An enzymatic assay of chloramphenicol coupled with fluorescence reaction

An alternative method for the assay of chloramphenicol using an enzymatic reaction coupled with a fluorescence detection system has been developed. Chloramphenicol was enzymatically acetylated by chloramphenicol acetyltransferase in the presence of acetyl-coenzyme A (acetyl-CoA) as the acetyl-donor, after which the liberated CoA-SH was derivatized with a fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. The assay was linear over the range of 2.5-40 micrograms/ml. Analytical recoveries of chloramphenicol at concentrations of 7.5 and 22.5 micrograms/ml, added to human serum, plasma, or a slightly hemolyzed serum, were in the range of 93.3 to 106.2%. The enzymatic assay was not affected by the presence of ten other antibiotics tested.