Fragmentation patterns
Clozapine metabolites
Clozapine
Several fragment ions were identified in the MS/MS experiments for clozapine.
Fragment ions at m/z 296, 282, 270, 268, 255, 243 and 227 were identified as the
fragmentation products of methylpiperazine. Fragment ion at m/z 208 is likely to be
originated from loss of C5H9N from methylpiperazine, together with a loss of HCl
(i.e. loss of HCl from the ion at m/z 243). Fragment ion at m/z 192 was originated
similarly by loss of HCl from the ion at m/z 228. Fragment ion at m/z 101 was
identified as charged methylpiperazine, and ion at m/z 84 via further loss of NH3 from
the ion at m/z 101.
RM1
Metabolite RM1 was identified as cysteine conjugate of clozapine. Fragment ion at
m/z 359 had lost cysteine by scission of the S-CH2 bond (loss of C3H5NO2) and
fragment ion at m/z 302 had lost C3H7N from fragment ion at m/z 359. Fragment ion
at m/z 327 was intact clozapine.
RM2
Metabolite RM2 was identified as dechlorinated glutathione conjugate. Fragment ion
at m/z 469 resulted from the loss of pyroglutamate (i.e. loss of C5H7NO3 = 129 u),
and fragment ion at m/z 412 from additional loss of C3H7N from the
methylpiperazine. Fragment ion at m/z 268 resulted from the loss of glutathione by
the S-CH2 bond and C3H7N from the methylpiperazine. Fragment ion at m/z 515
resulted from the loss of C5H9N from the methylpiperazine.
RM3 & RM4
Based on accurate masses, metabolites RM3 and RM4 were identified as glutathione
conjugates of clozapine and they had very similar fragmentation pattern. For RM3,
fragment ion at m/z 549 resulted from the loss of C5H9N from the methylpiperazine.
Fragment ion at m/z 503 resulted from loss of pyroglutamate (-129 u) from
glutathione and the ion at m/z 359 from loss of glutathione moiety except the sulphur.
Fragment ion at m/z 446 resulted from loss of pyroglutamate (m/z 503) combined
with the loss of C3H7N from methylpiperazine, while the ion at m/z 302 resulted via
loss of C3H7N from methylpiperazine from the m/z 359. Fragment ion at m/z 84 was
from the methylpiperazine and was the same as in clozapine.
RM5 & RM6
GSH-conjugates RM5 and RM6 were formed via addition of one extra oxygen atom
to clozapine together with the glutathione conjugation reaction and had similar
fragmentation patterns. In the MS/MS experiments, fragment ions at m/z 630, 519,
302, 117, 99 and 70 were identified for both metabolites. Ion at m/z 630 resulted from
cleavage of water, ion at m/z 519 from cleavage of pyroglutamate from glutathione,
and fragment ion at m/z 501 (for RM6 only) originated from combination of these
two fragmentation reactions. Ions at m/z 117, 99 and 70 were fragments from
methylpiperazine and revealed that biotransformation site for the extra oxygen was
located in the methylpiperazine-ring. Fragment ion at m/z 302 resulted from cleavage
of the S-CH2 bond and the loss of C3H7NO from the methylpiperazine. It indicates
that the oxygen is most likely close to the tertiary nitrogen in the methylpiperazine.
This is supported by fragment ion at m/z 444 in the spectrum of RM6, resulting from
the loss of C3H9NO from the methylpiperazine and a loss of pyroglutamate.
M1
Stable metabolite M1 was formed via dealkylation by the loss of CH2CH2 from the
methylpiperazine-moiety. M1 produced fragment ions at m/z 270, 244, 227, 208 and
192, which all resulted from fragmentation of the partially degraded methylpiperazine
ring and were mostly the same than for clozapine.
M2
Stable metabolite M2 was formed via dealkylation by the loss of CH2. Also M2
produced fragment ions matching with clozapine, ie. fragments at m/z 296, 282, 270,
244, 227, 208 and 192, which all resulted from the fragmentation of piperazine part,
together with ions at m/z 87 and 70 that were identified as piperazine fragments.
M3
Metabolite M3 was formed via hydroxylation/oxidation and dehydrogenation, and
produced fragment ions at m/z 313, 296, 243, 227, 192, 113, 87 and 70 in the MS/MS
experiments. Ion at m/z 313 originated by loss of CO, while the ions at m/z 296, 227,
243 and 192 were the same as in the spectrum of clozapine. This fragmentation
localized the biotransformations to methylpiperazine ring, the ions at m/z 313 and 296
pointing to aldehyde formation in N-methyl. The fragment ion at m/z 113 also
supported the localization of biotransformations to methylpiperazine moiety.
Fragment ions at m/z 87 and 70 resulted from further fragmentation of the ion at m/z
113 and supported the identification.
M4
The accurate mass of metabolite M4 corresponded to clozapine with one additional
oxygen atom. In the MS/MS experiments for M4, fragment ions at m/z 312, 286, 243,
208, 105 and 101 were observed. Ion at m/z 243 that corresponded the m/z 227 of
clozapine together with additional oxygen, pointing that oxygen had attached to the
dibenzodiazepine structure. It is worth noticing that m/z 243 of M4 is not the same as
m/z of clozapine (confirmed by accurate mass data, see Fig 2). This was supported
also by the ions at m/z 312 and 286, as well as m/z 101 that was intact
methylpiperazine similarly than for clozapine. Ion at m/z 208 was a further loss of
chlorine from the ion at m/z 243. The ion at m/z 105 had a molecular structure
C7H5O, resulting from fragmentation of the non-chlorine containing aromatic ring,
together with additional oxygen and the carbon from the 7-membered heterocycle,
locating the site of the hydroxylation in M4 to the non-chlorine containing aromatic
ring.
M5
Metabolite M5 had the same accurate mass as M4 and produced a high number of
fragment ions in the MS/MS experiments. Fragment ions at m/z 296, 282, 268, 256,
243 and 227 resulted from fragmentation of methylpiperazine ring, and were mostly
the same as in clozapine and lacked oxygen atom, which indicated that oxygen was
attached to the N-methyl-part of the methylpiperazine ring. Fragment ions at m/z 208
and 192 were also the same as in the spectrum of clozapine. Fragment ion at m/z 299
resulted from loss of C2H4O from methylpiperazine. Fragment ion at m/z 117 was
methylpiperazine ring with additional oxygen atom, and fragment ions at m/z 99, 85,
82 and 70 resulted from the further fragmentation of the ion at m/z 117. The
fragmentation pattern of the M5 suggests that the oxygen atom is attached to the
methylpiperazine ring and is most likely N-oxide of clozapine.
Ticlopidine metabolites
Ticlopidine
The fragmentation pattern of ticlopidine was investigated in the MS/MS experiments
and two fragmentation pathways were identified. The most abundant fragments at m/z
154 and 125, and minor fragments at m/z 99 and 89 all included the chlorine
substituted phenyl. Less abundant fragment ions at m/z 138 and 111 were from the
tetrahydrothionopyridine moiety. Fragment at m/z 228 resulted from the loss of HCl.
RM1
The mass of RM1 corresponded to oxidation + GSH conjugation. Several fragment
ions were observed in the MS/MS experiments, the most interesting being at m/z 539,
458 and 410. The ion at m/z 539 resulted from the loss of sulfoxide (SO), the ion at
m/z 458 from the loss of pyroglutamate from GSH moiety, and the ion at m/z 410
from the combination of these. Fragment ion at m/z 296 resulted from the cleavage of
the glutathione from S-CH2 bond (loss of C10H15N3O6) and loss of water. Fragment
ion at m/z 280 results from the loss of entire glutathione, including the sulphur atom.
Fragment ion at m/z 232 had additionally lost a sulfoxide. Fragment ions at m/z 154,
125 and 111 were the same as in the spectrum of ticlopidine.
RM2
RM2 had an identical mass to that of RM1, it also formed fragment ions at m/z 410
and 232 in the MS/MS experiments, suggesting that also RM2 was formed via Soxide, which was supported by the existence of fragment ion at m/z 386. Fragment
ions at 296, 154 and 125 were the same as above.
RM3
Also the mass of RM3 corresponded to oxidation + GSH conjugation. RM3 produced
a fragment ion at m/z 569 by loss of water, ion at m/z 458 by loss of pyroglutamate,
and ion at m/z 440, which resulted from both of these reactions. Fragment ion at m/z
280 resulted from the loss of whole glutathione moiety, and ion at m/z 262 with an
additional loss of water. Fragment ions at m/z 154, 125, 111 and 89 were the same as
in the spectrum of ticlopidine. Fragment ion at m/z 287 resulted by the loss of
C10H14ClN + H2O from the tetrahydrothionopyridine moiety and C5H7NO3
(pyroglutamate) from the glutathione. Any of the core-structure fragment ions did not
include the extra oxygen, so that there is no absolute confirmation for the oxidation
site, but most likely it is located in the thiophene-moiety, so that m/z 111 formed like
for ticlopidine, but with extra loss of water.
RM4
The mass of RM4 corresponded to oxidation + GSH conjugation and MS/MS
experiments produced fragment ions at m/z 458, 440 and 280, which were the same as
in the spectrum of RM3. Fragment ions at m/z 154, 125, 111 and 89 are the same as in
the spectrum of ticlopidine. Fragment at m/z 184 resulted from the loss of chlorine
substituted phenyl and C9H15N3O7 from glutathione. Fragment at m/z 287 resulted
from the loss of C8H10ClN + H2O from ticlopidine part and C5H7NO3 (pyroglutamate)
from glutathione. Fragment ion at m/z 434 resulted from the loss of C8H8ClN and
fragment ion at m/z 416 from additional loss of water.
M1 & M2
Stable metabolites M1 and M2 were formed via mono- and di-dehydrogenations,
respectively. Both of these metabolites formed fragment ions at m/z 125, 99 and 89 in
MS/MS experiments, which were the same as for ticlopidine. Ion at m/z 136 was
detected for M1, which corresponded to the ion at m/z 138 for ticlopidine with
dehydrogenation.
M3
M3 was formed via hydroxylation with two additional dehydrogenations. Most
abundant fragment ion at m/z 125 proved that the reactions occur in the
tetrahydrothionopyridine structure, but the exact position was not revealed. Fragment
ion at m/z 240 was due to loss of chlorine and was not informative.
M4
Metabolite M4 was formed via addition of one oxygen. The most intense fragment
ions were at m/z 262, 154 and 125. Again, ions at m/z 154 and 125 point that the
oxygen is attached to the tetrahydrothionopyridine structure. Fragment ion at m/z 262
is resulted from the loss of water. The high abundance of m/z 262 indicates that the
oxygen is in the aliphatic six-membered ring.
M5
Metabolite M5 was also formed via addition of one oxygen and produced fragment
ions at m/z 262, 170, 154, 138, 125, 111 and 89. Fragment ion at m/z 125 proved once
again that the oxygen is not attached to the chlorine substituted phenyl, and ion m/z
111 pointed that the oxygen is not attached to the tiophene ring either. Fragment ion
at m/z 262 resulted from loss of water, and fragment ion at m/z 138 from the loss of
C7H7OCl. Accurate mass of m/z 154 matches to structure [C7H8NOS]+, which
together with m/z 170 and previously mentioned fragments suggests that M5 is Noxide.
M6
M6 was also formed via addition of one oxygen atom, but only fragments at m/z 125
and 89 were identified, which suggests that the oxygen is not attached to the chlorine
substituted phenyl. The lack of water cleavage, on the contrary to metabolites M4 and
M5, indicates that the oxygen is probably aromatic.
M7
M7 was formed via hydroxylation and hydrogenation. Fragment ion at m/z 125
proved that neither of these modifications had occurred in the chlorine substituted
phenyl ring. Additionally, the fragment ion at m/z 154 proved that it was not an Noxide. Hydrogenation have most likely occurred either of the double bonds in the
tiophene ring, or then the structure contains opening of some of the rings.
M8
M8 was formed from hydroxylation reaction. Metabolite M8 produced fragment ion
m/z 278, resulting from loss of water. Fragment ions at m/z 170 and 138 resulted from
the tetrahydrothionopyridine structure and both contained two oxygen atoms. This
suggests that both oxygens are attached to the tetrahydrothionopyridine structure.
Fragment ion at m/z 96 contained nitrogen and one oxygen, giving further
confirmation that at least one oxygen was close to the nitrogen atom.
M9
Also the mass of M9 corresponded to ticlopidine with two oxygen atoms. The most
abundant fragment of metabolite M9 was ion at m/z 125, proving that neither of the
oxygen atoms were attached to the chlorine substituted phenyl. Fragments at m/z 154
and 89 supported this conclusion. Fragment at m/z 278 was the same as in M8.
Fragment at m/z 155 resulted from the loss of chlorine substituted phenyl and
nitrogen. Fragment at m/z 137 resulted with additional loss of water.
Citalopram metabolites
Citalopram
In the MS/MS experiments, 5 fragment ions were produced, which were useful in the
identification of the metabolites. For citalopram itself, fragment at m/z 307 resulted
from cleavage of water, fragment ion at m/z 280 resulted from loss of N(CH 3)2 group,
and fragment ion at m/z 262 resulted via further loss of water from m/z 280. Fragment
ion at m/z 116 contained the cyanide-substituted benzene, and fragment ion at m/z
109 the fluorine-substituted benzene.
M1
Metabolite M1 was formed via N-demethylation and it produced the fragment ions at
m/z 280, 262, 116 and 109. In addition, it formed fragment ion at m/z 293, which
resulted from the loss of NCH3.
M2
Metabolite M2 was identified as doubly demethylated citalopram. M2 produced
fragment ions at m/z 279, 280, 262, 116 and 109 in the MS/MS experiments.
Fragment ions at m/z 280, 262, 116 and 109 were the same as for citalopram, and the
ion at m/z 279 resulted from the loss of NH2.
M3
The mass of metabolite M3 matched with citalopram with one additional oxygen, and
in the MS/MS experiments fragment ions at m/z 323, 280, 262, 116 and 109 were
detected, being all the same than in the spectrum of citalopram.
Table S1. Accurate masses for fragment ions.
Clozapine
P
Fragment formula
C17H15N3Cl
C16H13N3Cl
C15H13N3Cl
C15H11N3Cl
C14H10N3Cl
C13H10N3Cl
C13H8N2Cl
C13H10N3
C13H8N2
C5H13N2
C5H10N
m/z calc.
296.0955
282.0793
270.0798
268.0642
255.0563
243.0563
227.0376
208.0875
192.0687
101.1079
84.0813
m/z obs. mDa
296.0956
0.1
282.0784 -0.9
270.0800
0.2
268.0643
0.1
255.0559 -0.4
243.0568
0.5
227.0380
0.4
208.0886
1.1
192.0690
0.3
101.1079
0.0
84.0814
0.1
RM1
C18H20N4SCl
C18H20N4Cl
C15H13N3SCl
359.1097 359.1114
327.1676 327.1696
302.0519 302.0502
1.7
2.0
-1.7
RM2
C23H27N6O6S
C23H29N6O3S
C20H22N5O3S
C15H14N3S
515.1713
469.2022
412.1438
268.0908
515.1714
469.2003
412.1444
268.0893
0.1
-1.9
0.6
-1.5
RM3
C23H26ClN6O6S
C23H28ClN6O3S
C20H21ClN5O3S
C18H20N4SCl
C15H13N3SCl
C4H6NO
549.1323
503.1632
446.1054
359.1097
302.0519
84.0449
549.1315
503.1635
446.1056
359.1092
302.0489
84.0450
-0.8
0.3
0.2
-0.5
-3.0
0.1
RM4
C23H28N6O3SCl
C20H21ClN5O3S
C18H20N4SCl
C15H13N3SCl
C4H6NO
503.1632 503.163
446.1054 446.1045
359.1097 359.109
302.0519 302.0513
84.0449 84.0451
-0.2
-0.9
-0.7
-0.6
0.2
RM5
C28H33N7O6SCl
C23H28N6O4SCl
C15H13N3SCl
C5H13N2O
C5H11N2
C4H8N
630.1902
519.1581
302.0519
117.1028
99.0921
70,0657
630.1893
519.1576
302.0501
117.1032
99.0922
70,0658
-0.9
-0.5
-1.8
0.4
0.1
0.1
RM6
C28H33N7O6SCl
C23H28N6O4SCl
C23H26N6O3S
C20H19N5O3SCl
C5H13N2O
C5H11N2
630.1902
519.1581
501.1476
444.0897
117.1028
99.0917
630.1886
519.1565
501.1448
444.0872
117.1026
99.0922
-1.6
-1.6
-2.8
-2.5
-0.2
0.5
C4H8N
70,0657
70,0659
0.2
M1
C15H13N3Cl
C13H11N3Cl
C13H8N2Cl
C13H10N3
C13H8N2
270.0798
244.0642
227.0376
208.0875
192.0687
270.0799
244.0643
227.0373
208.0899
192.069
0.1
0.1
-0.3
2.4
0.3
M2
C17H15N3Cl
C16H13N3Cl
C15H13N3Cl
C13H11N3Cl
C13H8N2Cl
C13H10N3
C13H8N2
C4H11N2
C4H8N
296.0955
282.0793
270.0798
244.0642
227.0376
208.0875
192.0687
87.0922
70.0657
296.0962
282.0769
270.0804
244.0645
227.0379
208.0889
192.0693
87.0922
70.0657
0.7
-2.4
0.6
0.3
0.3
1.4
0.6
0.0
0.0
M3
C17H18N4Cl
C17H15ClN3
C15H13N3Cl
C13H8N2Cl
C13H8N2
C5H9N2O
C4H11N2
C4H8N
313.1220
296.0955
270.0798
227.0376
192.0687
113.0709
87.0922
70.0651
313.1218
296.0955
270.0799
227.0382
192.0692
113.0709
87.0927
70.0656
-0.2
0.0
0.1
0.6
0.5
0.0
0.5
0.5
M4
C17H15N3OCl
C15H13N3OCl
C13H8N2Cl
C13H8N2O
C7H5O
C5H13N2
312.0904
286.0747
243.0325
208.0637
105.0340
101.1079
312.0878
286.0746
243.0341
208.064
105.0343
101.1076
-2.6
-0.1
1.6
0.3
0.3
-0.3
M5
C16H16N4Cl
C17H15N3Cl
C16H13N3Cl
C14H11N3Cl
C13H10N3Cl
C13H8N2Cl
C13H8N2
C5H13N2O
C5H11N2
C4H9N2
C5H8N
C4H8N
299.1063
296.0955
282.0798
256.0642
243.0563
227.0376
192.0687
117.1028
99.0922
85.0766
82.0657
70.0657
299.1060
296.0952
282.0797
256.0641
243.0559
227.0378
192.0688
117.1028
99.0921
85.0766
82.0655
70.0657
-0.3
-0.3
-0.1
-0.1
-0.4
0.2
0.1
0.0
-0.1
0.0
-0.2
0.0
Ticlopidine Fragment formula
P
C14H15ClNS
C14H13ClNS
m/z calc. m/z obs. mDa
228.0847 228.0850
0.4
154.0418 154.0426
0.3
C14H16NOS
C13H12SCl
C14H14NS
C8H9NCl
C7H8NS
138.0372 138.0378
125.0153 125.0161
111.0263 111.0272
99.0002 99.0007
89.0391 89.0397
0.3
0.4
0.3
0.8
0.7
RM1
C24H32N4O6SCl
C19H25N3O4S2Cl
C19H25N3O3SCl
C16H24N3O6S
C14H15NS2Cl
C14H15NOSCl
C14H15NCl
C8H9NCl
C7H6Cl
C6H7S
539.1731
458.0975
410.1305
386.1386
296.0334
280.0563
232.0893
154.0418
125.0158
111.0268
539.1744
458.0949
410.1324
386.1381
296.0334
280.0564
232.0883
154.0414
125.0154
111.0272
1.3
-2.6
1.9
-0.5
0.0
0.1
1.0
-0.4
-0.4
0.4
RM2
C19H25N3O4S2Cl
C19H25N3O3SCl
C16H24N3O6S
C14H15NS2Cl
C14H15NCl
C8H9NCl
C7H6Cl
458.0975
410.1305
386.1386
296.0334
232.0893
154.0418
125.0158
458.0945
410.1264
386.1396
296.0312
232.0872
154.0416
125.0154
-3.0
-4.4
1.0
-2.2
-2.1
-0.2
-0.4
RM3
C24H30N4O6S2Cl
C19H25N3O4S2Cl
C19H23N3O3S2Cl
C14H15NS2Cl
C11H15N2O3S2
C14H15NOSCl
C14H12ClNS
C8H9NCl
C7H6Cl
C6H7S
C7H5
569.1295
458.0975
440.0864
296.0334
287.0524
280.0563
262.0457
154.0418
125.0153
111.0263
89.0391
569.1276
458.0985
440.0881
296.0352
287.0513
280.0555
262.0441
154.0431
125.0159
111.027
89.0397
-1.9
1.0
1.7
1.8
-1.1
-0.8
-1.6
1.3
0.7
0.9
0.4
RM4
C19H25N3O4S2Cl
C19H23N3O3S2Cl
C16H24N3O7S2
C16H22N3O6S2
C11H15N2O3S
C14H15NOSCl
C8H10NS2
C8H9NCl
C7H6Cl
C6H7S
C7H5
458.0975
440.0864
434.1056
416.0950
287.0524
280.0563
184.0255
154.0418
125.0153
111.0263
89.0391
458.0880
440.0865
434.1073
416.0959
287.0530
280.0577
184.0254
154.0425
125.0157
111.0239
89.0401
1.6
0.1
1.7
0.9
0.6
1.4
-0.1
0.7
0.7
0.9
0.4
M1
C7H6NS
136.0221 136.0224
0.3
C7H6Cl
C5H4Cl
C7H5
125.0153 125.0160
99.0002 99.0006
89.0391 89.0394
0.7
0.4
0.3
M2
C7H6Cl
C5H4Cl
C7H5
125.0153 125.0159
99.0002 99.0006
89.0391 89.0397
0.6
0.4
0.6
M3
C14H10NOS
C7H6Cl
240.0483 240.0482
125.0153 125.0158
-0.1
0.5
M4
C14H13ClNS
C14H10ClNS
C8H9NCl
C7H6Cl
C7H5
262.0452
260.0301
154.0418
125.0153
89.0391
262.0450
260.0306
154.0424
125.0159
89.0394
-0.2
0.5
0.6
0.6
0.4
M5
C14H13ClNS
C8H9ClNO
C7H8NOS
C7H8NS
C7H6Cl
C6H7S
C7H5
262.0452
170.0367
154.0321
138.0372
125.0153
111.0263
89.0391
262.0459
170.0375
154.0330
138.0379
125.0160
111.0272
89.0395
0.7
0.8
0.9
0.7
0.7
0.9
0.4
M6
C7H6Cl
C7H5
125.0153 125.0161
89.0391 89.0399
0.8
0.8
M7
C8H9NCl
C7H6Cl
154.0424 154.0426
125.0158 125.0158
0.2
0.0
M8
C14H13NOSCl
C7H8NO2S
C7H8NO2
C6H8NS
C5H6NO
278.0406
170.0276
138.0555
126.0377
96.0449
278.0403
170.0275
138.0556
126.0372
96.0452
-0.3
-0.1
0.1
-0.5
0.3
M9
C14H13NOSCl
C7H7O2S
C7H5OS
C7H6Cl
C7H5
278.0406
155.0167
137.0061
125.0158
89.0391
278.0406
155.0168
137.0061
125.0157
89.0397
0.0
0.1
0.0
-0.1
0.6
Citalopram Fragment formula
P
C20H20N2F
C18H15NOF
C18H13NF
C8H6N
C7H6F
m/z calc.
307.1611
280.1138
262.1032
116.0500
109.0454
m/z obs. mDa
307.1614
0.3
280.1136 -0.2
262.1032
0.0
116.0504
0.4
109.0459
0.5
M1
C19H19N2OF
C19H18N2F
C18H15NOF
C18H13NF
C8H6N
311.1560
293.1454
280.1138
262.1032
116.0500
311.1567
293.1458
280.1154
262.1041
116.0504
0.7
0.4
1.6
0.9
0.4
M2
C18H15NOF
C18H16N2F
C18H13NF
C8H6N
C7H6F
280.1138
279.1298
262.1032
116.0500
109.0454
280.1142
279.1299
262.1033
116.0501
109.0455
0.4
0.1
0.1
0.1
0.1
M3
C20H22N2O2F
C20H20N2OF
C18H15NOF
C18H13NF
C8H6N
341.1665
323.1560
280.1138
262.1032
116.0500
341.1672
323.1560
280.1143
262.1036
116.0504
0.7
0.0
0.5
0.4
0.4