Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
41, Number 8, September 2003, pp. 398-404
Online Hydrogen-Deuterium Exchange and a Tandem-Quadrupole Time-of-Flight
Mass Spectrometer Coupled with Liquid Chromatography for Metabolite Identification
in Drug Metabolism
A.-E.F. Nassar
This work was initiated at Bayer Pharmaceutical, 400 Morgan Lane, West Haven,
CT 06156.
A method for metabolite identification in drug discovery and
development utilizing online hydrogen-deuterium (H-D) exchange and a tandem-quadrupole
time-of-flight mass spectrometer coupled with liquid chromatography has been
developed for the first time. For purposes of evaluating this method, nimodipine
is incubated with rat, dog, monkey, and human hepatic microsomes over a 60-min
period. This procedure involves the replacement of H2O with D2O in the mobile
phase. The equilibration time required to change (switch) from H2O in the mobile
phase into D2O is approximately 10 min. There is no significant difference
between the retention times in H2O or D2O mobile phase. The results show that
the separations of nimodipine and its metabolites and the subsequent H-D exchanges
were achieved online. This leads to the identification of nimodipine metabolites
formed in vitro. Five metabolites of nimodipine are identified and characterized.
These metabolites are formed by dehydrogenation, demethylation of methoxy group,
cleavage of the ester groups by hydrolysis or oxidation, and hydroxylation
of methyl groups. The combination of the H-D exchange process and the assay
system is found to be a powerful tool for the study of metabolite identification.
The method appears to be highly desirable for identification of metabolites
produced by dehydrogenation, oxidation, and dealkylation, which are discussed
herein. This method also greatly enhances throughput, which in turn facilitates
the ability to rapidly provide metabolite elucidation.
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