterinary antibiotics in the aquatic and terrestrial environment. Ecol. Indic. 2008, 8, 1−13. (8) U.S. Government Publishing Office (GPO). Topical Use of Nitrofurans Banned by FDA; GPO: Washington, D.C., 2017; https:// www.gpo.gov/fdsys/browse/collection.action?collectionCode= FR&browsePath=2002. (9) Verdon, E.; Hurtaud-Pessel, D.; Sanders, P. Evaluation of the limit of performance of an analytical method based on a statistical calculation of its critical concentrations according to ISO standard 11843: Application to routine control of banned veterinary drug residues in food according to European Decision 657/2002/EC. Accredit. Qual. Assur. 2006, 11, 58−62. (10) Antunes, P.; Machado, J.; Peixe, L. Illegal use of nitrofurans in food animals: Contribution to human salmonellosis? Clin. Microbiol. Infect. 2006, 12, 1047−1049. (11) Cunha, B. A. ANTIBIOTIC SIDE EFFECTS. Med. Clin. North Am. 2001, 85, 149−185. (12) Fraunfelder, F. T.; Bagby, G. C.; Kelly, D. J. Fatal Aplastic Anemia Following Topical Administration of Ophthalmic Chloramphenicol. Am. J. Ophthalmol. 1982, 93, 356−360. (13) Ahmadizadeh, M.; Esmailpoor, M.; Goodarzi, Z. Effect of Phenobarbital on Chloramphonicol-Induced Toxicity in Rat Liver and Small Intestine. Iran. J. Basic Med. Sci. 2013, 16, 1282−1285. (14) Hoogenboom, L. A.; Berghmans, M. C.; Polman, T. H.; Parker, R.; Shaw, I. C. Depletion of protein-bound furazolidone metabolites containing the 3-amino-2-oxazolidinone side-chain from liver, kidney and muscle tissues from pigs. Food Addit. Contam. 1992, 9, 623−630. (15) Radovnikovic, A.; Moloney, M.; Byrne, P.; Danaher, M. Detection of banned nitrofuran metabolites in animal plasma samples using UHPLC−MS/MS. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2011, 879, 159−166. (16) Hoogenboom, L. A. P.; van Bruchem, G. D.; Sonne, K.; Enninga, I. C.; van Rhijn, J. A.; Heskamp, H.; Huveneers-Oorsprong, M. B. M.; van der Hoeven, J. C. M.; Kuiper, H. A. Absorption of a mutagenic metabolite released from protein-bound residues of furazolidone. Environ. Toxicol. Pharmacol. 2002, 11, 273−287. (17) Verdon, E.; Couedor, P.; Sanders, P. Multi-residue monitoring for the simultaneous determination of five nitrofurans (furazolidone, furaltadone, nitrofurazone, nitrofurantoine, nifursol) in poultry muscle tissue through the detection of their five major metabolites (AOZ, AMOZ, SEM, AHD, DNSAH) by liquid chromatography coupled to electrospray tandem mass spectrometry-in-house validation in line with Commission Decision 657/2002/EC. Anal. Chim. Acta 2007, 586, 336−47. (18) Kaufmann, A.; Butcher, P.; Maden, K.; Walker, S.; Widmer, M. Determination of nitrofuran and chloramphenicol residues by high resolution mass spectrometry versus tandem quadrupole mass spectrometry. Anal. Chim. Acta 2015, 862, 41−52. (19) Park, M. S.; Kim, K. T.; Kang, J. S. Development of an analytical method for detecting nitrofurans in bee pollen by liquid chromatography−electrospray ionization tandem mass spectrometry. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2017, 1046, 172−176.

method, and quantitation. Our laboratory conducted further investigation into one of the violative samples of frog legs. This composite sample was initially made from 20 lots of frog legs collected from a warehouse. Our analysis revealed that 17 of the 20 samples had been heavily contaminated with (0.1−19 ng/g) AOZ and chloramphenicol (0.8−140 ng/g). In conclusion, a UHPLC−MS/MS method for the detection, quantitation, and confirmation of nitrofurans and phenicols in several seafood matrices was developed and validated for use in analyzing regulatory samples. This method produces improvements in accuracy on those previously reported in the literature, with a derivatization yield of >99%. While underivatized nitrofuran metabolites and their isotopically labeled internal standards were used for fortifications (spikes), commercially sourced derivatized nitrofuran metabolites and their isotopically labeled internal standards were used for building an in-solvent calibration curve, which is the key improvement that led to the improved accuracy that we report. For nitrofurans, chloramphenicol, and florfenicol, the reproducibility errors (CV) and SDs were lower than 10% and the recoveries corrected with internal standards were between 90 and 100% for nitrofuran metabolites and between 85 and 110% for chloramphenicol. The absolute recovery, calculated in a matrix-matched calibration curve, was found to be between 60 and 70% for the nitrofuran metabolites and between 70 and 80% for the phenicols. Finally, this validated method was successfully used to analyze and identify violations in imported seafood samples, which unequivocally demonstrates the ability of the method to extract and quantify varying levels of incurred residues.

■

AUTHOR INFORMATION

Corresponding Author

*Telephone: +1-804-335-3119. E-mail: fadialdeek@eurofinsus. com. ORCID

Fadi Aldeek: 0000-0002-1652-3952 Present Address

† Fadi Aldeek: Eurofins Lancaster Laboratories Professional Scientific Services (PSS) Insourcing Solutions, 601 East Jackson Street, Richmond, Virginia 23219, United States.

Funding

The authors thank the Food Emergency Response Network for financial support. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Rebecca Kitlica, Carion Haynes, and Amy Brown from the Department of Agriculture and Consumer Services (FDACS), Tallahassee, FL, for their assistance with standards and sample preparation and homogenization. The authors also thank Jo-Marie Cook, Matthew Standland, and Mark Crosswhite for many helpful discussions.

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REFERENCES

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