Liquid chromatography/tandem mass spectrometry analysis of chloramphenicol in cooked crab meat.

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method is described for the extraction, cleanup, determination, and confirmation of chloramphenicol (CAP) in cooked crab meat. The method involves pulverization of cooked crab meat with dry ice; extraction of the CAP into ethyl acetate (EtOAc); evaporation (by N2) of the EtOAc; addition of methanol, aqueous NaCl, and heptane; extraction of the lipids into the heptane, followed by extraction of the aqueous phase with EtOAc; evaporation (by N2) of the EtOAc; dissolution into methanol-water; filtration; and separation/detection/confirmation using LC/MS/MS. Crab meat was fortified at 0.25, 0.50, and 1.0 ng/g (ppb) chloramphenicol. Average absolute recoveries were 67, 84, and 86%, respectively, with relative standard deviation values all less than 1%. Four daughter ions (m/z 152, 176, 194, and 257) were monitored off the m/z 321 precursor ion. Determination was based on a standard curve using the peak areas of the m/z 152 daughter ion (the base peak) for standard solutions equivalent to 0.10, 0.20, 0.50, and 1.0 ppb in tissue (made with control crab extract). A set of 6 matrix controls (unfortified crab meat) was also analyzed, in which no chloramphenicol was detected. For identification purposes, the ion ratios (of each daughter ion versus the base daughter ion) of the fortified crab versus those of the chloramphenicol standards agreed within 10% (relative) at fortified chloramphenicol concentrations of 0.25-1.0 ppb.

Multilaboratory validation of a method to confirm chloramphenicol in shrimp and crabmeat by liquid chromatography-tandem mass spectrometry.

An existing method for chloramphenicol (CAP) determination in shrimp using a gas chromatograph with electron capture detector was adapted for confirmation of CAP with a liquid chromatograph interfaced to a triple quadrupole mass spectrometer. CAP residues are extracted from tissue with ethyl acetate, isolated via liquid-liquid extraction, and concentrated by evaporation. Extracts are chromatographed by using a reversed-phased column and analyzed by electrospray negative mode tandem mass spectrometry. Four product ions (m/z 152, 176, 194, and 257) of precursor m/z 321 were monitored. Moving from gas chromatography to liquid chromatography-tandem mass spectrometry improved the sensitivity of the method greatly, enabling reliable confirmation of CAP residues at 0.3 microg/kg (ppb). The method meets confirmation criteria recommended by the U.S. Food and Drug Administration and 4-point identification criteria established by the European Union. With slight modifications to accommodate different equipment, the method was validated in 3 laboratories.