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| Orientation and Training Food and Drug Administration |
| DOCUMENT NO.: IV-09 | VERSION NO.:1.2 | Section 9 - Seafood Chemistry | EFFECTIVE DATE: 07/22/2004 | REVISED: 06/27/2008 |
9.3 Chemotherapeutics in Seafood
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researchers in ORA and may or may not be published in a journal or the AOAC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):
- Why does MG absorb energy in the visible range and LMG does not?
- Why does MG and LMG adsorb onto the propylsulfonic acid cation exchange
solid phase extraction (PRS-SPE) column during the extraction procedure?
- What is the elution solvent for the PRS-SPE column and why does it elute
the analytes?
- Why did the method developers choose a cyano (CN) analytical column?
- What effects would varying the composition (ratio of acetonitrile to acetate
buffer) of the mobile phase have on peak retention? Why?
- 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?
- Why is it important to "pre-treat” the mobile phase with
PbO2 prior to use?
- 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?
- 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
- 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.
- Roybal et.al. (1995). Journal of AOAC International, 78(2),
453-457.
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 CAP-producing biota in their environment, are available for
view at http://intranet.cfsan.fda.gov/OC/pages/panda.htm.
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
- Why is it OK 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?
- What is the advantage of performing shrimp homogenization using dry ice?
What precautions should be taken?
- Why are standards made up in blank matrix extract?
- What are criteria for successful confirmation of the presence of CAP in
crab meat?
- How does one best use mass spectral data to mathematically confirm the
presence of a target analyte?
D. References
CFSAN lists approved chloramphenicol LC/MS methods (for both seafood and honey)
on their website: http://www.cfsan.fda.gov/~frf/capintro.html .
These methods were developed within ORA, primarily at the Pacific Regional
Laboratory Northwest (Seattle) and the Animal Drugs Research Center (Denver).
Check with your laboratory management or CPGM 7301.018 for which method to
use based on the equipment available in your laboratory. As of FY 2004, the
following methods were included for use in regulatory analyses:
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.
- Roybal, J. E. (1998) Chloramphenicol and related drugs, in Turnipseed & Long
(Eds.), Analytical procedures for drug residues in food of animal origin.
West Sacramento, CA: Science Technology System.
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
- 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
- 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.
- 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.
- 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.
- 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.
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