Research Highlights on Prohibited Antimicrobial Agents Used in Seafood

Research notes were compiled by Yun-Hwa Hsieh, Ph.D., Florida State University

Chloramphenicol

Background:

Chloramphenicol (CAP) is a broad spectrum antibiotic isolated in 1947 from Streptomyces venezuelae. It has been used since the 1950s to combat serious human infections and is commonly used in food producing animals to enhance the production. CAP is harmful for humans because it can cause aplastic anemia, which could lead to leukemia. The CAP-induced aplastic anemia is irreversible and not dose-dependent. Therefore, it has not been possible to identify a safe level of human exposure to CAP. The International Agency for Research on Cancer (IARC) in 1990 considered CAP as probably carginogenic to humans. CAP also induce gray baby syndrome, a pathologic situation that could be fatal up to 40% of the time. CAP affect newborn babies as it can pass through the placental barrier and excreted in breast milk (3).


CAP is highly lipid soluble, small, and unbound to protein. The bioavailability of CAP is very high after oral administration. It diffuses readily into all tissues of body including brain, but at different concentrations (10). The parent drug is rapidly metabolized by conjugation with glucuronic acid in liver. CAP is also degraded by intestinal bacteria into dehydrochloramphenicol and undergoes nitroreduction, which can cause the single strand breaks of DNA and induction of faulty chromatid exchange in bone marrow
(http://www.usp.org/pdf/EN/veterinary/chloramphenicol.pdf).


CAP was banned for use in food-producing animals in USA and Europe. A "zero tolerance" has been established by the Food, Drug Administration (FDA) for CAP residues in both domestic and imported animal products (2) However, there is still possibility of use of CAP in sea food products due to its broad spectrum of activity and low cost. Some states detected CAP in frozen seafood (shrimp, crabs and crayfish) imported from China and other southeast Asian countries.


Assay Methods:

Several analytical methods have been developed for the detection and quantification of CAP in seafood samples. They include liquid chromatography (LC), gas chromatography (GC), mass spectrometry (MS), and tandem MS-based techniques (3, 6, 9). The FDA approved methods for detection of CAP in seafood are HPLC-MS, GC-ECD (electron capture detector), GC-MS with a detection limit of 5 parts per billion (5 ppb) level in the sea foods. Canada and the European Union have refined their methods to detect even the lower levels and should have taken action on food products imported from China and Vietnam, which are report to be contaminated with CAP. Recently, the methodology has been refined and sensitivity of method is increased to detect the CAP at 1 ppb and is further modifying the methods to detect 0.3 ppb, which will place the US methodology in line with Canada and European Union.
(http://wwwfda.gov/bbs/topics/NEWS/2002/NEW00815.html).


Although the more recent LC/MS/MS methods have improved the sensitivity of the method, enabling reliable confirmation of CAP residues at <1 ppb levels (4, 7), these instrumental methods require expensive instruments and high skills for operation, and sample analysis is time consuming. Therefore, development of rapid immunoassays has become attractive because they offer the capability for a rapidly detection of CAP residue at trace levels without laborious sample preparation procedures.

 

Several immunoassays including radio immunoassay, enzyme immunoassays, and chemiluminescence immunoassay (5) have been reported in the literature. The reported detection limits are usually depending on the types and size of sample. The first known enzyme immunoassay for the detection and quantification of CAP was reported in 1984 (1). This assay uses rabbit anti-CAP antibody, however, shows cross reactivities with CAP sodium succinate and thiamphenicol, an experimental antibiotic similar in structure to CAP.


Enzyme-linked immunosorbent assay (ELISA) has been used to screen the presence of CAP in 10 kinds of matrices, including seafood, meat, honey, etc. and compared the results with different chromatography methods. The techniques employed were able to detect CAP residue at the level of 0.1-10 μg/kg in samples (8).


Biacore has developed a Qflex® kit Chloramphenicol that provides rapid, sensitive, and reliable automated detection of chloramphenicol residues in shellfish. The limit of detection of kits for chloramphenicol residues is 0.04 ppb in shellfish
(http://www.biacore.com/food/products/biacore_q/qflex_kits/index.html?backurl=/food/food_analysis/index.html).


References:

  1. Campbell GS, Mageau RP, Schwab B & Johnston RW. 1984. Detection and quantitation of chloramphenicol by competitive enzyme-linked immunoassay. Antimicrob Agents Chemother 25(2):205-211.
  2. FDA. 1982. In Code of federal regulations. Title 21 - Food and drugs. Office of the Federal Register National Archives and Records Service, General Services Administration Washington, D.C.: U.S. Government Printing Office. p. 404-414.
  3. Gikas E, Kormali P, Tsipi D & Tsarbopoulos A. 2004. Development of a rapid and sensitive SPE-LC-ESI MS/MS method for the determination of chloramphenicol in seafood. J Agric Food Chem 52(5):1025-1030.
  4. Hammack W, Carson MC, Neuhaus BK, Hurlbut JA, Nochetto C, Stuart JS, Brown A, Kilpatrick D, Youngs K, Ferbos K & Heller DN. 2003. Multilaboratory validation of a method to confirm chloramphenicol in shrimp and crabmeat by liquid chromatography-tandem mass spectrometry. J AOAC Int 86(6):1135-1143.
  5. Lin S, Han SQ, Liu YB, Xu WG & Guan GY. 2005. Chemiluminescence immunoassay for chloramphenicol. Anal Bioanal Chem 382(5):1250-1255.
  6. Pfenning AP, Roybal JE, Rupp HS, Turnipseed SB, Gonzales SA & Hurlbut JA. 2000. Simultaneous determination of residues of chloramphenicol, florfenicol, florfenicol amine, and thiamphenicol in shrimp tissue by gas chromatography with electron capture detection. J AOAC Int 83(1):26-30.
  7. Rupp HS, Stuart JS & Hurlbut JA. 2005. Liquid chromatography/tandem mass spectrometry analysis of chloramphenicol in cooked crab meat. J AOAC Int 88(4):1155-1159.
  8. Shen HY, and Jiang HL. 2005. Screening, determination and confirmation of chloramphenicol in seafood, meat and honey using ELISA, HPLC-UVD, GC-ECD, GC-MS-EI-SIM and GCMS-NCI-SIM methods. Anal. chim. Acta 535:33-41.
  9. Takino M, Daishima S & Nakahara T. 2003. Determination of chloramphenicol residues in fish meats by liquid chromatography-atmospheric pressure photoionization mass spectrometry. J Chromatogr A 1011(1-2):67-75.
  10. Yunis AA. 1988. Chloramphenicol: relation of structure to activity and toxicity. Annu Rev Pharmacol Toxicol 28:83-100.

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Fluoroquinolone

Background:

Fluoroquinolones are a subset of broad based antibiotics in the quinolone family. The majority of the clinical use of quinolones is in the form of fluoroquinolones. The base chemical in quinolones is nalidixic acid. The fluoroquinolone variety has a fluoro group attached to the central ring structure that makes up nalidixic acid.


Some varieties of fluoroquinoles include (3):

Enrofloxacin (veterinary)
Flumequine
Ciprofloxacin (Cipro, Ciproxin) (Human)
Lomefloxacin (Maxaquin)
Norfloxacin (Noroxin, Quinabic, Janacin)
Ofloxacin (Floxin, Oxaldin, Tarivid)
Levofloxacin (Cravit, Levaquin, Quixin)
Grepafloxacin (Raxar)
Sparflaxacin (Zagam)
Trovafloxacin (Trovan)
Gemifloxacin (Factive)
Moxifloxacin (Avelox)

Quinolones were discovered in 1962 and were originally used to treat urinary tract infections. Over time several generations of antibacterial drugs were developed including fluoroquinolones. The first fluoroquinolone developed was flumequine. The first generation was found to be toxic. Eventually other generations were developed that were useful on gram-positive and gram-negative bacteria (1).


The mechanism by which fluoroquinolones works is by blocking DNA gyrase which is a topoisomerase II enzyme. By blocking gyrase it prevents bacteria DNA replication. By interfering with DNA replication fluoroquinolones actively kill bacteria. Since it can enter cells very easily fluoroquinolones are often useful in treating intracellular bacteria (7).


Use of fluoroquinoles on various infections was once deemed safe. Fluoroquinolones have been used successfully to treat salmonellosis, are commonly the recommended agent of first choice and have also proven useful for the treatment of infections caused by multiple antibiotic-resistant (MAR) strains (7). Side effects became more evident in the 1980’s and use of fluoroquinolones has become an important public concern. Widespread use in veterinary medicine in Europe is blamed for many fluroquinolone-resistant bacteria strains. Quinolone-resistant Salmonella can also be cross-resistant to other agents including chloramphenicol and tetracycline (7).


Common issue today is with drug residues in animal food products. Ciprofloxacin was found in milk samples as well as other animal products including fish. Generally it is formed as a metabolite of enrofloxacin which has a long depletion time in fish edible tissues (6).


Assay Methods:

Liquid chromatography (LC) has been the most commonly used method for determining fluoroquinolones in seafood. Other methods used are gas chromatography (GC), capillary electrophoresis (CE) as well as high performance thin-layer chromatography (HPTLC). More recent chromatographic separation techniques for determination and confirmation of the identitity of fluoroquinolones in seafood are paired with mass spectroscopy (MS) or tandem MS detection. The detection limit could be as sensitive as 1-3 mg/kg. It should be noted that the recovery of various fluoforquinolone compounds in different sample matrices varies (4, 5, 8).


Immunoassay techniques have been developed rescently to rapidly screen fluoroquinolone levels in foods. Generic and specific enzyme linked-immunosorbent assays (ELISA) for both the quinolones and fluoroquinolones have also been developed that uses the cross-reactivity of an antibody raised against norfloxacin linked to ovalbumin to detect nine different drugs in these classes (2). Detection of up to 15 fluoroquinolones including the base compound nalidixic acid in most of the animal tissues is less than 10 mg/kg or less (2, 3).


References:

  1. Ball P. 2000. Quinolone generations: natural history or natural selection? J Antimicrob Chemother 46 Suppl T1:17-24.
  2. Bucknall S, Silverlight J, Coldham N, Thorne L & Jackman R. 2003. Antibodies to the quinolones and fluoroquinolones for the development of generic and specific immunoassays for detection of these residues in animal products. Food Addit Contam 20(3):221-228.
  3. Huet AC, Charlier C, Tittlemier SA, Singh G, Benrejeb S & Delahaut P. 2006. Simultaneous determination of (fluoro)quinolone antibiotics in kidney, marine products, eggs, and muscle by enzyme-linked immunosorbent assay (ELISA). J Agric Food Chem 54(8):2822-2827.
  4. Johnston L, Mackay L & Croft M. 2002. Determination of quinolones and fluoroquinolones in fish tissue and seafood by high-performance liquid chromatography with electrospray ionisation tandem mass spectrometric detection. J Chromatogr A 982(1):97-109.
  5. Juan-Garcia A, Font G & Pico Y. 2006. Determination of quinolone residues in chicken and fish by capillary electrophoresis-mass spectrometry. Electrophoresis 27(11):2240-2249.
  6. Lucchetti D, Fabrizi L, Guandalini E, Podesta E, Marvasi L, Zaghini A & Coni E. 2004. Long depletion time of enrofloxacin in rainbow trout (Oncorhynchus mykiss). Antimicrob Agents Chemother 48(10):3912-3917.
  7. Piddock LJ. 2002. Fluoroquinolone resistance in Salmonella serovars isolated from humans and food animals. FEMS Microbiol Rev 26(1):3-16.
  8. Schneider MJ, Vazquez-Moreno L, Bermudez-Almada Mdel C, Guardado RB & Ortega-Nieblas M. 2005. Multiresidue determination of fluoroquinolones in shrimp by liquid chromatography-fluorescence-mass spectrometry. J AOAC Int 88(4):1160-1166.

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Malachite Green

Background:

Malachite green (MG), also called aniline green, basic green 4, diamond green B, or victoria green B, is a toxic triphenylmethane dye primarily used as a dyeing agent of textiles. MG is also used as a biological stain for microscopic analysis of cell and tissue samples. Leuco-malachite green (LMG), a reduced product and major metabolite of MG, is used as a detection agent for latent blood in criminalistics. Because of its ecto-parasiticide, fungicide and antiseptic properties MG was introduced in aquaculture industry in 1993 ( 5).


After administration, MG is readily absorbed by fish and metabolically reduced to the leucomalachite green (LMG) which accumulates in fatty tissue of the fish. The majority of persistent residues present in fish are, therefore, in the form of LMG. LMG is lipophilic and can remain in fatty tissue for extended periods of time (9). Numerous incidences of MG misuse in aquaculture have occurred, and analytical methods are needed to monitor for low levels of this drug in fish tissue.


MG is related structurally to other carcinogenic triphenylmethane dyes: gentian violet, a thyroid and liver carcinogen in rodents, and pararosaniline, a bladder carcinogen in humans. Animal study indicated that LMG causes a greater number and more severe changes than MG. Examination of DNA and liver extracts indicated that the compounds may be metabolized in a manner similar to carcinogenic aromatic amines. Therefore, The main concern over the use of MG is due to the potential for consumer exposure to this suspected mutagen and teratogen based on its structural similarity to known carcinogens (6, 7).


For these reasons, MG and LMG are bannd as an aquaculture veterinary drug in many countries including the United States, Canada and the European Union (3). Although MG is not approved for use by U.S. Food and Drug Administration (FDA), the use of MG in worldwide aquaculture still continues because of low cost, availability and high efficacy (8).


Assay Methods:

Both MG and LMG are prohibited in seafood. Method should detect both the chromatic and leuco forms of MG residues. The European Commission requires that methods be able to determine the sum of MG and LMG residues at the minimum performance limit of 2 ng/g (1). The method proposed by FDA does not distinguish between LMG and MG residues that may be present, but rather allows quantification of the sum of the residues. The method has been already validated in catfish, trout, tilapia, basa, and shrimp, and verified in other species (4).


Several analytical approaches for the determination of residues of MG have been published. Because MG has a strong chromophore at 618 nm and is positively charged, detection schemes are often based on liquid chromatography, such as HPLC, with visible absorbance or mass spectrometric detection. For colorless reduced leuco form in fish tissue, oxidation is required to convert the residue to MG for visible analysis (10). However, freeze-thaw cycles, and storage temperature may affect the recovery of both MG and LMG (2, 5)


Under current U.S. Food and Drug Administration (FDA) sample testing protocols, MG and LMG residues are able to be quantitatively determined and confirmed at a minimum level of 1 ng/g (3). These potocols are using quantitative LC-VIS method and confirmatory LC-MS method for the analysis of LMG and MG incorporating an in-situ oxidation.


Three competitive ELISA kits are currently available for detection of MG and LMG. The ELISA kits are an immunoassays intended for use in the analysis of MG and LMG in fish tissue. The analysis can be completed within 1-2 h that offers a major advantage over conventional chromatographic methods and suitable for screening large numbers of routine samples.


References:

  1. Commission Decision 2004/25/EC as regards the setting of minimum required performance limits (MRPLs) for certain residues in food of animal origin. Off. J. Eur. Union L6:38-39.
  2. Allen JL, Gofus JE & Meinertz JR. 1994. Determination of malachite green residues in the eggs, fry, and adult muscle tissue of rainbow trout (Oncorhynchus mykiss). J AOAC Int 77(3):553-557.
  3. Andersen WC, Roybal JE & Turnipseed SB. 2005. Liquid chromatographic determination of malachite green and leucomalachite green (LMG) residues in salmon with in situ LMG oxidation. J AOAC Int 88(5):1292-1298.
  4. Andersen WC, Turnipseed SB & Roybal JE. 2006. Quantitative and confirmatory analyses of malachite green and leucomalachite green residues in fish and shrimp. J Agric Food Chem 54(13):4517-4523.
  5. Bergwerff AA & Scherpenisse P. 2003. Determination of residues of malachite green in aquatic animals. J Chromatogr B Analyt Technol Biomed Life Sci 788(2):351-359.
  6. Culp SJ, Beland FA, Heflich RH, Benson RW, Blankenship LR, Webb PJ, Mellick PW, Trotter RW, Shelton SD, Greenlees KJ & Manjanatha MG. 2002. Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green. Mutat Res 506-507:55-63.
  7. Culp SJ, Blankenship LR, Kusewitt DF, Doerge DR, Mulligan LT & Beland FA. 1999. Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1 mice. Chem Biol Interact 122(3):153-170.
  8. Halme K, Lindfors E & Peltonen K. 2007. A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 845(1):74-79.
  9. Srivastava S, Sinha R & Roy D. 2004. Toxicological effects of malachite green. Aquat Toxicol 66(3):319-329.
  10. Turnipseed SB, Andersen WC & Roybal JE. 2005. Determination and confirmation of malachite green and leucomalachite green residues in salmon using liquid chromatography/mass spectrometry with no-discharge atmospheric pressure chemical ionization. J AOAC Int 88(5):1312-1317.

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Nitrofuran

Background:

Nitrofurans are synthetic antibiotics characterized by their basic chemical structure (nitrofuran ring) that are frequently used as veterinary drugs because of broad-spectrum antibacterial effect against gram positive and gram negative bacteria, and their role as growth promoter. Four most widely used nitrofurans are Furazolidone, Furaltadone, Nitrofurantoin, and Nitrofurazone (3).


Nitrofurans are banned for food producing animals by European Union (EU) and The US Food and Drug Administration (FDA). In the past, FDA permitted furazolidone aerosol powder and nitrofurazone topical powder as topical nitrofurans that can be used in cattle. The approval for nitrofuran drugs was withdrawn by FDA in 1992, followed by the prohibition of the extra label use of topical nitrofurans animal and human drugs in food-producing animals Due to the carcinogenic and mutagenic properties (1).


Nitrofuran compounds was used in Japan as antimicrobial agents in the processing of seafoods. Nitrofuran AF-3 (furylfuramide) and nitrofuran Z (nitrofuryl acrylamide) were two compounds approved by the Minister of the Welfare of Japan for use in the fish and fish-sausage industry (8). Now Japan has banned the use of nitrofurans and implemented zero tolerance for nitrofurans.


The tissue bound residues of nitrofurans are very stable against the common food processing techniques including cooking, baking, grilling and microwaving. Due to the insufficient assays for residual analysis and genotoxic and carcinogenic nature of nitrofurans, foods containing their residues at any concentration are not considered fit for human consumption. Patients showing hypersensitivity to nitrofurantoin show symptoms like sudden fever, malaise, cough, pleuritis, leucocytosis and sometimes eosinophilia. (2)


Since it has been banned from use in food animal production, furazolidone was found in imported poultry and prawns. Because nitrofurans are light sensitive and undergo rapid metabolism in animals, parent nitrofurans are not detected in most food products. The methods for detection of nitrofurans must be based on the detection of tissue-bound metabolized residues. The side chains of bound metabolites, such as 3-amino-2-oxzolidinone (AOZ) from furazolidone, 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) from furaltadone, 1-aminohydantoin (AHD) from nitrofurantoin, and semicarbazide (SEM) from nitrofurazone are more stable then their parent drug, hence used as targeted molecules for detection (3). This ban is commonly monitored by use of analytical methods to detect the AOZ (5).


Assay Methods:

Most of the analytical techniques used for the detection of nitrofurans are based on chromatograpy coupled with various detectors including high performance liquid chromatography (HPLC) with UV, diode array or electrochemical detection or Gas chromatography (GC) with electron capture detection system. Current methods are based on the acid hydrolysis of the bound residues of nitrofurans. AOZ could be released from the parent drug and protein bound residues under mild acidic conditions. Two different methods (i) solvent extraction and (ii) solid phase extraction have been used for the detection of nitrofurans. (4).


Since a definitive minimum required detection limit of 1 μg/kg is set in EU for nitrofuran drugs in poultry and aquaculture products, the laboratories testing for nitrofuran residues should have to reach at least this limit. A LC-tandem mass spectrometric method was developed by Chu and Lopez (3) for the determination of bound residues of nitrofuran drugs in shrimp with a detection limit of 1 ppb.


Recently, immunoassays have been developed for rapid and effective screening of tissue bound residues of nitrofurans. A 42-month project was launched by European Union called FoodBRAND (Food Bound Residues And Nitrofuran Detection) to improve the methods used for detection of nitrofuran antibiotics. One of the aims was to develop and validate immunoassay screening kits for the detection of bound residues of the nitrofurans (www.asfni.ac.uk/foodbrand).


Cooper et al (5) produced first polyclonal antibodies to detect AOZ, a stable metabolite from furazolidone. The antibody showed minimal cross reactivity with other metabolites of nitrofurans and significant cross reactivity with the parent drug furazolidone (35%). These antibodies were used for detection of AOZ in prawn tissue (6). The comparision of ELISA with LC-MS/MS showed a consistent result under estimation with 8 different prawn samples. The limit of detection for the assay was 0.1 mg/kg and the detection capablility was achieved at fortification between 0.4 and 0.7 mg/kg.


Since monoclonal antibody based assays are more specific, sensitive and continuous source of similar type of antibody, Diblikova et al (7) developed a monoclonal antibody based ELISA for AOZ quantification using simplified sample preparation. The assay was validated in different animal tissues (shrimp, poultry, beef and pork muscle). The detection capability of the assay was 0.4 mg/kg in shrimps. The monoclonal ELISA was also comparable with reference LC-MS/MS technique with excellent correlation within the concentration range of 0-32.1 μg/kg in the naturally contaminated shrimp samples.


References:

  1. FDA steps up seafood sampling. 2002. FDA Consumer 36(5):4.

    http://www.fda.gov/fdac/departs/2002/502_upd.html#seafood

     

  2. Back O, Liden S & Ahlstedt S. 1977. Adverse reactions to nitrofurantoin in relation to cellular and humoral immune responses. Clin Exp Immunol 28(3):400-406
  3. Chu PS & Lopez MI. 2005. Liquid chromatography-tandem mass spectrometry for the determination of protein-bound residues in shrimp dosed with nitrofurans. J Agric Food Chem 53(23):8934-8939.
  4. Conneely A, Nugent A & O'Keeffe M. 2002. Use of solid phase extraction for the isolation and clean-up of a derivatised furazolidone metabolite from animal tissues. Analyst 127(6):705-709.
  5. Cooper KM, Caddell A, Elliott CT, Kennedy DG. 2004. Production and characterization of polyclonal antibodies to a derivative of 3-amino-2-oxazolidinone, a metabolite of the nitrofurans furazolidon. Analytica Chimica Acta 520:79-86.
  6. Cooper KM, Elliott CT & Kennedy DG. 2004. Detection of 3-amino-2-oxazolidinone (AOZ), a tissue-bound metabolite of the nitrofuran furazolidone, in prawn tissue by enzyme immunoassay. Food Addit Contam 21(9):841-848.
  7. Diblikova I, Cooper KM, Kennedy DG, Franek M. 2005. Monoclonal antibody based ELISA for the quantification of nitrofurans metabolite 3-amino-2-oxazolidinone in tissues using a simplified sample preparation. Analytica Chimica Acta 540:285-292.
  8. Waters ME & Handy MK. 1969. Effect of nitrofurans and chlortetracycline on microorganisms associated with shrimp. Appl Microbiol 17(1):21-25.

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