3-amino-5-morpholinomethyl-2-oxazolidinone and furaltadon

Regulatory monitoring for nitrofuran drug residues in aquaculture products has largely focused on LC-MS/MS. In addition, there is a need for facile and high-throughput screening methods for monitoring programs. We evaluated the performance of Ridascreen (R-Biopharm) ELISA kits for nitrofuran drug residues in fish muscle, with verification by LC-MS/MS. Kits were available for 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholino-methyl-2-oxazolidinone (AMOZ) side-chains of furazolidone and furaltadone, respectively. We found good repeatability in fortified and incurred muscle samples, with RSDs ranging from 1.8% to 7.6%. Recoveries of AOZ and AMOZ from muscle fortified at levels of 0.5-2 ng/g ranged from 98% to 114%. Excellent selectivity was demonstrated. The minimum detection limits (MDLs) for AOZ and AMOZ in muscle were 0.05 and 0.2 ng/g, respectively. ELISA data were highly correlated with those of LC-MS/MS. Results of this study support the use of these kits as screening assays for nitrofuran residues in fish muscle.

Topics: Animals; Aquaculture; Calibration; Chromatography, Liquid; Drug Residues; Enzyme-Linked Immunosorbent Assay; Food Analysis; Furazolidone; High-Throughput Screening Assays; Ictaluridae; Morpholines; Nitrofurans; Oxazolidinones; Seafood; Tandem Mass Spectrometry

In this study, an indirect competitive enzyme-linked immunosorbent assay with chemiluminescent (CLELISA) detection for 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) was developed. A monoclonal antibody (MAb) against AMOZ was prepared through immunizing BALB/c mice with 4-carboxybenzaldehyle derivatized AMOZ (CPAMOZ), conjugated with bovine serum albumin (BSA) as antigen. The effects of the substrates luminol, p-iodophenol and urea peroxide on the performance of the assay were studied and optimized. In addition, the specificity of the MAb, estimated as the cross-reactivity values with 4-nitrobenzaldehyde derivatized AMOZ (NPAMOZ), CPAMOZ and AMOZ, was 100%, 27.45% and 0.18%, respectively. The sensitivity of the developed CLELISA was estimated as 50% inhibitory concentration (IC50) value (0.14μg/l) with a linear working range between 0.03 and 64μg/l, and a limit of detection of 0.01μg/l. The CLELISA described in this study was 5-fold more sensitive than the indirect competitive ELISA previously developed in our laboratory. Finally, this new CLELISA was compared with a commercial kit to detect NPAMOZ in spiked fish, shrimp, honey and egg samples. The recovery values from four spiked fish, shrimp, honey and egg samples with different concentrations of NPAMOZ in CLELISA were 92.1-107.7%. Thus, the immunoassay method described here has a broad detection range and high sensitivity and is a valid and cost-effective means for high throughput monitoring of residual AMOZ levels in fish, shrimps, honey and eggs with potential applications in other animal tissues.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Cattle; Cross Reactions; Drug Residues; Eggs; Enzyme-Linked Immunosorbent Assay; Female; Fishes; Food Contamination; Honey; Luminescent Measurements; Mice; Mice, Inbred BALB C; Morpholines; Nitrofurans; Oxazolidinones; Penaeidae

A heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of the furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) was developed. AMOZ was derivatised with 2-(4-formylphenoxy) acetic acid or 2-(3-formylphenoxy) acetic acid to obtain two novel immunizing haptens. The ability of these haptens in producing specific polyclonal antibodies against the nitrophenyl derivative of AMOZ (NPAMOZ) was compared with that of traditional immunizing haptens (derivatised AMOZ with 3-carboxybenzaldehyle or 4-carboxybenzaldehyle). The results indicated that the novel immunizing haptens were able to produce antibodies with almost a two-fold improvement in sensitivity of the ciELISA for NPAMOZ in comparison with the existing antibody based ELISAs. The differences in sensitivity were explained by the molecular modeling of the lowest energy conformations of NPAMOZ and the haptens. Another novel hapten, derivatised AMOZ with 2-oxoacetic acid, was synthesized and used as a heterologous coating hapten. The results showed that this strategy of using only a partial structure of the target molecule as the coating hapten was able to obtain a two to three-fold improvement in sensitivity. This study provided a modern approach for the development of an immunoassay with improved sensitivity for the metabolites of nitrofuran antibiotics.

Topics: Animals; Antibody Formation; Antibody Specificity; Chickens; Enzyme-Linked Immunosorbent Assay; Fishes; Haptens; Immunization; Immunoconjugates; Immunoglobulin G; Male; Models, Molecular; Morpholines; Nitrofurans; Oxazolidinones; Penaeidae; Rabbits; Reproducibility of Results; Swine

This report describes an enzyme-linked immunosorbent assay (ELISA) for tissue-bound metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and the application to residue analysis in cultured fish samples. The residue is monitored as a marker for the drug furaltadone. The assay enables the detection of protein bound AMOZ in the form of a 2-nitrophenyl derivative (2-NP-AMOZ) in sample supernatant or extract after acid hydrolysis and derivatization with o-nitrobenzaldehyde. Polyclonal rabbit antibodies were produced with a new immunogen hapten, 2-NP-HXA-AMOZ. The new ELISA had adequate analytical sensitivity (IC(50) value 0.325 μg kg(-1); limit of detection 0.1 μg kg(-1)) to determine a trace of AMOZ residue and had a high selectivity. Recoveries of AMOZ fortified at the levels of 0.1, 0.5 and 1.0 μg kg(-1) ranged from 89.8 to 112.5% with coefficients of variation of 12.4-16.2% over the range of AMOZ concentrations studied. The results obtained with the ELISA correlated well with those obtained by commercial test kits for 150 tested samples (r=0.984). The results suggest that the developed ELISA is a highly specific, accurate, and sensitive method suitable for high throughput screening for AMOZ residues.

Topics: Animals; Benzaldehydes; Biomarkers; Drug Residues; Enzyme-Linked Immunosorbent Assay; Fishes; High-Throughput Screening Assays; Inhibitory Concentration 50; Molecular Structure; Morpholines; Nitrofurans; Oxazolidinones; Sensitivity and Specificity

To determine 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) residues released from protein bound AMOZ in animal tissues.. Polyclonal and monoclonal antibodies were produced in this study. A rapid, sensitive, and specific competitive direct enzyme-linked immunosorbent assay (cdELISA) was developed.. Rabbit polyclonal antibodies were used in the optimized cdELISA method, and exhibited negligible cross-reactivity with other compounds structurally related to AMOZ. The IC(50) of the polyclonal antibody was 0.16 ng/mL. The method limit of detection in four different types of animal and fish tissues was less than 0.06 μg/kg. Recoveries ranged from 80% to 120% for fortified samples with the coefficient of variation values less than 15%. The results of the cdELISA method were in good agreement with the results from an established liquid chromatography-tandem mass spectrometry confirmatory method used for AMOZ residues.. The cdELISA method developed in the present study is a convenient practical tool for screening large numbers of animal and fish tissue samples for the the detection of released protein bound AMOZ residues.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Molecular Structure; Morpholines; Nitrofurans; Oxazolidinones

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.

Topics: Animals; Anti-Bacterial Agents; Anti-Infective Agents, Urinary; Chromatography, High Pressure Liquid; Chromatography, Liquid; Drug Residues; Food Contamination; Furazolidone; Hydantoins; Kidney; Liver; Mass Spectrometry; Morpholines; Muscle, Skeletal; Nitrofurans; Nitrofurantoin; Nitrofurazone; Oxazolidinones; Semicarbazides; Swine