Drug Metabolism in Drug Design and Development Basic Concepts and Practice

in-source fragmentation. This may lead to an overestimation of the aglycone
concentration if the chromatographic separation between the glucuronide
and aglycone is not achieved (Yan et al., 2003). APCI and APPI typically
result in in-source fragmentation of glucuronide to a much greater extent
than ESI.
Acyl-glucuronide metabolites, formed by conjugation of glucuronic acid
with a carboxylic acid moiety on a drug or metabolite, contain an ester group
that is susceptible to both hydrolysis and intramolecular acyl migration
(Stachulski et al., 2006). The unstable nature of acyl glucuronides makes
careful sample handling crucial. The common practice is to immediately cool
the sample on ice after collection and acidified to pH 2–4 to prevent hydrolysis
and minimize acyl migration.

11.7.2 N-Oxides

AmineN-oxides are thermally labile species that undergo decompositions in
many ways (Albini, 1993). Deoxygenation is often observed forN-oxides
during atmospheric pressure chemical ionization, and occurs due to thermal
energy activation in the vaporizer of the APCI source. Deoxygenation
does not occur during the softer ESI process and thereby may represent a
potential way to differentiateN-oxides from hydroxylated metabolites since
the latter usually do not undergo thermal deoxygenation (Ramanathan et al.,
2000).
In addition to deoxygenation,tert N-oxides containing an alkyl or benzyl
group on the N-oxide nitrogen also undergo an N- to O- rearrangement
(Meisenheimer arrangement), followed by elimination of an aldehyde (or a
ketone) through an internal hydrogen transfer (Scheme 11.5). This has been
observed under both APCI and APPI conditions (Ma et al., 2005). The
elimination of an aldehyde or a ketone results from thermal energy activation
at the vaporizer and is not induced by collisional activation. Significant in-
source fragmentation was observed in the APCI and APPI mass spectra of
clozapineN-oxide (Fig. 11.7). Notably, two major fragment ions atm/z313 and
327, observed in the APCI and APPI mass spectra, were not detected in the ESI
mass spectrum (Fig. 11.7). The fragment ion atm/z327 (MH+O) arose

N

+

R1

R2 H

O

N

O

H

R2

R1

R2

R1

O

N

H

SCHEME 11.5 Proposed mechanism of aldehyde or ketone elimination following
Meisenheimer rearrangement oftert-amineN-oxides.

350 APPLICATION OF LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY