from thermal energy activation in the vaporizer of the APCI and APPI sources.
The most prominent fragment ion (m/z 313) resulted from the loss of
formaldehyde. This fragment ion resulted from a thermally induced
Meisenheimer rearrangement with migration of theN-methyl group to the
corresponding N-alkoxylamine, followed by elimination of formaldehyde
through an internal hydrogen transfer. This fragmentation would not,
however, be expected from hydroxylated metabolites. Applying the
Meisenheimer rearrangement reaction scheme, this fragment can be used to
distinguish not onlyN-oxides from isomeric hydroxylated metabolites, but
potentially the exact site ofN-oxidation in a setting where multiple sites of
differing chemical environment are present. The fragment ion atm/z313 in
APCI and APPI indicates that the position ofN-oxygenation is at the nitrogen
atom of the piperazine ring where the methyl group is attached, and not in the
diazepine ring.
The tertiary amineN-oxides with ab-hydrogen in the alkyl substituted
group may also undergo Cope elimination to give a hydroxylamine and an
olefin. TheN-oxide metabolite of SC-57461 was found to be unstable in organic
m/z (Th)(^150) ClozapineN-oxide
300
450
600
750
Intensity (counts)
313.1210
327.1369343.1316
243.0535 268.0635 299.1056 325.1199
256.0629
245.0505
-O
m/z (Th)
120 140 160 180 200 220 240 260 280 300 320 340 360
300
600
900
1200
327.1347
343.1316
Intensity (counts) 325.1197
-O
120 140 160 180 200 220 240 260 280 300 320 340 360
300
600
900
343.1324
Intensity (counts) 325.1228
m/z (Th)
N
N
N
N
H
Cl
H 3 C O
120 140 160 180 200 220 240 260 280 300 320 340 360
-O
-CH 2 O
-CH 2 O
-H 2 O
-H 2 O
-H 2 O
313.1193
(a) APCI
(b) APPI
(c) ESI
FIGURE 11.7 (a) APCI, (b) APPI, and (c) ESI mass spectra of clozapineN-oxide.
Reprinted from Ma et al. (2005) with permission of the American Chemical Society.
CHARACTERIZATION OF UNSTABLE METABOLITES 351