Drug Metabolism in Drug Design and Development Basic Concepts and Practice

2005). An equimolar ratio of glutathione and^13 C 2 =^15 N-labeled glutathione
(stable-isotope label in glycine residue) was applied to trap reactive
metabolites from microsomal incubations. Unambiguous identification of
GSH-conjugates was realized by the presence of a unique doublet isotopic
peak with m/z difference of 3 Th in the mass spectra. Additionally,
in subsequent MS/MS experiments, the loss of 75 Da (glycine) and
129 Da (pyroglutamic acid) from GSH-adducts, and 78 Da and 129 Da
from stable-isotope-labeled GSH-adducts, substantiated the presence of
GSH-conjugates.
This approach was applied to characterize reactive metabolites of
acetaminophen. Acetaminophen is well known to form a reactive metabolite,
N-acetyl-p-benzoquinone imine (NAPQI, Scheme 11.9). When a 1 : 1 mixture
of natural GSH and stable-isotope-enriched^13 C 2 =^15 N-GSH was added to the
human liver microsmal incubations, the reactive metabolites/intermediates
were trapped by GSH. The sample was then analyzed using a CNL of 129 Da
to detect the glutathione conjugates in an LC/MS/MS experiment. The total
ion chromatogram included three major peaks and several other minor peaks;
but only one neutral loss scan MS/MS spectrum showed doublet peaks atm/z
457 and 460 Th with approximately equal intensity. This unique ion cluster
pattern suggested the formation of GSH-conjugates. In contrast, the CNL
mass spectra of other peaks did not show these unique doublet peaks,
indicating they were false positives. The formation of GSH-conjugates was
further confirmed by full scan MS/MS spectra (Fig. 11.8). The product ion
spectrum ofm/z457 showed major fragment ions atm/z382 and 328, resulting
from neutral losses of glycine (75 Da) and pyroglutamic acid (129 Da), while
the product ion spectrum ofm/z460 showed fragment ions atm/z382 and 331
resulting from losses of^13 C 2 =^15 N-labeled glycine (78 Da) and pyroglutamic
acid. This approach can be fully automated by employing isotopic pattern
recognition and data-dependent acquisition methods, which would greatly

OH

HN

O

O

N

O

OH

HN

O

S

N

H OH

O

O NH

NH 2

O

OH

O

Bioactivation GSH

SCHEME 11.9 Bioactivation of acetaminophen to form a reactive N-acetyl-
p-benzoquinone imine (NAPQI) and trapping of the electrophilic intermediate by GSH.

DETECTION AND CHARACTERIZATION OF REACTIVE METABOLITES 355