WO2008115580A2

WO2008115580A2 - Method for detecting nitrofuran

Info

Publication number: WO2008115580A2
Application number: PCT/US2008/003750
Authority: WO
Inventors: Say-Jong Law
Original assignee: Charm Sciences Inc
Current assignee: Charm Sciences Inc
Priority date: 2007-03-21
Filing date: 2008-03-21
Publication date: 2008-09-25
Anticipated expiration: 2009-09-21
Also published as: WO2008115580A3

Abstract

A method for detecting nitrofuran that includes rapid acid hydrolysis of sample to obtain a sample suitable for testing in less than 16 hours.

Description

Method for Detecting Nitrofuran

[0001] This application claims priority from U.S. Provisional Patent Application 60/919,325, filed March 21, 2007 (Method for Detecting Nitrofuran) and U.S. Provisional Patent Application 60/921,171, filed March 30, 2007 (Method for Detecting Nitrofuran), both of which are hereby incorporated by reference.

Background

[0002] Nitrofurans are synthetic broad-spectrum antibiotics used in animal, aquaculture and honey production. Nitrofurans are antibacterial, antiprotozoan and growth promoters. In animal studies the parent drugs and their metabolites showed carcinogenic and mutagenic characteristics. For that reason, nitrofuran use in the treatment of animals used for food production is prohibited. Despite such bans, residues continue to appear in the food supply. In particular, metabolites of nitrofurans can be tissue or protein bound resulting in residue remaining long after administration of the parent drug.

[0003] Common parent nitrofuran drugs, and their respective side chains (the R groups), include: furazolidone (side chain: 3-amino-2-oxazolidinone = AOZ), furaltadone (side chain: 3-amino-5-morpholinomethyl-2-oxazolidinone = AMOZ), nitrofurantoin (side chain: 1- aminohydantoin = AHD) and nitrofurazone (side chain: semicarbazide = SEM). The structures are as follows:

nitrofurantoin

nitrofurazone

furazolidone

[0004] Nitrofurans are rapidly metabolized. The in situ half-life can be less than two hours. During metabolism, the nitrofuran parent may be reduced, such as by one or more nitroreductases. In one such scenario, the basic nitrofuranyl moiety (the common portion attached to the R group), is transformed into a different chemical group, while the R group (on the right hand side of the nitrofuran parent structure) remains intact.

[0005] Several methods for determining the use of a nitrofuran parent drug are via indirect metabolite detection. Such methods include liquid chromatography with ultra violet detection (LC-UV), liquid chromatography with mass spectrometer detection (LC-MS), liquid chromatography with tandem mass spectrometer detection (LC -MS/MS) and immunodiagnostics. The metabolite can be protein bound and, therefore, a hydrolysis step is used to cleave the side chain. Those hydrolysis and derivatization reactions often require 16- 24 hours prior to sample detection.

Summary

[0006] An aspect is a method and test kit that includes a sample preparation step that allows rapid results - less than 16 hours - when testing for nitrofuran in biological samples. Sample preparation time can be reduced by decreasing the time for acid hydrolysis by one or a combination of: providing a concentration of NBA, or other aryl aldehyde with electron withdrawing group, effective to reduce time for acid hydrolysis to less than 16 hours; increasing incubation temperature to a level effective to reduce time for acid hydrolysis to less than 16 hours (for example above 37 degrees C, such as about 80 degrees C); and increasing amount or strength of acid used to a level effective to reduce time for acid hydrolysis to less than 16 hours.

[0007] An aspect includes reacting substituted or non-substituted 2-nitrobenzaldehyde (2- NBA) with the sample. Yet another aspect includes reacting substituted or non-substituted 4- nitrobenzaldehyde (4-NBA) with the sample. The resulting material is then tested for the presence of nitrofuran such as in an immunoassay or by any of a variety of liquid chromatography detection methods.

Substituted 4-NBA Substituted 2-NBA

X = electrically neutral, or non-electron donating substituent (s)

[0008] Other aspects include subjecting the sample to a rapid acid hydrolysis by reacting more than 10 millimolar (mM), for example 40 mM, of either 2-nitrobenzaldehyde (2-NBA) or 4-NBA (2-NBA and 4-NBA hereinafter referred to as NBA) with the sample in the presence of an acid, such as HCl. Concentration of acid can be approximately about 0.5 N' to about 2 N HCl. Aspects also include incubating the NBA/acid combination which can shorten the reaction time and maximize the yield of the product. Incubation temperature ranges include about 37 degrees C to about 100 degrees C. At above 37 degrees C, such as 50 degrees C, the sample can be ready for analysis in as little as about 2 hours. At more elevated temperatures, such as about 80 degrees C, the sample can be ready in as quickly as 15 minutes. Although many aspects and embodiments are discussed relative to the use of 2- NBA and 4-NBA, it will be appreciated that other NBA isomers may be useful and other aryl aldehydes with electron withdrawing groups can be useful.

[0009] U.S. Patent Application Serial Number 11/583236, filed October 18, 2006, hereby incorporated by reference, describes a variety of techniques, including particular immunogens, for producing antibodies with sensitivity to nitrofuran drugs, including protein bound metabolites. Those antibodies can be used after the sample preparation, including acid hydrolysis, described herein. Similarly, other antibodies with sensitivity to various nitrofuran drugs and metabolites may be useful.

Detailed Description

[0010] Embodiments include generating a 4-NP derivative of a nitrofuran. Other embodiments include generating a 2-NP derivative of a nitrofuran (4-NP derivative and 2-NP derivative hereinafter referred to as NP derivative). Yet other embodiments include generating a substituted or non-substituted NP derivative of a nitrofuran. The NP derivative can be used for detecting a nitrofuran in a sample, such as a food sample. In particular, proteinaceous samples, such proteinaceous samples in which the nitrofuran is present as a biomolecule nitrofuran metabolite, may require the sample to be hydrolyzed, such as by acid hydrolysis. The biomolecule could be a small molecular biochemical alone or conjugated further to a protein carrier, a peptide conjugated to a protein carrier, a protein, a nucleic acid, or a polysaccharide. The acid hydrolysis can improve detection in an immunoassay, or liquid chromatography, particularly when the sample contains protein and/or the nitrofuran is present as a protein bound metabolite. Higher concentrations of NBA greater than 1OmM, for example between 1OmM and 5OmM, such as about 40 mM, can help drive the acid hydrolysis at a faster rate. Other factors that can allow faster acid hydrolysis include increased incubation temperature and increased volume, strength and/or concentration of acid.

[0011] The following diagrams illustrate the preparation of the 4-NP derivative of each of furazolidone (FZD), nitrofurantoin (NFT) and furaltadone (FTD). As depicted, the acid hydrolysis with 4-NBA causes the various R₂ groups on the nitrofuran or nitrofuran metabolite, whether parent, metabolite or protein bound metabolite, to be replaced by the 4- NBA with the resulting product being the 4-NP derivative. The 4-NP derivative can be detected in the sample such as by using an immunoassay or liquid chromatography detection (e.g. UV, MS, MS/MS) with sensitivity to one or more of the 4-NP derivatives.

Generation of 4-NP-AOZ from furazolidone (FZD), furazolidone metabolites (FZDM) with or without being bound to protein.

Examples of R₂ group:

Furazolidone (FZD) Furazolidone metabolite (FZDM)

Protein or Peptide

Protein bound FZDM Generation of 4-NP-AHD from nitrofurantoin (NFT), nitrofurantoin metabolites (NFTM) with or without being bound to protein.

ves

Examples of R? group:

Nitrofurantoin (NFT) Nitrofurantoin metabolite (NFTM)

Protein or Peptide Protein bound NFTM Generation of 4-NP-AMOZ from furaltadone (FTD), furaltadone metabolites (FTDM) with or without being bound to protein.

Examples of R₂ group:

Furaltadone (FTD) Furaltadone metabolite (FTDM)

Protein or Peptide Protein bound FTDM [0012] Various immunoassay embodiments include ELISA formats.. Several ELISA formats are possible including adsorbing antibody to an inert surface, for example a 96-well polystyrene plate. After adsorption of the antibody, the surface is washed with a solution of an appropriate blocking agent, for example casein from non-fat dry milk powder. After sample preparation such as acid hydrolysis with NBA, sample can be added to the well. A nitrofuran tracer is also added to the plate. The tracer can be either nitrofuran metabolite or nitrofuran derivative directly labeled with an enzyme or indirectly, for example utilizing a biotin/avidin bridge. Analyte in the sample (nitrofuran metabolite) competes with tracer to bind to the immobilized (or coated) antibody. After washing, the surface is treated with a substrate that forms a colored product when contacted with the bound enzyme tracer. Color intensity is inversely related to the amount of analyte present. \ ■ [.^{■ ■} .

[0013] In another example of an ELISA format, an analyte competitor, such as S-linked nitrofuran metabolite-Cys-BSA or N-linked nitrofuran metabolite-BSA, is adhered to the surface of the 96-well plate. Enzyme labeled antibody, performed or added stepwise, can be mixed with the previously prepared sample (for example by the acid hydrolysis with NBA) and applied earlier or simultaneously to the plate. Uncaptured labeled antibody is then washed off the surface. After substrate is added the color development is observed. In this example, there is an inverse relationship between the color intensity and the amount of antigen present.

[0014] In a lateral flow test device, antibody can bind to the NP derivative in the sample preparation to form a complex. The method and device can utilize a membrane strip, such as a nitrocellulose strip. One example utilizes colloidal gold particles as a label bound to the antibody. The size of the particle can be adapted to the porosity of the membrane strip. The particles are preferably sufficiently small to be transported along the membrane by capillary action of a fluid sample. The number of particles present in the test strip may vary, depending on the size and composition of the test strip and the desired level of sensitivity of the assay. For example, using fewer particles may help increase test sensitivity.

[0015] Any one of a variety of labels may be employed including colloidal gold particles. Other useful labels include, but are not limited to, colloidal sulphur particles; colloidal selenium particles; colloidal barium sulfate particles; colloidal iron sulfate particles; metal iodate particles; silver halide particles; silica particles; colloidal metal (hydrous) oxide particles; colloidal metal sulfide particles; colloidal lead selenide particles; colloidal cadmium selenide particles; colloidal metal phosphate particles, colloidal metal ferrite particles, any of the above-mentioned colloidal particles coated with an organic or inorganic layer; protein or peptide molecules; liposomes; or organic polymer latex particles, such as polystyrene latex beads. Still other labels may also be used including, but not limited to, luminescent labels; fluorescent labels; or chemical labels, such as electroactive agents (e.g., ferrocyanide); enzymes; radioactive labels; or radiofrequency labels.

[0016] The test device can include a support strip and a sample-absorbing matrix, for example composed of a cellulosic, sponge-like material. Such a sample-absorbing matrix allows for absorbing an amount of the sample and can also filter unwanted substances from the sample prior to the sample contacting test reagents. The test device also can include a mobile-phase support attached to the support strip and in contact with the sample-absorbing matrix. In an example, a mobile-phase composition is disposed within or on the mobile-phase support and has one or more labeled receptors, such as one or more gold labeled antibodies with affinity to a nitrofuran and/or metabolite thereof. These kits may also include various combinations of polyclonal and monoclonal antibodies.

[0017] The mobile-phase composition can be applied prior to test operation, for example by spraying and drying onto a porous surface such as polyethylene membrane. A useful membrane is POREX® (POREX is a registered trademark of Porex Technologies Corp. of Fairburn, Georgia) membrane. When exposed to a sample, for example of fluid milk, honey extract, shrimp extract (such as shrimp homogenate), beef extract, pork extract, chicken extract, or other liquid or liquefied matrix, the mobile-phase composition can be carried in the sample flow together with the sample. In test operation the sample flows and the antibody binds to nitrofuran and/or metabolite present in the sample to form antibody-analyte complexes. Alternatively, the mobile phase can be combined with sample prior to application to the test strip or other solid support. In this alternative embodiment antibody can bind to nitrofuran and/or metabolite present in the sample prior to contact with the test strip.

[0018] In an example, the test strip includes a stationary-phase support strip, which may be part of the same strip as the mobile-phase composition, or on a separate strip in fluid flow contact with the first strip. The support strip has a first membrane end in contact with the mobile-phase composition and a second membrane end that may be in contact with an optional disposal zone. Lateral-capillary flow of the sample is from the first membrane end to the second membrane end.

[0019] The one or more test zones may include a binder, such as a representative analyte or analogue thereof, which captures unbound labeled receptor. One^* or more optional control zones may also be on the stationary-phase membrane. The control zone may contain receptor for the analyte receptor, for example, antibody to the particular receptor, such as anti-species antibody, for binding with both analyte-bound receptor and excess unbound ■ receptor. Alternatively, the control zone may be involved in an independent reaction that informs the user that the test is complete and includes consistent visual indicators, such as color development, for comparison to the test zone. The control zone can generate signal either on contact with sample or on contact with specific test material, such as labeled receptor, such as when the control zone includes an anti-species antibody or one of the several useful antibody binders known in the art including protein A, protein G or recombinant varieties of proteins A and G.

[0020] Examples of possible test zone binders include nitrofurans, nitrofuran metabolites, acrylonitrile derivatives of a nitrofuran, or analogues thereof and NP nitrofuran derivatives. For example NP-AOZ, NP-AHD, NP-AMOZ, nitrofurantoin metabolite (NFTM)-Cys- adduct, nitrofurazone metabolite (NFZM)-Cys adduct, furazolidone metabolite (FZDM)-Cys adduct, furaltadone metabolite (FTDM)-Cys adduct. Such binders can be either synthetically derived or, in some cases, the related naturally occurring species. Such a binder may be disposed on the test zone portion of the membrane for example by spraying. Prior to spraying, said binder can be conjugated to an attachment or carrier protein. Suitable attachment proteins are known to those skilled in the art to be proteins that bind readily to solid supports, such supports that include nitrocellulose. A useful attachment protein includes a carrier protein, i.e., a protein commonly used in conjunction with an immunogen, such as generally water soluble proteins with multiple accessible amino groups including albumin, e.g., bovine serum albumin (BSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH) and thyroglobulin (THG).

[0021] The lateral flow test device and method can also be in a sandwich assay format or an inhibition/competitive format. Multiple test zones can be utilized, for example to detect the presence of multiple analytes using multiple specific antibodies. In such multiple analyte tests, multiple control lines may or may not be required.

[0022] An embodiment includes a lateral flow assay that is capable of rapidly detecting one or more nitrofurans and/or metabolites such as tissue bound metabolites of nitrofurans in a sample. Such lateral flow assay formats can include those described in U.S. Patents 7,097,983; 6,319,466; 6,475,805; and 5,985,675, the teachings of which are incorporated herein by reference.

[0023] Lateral flow test results can be interpreted visually or by use of a reader, or analyzer, such as a ROSA® reader (ROSA is a registered trademark of Charm Sciences, Inc. Lawrence, MA). Other reader/analyzer examples include fluorometers, luminometers, bar code readers, radiation detectors (such as scintillation counters), UV detectors, infrared detectors, electrochemical detectors or optical readers, such as spectrophotometers. The reader can be used to distinguish between one or more test zones and one or more control zones or simply to determine a relative change in the test zone. In one embodiment the reader is a ROSA reader. In a particular embodiment, the analyzer is an optical reader, e.g., the reader described in U.S. Patent No. 6,124,585, issued September 26, 2000, hereby incorporated by reference.

[0024] In an embodiment not utilizing a lateral flow test strip, a radiolabeled tracer can be employed with the antibody. For example, in a method known as the Charm II method, an IGGSORB® tablet (IGGSORB is a registered trademark of The Enzyme Center, Lawrence, Massachusetts) containing a lyophilized preparation of protein A fixed to the cell walls of inactivated Staphylococcus aureas, is added to a test tube along with 300 μL of deionized water. An appropriate amount of antibody is added to the tube and the tube is mixed. If required, an amount of buffer, for example 5ml of MSU-EB (from Charm Sciences, Inc.) is added to the tube along with 5 μL of appropriate radiolabeled tracer and the tube is again mixed. The tube is incubated for five minutes at 40⁰C and then centrifuged for 5 minutes at 3400rpm. The supernatant is poured off and scintillation fluid is added for tracer detection. EXAMPLES Example 1

Deriving 4-NPAOZ. 4-NPAMOZ and 4-NP-AHD from Shrimp [0025]

1Og of thawed, peeled, raw shrimp/ meat (no shells/heads, etc) tissue was added directly into a blender. 3OmL of deionized (DI) water was then added to the shrimp tissue and the mixture grinded on high speed for 40 seconds (± 5 seconds). 2.5 grams of the homogenate was added to a test tube with ImL of 1.0 N HCl and 50 uL of 4OmM 4-NBA. The test tubes were capped, shaken by hand to loosen the mixture, vortexed (1Ox) and incubated for 15min at 80⁰C. The cap was removed ImL of neutralization solution (IM K₂HPO4 and 0.8N NaOH) was added. The test tubes were again capped, shaken by hand and vortexed (1Ox). 2mL of ethyl acetate was then added, the test tubes capped, shaken by hand and then vortexed for 30 seconds. The mixture was then centrifuged at 250 g for 1 minute. 1.0 mL was removed from the upper organic layer and transferred to another 13 mm test tube. The organic extract was dried by placing the tube in a 55-60⁰C heating block with air flow directed into the tubes. Drying time was about 10 minutes. 0.4mL n-heptane was added to the dried residue, the tubes mixed. 0.4 ml of Sample buffer (1OmM sodium phosphate with 0.05% Tween 20, pH 7.2) was then added and the mixture vortex (1Ox) and centrifuged at 250 g for 5 minutes. 300 uL was removed from the lower aqueous layer and transferred to an Eppendorf tube for ELISA in a 100 μl sample well.

Example 2(a) Synthesis of 4-NP-AOZ

4-NBA AOZ 4-NP-AOZ

[0026] A solution of AOZ (12.4 mg, 0.12 mmole) in 0.6 ml of acetonitrile was mixed with a solution of 4-nitrobenzaldehyde (4-NBA, 22 mg, 0.15 mmole) in 0.6 ml of acetonitrile and treated with 0.1 ml of 1 N HCl. The reaction mixture was stirred at room temperature overnight and filtered. The solid was recrystallized from the mixture of five parts acetonitrile to three parts ethyl ether (acetonitrilerethyl ether 5:3), filtered and air-dried to give a white solid (6.7 mg, 0.03 mmole). Thin layer chromatography showed a homogenous spot (Rf = 0.44) when developed with ethyl acetate. HPLC analysis of the product on a PLRP-S, 5 um, 100 A⁰, 4 x 150 mm column eluted isocratically with a solvent mix of 20 mM K₂HPO₄ pH 9.0:acetonitrile 60:40 showed a single peak with retention time of 6.9 +/- 0.2 min. The pure compound showed a UV/vis spectrum with a major peak absorbance around 323 nm and a minor peak absorbance around 230 nm in the same solvent system.

Example 2b Synthesis of 4-NP-AMOZ

[0027] A solution of AMOZ (30.2 mg, 0.15 mmole) in 0.75 ml of acetonitrile was mixed with a solution of 4-NBA (27.2 mg, 0.18 mmole) in 0.75 ml of acetonitrile and treated with 0.12 ml of 1 N HCl. The reaction mixture was stirred at room temperature overnight and concentrated to induce crystallization. The suspension was filtered and the yellow solid retentate was washed with 1 ml of acetonitrile and air-dried to give the desired product (20.8 mg, 0.062 mmole). Another crop of product (15.8 mg, 0.047 mmole) was obtained by recrystallization from the filtrate. HPLC analysis was carried out as in the analysis of 4-NP- AOZ, showing a single peak with retention time of 6.6 min. The pure compound showed similar UV/vis spectrum with a major peak absorbance around 323 nm and a minor peak absorbance around 230 nm in the same solvent system, characteristic of the common 4- nitrophenyl-hydrazone chromophore present.

Example 3 Evidence of conversion of pure furazolidone to 4-NP-AOZ

[0028] Generation of 4-NP-AOZ from pure furazolidone was carried out in similar manner as described in Example 1. The procedure was as follows:

[0029] A solution of furazolidone (FZD, 10 ug, 0.044 umole) in 0.5 ml of 1% DMF/water solution was treated with 1 ml of 1 N HCl and 50 ul of 40 mM 4-NBA (2 umole) in DMSO. The solution was heated on a heating block at 80⁰C for 15 min, neutralized with 1 ml of 1 M K₂ HPO₄/O.8 N NaOH and extracted with 2 ml of ethyl acetate. One ml of solvent extract was withdrawn and dried in 6O⁰C water bath under air flow for 10 minutes. The dried residue was first dissolved with 50 ul of acetonitrile and then diluted with 450 ul of water to give a sample solution A for HPLC analysis. Two fifths (200 ul) of the sample solution was injected onto an HPLC system equipped with a photodiode array detector and driven by software which is capable of system control, monitoring, spectral analysis of the peaks and data processing/reporting. The HPLC column was a reverse phase column (PLRP-S, 5 urn,

100 A°, 4 x 150 mm) as described in Example 2. [0030] The HPLC chromatogram was monitored at 323 nm for the presence of 4-NP-AOZ which has a major peak absorbance at 323.9 nm and a minor peak absorbance at 231.6 nm. As negative control, two other sample solutions (B and C) were prepared in the same manner except without the addition of FZD or 4-NBA, respectively. FZD has peak absorbance at 366.6 and 261.0 nm. Its absorbance at 323 nm is about !Λ as intense as the 366.6 nm peak. 4- NBA has peak absorbance at 266.9 nm. Its absorbance at 323 nm is about 6% of the peak intensity.

[0031] The resulting HPLC chromatograms showed the following:

[0032] Sample solution A chromatogram contained predominantly the 4-NP-AOZ peak (6.9 min) and the excess of 4-NBA (13.3 min). Very little of unreacted FZD (peak 4.20 min) was detected.

[0033] Sample solution B chromatogram contained essentially only the 4-NBA peak at 13.2 minutes was detectable in the sample solution B chromatogram. No FZD and 4-NPAOZ peaks were detectable indicating that no 4-NP-AOZ can be generated in the absence of FZD.

[0034] Sample solution C chromatogram contained a peak at 4.18 minutes (FZD). A completely flat baseline was observed beyond 5.5 min of elution indicating that no 4-NP- AOZ can be generated in the absence of 4-NBA.

Claims

1. A method for determining that an animal has contacted a nitrofuran comprising detecting a nitrofuran metabolite in a biological sample derived from the animal, said detecting comprising the steps of:

a) hydrolyzing a biomolecule metabolite in the sample, said hydrolyzing comprising reacting the sample with a nitrobenzaldehyde to produce a NP derivative of said nitrofuran metabolite; and b) contacting the sample with an antibody, the antibody having an affinity to the NP derivative of said nitrofuran metabolite, . ...

characterized in that a concentration of nitrobenzaldehyde comprises greater than 10 millimolar.

2. The method of claim 1 wherein the antibody is produced using an immunogen comprising a synthetically derived protein bound metabolite of said nitrofuran.

3. The method of any one of claims 1 and 2 wherein the antibody is produced using an immunogen comprising an N-linked derivative of a nitrofuran.

4. The method of any one of claims 1 and 2 wherein the antibody is produced using an immunogen comprising an S-linked derivative of a nitrofuran.

5. The method of claim 1 further characterized in that said hydrolyzing comprises incubating at a temperature between 37 degrees C and about 100 degrees C.

6. The method of claim 1 further characterized in that said hydrolyzing comprises incubating at above 37 degrees C.

7. The method of claim 1 further characterized in that said hydrolyzing comprises incubating at about 80 degrees C.

8. The method of claim 1 wherein the biological sample comprises a shrimp homogenate.

9. The method of claim 1 wherein the concentration of nitrobenzaldehyde comprises 40 millimolar.

10. Then method of claim 1 wherein the nitrobenzaldehyde comprises a 4-nitrobenzaldehyde.

11. The method of any one of claims 1-10 wherein the hydrolyzing comprises reacting the sample with a substituted nitrobenzaldehyde.

12. The method of any one of claims 1-10 wherein the hydrolyzing comprises reacting the sample with a non-substituted nitrobenzaldehyde.

13. A method for determining that an animal has contacted a nitrofuran comprising detecting a nitrofuran metabolite in a biological sample derived from the animal, said detecting comprising the steps of:

a) hydrolyzing a biomolecule metabolite in the sample, said hydrolyzing comprising reacting the sample with a nitrobenzaldehyde to produce a NP derivative of said nitrofuran metabolite; and b) performing liquid chromatography detection on said NP derivative,

14. The method of claim 13 further characterized in that said hydrolyzing comprises incubating at a temperature between 37 degrees C and about 100 degrees C.

15. The method of claim 13 further characterized in that said hydrolyzing comprises incubating at above 37 degrees C.

16. The method of claim 13 further characterized in that said hydrolyzing comprises incubating at about 80 degrees C.

17. The method of claim 13 wherein the biological sample comprises an animal homogenate.

18. The method of claim 13 wherein the hydrolyzing occurs in less than 2 hours.

19. The method of claim 13 wherein the hydrolyzing occurs in about 15 minutes.

20. Then method of claim 13 wherein the nitrobenzaldehyde comprises a 4- nitrobenzaldehyde.

21. The method of any on of claims 13-20 wherein performing the liquid chromatography is in conjunction with using a mass spectral detection.

22. A method for determining that an animal has contacted a nitrofuran comprising detecting a nitrofuran metabolite in a biological sample derived from the animal, said detecting comprising the steps of:

a) hydrolyzing a biomolecule metabolite in the sample, said hydrolyzing comprising reacting the sample with nitrobenzaldehyde to produce an NP derivative of said nitrofuran metabolite; and b) contacting the sample with an antibody, the antibody having an affinity to the NP derivative of said nitrofuran metabolite,

characterized in that said hydrolyzing comprises incubating at a temperature above 37 degrees C.

23. The method of claim 22 further characterized in that the concentration of nitrobenzaldehyde comprises greater than 10 millimolar.

24. The method of claim 22 wherein the concentration of nitrobenzaldehyde comprises 40 millimolar.

25. The method of claim 22 wherein the antibody is produced using an immunogen comprising a synthetically derived protein bound metabolite of said nitrofuran.

26. The method of any one of claims 22-25 wherein the antibody is produced using an immunogen comprising an N-linked derivative of a nitrofuran.

27. The method of any one of claims 22-25 wherein the antibody is produced using an immunogen comprising an S-linked derivative of a nitrofuran.

28. The method of claim 22 further characterized in that said hydrolyzing comprises incubating at a temperature between about 37 degrees C and about 100 degrees C.

29. The method of claim 22 further characterized in that said hydrolyzing comprises incubating at about 80 degrees C.

30. The method of claim 22 wherein the biological sample comprises a shrimp homogenate.

31. Then method of claim 22 wherein the nitrobenzaldehyde comprises a 4- nitrobenzaldehyde.

32. A method for determining that an animal has contacted a nitrofuran comprising detecting a nitrofuran metabolite in a biological sample derived from the animal, said detecting comprising the steps of:

a) hydrolyzing a biomolecule metabolite in the sample, said hydrolyzing comprising reacting the sample with a nitrobenzaldehyde to produce an NP derivative of said nitrofuran metabolite; and b) performing a liquid chromatography detection on said NP derivative,

characterized in that said hydrolyzing comprises incubating at greater than 37 degrees C.

33. The method of claim 32 further characterized in that the nitrobenzaldehyde concentration is greater than 10 millimolar.

34. The method of claim 32 further characterized in that said hydrolyzing comprises incubating at a temperature between about 37 degrees C and about 100 degrees C.

35. The method of claim 32 further characterized in that said hydrolyzing comprises incubating at a temperature of about 80 degrees C.

36. Then method of claim 32 wherein the nitrobenzaldehyde comprises a 4- nitrobenzaldehyde.

37. A method for determining that an animal has contacted a nitrofuran comprising detecting a nitrofuran metabolite in a biological sample derived from the animal, said detecting comprising hydrolyzing a biomolecule metabolite in the sample, said hydrolyzing comprising reacting the sample with a nitrobenzaldehyde to produce an NP derivative of said nitrofuran metabolite, wherein the combination of either or both incubation temperature of greater than 37 degrees C and a nitrobenzaldehyde concentration of greater than 10 mM, is effective to allow an acid hydrolysis to occur in less than 16 hours.