Assay Methods

Zero Tolerance

Since maximum residue limits for chloramphenicol and nitrofurans in foods have not been set, these particular broad spectrum antibiotics are regulated in the United States and European Union by a policy of “zero tolerance.” Governmental detection standards for zero tolerance vary among countries. (Anonymous, 2002.)

Current US Food and Drug Administration Detection Levels of Prohibited Antimicrobial Agents in Seafood

Chloramphenicol	0.3 ppb
Nitrofurans	1.0 ppb
Fluoroquinolones	5.0 ppb
Malachite green	1.0 ppb
Quinolones	10 ppb (Oxolinic Acid) and 20 ppb (Flumequine)
Ivermectin	10 ppb
Oxytetracycline	2.0 ppm

Source: Collette, 2006.

The following drugs (both animal and human), families of drugs, and substances are prohibited for extra-label uses in all food-producing animals:

Chloramphenicol Clenbuterol Diethylstilbestrol (DES) Dimetridazole Ipronidazole Other nitroimidazoles Furazolidone, Nitrofurazone, other nitrofurans Sulfonamide drugs in lactating dairy cattle (except approved use of sulfadimethoxine, sulfabromomethazine, and sulfaethoxypyridazine) Fluoroquinolones; and Glycopeptides

Source: US Food and Drug Administration, Center for Veterinary Medicine, 2002

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Prohibited Antimicrobial Agent Residue Analysis.

Residue analysis typically involves both screening and confirmatory methods. Highly specific quantitative methods for identifying antibiotic residues include the use of liquid chromatography (LC), high-performance liquid chromatography (HPLC), liquid chromatography with mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS). Current qualitative screening methods for antibiotics are microbiological tests, immunoassays, and receptor assay systems. Ideal methods for antibiotic residue analysis should have the quality attributes of accuracy, precision, sensitivity, simplicity, specificity, and wide applicability. They should be able to measure a wide range of veterinary drug residues – including metabolites and bound residues – directly in the food. (Weifen, 2002.)

 

Although, the chromatographic methods showed their effectiveness in detecting of these drug residues (MG and LMG), they have several disadvantages, such as multi-step sample preparation, long analysis time, need of expensive equipment, high skill for operation, and being impossible to use these methods in routine field screening. It requires thorough clean up of sample to obtain reliable results.

Enzyme-linked immunosorbent assay (ELISA) testing system is a popular and near ideal rapid assay system; it can be used in the field and as a rapid test for screening large number of routine samples. However, the detection limit, sensitivity, and reliability of the assay depend on the quality of the antibody used in a particular assay kit. If the antibody has some cross reactivity with similar molecules, with low affinity with the antigen, then the assay may have low sensitivity and obtain either false negative or false positive results or both. If the antibody used is highly specific and has high affinity to the target antigen, the assay may be highly sensitive and very reliable. One company’s assay may not be as good as a similar assay developed by a different company in terms of certain metabolites, if they use different antibodies. Therefore, ELISA should be evaluated case-by case, and cannot be generalized for all antibodies used or all metabolites from the same parent drug. It is important to read the manufacturer’s information regarding the specific detecting compound(s), the cross-reactivity of the assay and the detection limit of the assay. It is also recommended to compare several commercial ELISA assays and validate the assay internally with the samples to be analyzed (Hsieh, 2007).

Most of the commercially available immunoassays are competitive enzyme-linked immunosorbent assays (Competitive ELISA) which are suitable for a quantitative detection of the target drug residues (antigens). The basic principle of Competitive ELISA is based on binding of limited number of antigen-specific antibody molecules in liquid phase to solid phase bound antigen and is competitively inhibited by free liquid phase antigen in the sample under assay. The immobilized antibody is then revealed by using an enzyme-linked secondary antibody, which is subsequently reacting with an appropriate substrate for color development. The enzyme activity visualized as color intensity is measured by spectrophotometry and is inversely proportional to the concentration of antigen in the sample.

In general, chromatographic and immunoassays are complimentary methods but cannot replace each other. Immunoassay such as ELISA often picks up a group of related compounds (isoforms of the same compound or closely related compound or metabolites), but chromatography techniques separates and identifies individual ones. ELISA is best suited for rapid screening, while LC, HPLC and LC/MS and LC/MS/MS are intended for confirmation and identification. (Hsieh, 2007)

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Chloramphenicol

Research notes on chloramphenicol

US Food and Drug Administration Methods for Detection of Chloramphenicol Residue in Seafood

Shrimp:
• Determination of Chloramphenicol Residues in Shrimp and Crab Tissues by Electrospray Triple Quadrupole LC/MS/MS, July 2003 - US FDA Laboratory Information Bulletin No. 4306, June 2003 No. 6* (16 p.) - US FDA
• LC/MS/MS Analysis of Chloramphenicol in Shrimp, November 2002 - US FDA Laboratory Information Bulletin No. 4290, September 2002 No. 9* (18 p.) - US FDA

Crab:
• LC/MS/MS Analysis of Chloramphenicol in Crab Meat, July 2003 - US FDA Laboratory Information Bulletin No. 4302, April 2003 No. 4* (11 p.) - US FDA
• Determination of Chloramphenicol Residues in Shrimp and Crab Tissues by Electrospray Triple Quadrupole LC/MS/MS, July 2003 - US FDA Laboratory Information Bulletin No. 4306, June 2003 No. 6* (16 p.) - US FDA

Crawfish:
• LC/MS/MS Analysis of Chloramphenicol in Crawfish Meat, November 2002- US FDA Laboratory Information Bulletin No. 4303, April 2003 No. 4* (10 p.) - US FDA

For additional information on the US FDA methods, please contact: Dr. William R. Jones, Office of Seafood, E-mail: wjones1@cfsan.fda.gov, Telephone: (301) 436-1422

Rapid Assay Kits for Detection of Chloramphenicol Residue in Seafood

1. Charm II Amphenicols/Chlorampenicol amphenicols: 5 ppb, chlorampenicol: 0.2 ppb
2. Chloramphenicol EIA Kit (Antix Biotech Inc.) 0.05 ppb
3. Chloramphenicol EIA Test (Euro-Diagnostica) 0.02 ppb
4. Chloramphenicol, ELISA kit (Abraxis) 0.01 ppb
5. I'screen CAP (Tecna) 0.025 ppb
6. MaxSignal™ Chloramphenicol (CAP) ELISA Test Kit 0.025 ppb
7. Qflex® Kit Chloramphenicol (Biacore) 0.04 ppb
8. RIDASCREEN® Chloramphenicol 6.25 ppt
9. Transia® Plate Chloramphenicol (Biocontrol) 0.02 ppb

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Fluoroquinolones

Research notes on Fluroquinolones

US Food and Drug Administration Methods for Fluoroquinolone Residue Detection in Seafood

• Concurrent Determination of four Fluoroquinolones; Ciprofloxacin, Enrofloxacin, Sarafloxacin and Difloxacin in Atlantic Salmon Tissue by LC with Fluorescence Detection (determinative method for all Fluoroquinolones and all species), October 24, 2003* (14 p.)
• Confirmation of Fluoroquinolone Residues in Salmon and Shrimp Tissue by LC/MS: Evaluation of single Quadrupole and Ion Trap Instruments: Laboratory Information Bulletin 4298 (confirmatory testing for all Fluoroquinolones and all species), March 2003* (20 p.)
• Confirmation of Fluoroquinolones in Catfish Tissue by Electrospray LC/MS: Laboratory Information Bulletin 4108, August 1997* (16 p.)

Rapid Assay Kits for Fluoroquinolone Residue Detection in Seafood

1. Enrofloxacin ELISA Test Kit (Gentaur) 0.5 ppb
2. Flumequin ELISA (Gentaur) 0.5 ppb
3. Fluoroquinolones EIA (Euro-Diagnostica) 0.3 ppb
4. Fluoroquinolones Plate ELISA Kit (Abraxis) 10 ppb
5. MaxSignal™ Fluroquinolones ELISA Test Kit (BIOO Scientific) 1 ppb

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Malachite Green (MG), Leuco-Malachite Green (LMG) and Crystal Violet

Research notes on Malachite Green and Leuco-Malachite Green

US Food and Drug Administration Methods for MG, LMG and Crystal Violet Residue Detection in Seafood

• Determination and Confirmation of Leucomalachite Green in Salmon using No-Discharge Atmospheric Pressure Chemical Ionization LC-MSⁿ: Laboratory Information Bulletin 4333, 2004, p. 13*.
• Determination of Malachite Green and Leucomalachite Green in Salmon with In-Situ Oxidation and Liquid Chromatography with Visible Detection: Laboratory Information Bulletin 4334 2004, p. 13*
• Amendment: Application of Laboratory Information Bulletins 4333 and 4334 for the Determination of Leucomalachite Green and Malachite Green Residues in Catfish, Basa, Tilapia, Trout, and Shrimp, 2005*
• Quantitative and Confirmatory Analyses of Malachite Green and Leucomalachite Green Residues in Fish and Shrimp: Laboratory Information Bulletin 4363, 2005, p. 25*.
• Quantitative and Confirmatory Analyses of Crystal Violet (Gentian Violet) and Brilliant Green in Fish: Laboratory Information Bulletin 4395, 2007

Rapid Assay Kits for MG, LMG and Crystal Violet Residue Detection in Seafood

1. ELISA Kit for Malachite Green (Abraxis) .01 ppb
2. Leucomalachite Green ELISA Kit (Glyconex) .05 ppb
3. Malachite Green EIA Kit (Euro-Diagnostica) 0.2 ppb
4. MaxSignal™ Crystal Violet/LCV ELISA Test Kit (Bioo Scientific) 0.1 ppb
5. MaxSignal™ Malachite Green ELISA Test Kit (BIOO Scientific) 0.1 ppb
6. MG ELISA Kit (Glyconex) .05 ppb

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Nitrofurans

Research on Nitrofurans

US Food and Drug Administration Method for Nitrofuran Residue Detection in Seafood

• Detection of Nitrofuran Metabolites in Shrimp, April 1, 2004

Rapid Assay Kits for Nitrofuran Metabolites AOZ and AMOZ Residue Detection in Seafood

1. Charm AOZ (ELISA) 0.3 ppb
2. Charm AMOZ (ELISA) 0.5 ppb
3. MaxSignal™ Furaltadone (AMOZ) ELISA Test Kit (Bioo Scientific) 0.1 ppb
4. MaxSignal™ Furazolidone (AOZ) ELISA Test Kit (Bioo Scientific) 0.1 ppb
5. RIDASCREEN® Nitrofuran (AMOZ) ~0.2 ppb
6. RIDASCREEN® Nitrofuran (AOZ) ~0.1 ppb

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References

1. Anonymous. 2002. GAA Reviews Antibiotics Issues at Shrimp Summit. Global Aquaculture Advocate. April. p. 5.
2. Collette, R. 2006. Personal communication.
3. Hsieh, Y-H.P. 2007. Personal Communication.
4. US Food and Drug Administration, Center for Veterinary Medicine. 2002. Reminder -- Extra-Label Use of Fluroquinolones Prohibited. CVM Update. http://www.fda.gov/cvm/CVM_Updates/noeluflq.htm (August 31, 2007)
5. Weifen, W. and L. Hong. 2002 Determining Antibiotic Residues in Food: Current and Future Methods. October p. 77-78.

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