The Research For A Determinaton Method On Metabolites Of Nitrofurans

Nitrofurans drugs (NFS) have been widely used in livestock and aquatic products for the treatment and prevention the bacteria and the original biological cause various gastrointestinal infection, also as growth promoter. Because of NF potentially carcinogenic and mutagenic efFects on human health, the use of these compounds in food-producing animals is prohibited in various countries, except for topical applications.This paper provides an overview of nitrofuran toxicity, metabolism, sample preparation methods, instrumental methods. High performance liquid chromatography with a fluorescence detector (HPLC-FLD) method and tandem mass spectrometry were developed for the simultaneous determination of metabolites of four nitrofuran drugs in pork and shrimp muscle, respectively. The mass spectra of the new derivative metabolites have been analyzed and the fragmentation patterns have been studied. And fluorescence spectrum determination of dissociation constant of naphthol of a new schiffbase has also been developed. Main work as follows:1. High performance liquid chromatography with a fluorescence detector for the determination of nitrofuran metabolites in pork and shrimp muscleA simple and sensitive high performance liquid chromatography with a fluorescence detector (HPLC-FLD) method has been developed for the simultaneous determination of metabolites of four nitrofuran drugs (furazolidone, furaltadone, nitrofurantoin and nitrofurazone) in pork and shrimp muscle. The method involves the acid hydrolysis of the protein-bound drug metabolites and the conjugation of the released side chains with a novel fluorescence agent2-hydroxy-l-naphthaldehyde. After the liquid-liquid extraction and effective separation of the derivatives on a YMC-Pack PolymerCig column at40℃under alkaline conditions, the high fluorescence intensity of these derivatives at emission wavelength Xem=463nm enables their first simultaneous determination in pork muscle at concentrations as low as1μg/kg. The method is validated using blank pork and shrimp muscle fortified with all four metabolites at0.5,1.0and2.0μg/kg. Recoveries are87.4～107%with relative standard deviations of<8.5%for all four metabolites.2. Determination of nitrofuran metabolites in pork and shrimp by liquid chromatography tandem mass spectrometryA liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) method is presented for the simultaneous analysis of the metabolites of four nitrofuran veterinary drugs, AOZ, AMOZ, AH, and SEM in pork and shrimp samples. The method adopts a combined hydrolysis of protein-bound drug metabolites and derivatization of the resulting metabolites with2-hydroxy-l-naphthaldehyde (HN) during two hours incubation, Potassium chloridewas used to precipitate proteins, followed by a liquid-liquid extraction. The target compounds were identified and quantitatively determined by high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry (ESI/MS/MS) operated in multiple reaction monitoring mode. The overall corrected recoveries ranged between85.6and106%, with relative standard deviations (RSDs) of2.3-8.0%for the entire procedure.3. Fluorescence spectrum determination of dissociation constant of naphthol of a new schiff baseThe compound of2-hydroxy-1-naphthalene formaldehyde5-hydroxytry-ptophan, a new Schiff base, is easily prepared by reacting2-hydroxy-l-naphthalene formaldehyde (2HN) and5-hydroxytryptophan (5HTP) in ethanol.2HN5HTP was characterized by means of infrared absorption spectrum (IR), ultraviolet-visible absorption spectrum (UV-vis), fluorescence spectrum (FS), nuclear magnetic resonance (NMR) techniques. Dissociation constant pKa=9.44for naphthol of2HN5HTP has been obtained in aqueous solution at25℃based on FS.pKa=9.16,9.05,8.76, and8.63have also been obtained in different mixed solvent (1+1) of water and organic solution (MeOH, EtOH, MeCN and DMK).Furthermore, pKa. for naphthol of2HN5HTP is further validated by UV-vis, and the results show very good agreement with those of FS.