Drug Metabolism in Drug Design and Development Basic Concepts and Practice

11.4 TECHNIQUES FOR IMPROVING METABOLITE DETECTION

AND IDENTIFICATION

11.4.1 Chemical Derivatization

Chemical derivatization used in metabolite identification is a process of adding
ionizable groups onto molecules that are difficult to ionize to facilitate their
detection by mass spectrometry (Liu and Hop, 2005). In the pre-API era,
chemical derivatization was aimed at improving volatility of compounds,
which were difficult to transfer to the gas-phase for analysis. Since the
development of API techniques, derivatization is not very frequently required.
Nonetheless, chemical derivatization of very polar, small metabolites is
sometimes advantageous. It reduces their polarity and increases their molecular
weight thus making them more amenable to LC separation while minimizing
potential mass spectral interferences from low molecular weight endogenous
material present in the sample (Dalvie and O’Donnell, 1998).
Chemical derivatization can also be very useful for metabolite structural
confirmation. Two oxidative metabolites, M4 and M7, were detected in the
incubation of pioglitazone with dog liver microsomes. LC/MS/MS analysis
indicated the introduction of a hydroxyl group on the 2-(5-ethyl-2-pyridyl)
ethyl moiety (Shen et al., 2003). In order to assign the exact site of
hydroxylation, chemical oxidation by Jones reagent was applied to distinguish
the terminal hydroxyl (M7) fromv-1 hydroxyl (M4, Scheme 11.1). Oxidation
of M4 would give rise to a ketone derivative that is two mass units lower than
M4. On the contrary, M7 would give rise to a carboxylic acid derivative
following oxidation, which is 14 mass units higher than M7 itself. This
approach was applied to unequivocally distinguish the two isomeric
metabolites (Liu and Hop, 2005).
SinceN-oxide metabolites have the same elemental composition as those
metabolites resulting from hydroxylation, differentiation by mass spectrometry

S

NH

N O

O

O

S

NH

N O

O

O

OH

S

NH

N O

O

O

HO

S

NH

N O

O

O

O

S

NH

N O

O

O

O

HO

K 2 Cr 2 O 7

K 2 Cr 2 O 7

H 2 S 2 O 4

H 2 S 2 O 4

134

150

150

148

164

Pioglitazone, m/z 357

M4, m/z 373

M7, m/z 373

m/z 371

m/z 387

SCHEME 11.1 Oxidation of M4 and M7 of pioglitazone using Jones reagent for
structural characterization.

TECHNIQUES FOR IMPROVING METABOLITE DETECTION 339