Diazepam metabolites in the rat: Characterization by high-resolution mass spectrometry and nuclear magnetic resonance

This report illustrates the usefulness of combining thin-layer chromatography for metabolite separation and purification with high-resolution mass spectrometry and nuclear magnetic resonance for metabolite characterization. Four metabolites of diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) were identified in the intestinal tract of one rat that had been injected intraperitoneally with 25 mg of 3H-diazepam. These are the glucuronide and/or sulfate conjugates of 3-hydroxy diazepam (metabolite A), and the conjugates of the 5-hydroxyphenyl analogues of diazepam, 3-hydroxy diazepam, and N-desmethyl diazepam (metabolites B, C, and D, respectively). In each of the phenolic metabolites (B, C, and D) the hydroxyl function is in the para position.