Determination Of Nitrofuran Metabolites Research Articles

Determination of nitrofuran metabolites and nifurpirinol in animal tissues and eggs by ultra-high performance liquid chromatography-tandem mass spectrometry validated according to Regulation (EU) 2021/808

In this work, an analytical method for the determination of eight non-allowed nitrofurans, including nifurpirinol and the metabolites of furazolidone, furaltadone, nitrofurantoin, nitrofurazone, nifursol, nitrovin, and nifuroxazide in animal tissues, including muscle (poultry, bovine, ovine, porcine, rabbit, and fish), kidney (bovine, ovine, porcine), and eggs, has been developed and validated according to Regulation (EU) 2021/808. The method was based on derivatization with 2-nitrobenzaldehyde in acid medium, followed by vortex-assisted liquid-liquid extraction and solid phase extraction for sample purification prior to ultra-high performance liquid chromatography-tandem mass spectrometry. Under selected conditions, the method was validated showing satisfactory relative matrix effects (CV ≤ 20 %), linearity (R2 ≥ 0.98), trueness (≤20 %, expressed as bias), accuracy (83–120 %), repeatability (1.7–19.9 %), reproducibility (1.9–25.7 %), specificity (blank signal ≤30 % at the LCL), and ruggedness. The decision limit for confirmation (CCα) for the target analytes ranged from 0.27 to 0.35 μg kg−1, all below the current reference point for action (RPA) of 0.5 μg kg−1 for the studied compounds. This validated method is currently accredited according to UNE-EN ISO/IEC 17025 by the Spanish National Accreditation Body (ENAC) to be implemented for official control analyses in the Public Health Laboratory of Valencia (Spain).

Determination of nitrofuran metabolites in muscle by LC-MS/MS and LC-MS/MS/MS: Alternative validation according to Commission Implementing Regulation (EU) 2021/808

Nitrofurans are banned in food of animal origin in the European Union. A rugged liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and LC-MS/MS cubed (LC-MS/MS/MS) analytical method allowing the confirmation of five metabolites of nitrofuran antibacterial agents, furaltadone, furazolidone, nifursol, nitrofurantoin and nitrofurazone in animal muscle tissue was developed and validated. The validation of 1-aminohydantoin (AHD), 3-amino-5-methyl-morpholino-2-oxazolidinone (AMOZ), 3-amino-2-oxazolidinone (AOZ), 3,5-dinitrosalicylic acid hydrazide (DNSH) and semicarbazide (SEM) was carried out according to Commission Implementing Regulation (EU) 2021/808, by the adoption of a matrix-comprehensive in-house validation concept and the standard addition calibration. Validation was successfully performed, starting from 0.4 µg kg−1. Decision limit (CCα), recovery, repeatability, within-laboratory reproducibility and uncertainty of measurement were determined together with a factorial effect analysis. The procedure was verified through the analysis of two certified reference materials. The sample preparation consisted in acid hydrolysis and derivatization steps, followed by a liquid-liquid extraction of the analytes with ethylacetate.

Rapid multi-residue LC-MS/MS determination of nitrofuran metabolites, nitroimidazoles, amphenicols, and quinolones in honey with ultrasonic-assisted derivatization – magnetic solid-phase extraction

A rapid multi-residue LC-MS/MS method for the identification and determination of banned veterinary drugs in honey was developed. A total of 31 investigated veterinary drugs belonging to 4 classes including nitrofurans metabolites, nitroimidazoles, amphenicols, and quinolones were quantified by LC-MS/MS with ESI using one single injection. The sample preparation included treatment with 5-nitro-2-furaldehyde (5-NFA) in a thermostated ultrasonic bath (80 °C, 0.5М НСl, 20 min) to liberate matrix-bound residues of nitrofurans. Magnetic hypercrosslinked polystyrene (HCP/Fe3O4) was proposed for the solid-phase extraction and clean-up of target analytes prior to LC-MS/MS analysis. To evaluate and validate the performance of method, the criteria of the Decision (EC) no 2002/657 were applied. The LOQs of the examined analytes range from 0.3 to 1 μg kg−1, which indicates good sensitivity to quantify the target compounds in honey. The recoveries of veterinary drugs from 1 g of honey with 50 mg of the sorbent are 97–109% for nitrofuran metabolites, 84–115% for nitroimidazoles, 86–103% for amphenicols, and 97–118% for quinolones. The relative standard deviations of intra-day and inter-day precision analyses (RSD) are less than 16%. This methodology was applied to real honey samples and trace levels of some veterinary drugs were detected.

Hydrophobic carboxymethyl cellulose as a clean-up sorbent in the determination of nitrofuran metabolites in animal-fat samples

Hydrophobic-CMC biopolymers were fabricated for the removal of fat from food sample matrices. The CMCs were synthesized via the esterification of CMC with three alcohols with carbon chains of different lengths, in the presence of sulfuric acid.

Determination of nitrofuran metabolites in meat products by UHPLC-fluorescence with ultrasonic-assisted derivatization

A rapid and sensitive analytical method has been proposed to monitor simultaneously four nitrofuran metabolites (NFMs) in meat products by ultra-high performance liquid chromatography–fluorescence detection (UHPLC-FLD). These NFMs were derivatized by 4-(N,N diphenylamino)benzaldehyde (DABA) by thermostatic ultrasonication within 20 min. The results confirmed that the four NFMs derivatives had excellent fluorescence response at specific excitation (λex = 260 nm) and emission (λem = 500 nm) wavelengths, and all NFMs derivatives could be separated entirely on the reversed-phase Syncronis C18 column within 10 min. The limits of detection (LOD) and quantitation (LOQ) in this new method were 0.21 to 0.28 and 0.69 to 0.92 μg/kg, respectively. The four NFMs displayed excellent linearity (R > 0.998), and the recovery rate was satisfactory, ranging from 91.6% to 104.0%. This method successfully determined the NFMs in fish, shrimp and chicken. The method is highly sensitive and accurate, therefore, it may have potential for detecting four NFMs residues in other foodstuffs.

Determination of Nitrofuran Metabolites in Fish by Ultraperformance Liquid Chromatography-Photodiode Array Detection with Thermostatic Ultrasound-Assisted Derivatization.

Nitrofuran (NF) is a class of broad-spectrum antibiotics that are used illegally in animal feeding. NF and its metabolites have proven to pose potential risk to human health. To address the current analytical needs to quantify low levels of NF metabolites in animal foods, a sensitive method was developed for simultaneous detection of four NF metabolites in fish products by an ultraperformance liquid chromatography-diode array detector (UPLC-DAD). With 2-nitrobenzaldehyde (2-NBA) as the derivatizing reagent, the metabolites were hydrolyzed and derivatized under the assistance of thermostatic ultrasound. Compared with the current detection methods, the time of the derivatization reaction has been shortened from 16 to 2 h. The relative coefficient of four NF metabolite derivatives reached more than 0.998, with excellent linear relationship. The limits of detection (LODs) and limits of quantification (LOQs) of six repeated determinations reached 0.25–0.33 and 0.80–1.10 μg/kg, respectively. For all four NF metabolites, the limit of detection of the method was below the minimum required performance limit (MRPL) of 1.0 μg/kg, which makes it compatible with the EU requirements. The recoveries ranging from 89.8 to 101.9% with relative standard deviation below 6.5% were obtained for all of the NF metabolites. What’s more, this method was successfully applied for the determination of four NF metabolites in the fish products. As a promising approach, this method could also be extended for the quantitation of NF metabolites in aquaculture and poultry products.

Determination of four nitrofuran metabolites in gelatin Chinese medicine using dispersive solid phase extraction and pass-through solid phase extraction coupled to ultra high performance liquid chromatography-tandem mass spectrometry

This study established a validated analytical method for the first time on the determination of nitrofuran metabolites, including semicarbazide (SEM), 1-aminohydantoin (AHD), 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholinomethyl-2-oxazolinone (AMOZ) in gelatin Chinese medicine. A C18 column with the mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate in water was used to separate these nitrofuran metabolites. The limit of detection of SEM, AHD, AOZ and AMOZ were found to be 0.2 µg/kg, 0.3 µg/kg, 0.2 µg/kg and 0.2 µg/kg, whereas their limit of quantification were 0.6 µg/kg, 0.8 µg/kg, 0.6 µg/kg and 0.5 µg/kg. These nitrofuran metabolites exhibited a good linear standard curve (regression coefficients above 0.99) with a concentration range of 2 µg/L to 100 µg/L. Regarding extraction procedure, gelatin Chinese medicine was pre-treated with pepsin and then extracted using 5% formic acid (v/v) in acetonitrile. The resultant extract was purified through dispersive solid phase extraction using 1000 mg anhydrous sodium sulfate, 300 mg octadecyl carbon silica gel sorbent absorbent and 500 mg ethylenediamine-N-propyl carbon silica gel absorbent, and then further purified on Oasis PRiME HLB cartridges. The matrix effect was effectively eliminated after the clean-up procedure as confirmed by comparing the ratio of standard curves prepared by standards dissolved in both matrix solvent and 5 mmol/L ammonium acetate in water: acetonitrile (95:5, v/v). The recoveries of these nitrofuran metabolites under the 1 µg/kg, 2 µg/kg and 10 µg/kg spiking levels were between 77.4% and 95.6%. These metabolites after the extraction were stable at 4 °C for 24 h. The validated method was used to analyze the residue level of these nitrofuran metabolites in 25 gelatin Chinese medicines. Results showed that only one Colla Corii Asini sample contained SEM (2.52 µg/kg) and AOZ (6.27 µg/kg), whereas one Testudinis Carapacis et Plastri sample had SEM (1.27 µg/kg) and AMOZ (9.53 µg/kg).

Determination of Nitrofuran Metabolites in Muscular Tissue by High-Performance Liquid Chromatography with Mass Spectrometric Detection

We developed a procedure for determining residual amounts of four nitrofuran metabolites (3‑amino-2-oxazolidinone, 3-amino-5-methylmorpholino-2-oxazolidinone, 1-aminohydantoin hydrochloride, and semicarbazide hydrochloride) in muscle tissue in the form of 2-nitrophenyl derivatives by HPLC with selected reaction monitoring mass spectrometric detection. The effect of the matrix suppression of ionization is shown. The selectivity of extraction is increased by optimizing the sample preparation procedure, and the effect of organic impurities on the result measurements is minimized. The lower limit of the analytical range is 10 μg/kg for semicarbazide hydrochloride and 1 μg/kg for the remaining nitrofuran metabolites.

Determination of nitrofuran metabolites in marine products by high performance liquid chromatography–fluorescence detection with microwave-assisted derivatization

A sensitive method for simultaneous detection of four nitrofuran metabolites (3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 3-amino-morpholinomethyl-2-oxazolidinone (AMOZ) and 1-aminohydantoin (AH)) in marine products.

超高效液相色谱–质谱法测定动物源食品中硝基呋喃类代谢物残留量的方法验证

超高效液相色谱–质谱法测定动物源食品中硝基呋喃类代谢物残留量的方法验证

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 nitrofuran metabolites in shrimp by high performance liquid chromatography with fluorescence detection and liquid chromatography–tandem mass spectrometry using a new derivatization reagent

A high performance liquid chromatography with fluorescence detection (HPLC–FLD) method for the simultaneous determination of total nitrofuran metabolite residues (furazolidone, furaltadone, nitrofurantoin, and nitrofurazone) in shrimp was developed. The method involves the acid hydrolysis of protein-bound metabolites, followed by the derivatization of the freed metabolites with the new fluorescent derivatization reagent 2-hydroxy-1-naphthaldehyde (HN) and subsequent liquid–liquid extraction (LLE). Separation is achieved on a YMC-Pack Polymer C18 column under alkaline conditions, and the high fluorescence intensity of the derivatives at an emission wavelength Em=463nm (Ex=395nm) enables, for the first time, their simultaneous determination in shrimp at concentrations as low as 1μg/kg by HPLC–FLD. The method was validated using blank shrimp fortified with all four metabolites at 0.5, 1.0 and 2.0μg/kg. Recoveries were >87% with relative standard deviations of <8.1% for all four metabolites. Furthermore, the results obtained by HPLC–FLD were in very good agreement with those obtained by LC–MS/MS analysis.

Determination of nitrofuran metabolites in seafood by ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry

A new analytical method has been developed for the simultaneous determination of 4 nitrofuran metabolites in seafood by ultra-high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UHPLC–QqQ-MS/MS). The extraction procedure was based on a simultaneous acidic hydrolysis and derivatization using 2-nitrobenzaldehyde (2-NBA), followed by a solid-phase extraction (SPE). Recovery was studied by spiking blank samples at two concentration levels (1 and 10μg/kg) and recoveries ranged from 73 to 100% and 79 to 103%, respectively. Precision values, expressed as relative standard deviation (RSD) were ≤19% and ≤23% for intra-day and inter-day precision, respectively. Linearity was studied in the range 1–50μg/kg and the obtained determination coefficients (R2) were ≥0.9900 for all compounds. Limits of detection (LODs) for the derivatized nitrofuran metabolites were 0.5–0.8μg/kg and limits of quantification (LOQs) were established at 1μg/kg, whereas decision limit (CCα) and detection capability (CCβ) ranged from 1.5 to 2.6μg/kg and 1.6 to 3.1μg/kg, respectively. Finally, the method was applied to real food samples, but nitrofurans were not found.

Determination of nitrofuran metabolites in poultry muscle and eggs by liquid chromatography-tandem mass spectrometry

The use of nitrofurans in food-producing animals has been banned in EU. Detection of the protein-bound nitrofuran metabolites is the best approach to evaluate their utilization. A fast, sensitive and reliable LC–MS–MS method is presented to analyze simultaneously the metabolites of four commonly used nitrofuran drugs, furazolidone, furaltadone, nitrofurazone and nitrofurantoin. The sample clean up was performed by a single liquid–liquid extraction step, after a hydrolysis and derivatisation process. Separation of the molecules was performed by liquid chromatography in a C18 column (100 mm × 2.1 mm, 4 μm) at room temperature. The quantitative and confirmatory determination of these metabolites was performed by multiple reactions monitoring (MRM). Limits of quantification of 0.5 ng g −1 were achieved and the total analysis was accomplished in 5 min. This protocol has been applied to identify contaminated samples of poultry muscle and egg products.