DETERMINATION OF CHLORAMPHENICOL AND TWO METABOLITES IN CRAB BY LC-MS/MS  

Edward L.E. Jester*, Harold A. Flores Quintana, Kathleen R. El Said, Ann Abraham, and Ronald A. Benner, Jr.
 
U.S. Food and Drug Administration
Division of Seafood Science and Technology
1 Iberville Drive
Dauphin Island, AL 36528-0158, USA
edward.jester@fda.hhs.gov

Chloramphenicol (CAP) is a highly effective, broad-spectrum antibiotic, with a history of usage in food animals, including aquaculture species.  Its use has been associated with numerous toxic effects in humans, including bone marrow depression which can cause potentially fatal aplastic anemia.  Due to safety issues, CAP is banned from food animal use in the United States (U.S.), and many other countries.  The presence of CAP residues in aquaculture products is a major concern for the U.S. Food and Drug Administration, particularly with increases in imports, since it continues to be used for food animal disease treatment in some countries.  

CAP is rapidly eliminated following exposures in animals.  Depletion and metabolism studies of CAP in seafood have been limited. Currently, validated confirmatory analytical methods are available for monitoring CAP, but not its biomarkers.  For the purpose of identifying an appropriate marker residue for CAP exposure, we exposed crab to CAP, and identified the metabolites CAP-base (CAP-B) and CAP-alcohol (CAP-OH) as viable biomarkers.  

We present a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for CAP, CAP-B, and CAP-OH in crab muscle.  A single LC-MS/MS polarity switching, ESI (±) method for simultaneous analysis of CAP, CAP-B, and CAP-OH has been developed on a Waters Xevo TQ MS.  Separation of analytes was achieved in reverse phase mode on a Phenomenex Kinetex 1.7 µm PFP column (50 x 2.1 mm) in 5 min.  Retention times for CAP-OH, CAP, and CAP-B are 1.5, 1.9, and 2.2 min, respectively.  External standard calibration curves (0.125 to 4 ng/mL) were prepared from CAP, CAP-B, and CAP-OH reference material, with obtained R2 values of 0.9989, 0.9999, and 0.9999, respectively.  Interday repeatability of CAP, CAP-B, and CAP-OH standards (n = 7) ranged from 1 to 18%, with average repeatability of 12, 4, and 7%, respectively.