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Volume IV - 9.3 Chemotherapeutics in Seafood

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Orientation and Training

Food and Drug Administration

DOCUMENT NO.:
IV-09
VERSION NO.: 1.5

Section 9 - Seafood Chemistry

EFFECTIVE DATE: 07-22-04REVISED: 02-14-13

 

Over the past several years, there has been a significant increase in the commercial production, and consumption of aquacultured products.  As this industry grows, so does the use of approved and non-approved chemicals.  Fish or seafood raised in a controlled environment can be better protected from catching wild parasites and fed to promote growth.  On the other hand, fish/seafood raised in a high-density setting can cause quick exchange of disease, resulting in the need to pro-actively or actively treat with drugs.  The use of non-approved chemical compounds on aquaculture products, or the misuse of approved chemicals, may have an impact on the safety of consumers.  The main public health concern is with antibiotic residues finding their way into the environment (by pond run-off, surviving sewage treatment, etc.) and influencing the development of antibiotic-resistant strains of bacteria; or by the chronic ingestion of antibiotics in our diet giving rise to antibiotic-resistant strains of bacteria.

The FDA routinely monitors domestic and imported aquaculture and seafood products for drug residues under Compliance Program Guidance Manual 7304.018 "Chemotherapeutics in Seafood." The drugs (and species) are on surveillance status and may change from year to year. The analytical methods are developed primarily by researchers in ORA and may or may not be published in a journal or the AOAC Official Methods of Analysis, but only circulated by DFS or published in FDA’s Laboratory Information Bulletin. However, the compliance program will serve as guidance on which method is correct to use. For the listed specie-drug combinations, a determinative analytical method is specified, but only some have an additional confirmation method listed (typically LC or GC with MS). The training methods discussed in this section focus on the determinative methods only. Training for the associated confirmation methods may be done on an individual basis. The chemistry involved with these methods are some of the most difficult the analyst will find in the field laboratory because of the challenge of finding residue levels of analyte within a complex matrix. These methods are considered intermediate and advanced training.

 9.3.1 Malachite/Leucomalachite Green [Advanced]

A.  Background

Malachite green is a cationic triphenylmethane dye used as a topical fungicide in the aquaculture industry to control the growth of fungi on fish and incubating eggs.  Studies have shown that malachite green excretes rapidly, but the base, leucomalachite green, has a long residence time in muscle tissue.  This exercise instructs the trainee to determine the amount of malachite green present in a sample of catfish tissue. 

B.  Exercise

Review all references. The trainer will provide a sample that has been fortified with 10 ppb with malachite green and leucomalachite green.  Determine the amount of malachite green and leucomalachite green present, using the method described in Compliance Program 7304.018.  In addition to the training sample, prepare and take through the method a fortified sample (20 ppb) and a negative control sample.  Determine the % recovery of the fortified sample.  Report all findings on an analytical worksheet.

C.  Questions

Consider the chemical structures of malachite green (MG) and leucomalachite green (LMG):

  1. Why does MG absorb energy in the visible range and LMG does not?
  2. Why does MG and LMG adsorb onto the propylsulfonic acid cation exchange solid phase extraction (PRS-SPE) column during the extraction procedure?
  3. What is the elution solvent for the PRS-SPE column and why does it elute the analytes?
  4. Why did the method developers choose a cyano (CN) analytical column?
  5. What effects would varying the composition (ratio of acetonitrile to acetate buffer) of the mobile phase have on peak retention? Why?
  6. The detector is set at 618 nm, the maximum Absorbance wavelength for MG, which is blue-green in color. How is it LMG, which is colorless, is also detected at this wavelength?
  7. Why is it important to "pre-treat" the mobile phase with PbO2 prior to use?
  8. The method System Suitability requirements state that the LMG peak height should be >90% of that for MG for equivalent weight (ng) injected. What is the probable cause for failure to meet this requirement?
  9. A chromatogram for an injection of a LMG/MG standard shows two peaks, but they are misshapen: broad, with shoulders, or "fronting". What could be causing this?

D.  References

  1. U.S. Food & Drug Administration, Center for Food Safety and Applied Nutrition. Compliance program guidance manual, Compliance Program 7304.018, Chemotherapeutics in seafood. Washington DC: U.S. Government Printing Office.
  2. LIB 4363, Quantitative and Confirmatory Analyses of Malachite Green and Leucomalachite Green Residues in Fish and Shrimp, November 2005.

 9.3.2 Chloramphenicol [Intermediate]

A. Background

Chloramphenicol (CAP) is a powerful antibiotic often reserved for cases of resistant bacterial infections in humans; and in rare cases is implicated in the development of aplastic anemia – thus a possible danger in its therapeutic use or by exposure as an unexpected food adulterant. It is also effective against common shellfish diseases, thus its use in veterinary practice. Testing of shrimp, crab, or crayfish for chloramphenicol has waxed and waned over the years. Originally CAP testing was strictly a GC/MS method, but now ORA utilizes several different LC/MS/MS methods for simultaneous determination and confirmation. Regulatory testing is based on the premise that the presence of CAP in seafood is by human addition and may render a food unsafe.  Declarations from experts in the field, stating that CAP is not being absorbed by the shrimp from indigenous.

B. Exercise

Review all references. The trainer will provide a homogenized shrimp sample that has been fortified at 0.3 ppb with CAP, as well as additional blank homogenized shrimp meat for use as described below. (The trainee should also gain practice with the dry-ice homogenization process by assisting an experienced analyst.) Determine the amount of CAP present, using a method described in Compliance Program 7304.018. In addition to the training sample, prepare and take through the method a fortified sample (0.3 ppb or as appropriate) and a negative control sample. Determine the % recovery of the fortified sample. Report all findings on an analytical worksheet.

C. Questions

  1. Why is it permissible to use plastic centrifuge tubes plus aspiration for liquid-liquid extraction in the Rupp and Stuart methods (see below) versus traditional separatory funnels, such as in the Neuhaus method? What are the advantages?
  2. What is the advantage of performing shrimp homogenization using dry ice? What precautions should be taken?
  3. Why are standards made up in blank matrix extract?
  4. What are criteria for successful confirmation of the presence of CAP in crab meat?
  5. How does one best use mass spectral data to mathematically confirm the presence of a target analyte?

D. References

Check with your laboratory management or CPGM 7301.018 for which method to use based on the equipment available in your laboratory. The following methods were included for use in regulatory analyses but not limited to:

Shrimp: 

Crab: 

Crawfish: 

Dry ice homogenization: 

  • Bunch, E. A., et al. "Homogenous sample preparation of raw shrimp with the aid of dry ice, " JAOAC Int., 78:883-887. 

Other:

  • U.S. Food & Drug Administration, Center for Food Safety and Applied Nutrition. Compliance program guidance manual, Compliance Program 7304.018, Chemotherapeutics in seafood. Washington DC: U.S. Government Printing Office.

 9.3.3 Oxolinic Acid [Intermediate]

A. Background

Oxolinic acid (OA) is a quinolone antibacterial useful in the treatment or prevention of aquaculture diseases.  It is reported that OA residues may persist in fish many days post dosing, especially in the skin and bones.  Cooking may release OA residues from bone and skin into the muscle and water. (Zomer & Charm, 1998).

B. Exercise

Review references. The trainer will provide a homogenized salmon sample that has been fortified at 20 ppb with OA, as well as additional blank homogenized salmon meat for use as described below.  (The trainee should also gain practice filleting a salmon by assisting an experienced analyst.)  Determine the amount of OA present, using the determinative method described in Compliance Program 7304.018. In addition to the training sample, prepare and take through the method a fortified sample (20 ppb or as appropriate) and a negative control sample. Determine the % recovery of the fortified sample. Report all findings on an analytical worksheet.

C. Questions

  1. State the general difference between oxolinic acid and oxalic acid.  In which part of the method is oxalic acid used and what is its function?

D. References

  1. Larocque, L., et al. (1991). Determination of oxolinic acid residues in salmon muscle tissue by liquid chromatography with fluorescence detection. J. AOAC Int. 74(4):608-611.
  2. Pfenning, A.P., et al. (July 1996). Confirmation of incurred residues of flumequine, nalidixic, oxolinic, and piromidic acids in shrimp and salmon. FDA Laboratory Information Bulletin, No. 4039.
  3. U.S. Food & Drug Administration, Center for Food Safety and Applied Nutrition. Compliance program guidance manual, Compliance Program 7304.018, Chemotherapeutics in seafood. Washington DC: U.S. Government Printing Office.
  4. Zomer, E., and Charm, S. E. (1998) Quinolones, in Turnipseed & Long (Eds.), Analytical procedures for drug residues in food of animal origin. West Sacramento, CA: Science Technology System.