Nitrofuran Metabolites Research Articles

New Method of Analysis of Nitrofurans and Nitrofuran Metabolites in Different Biological Matrices Using UHPLC-MS/MS.

IntroductionThe major difficulty in analysis of nitrofurans in feed, feed water, and food of animal origin is that nitrofurans have low molecular weights and fast metabolism. The principal goal of this study was to prepare a procedure for the determination of nitrofurans and their metabolites by a single method in different types of feed, feed water, and food of animal origin.Material and MethodsTwo-gram samples were subjected to hydrolysis and derivatisation processes by addition of hydrochloric acid and 2-nitrobenzaldehyde. After incubation the sample was purified by solid phase extraction technique. Nitrofurans were analysed using ultra-high-pressure liquid chromatography-MS/MS (UHPLC-MS/MS).ResultsThe results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC regarding apparent recoveries (88.9%–107.3%), repeatability (2.9%–9.4%) and within-laboratory reproducibility (4.4%–10.7%).ConclusionThe method can be successfully applied to monitor nitrofurans and their metabolites in different matrices.

Quantitation and Confirmation of Chloramphenicol, Florfenicol, and Nitrofuran Metabolites in Honey Using LC-MS/MS.

This paper describes a rapid and robust method utilizing a single liquid-liquid extraction for the quantitation and confirmation of chloramphenicol, florfenicol, and nitrofuran metabolites in honey. This methodology combines two previous extraction methods into a single extraction procedure and utilizes matrix-matched calibration standards and stable isotopically labeled standards to improve quantitation. The combined extraction procedure reduces the average extraction time by >50% when compared with previously used procedures. The drug residues were determined using two separate LC-tandem MS conditions. Validation of all the analytes was performed, with average quantitation ranging from 92 to 105% for all analytes and the RSDs for all analytes being ≤12%.

Comparative Characteristics of Physical, Chemical, and Biochemical Methods for Determining Nitrofuran Metabolites in Food

Background. Nitrofurans belong to a class of synthetic antibiotics, which are widely used in various livestock sectors due to their high effectiveness in control over infectious diseases and as a growth activator. Nitrofurans are rapidly metabolized compounds, but the metabolites of these drugs are able to bind to protein structures and accumulate in the body of animals. Nitrofuran metabolites show carcinogenic properties, therefore it is necessary to control their content in food products of animal origin. Objective. The aim of the paper is to summarise modern methods for nitrofurans analysis. Methods. The modern methods of sample preparation of food products of animal origin for extracting nitrofuran metabolites and methods for detecting these analytes are systematized and analyzed. Results. Modern analysis methods have a sufficient sensitivity level of nitrofurans determination in the form of final products – furaltadone and furazolidone metabolites. There are several methods of detection, among which are chro­matographic methods and enzyme immunoassay. Conclusions. Analysis of the literature has shown that use of combined techniques combining selective removal of nitrofuran metabolites from the sample matrix and their quantification, allows screening and confirmatory test for the presence of nitrofurans in food and also allows detecting analytes at 1 mg/kg.

Розробка та оцінка придатності методу визначення нітрофуранів в меді за допомогою рідинної хроматографії високого тиску – тандемної мас-спектрометрії (UPLC-MS-MS)

<p><span lang="UK">Over the past decade, Ukraine has been one of the leaders in exporting honey to EU countries. The main obstacle to increasing the export of Ukrainian honey to EU countries is the discrepancy of honey safety indicators with the requirements of importing countries. This is due to the use of a significant number of drugs with antimicrobial spectrum of action in the treatment and prevention of diseases of bees, the remains of which fall into honey. In domestic honey, according to recent data, the remains of such groups of antibiotics and antimicrobial agents as chloramphenicol, nitrofuran, nitroimidazole, sulfanilamides, tetracyclines and aminoglycosides are most commonly found.</span><span lang="EN-US">The nitrofurans, which are quite stable, can be stored in honey for a long time and are not destroyed even at high temperatures. Therefore, the urgent question remains the development and introduction into practice of laboratory analysis of a sensitive and reliable method for determining the residual amounts of nitrofurans in honey.The method developed by us allows us to determine the residual amounts of metabolites of nitrofurans in honey, namely: furazolidone derivative - 3-amino-2-oxazolidinone (AOZ), furaltadone-3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), nitrofurase-semicarbazide SEM) and nitrofurantoin-1-aminohydandomine (AHD).The use of drugs nitrofuran number in the treatment and prevention of infectious diseases of bees involves the receipt of their metabolites in honey in the human body.The conducted studies revealed that nitrofurantoin (38% of honey samples) was used most often in beekeeping, followed by fureladone (24%), while nitrofurase and furazolidone were used equally in 19% of honey samples, respectively.The conducted studies revealed 4 metabolites of nitrofurans in natural honey, namely the metabolite furazolidone 3-amino-2-oxazolidinone (AOZ), nitrofurase-semicarbazide (SEM), furaltadone-3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), and nitrofurantoin - 1-aminohydandomine (AHD).The content of 3-amino-2-oxazolidinone (AOZ) and semicarbazide (SEM) in honey exceeds the MDR by the norms of Ukraine. According to EU norms, the content of 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and 1-aminohydinotin (AHD) in honey exceeds MDR and the semicarbazide content (SEM) permissible concentration.</span></p>

Comparative Characteristics of Physical, Chemical, and Biochemical Methods for Determining Nitrofuran Metabolites in Food

Background. Nitrofurans belong to a class of synthetic antibiotics, which are widely used in various livestock sectors due to their high effectiveness in control over infectious diseases and as a growth activator. Nitrofurans are rapidly metabolized compounds, but the metabolites of these drugs are able to bind to protein structures and accumulate in the body of animals. Nitrofuran metabolites show carcinogenic properties, therefore it is necessary to control their content in food products of animal origin.Objective. The aim of the paper is to summarise modern methods for nitrofurans analysis.Methods. The modern methods of sample preparation of food products of animal origin for extracting nitrofuran metabolites and methods for detecting these analytes are systematized and analyzed.Results. Modern analysis methods have a sufficient sensitivity level of nitrofurans determination in the form of final products – furaltadone and furazolidone metabolites. There are several methods of detection, among which are chro­matographic methods and enzyme immunoassay.Conclusions. Analysis of the literature has shown that use of combined techniques combining selective removal of nitrofuran metabolites from the sample matrix and their quantification, allows screening and confirmatory test for the presence of nitrofurans in food and also allows detecting analytes at 1 mg/kg.

Simultaneous detection of four nitrofuran metabolites in honey by using a visualized microarray screen assay.

A visualized microarray sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics. Using a multiplexed approach, metabolites of four main nitrofuran antibiotics can be detected simultaneously. Individual antigens were spotted onto 96-well plates. An indirective competitive assay format, with visualized signal response, was employed. An extraction method, based on derivatization with 2-nitrobenzaldehyde and partition into ethyl acetate, was used for screening. The limits of detection were 0.10, 0.04, 0.04, and 0.10ngg-1 for 3-amino-5-morpholino-2-oxazolidone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), and 1-aminohydantoin (AHD), respectively. The recovery rate ranged from 78% to 93% for the four targets. In addition, this method was easy to operate with low detection cost and fast speed. This microarray method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety monitoring.

Occurrence and levels of nitrofuran metabolites in sea cucumber from Dalian, China

ABSTRACTThe occurrence and levels of nitrofuran metabolites (NFMs) in sea cucumber (SC) from Dalian, China, are reported. Four metabolites including 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM) and 1-aminohydantoin (AHD) in different SC products (fresh, instant and dry salted SCs) were measured. The frequency of occurrence for NFMs in all SC samples was 42.7%. The total NFM concentrations ranged from non-detectable to 64.6 ng g–1, with a mean of 3.59 ng g–1. AOZ and SEM were the dominant congeners, accounting for 40.1% and 59.1% of the total NFMs, respectively. The concentrations and patterns varied among different regions. Higher levels of NFMs were found in the fresh SC products, and the order for the average concentration of ∑4NFM was fresh > dry salted > instant.

Analysis of banned veterinary drugs and herbicide residues in shellfish by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and gas chromatography-tandem mass spectrometry (GC/MS/MS)

Seafood safety is a crucial public health concern for consumers. In this study, we applied a validated method to analyze the residue of banned veterinary drugs in shellfish, namely chloramphenicol, malachite green, leucomalachite green, and nitrofuran metabolites; additionally, the QuEChERS method was employed to detect 76 herbicides by LC/MS/MS and GC/MS/MS. In total, 42 shellfish samples, which included hard clams, freshwater clams, and oysters, were collected from aquafarms and production areas in Taiwan during 2012. Our results revealed 3.8ng/g of chloramphenicol in one hard clam, 19.9–32.1ng/g of ametryn in two hard clams, 16.1–60.1ng/g of pendimethalin in four hard clams, and 17.0ng/g of mefenacet in one oyster, indicating that 19.1% of the samples contained residues from banned veterinary drugs and pesticides. These data can be used to monitor the residue of veterinary drugs and pesticides in aquatic organisms and as a reference for food safety.

Multi-class method for the determination of nitroimidazoles, nitrofurans, and chloramphenicol in chicken muscle and egg by dispersive-solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry

This study describes the development of a multiresidue method for the efficient identification and quantification of nitroimidazoles, nitrofurans, and chloramphenicol in chicken and egg. After derivatization of nitrofuran metabolites, dispersive-solid phase extraction was used for the extraction of target analytes. An optimization strategy involved the selection of sorbents and extraction solutions for dispersive-solid phase extraction in order to achieve acceptably high recoveries and reduce co-extractives in the final extracts. Analytes were determined by ultra-high performance liquid chromatography-tandem mass spectrometry, in one single injection with a chromatographic run time of 7.5min. Mean recoveries ranged from 86.4% to 116.7% and interday precision was lower than 18%. The limits of quantification were between 0.1 and 0.5μg/kg, which were satisfactory to support surveillance monitoring. Finally, the method was applied to real samples, and metabolite of furazolidone, metronidazole and its metabolite, dimetridazole and its metabolite were detected in both chicken and egg samples.

Lateral flow biosensor for multiplex detection of nitrofuran metabolites based on functionalized magnetic beads.

The use of potential mutagenic nitrofuran antibiotic in food animal production has been banned world-wide. Common methods for nitrofuran detection involve complex extraction procedures. In the present study, magnetic beads functionalized with antibody against nitrofuran derivative were used as both the extraction and color developing media in lateral flow biosensor. Derivatization reagent carboxybenzaldehyde is firstly modified with ractopamine. After reaction with nitrofuran metabolites, the resultant molecule has two functional groups: the metabolite moiety and the ractopamine moiety. Metabolite moiety is captured by the antibody that iscoated on magnetic beads. This duplex is then loaded onto biosensor and ractopamine moiety is further captured by the antibody immobilized on the test zone of nitrocellulose membrane. Without tedious organic reagent-based extraction procedure, this biosensor was capable of visually detecting four metabolites simultaneously with a detection limit of 0.1μg/L. No cross-reactivity was observed in the presence of 50μg/L interferential components. Graphical abstract Derivatization of nitrofuran metabolites (AHD, AOZ, SEM, or AMOZ) and LFA detection of the derivative products.

Validation of a Rapid Quantitative Method for the Residues of Nitrofuran Metabolites in Loach by Accelerated Solvent Extraction and HPLC Triple Quadrupole Mass Spectrometry

Validation of a Rapid Quantitative Method for the Residues of Nitrofuran Metabolites in Loach by Accelerated Solvent Extraction and HPLC Triple Quadrupole Mass Spectrometry

Development and Validation of Confirmatory Method for Analysis of Nitrofuran Metabolites in Milk, Honey, Poultry Meat and Fish by Liquid Chromatography-Mass Spectrometry

Abstract In this study we have devoloped and validated a confirmatory analysis method for nitrofuran metabolites, which is in accordance with European Commission Decision 2002/657/EC requirements. Nitrofuran metabolites in honey, milk, poultry meat and fish samples were acidic hydrolised followed by derivatisation with nitrobenzaldehyde and liquid-liquid extracted with ethylacetate. The quantitative and confirmative determination of nitrofuran metbolites was performed by liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) in the positive ion mode. In-house method validation was performed and reported data of validation (specificity, linearity, recovery, CCα and CCβ). The advantage of this method is that it avoids the use of clean-up by Solid-Phase Extraction (SPE). Furthermore, low levels of nitrofuran metabolites are detectable and quantitatively confirmed at a rapid rate in all samples.

Determination of nitrofuran metabolites in shrimp muscle by liquid chromatography-photo diode array detection

The use of nitrofurans on any animal in the European Union (EU) and any animal or animal food products intended for export to the EU was banned in 1993 (except furazolidone which was banned in 1995) due to the carcinogenicity of the parent drugs and their metabolites. Thereafter, the screening of food of animal origin for nitrofurans and their metabolites became mandatory for all exports to the EU. This paper describes a High Performance Liquid Chromatography – Diode Array Detection (HPLC-DAD) method to detect tissue bound nitrofuran metabolites, namely 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholino-methyl-1,3-oxazolidinone (AMOZ), semicarbazide (SEM) and 1-aminohydantoin (AHD). The bound residues were hydrolysed and derivatized into the corresponding nitrophenyl derivatives (NPAOZ, NPAMOZ, NPSEM and NPAHD) with 2-nitrobenzaldehyde and analysed by HPLC-UV at 275 nm. The linearity of the photometric detector response to the four derivatized nitrofuran metabolites was verified using matrix-matched calibration standards in the range of 1–20 μg/kg and the average correlation coefficients for NPAOZ, NPAMOZ, NPSEM and NPAHD were 0.9960, 0.9951, 0.9984 and 0.9993, respectively. The decision limit of the method was below the Minimum Required Performance Limit (MRPL) of 1 μg/kg, for all four nitrofuran metabolites, which makes it compatible with the EU requirements. The average recoveries for (fortified samples between 1 and 5 μg/kg) AOZ, AMOZ, SEM and AHD were 107%, 107%, 115% and 114%, respectively. This is the first report of a HPLC-DAD method detecting nitrofuran metabolites below the MRPL according to our understanding. This method has been successfully used with aquaculture and poultry products; however, currently it is validated according to EU guidelines only for shrimp muscle tissue.

Determination of nitrofurans metabolites residues in aquatic products by ultra-performance liquid chromatography–tandem mass spectrometry

A method was described for monitoring four nitrofuran metabolites including 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ), 3-amino-2-oxazolidone (AOZ), 1-amino-hydantoin (AHD) and semicarbazide (SEM) in aquatic products. The analytes were quantified by ultra-performance liquid chromatography–tandem mass spectrometry operating in positive ion multiple monitoring mode (MRM) after 2-nitrobenzaldehyde derivatization. The matrix calibration curve was established with correlation coefficient (R2>0.99) in the range of 1–100ngml−1. Limit of detection and limit of quantification for all analytes were 0.5 and 1.5μgkg−1, respectively. Recovery rates and relative standard deviations ranged from 88% to 112% and 2% to 4%, respectively. The validated method was successfully applied to detect nitrofuran metabolites in 120 fish samples. The analytes were detected in 6/16 species of fish samples, with a total detection rate of 6.5%. AOZ was most frequently detected (8.3%), followed by AMOZ (7.5%), AHD (5.0%) and SEM (5.0%). The method is proposed for monitoring nitrofuran metabolites in aquatic products.

Development and validation of a modified QuEChERS protocol coupled to LC–MS/MS for simultaneous determination of multi-class antibiotic residues in honey

LC–MS/MS assay was developed and validated according to EU guidelines for determination of nitrofuran metabolites and nitroimidazole residues in honey. Crude samples were acid-treated to liberate matrix-bound residues and a modified QuEChERS protocol was employed. Nitrofurantoin, furazolidone, furaltadone and nitrofurazone were determined via analysis of their metabolites AHD, AOZ, AMOZ and SEM, respectively while nitroimidazole residues; ronidazole (RNZ) and dimetridazole (DMZ) were determined directly. For all analytes, neat standard calibration curves, after correction for matrix effect were successfully employed. Decision limit (CCα) and detection capability (CCβ) were below the MRPL for nitrofurans (1.00μgkg−1) and the recommended concentration for nitroimidazole (3.00μgkg−1), respectively. The CCα, CCβ, percentage recovery and CV% ranges were 0.12–0.74μgkg−1, 0.21–1.27μgkg−1, 90.96–104.80% and 2.65–12.58%, respectively. This work is part of the national initiative for establishing a national monitoring program for drug residues in Egyptian honey.

Preparation and characterisation of certified reference materials for furazolidone and nitrofurazone metabolites in prawn

Nitrofuran residues in food, particularly intensively farmed prawns, were identified as an important trade and health issue. No certified reference materials were available to allow laboratories to demonstrate satisfactory performance of their analytical methods. Freeze-dried prawn reference materials MX012A and MX012B were prepared at the National Measurement Institute, Australia, from blank and contaminated prawn tissue. A high-accuracy, exact-matching isotope dilution mass spectrometry method was developed for the analysis of two nitrofuran metabolites in the prawn materials. Residues were extracted by acid hydrolysis and derivatisation with 2-nitrobenzaldehyde, extracts were purified by liquid–liquid extraction followed by solid-phase extraction, and instrumental analysis was by ultra-high-pressure liquid chromatography with electrospray tandem mass spectrometry (UHPLC-ESI-MSMS). The method was used to certify the reference materials for mass fraction of 3-amino-2-oxazolidinone (AOZ) and semicarbazide (SEM), including the preparation of detailed measurement uncertainty budgets. MX012A contains AOZ at (137.5 ± 8.5) ng/g of freeze-dried material, and MX012B contains AOZ at (30.2 ± 1.8) ng/g and SEM (fortified) at (70.3 ± 3.1) ng/g. When reconstituted as directed on the certificate, the materials contain AOZ at (20.6 ± 1.3) ng/g and (4.53 ± 0.27) ng/g (MX012A and MX012B, respectively) and SEM at (10.5 ± 0.5) ng/g (MX012B). The individual components contributing to the measurement uncertainty estimates were the mass fractions assigned to calibration standards, gravimetric mass measurements, precision of the analytical method, reproducibility, long-term storage stability of the material at −20 °C, stability of the material during transportation, and potential bias due to extraction efficiency of the incurred analyte and matrix interference.

Quantitative Analysis of Nitrofuran Metabolites and Chloramphenicol in Shrimp Using Acetonitrile Extraction and Liquid Chromatograph-Tandem Mass Spectrometric Detection: A Single Laboratory Validation.

A method was developed and validated for the simultaneous analysis of chloramphenicol and nitrofuran metabolites in shrimp according to the guideline established by the U.S. Food and Drug Administration Office of Foods and Veterinary Medicine. The extraction steps following the overnight hydrolysis and derivatization are simpler than the conventional ethyl acetate extraction method. The main steps are neutralization of hydrolysates, addition of acetonitrile for extraction, and salting out of organic phase from the acetonitrile-aqueous mixture. Extracts are analyzed for chloramphenicol and nitrofuran metabolites by LC-MS/MS in a single injection with polarity switching between the positive electrospray ionization (ESI) mode for the nitrofurans and the negative ESI mode for chloramphenicol. Recoveries calculated using an extracted matrix calibration curve and labeled internal standards for chloramphenicol and nitrofurans ranged from 98.6 to 109.2% with RSDs less than 18%. This method that combines the analysis of chloramphenicol with the nitrofurans was shown to generate analytical results similar to those obtained using the individual drug-class analytical methods currently used for the analysis of chloramphenicol or nitrofurans in shrimp.

Simultaneous Determination of Nitrofuran Metabolites and Chloramphenicol in Shrimp with a Single Extraction and LC-MS/MS Analysis.

A method has been developed to quantify the nitrofuran metabolites 3-amino-5-morphorinomethyl-1,3-oxazolidinone, 3-amino-oxazolidinone, 1-aminohydantoin, and semicarbazide, as well as chloramphenicol in shrimp with a single extraction procedure followed by LC-MS/MS analysis. Dynamic selected reaction monitoring with positive and negative ionization mode switching was used. The method LODs were 0.5 ng/g for the nitrofuran metabolites and 0.3 ng/g for chloramphenicol.

Quantitation of Chloramphenicol and Nitrofuran Metabolites in Aquaculture Products Using Microwave-Assisted Derivatization, Automated SPE, and LC-MS/MS.

This paper describes a rapid and robust method utilizing microwave-assisted derivatization, automated SPE, and LC-MS/MS for the quantitation and confirmation of chloramphenicol (CAP) and nitrofuran metabolites in various aquaculture matrixes. The use of equipment presented in this work allowed extractions to be completed on average within 6 h, with quantitation accuracy ranging from 89 to 107% and RSD≤8.3%. The demonstrated detection limits for all the nitrofuran metabolites of interest in three different matrixes were ≤0.06 ng/g, with a quantitation limit of ≤0.2 ng/g. Additionally, the method exhibited a CAP detection limit for all matrixes≤0.01 ng/g and an LOQ of ≤0.03 ng/g.

Fortified versus incurred residues: extraction of furazolidinone metabolite from prawn.

Most methods reported in the literature for determination of nitrofuran metabolites use the same extraction and derivatisation conditions to hydrolyse and extract the protein-bound residues from animal tissue. While undertaking certification of reference materials for nitrofuran metabolites in freeze-dried prawn, it was found that these conditions are satisfactory for recovery of spiked residues; however, extraction efficiencies of incurred furazolidinone could be substantially increased by further optimisation of the extraction conditions. The availability of a suitable certified reference material allows laboratories to ensure that their method is optimised for incurred residues.