( ood cell production in bone marrow lead...)Simultaneously characterizing and quantifying chloramphenicol and its metabolite using LC/MS/MS

Quantification of chloramphenicol and its metabolites in its metabolic stability study using rat liver S9 is described. With the advent of liquid chromatography/mass spectrometry and information dependent data acquisition function in mass spectrometry, it has become possible to quantify chloramphenicol and its metabolites and characterize them at the same time. This was once impossible to be analyzed in a sample within a single chromatographic run.

Materials and methods

Biological Methods: Chloramphenicol was incubated with rat liver S9 fractions at a substrate concentration of 50 micro molar and a protein concentration of 1 mg/ml in 100 mM potassium phosphate buffer at pH 7.4. Reactions were initiated by the addition of nicotinamide adenine dinucleotide phosphate reduced form (NADPH), uridine diphosphoglucuronic acid (UDPGA), and adenosine phosphate phosphosulfate (APPS) to give the final cofactor concentrations of 2 mM, 4 mM, and 4 mM, respectively. Incubation mixtures (2 mL total volume) were shaken in a water bath kept at 37C. An aliquot of 200 micro liter incubation solutions was sampled at t=0, 10, 20, 30, 40, 50, and 60 minutes. The reactions were terminated by adding two volumes of ice-cold acetonitrile. The samples were then vortex mixed and centrifuged. The supernatants were dried and the residues reconstituted in a 100 micro liter of 0.1% formic acid in water and acetonitrile (95:5, v/v). Ten micro liters was injected onto HPLC.

HPLC/MS: LC/MS analyses of chloramphenicol in vitro samples were carried out by coupling a Shimadzu 10AD system to a SCIEX Q TRAP^TM LC/MS/MS System. The HPLC eluent was introduced to the mass spectrometer using a TurboIonSpray. T
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