Determinations of residual furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in fish feeds by HPLC-UV and LC-MS/MS, respectively

The antibacterial drug furazolidone belonging to the group of nitrofuran antibacterial agents has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle, and farmed fish in China. During application a large proportion of the administered drug may reach the environment directly or via feces. Although the use of furazolidone is prohibited in numerous countries, there are indications of its illegal use. It is known that furazolidone can be rapidly metabolized to 3-amino-2-oxazolidinone (AOZ) in the body of the target organism. In this study, a total of 21 fish feed samples, including 17 commercial fish feeds from local markets in China (representing 15 different formulations) and 4 fish feeds obtained from Germany and Turkey, respectively, are analyzed to determine whether the drug is still illegally used or commercially available feeds are contaminated by this drug. High-performance liquid chromatography (HPLC) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods have been implemented to determine furazolidone and its metabolite AOZ in fish feeds containing animal protein, respectively. An efficient and convenient cleanup method for the determination of furazolidone in fish feeds is developed, and a simple cleanup method for the determination of AOZ is used. Method recoveries for samples used were determined as 87.7-98.3% for furazolidone at two spike levels of 2.0 and 5.0 ng g(-1) and as 95.6-102.8% for AOZ at spike levels of 0.4 and 0.8 ng g(-1). Limits of detections were 0.4 ng g(-1) for furazolidone and 0.05 ng g(-1) for AOZ. The established methods are therefore suitable for the determination of furazolidone and its metabolite AOZ in fish feeds at trace contamination levels. Using the established methods, all fish feed samples have been proved to be furazolidone negative; however, AOZ is tested in 16 of 17 fish feeds obtained from local markets in the Hubei province of China, with a positive rate as high as 94.1%.

Residues of enrofloxacin, furazolidone and their metabolites in Nile tilapia (Oreochromis niloticus)

The residues of enrofloxacin and its metabolite in Nile tilapia (Oreochromis niloticus) were studied after oral dose of 50 mg/kg for 7 days. To find the differences between Nile tilapia and Chinese shrimp (Penaeus chinensis), the residues of enrofloxacin in P chinensis were also studied under the same conditions. The results showed that enrofloxacin metabolized into ciprofloxacin in both Nile tilapia and P chinensis, the maximal concentration of enrofloxacin in muscle, liver and plasma of Nile tilapia were 3.61 mu g/g, 5.96 mu g/g, 1.25 mu g/ml respectively, and ciprofloxacin in muscle was 0.22 mu g/g. The maximal concentration of enrofloxacin and ciprofloxacin in P chinensis were 1.68 mu g/g and 0.07 mu g/g respectively. The predicted withdrawal time for Nile tilapia was 22 days, and P. chinensis was 12 days under our experiment conditions. The residues of fitrazolidone [3-(5-nitrofurfurylidenamino)-2-oxazolidinone] and its main metabolite 3-amina-2-oxazolidinone (AOZ) in Nile tilapia were first determined by HPLC/MS. Results showed that after oral dose of 30 mg/kg for 7 days, the maximum concentration of farazolidone in Nile tilapia was 413 mu g/kg after 6 h, whereas AOZ residue reached its maximum (31 mu g/kg) right after stopping treatment. In contrast to the high metabolic rate of furazolidone, AOZ was very difficult to eliminate in vivo, thus the withdrawal time of furazolidone in Nile tilapia was 22 days at least. (c) 2005 Elsevier B.V. All rights reserved.

Detection of 3-amino-2-oxazolidinone (AOZ), a tissue-bound metabolite of the nitrofuran furazolidone, in prawn tissue by enzyme immunoassay

Furazolidone, a nitrofuran antibiotic, is banned from use in food animal production within the European Union. Increasingly, compliance with this ban is monitored by use of analytical methods to detect a stable tissue-bound metabolite, 3-amino-2-oxazolidinone (AOZ). Widespread use of furazolidone in poultry and prawns imported into Europe highlighted the urgent need for development of nitrofuran immunoassay screening tests. The first enzyme-linked immunoabsorbant assay for detection of AOZ residues in prawns (shrimps) is now described. Prawn samples were derivatized with o-nitrobenzaldehyde, extracted into ethyl acetate, washed with hexane and applied to a competitive enzyme immunoassay based on a rabbit polyclonal antiserum. Assay limit of detection (LOD) (mean+3 s) calculated from the analysis of 20 known negative cold and warm water prawn samples was 0.1 mug kg(-1). Intra- and interassay relative standard deviations were determined as 18.8 and 38.2%, respectively, using a negative prawn fortified at 0.7 mug kg(-1). The detection capability (CCbeta), defined as the concentration of AOZ at which 20 different fortified samples yielded results above the LOD, was achieved at fortification between 0.4 and 0.7 mug kg(-1). Incurred prawn samples (n=8) confirmed by liquid chromatography coupled with tandem mass spectrometry detection to contain AOZ concentrations between 0.4 and 12.7 mug kg(-1) were all screened positive by this enzyme-linked immunoabsorbant assay. Further data are presented and discussed with regard to calculating assay LOD based on accepting a 5% false-positive rate with representative negative prawn samples. Such an acceptance improves the sensitivity of an ELISA and in this case permitted an LOD of 0.05 mug kg(-1) and a CCbeta of below 0.4 mug kg(-1).

Production and characterisation of monoclonal antibodies for the detection of AOZ, a tissue bound metabolite of furazolidone

3-amino-2-oxazolidinone (AOZ) is a tissue bound toxic metabolite derived from the nitrofuran antibiotic, furazolidone. AOZ is detected in the derivatised form of 3-{[(2-nitrophenyl) methylene]amino}-2-oxazolidinone (NP AOZ). 3-{[( 3- carboxyphenyl)-methylene]amino-2-oxazolidinone (CP AOZ) was used as the immunising hapten for the production of monoclonal antibodies against NP AOZ. Monoclonal antibodies were produced using hybridomas from the fusion of murine myeloma cells and spleen cells isolated from BALB/c mice immunised with CP AOZ-ethylenediamine-human serum albumin (CP AOZ-ed-HSA). The antibody production in ascitic fluids from clones 3B8/2B9 and 2D11/A4 was monitored during a 16 month period. Repeated cultures of these hybridomas, followed by injection into mice and cloning did not change the assay parameters. Clone 2D11/A4 exhibited long term stability in antibody production throughout the experiment whereas clone 3B8/2B9 demonstrated variability in particular antibody yields whilst retaining assay sensitivity. Reasons for this production variability in clones are discussed. In an optimised direct ELISA format, the antibodies exhibited a 50% binding inhibition in the range of 0.52-1.15 ng/ml with NP AOZ (0.22-0.50 ng/ml, respective AOZ equivalents) and showed high specificity towards this analyte. The sensitivity of monoclonal antibodies incorporated into the ELISA is compatible with the European Union MRLP and is currently in use for routine analysis.

Ensaio terapêutico na infecção por Giardia muris em camundongo com metronidazole, tinidazole, secnidazole e furazolidone

Foi testado in vivo a sensibilidade de Giardia muris a quatro drogas comumente usadas no tratamento da giardíase humana. Foram utilizados 7 grupos de animais, com 12 camundongos cada, sendo que o grupo controle recebeu apenas solução salina 0,15M (0,5ml/animal). Os demais grupos receberam em dose única: metronidazole e furazolidone (500mg/kg), tinidazole e secnidazole (200mg/kg). A eficácia das drogas foi avaliada através da contagem de cistos nas fezes e pela ausência de trofozoítos no intestino. O metronidazole foi a droga mais eficaz. Os cortes histológicos mostraram diferenças entre o padrão da mucosa intestinal de animais normais e parasitados. No entanto, não se observou diferença entre o padrão de mucosa de animais infectados tratados e não tratados, o que sugere que estas alterações podem ser causadas pelo parasito e não pelas drogas.

呋喃唑酮对粘细菌性鱼病的防治试验

<正> 呋喃唑酮(痢特灵,Furazolidone,Furoxone)是广泛用于治疗人体细菌性痢疾的有效药物。其物理性状为黄色粉状、无味、难溶于水。据文献报导,它有广谱的抗菌作用,5～10微克/毫升能抑制一般的革兰氏阳性和阴性细菌,20～50微克/毫升有杀菌作用,对抗原虫也有一定的效果。它的作用机理,可能是抑制乙酰辅酶A而干扰了细菌糖代谢的早期阶段。我国鱼类细菌性鱼病流行较广,危害也较大,虽然多年来曾使用多种药物治疗,但因长期使用,产生抗药性(如抗菌素等)或药物保管和使用方法的问题,效果均不甚理想。为此,

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400 mg kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Monoclonal antibody-based ELISA for the quantification of nitrofuran metabolite 3-amino-2-oxazolidinone in tissues using a simplified sample preparation

A sensitive and specific monoclonal ELISA for the determination of tissue bound furazolidone metabolite 3-amino-2-oxazolidinone (AOZ) is described. The procedure enables the detection of AOZ in matrix supernatant after homogenisation, protease treatment, acid hydrolysis and derivatisation of AOZ released from the tissue by o-nitrobenzaldehyde. The formed p-nitrophenyl 3-amino-2-oxazolidinone (NPAOZ) is determined by ELISA calibrated with matrix-matched standards in the concentration range of 0.05-5.0 mu g l(-1). The assay was validated according to criteria set down by Commission Decision 2002/657/EC for the performance and validation of analytical methods for chemical residues. Detection capability, set on the basis of acceptance of no false negative results, was 0.4 mu g kg(-1) for shrimp, poultry, beef and pork muscle. This sensitivity approaches the established confirmatory LC-MS/MS able to quantify tissue-bound AOZ at levels as low as 0.3 mu g kg(-1). An excellent correlation of results obtained by ELISA and LC/MS-MS within the concentration range 0-32.1 mu g kg(-1) was found in the naturally contaminated shrimp samples (r = 0.999, n = 8). A similar con-elation was found for the incurred poultry samples within the concentration range of 0-10.5 mu g kg(-1) (r = 0.99, n = 8). (c) 2005 Elsevier B.V All rights reserved.

Residues of nitrofuran antibiotic parent compounds and metabolites in eyes of broiler chickens

Nitrofuran antibiotic residues in food continue to be of international concern. The finding of sources of semicarbazide (SEM), other than through the misuse of nitrofurazone, present a challenge to the use of SEM as a definitive marker residue for this drug. Detection of intact (parent) nitrofurazone would avoid confusion over the source of SEM residues. Broiler chickens were fed sub-therapeutic nitrofuran-containing diets and their tissues were analysed for parent compounds and metabolites by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS). Depletion half-lives in muscle were longer for tissue-bound metabolite residues, 3.4 days - 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) - to 4.5 days (SEM), than total metabolite residues, 2.0 days (AOZ) to 3.2 days (SEM). Metabolite concentrations were higher in eyes than in muscle. Metabolite half-lives in eyes ranged from 8.5 days (1-aminohydantoin (AHD)) to 20.3 days (SEM). Nitrofuran parent compounds were also detected in eyes. Furaltadone was detected in single eyes after 21 days' withdrawal of a 6 mg kg -1 furaltadone diet. When 50 eyes from broilers containing metabolites in muscle close to the 1 µg kg -1 minimum required performance level (MRPL) were pooled into single samples, 1.2 ng of furazolidone and 31.1 ng of furaltadone were detected, but nitrofurazone was not detected due to the long depletion half-life of SEM in muscle. Further studies are required to improve LC-MS/MS nitrofurazone sensitivity and refine the sample size necessary to use nitrofurazone detection in pooled eyes as a complement to SEM detection in muscle.

Elétrodo de pasta de carbono molecularmente impresso para deteção voltamétrica de Furazolidona

A furazolidona é uma substância ativa do medicamento Giarlam que contém um espetro anti-bacteriano relativamente amplo e que é frequentemente usado para tratar certas doenças bacterianas e protozoárias no homem. A maioria dos fármacos exige uma dosagem que garanta os níveis de segurança e eficácia de atuação. A necessidade de dosear os medicamentos e os seus metabólitos exige o desenvolvimento constante de métodos analíticos eficientes. Neste trabalho desenvolveu-se um novo sensor eletroquímico para a deteção da furazolidona, baseado num elétrodo de pasta de carbono modificado com um polímero molecularmente impresso. A procura de novos materiais que permitam uma melhor seletividade e sensibilidade aos sistemas de deteção é especialmente importante no desenvolvimento de métodos analíticos. Os polímeros molecularmente impressos enquadram-se nesse perfil e o seu uso tem vindo a ser cada vez mais frequente como ferramenta importante em química analítica. Assim, sintetizou-se um polímero com cavidades seletivas para a Furazolidona. Este polímero foi, misturado com grafite e perafina de modo a produzir uma pasta de carbono. Uma seringa de plástico foi usada como suporte da pasta de carbono. O comportamento eletroquímico do sensor foi avaliado e diversas condições de utilização foram estudadas e otimizadas. O sensor apresenta um comportamento linear entre a intensidade do pico e a concentração numa gama de concentrações entre 1 e 100 μM, um limite de deteção de 1 μM e uma precisão (repetibilidade) inferior a 7%. A aplicabilidade do sensor fabricado em amostras complexas foi avaliada pela deteção do fármaco em amostras de urina.

Multiple Antibiotic Resistances of Vibrio Isolates from Coastal and Brackish Water Areas

An experiment was designed to assess the occurrence of multiple antibiotic resistances in Vibrio sp from different (brackish and marine) environments. Water samples front nine marine landing sites and two coastal inland aquaculture farms were screened for the Vibrio spp and assessed their resistance to twenty-two different antibiotics, which arc commonly encountered in the aquatic ecosystem. Tissue samples (shrimp, mussel and sepia) were tested from the sampling site with highest antibiotic resistance. Of' the total 119 Vibrio isolates, 16. 8% were susceptible to all antibiotics. Of the resistant (83.19%) Vibrio strains, 30.3% were resistant against three antibiotics, 55.5% were resistant against 4-10 antibiotics, 14.14% were resistant against more than 10 antibiotics and 54% have shown multiple antibiotics resistance (MAR). Antibiotic resistance index was higher in Coastal 3, 6, Aqua farm 2 in isolates from water samples and all the tissues tested. Interestingly, incidence of antibiotic resistance in isolates from water samples was comparatively lower in aquaculture farms than that observed in coastal areas. Highest incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rilanipicin and Streptomycin and lowest against Chloramphenicol, Tetracycline, Chlortetracycline, Furazolidone, Nalidixic acid, Gentamycin Sulphafurazole, Trimcthoprinr, Neomycin and Amikacin irrespective of the sampling sites. Results from various tissue samples collected from the sites of highest antibiotic resistance indicated that antibiotic resistance Vibrio spp collected from fish and tissue samples were higher than that of water samples. Overall results indicated that persistent use of antibiotics against diseases in human beings and other life forms may pollute the aquatic system and their impact on developing antibiotic resistant Vibrio sp may be a serious threat in addition to the use of antibiotics in aquaculture farms.

Multiple Antibiotic Resistances of Vibrio Isolates from Coastal and Brackish Water Areas

An experiment was designed to assess the occurrence of multiple antibiotic resistances in Vibrio sp from different (brackish and marine) environments. Water samples front nine marine landing sites and two coastal inland aquaculture farms were screened for the Vihrio spp and assessed their resistance to twenty-two different antibiotics, which arc commonly encountered in the aquatic ecosystem. Tissue samples (shrimp, mussel and sepia) were tested from the sampling site with highest antibiotic resistance. Of' the total 119 Vihrio isolates, 16. 8'7(, were susceptible to all antibiotics. Of the resistant (83.19%) Vibrio strains, 30.3% were resistant against three antibiotics, 55.5% were resistant against 4-10 antibiotics, 14.14% were resistant against more than 10 antibiotics and 54% have shown multiple antibiotics resistance (MAR). Antibiotic resistance index was higher in Coastal 3, 6, Aqua farm 2 in isolates from water samples and all the tissues tested. Interestingly, incidence of antibiotic resistance in isolates from water samples was comparatively lower in aquaculture farms than that observed in coastal areas. Highest incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rilanipicin and Streptomycin and lowest against Chloramphenicol, Tetracycline, Chlortetracycline, Furazolidone, Nalidixic acid, Gentamycin Sulphafurazole, Trimcthoprinr, Neomycin and Amikacin irrespective of the sampling sites. Results from various tissue samples collected from the sites of highest antibiotic resistance indicated that antibiotic resistance Vibrio spp collected from fish and tissue samples were higher than that of water samples. Overall results indicated that persistent use of antibiotics against diseases in human beings and other life forms may pollute the aquatic system and their impact on developing antibiotic resistant Vibrio sp may be a serious threat in addition to the use of antibiotics in aquaculture farms.

Multiple Antibiotic Resistances of Vibrio Isolates from Coastal and Brackish Water Areas

An experiment was designed to assess the occurrence of multiple antibiotic resistances in Vibrio sp from different (brackish and marine) environments. Water samples from nine marine landing sites and two coastal inland aquaculture farms were screened for the Vibrio spp and assessed their resistance to twenty-two different antibiotics, which are commonly encountered in the aquatic ecosystem. Tissue samples (shrimp, mussel and sepia) were tested from the sampling site with highest antibiotic resistance. Of the total 119 Vibrio isolates, 16. 8% were susceptible to all antibiotics. Of the resistant (83.19%) Vibrio strains, 30.3% were resistant against three antibiotics, 55.5% were resistant against 4-10 antibiotics, 14.14% were resistant against more than 10 antibiotics and 54% have shown multiple antibiotics resistance (MAR). Antibiotic resistance index was higher in Coastal 3, 6, Aqua farm 2 in isolates from water samples and all the tissues tested. Interestingly, incidence of antibiotic resistance in isolates from water samples was comparatively lower in aquaculture farms than that observed in coastal areas. Highest incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rifampicin and Streptomycin and lowest against Chloramphenicol, Tetracycline, Chlortetracycline, Furazolidone, Nalidixic acid, Gentamycin Sulphafurazole, Trimethoprirn, Neomycin and Amikacin irrespective of the sampling sites. Results from various tissue samples collected from the sites of highest antibiotic resistance indicated that antibiotic resistance Vibrio spp collected from fish and tissue samples were higher than that of water samples. Overall results indicated that persistent use of antibiotics against diseases in human beings and other life forms may pollute the aquatic system and their impact on developing antibiotic resistant Vibrio sp may be a serious threat in addition to the use of antibiotics in aquaculture farms.