JOURNAL OF MASS SPECTROMETRY

J. Mass Spectrom. 2003; 38: 555–572

Published online 25 April 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.472

Structural characterization and isomer differentiation

of chalcones by electrospray ionization tandem mass
spectrometry
Junmei Zhang and Jennifer S. Brodbelt∗
Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA

Received 15 November 2002; Accepted 9 February 2003

A series of chalcones were characterized by electrospray ionization tandem mass spectrometry (MSn ).
Several ionization modes were evaluated, including protonation, deprotonation and metal complexation,
with metal complexation being the most efficient. Collision-activated dissociation (CAD) was used to
characterize the structures, and losses commonly observed include H2 , H2 O, CO and CO2 , in addition
to methyl radicals for the methoxy-containing chalcones. CAD of the metal complexes, especially [CoII
(chalcone — H) 2,2 -bipyridine]+ , allowed the most effective differentiation of the isomeric chalcones with
several diagnostic fragment ions appearing upon activation of the metal complexes. MSn experiments
were performed to support identification of some fragment ions and to verify the proposed fragmentation
pathways. In several cases, MSn indicated that specific neutral losses occurred by stepwise pathways, such
ž ž
as the neutral loss of 44 u as CH3 and HCO , or CH4 and CO, in addition to CO2 . Copyright  2003 John
Wiley & Sons, Ltd.

KEYWORDS: chalcone structural characterization; isomer differentiation; electrospray ionization mass spectrometry;
collision-activated dissociation; metal complexation

INTRODUCTION precursors of other flavonoids. Chalcones are first synthe-

Owing to their antioxidant abilities and emerging chemo-
preventive properties against heart disease, aging and
cancer,1 – 3 flavonoids have become the focus of increas-
ing numbers of research studies. Flavonoids are a large
group of phytochemicals that have the general structure of a
15-carbon skeleton, which consists of two phenyl rings con-
nected by a three-carbon bridge.4,5 More than 4000 known
flavonoids are classified into subgroups, including flavone,
flavanone, flavonol, isoflavonoid, anthocyanidin and chal-
cone. Chalcones, differing from all other flavonoids by the
absence of the C ring, are still considered a subclass of
flavonoids.6 Chalcones, along with retrochalcones and dihy-
drochalcones, in fact have an open structure and a carbon
skeleton numbered in a way different from other flavonoids
(Fig. 1). Chalcones have shown good physicochemical and
biological activities similar to other flavonoids, includ-
ing some antibacterial,7 antifungal,7 – 9 anti-inflammatory,10
antimicrobial,11 antitumor,12,13 and anticancer14 properties.
Although considered as minor flavonoids, chalcones play a
key role in flavonoid biosynthesis5,15,16 because they are the
Ł Correspondence to: Jennifer S. Brodbelt, Department of Chemistry
and Biochemistry, University of Texas, Austin, Texas 78712, USA.
E-mail: jbrodbelt@mail.utexas.edu
Contract/grant sponsor: National Institutes of Health;
Contract/grant number: NIH RO1 GM63512.
Contract/grant sponsor: Welch Foundation; Contract/grant
number: F-1155.
sized in plants by chalcone synthase (CHS) and then cyclized
to other flavonoids by chalcone isomerase (CHI). Since the
chemopreventive properties of flavonoids (including chal-
cones) depend on both the different functional groups and
their relative positions, it is important to be able to charac-
terize the structures and differentiate the isomers.
Chalcones are commonly found in licorice (liquorice) and
apple seeds.6 Native chalcone glycosides tend to transform
to flavanone glycosides during extraction. Acid hydrolysis,
as is used prior to high-performance liquid chromatography
(HPLC) in many routine analyses for dietary flavonoids,
converts the chalcones to the corresponding flavanones. This
isomerization has been evaluated in solution,17 – 19 and also
by theoretical calculations.20
Although mass spectrometry has been involved in
chalcone structural characterization since the early 1960s,
most studies have focused on electron ionization21 – 29 with
one report of chemical ionization,30 field desorption31 and a
few on fast atom bombardment studies.32,33 Special attention
has been given to 20 -hydroxychalcones21,29 because of
their different fragmentation patterns from other chalcones.
The cyclization of chalcones to flavanones also has been
observed in the gas phase.21 – 23 It was noticed that the
mass spectra of chalcones bearing a 20 -hydroxyl functional
group were nearly identical with those of the isomeric
flavanones. Isomerization was first proposed and later
supported by experimental data.21 Based on experiments
using the relatively energetic electron ionization method,

Copyright  2003 John Wiley & Sons, Ltd.

556 J. Zhang and J. S. Brodbelt

Chalcones Flavanones

3′
O 2′ 4′
6 2′ 1
8 B
1 1′ O 5′
5 3′ 2
7
A 6′
B A C
4 2 6′ 4′
6 3
3 5′ 5 4
O

Chalcone Mw -OH -OCH3 -CH3

1 224 2′ H3CO O
2 240 4,2′
3 240 2′, 4′
4 240 2, 2′
5 240 3, 2′ OH O
6 240 2′, 5′ FA (Mw 270)
7 254 2′ 4′
8 254 2′ 4
9 256 4, 2′,5′ OH
10 268 2′ 4 5′
11 270 2′, 6′ 4′ OH
12 270 2, 2′ 3
13 270 2′, 4′ 4 HO O
14 284 2′ 3, 4
15 284 2′ 3′, 4′
16 284 2′ 4, 4′
17 284 2′ 2, 4′
18 288 3,4, 2′, 4′, 6′ OH O
19 298 2′ 3, 4 5′ FB (Mw 288)
20 298 4, 2′,5′

Figure 1. Chalcone and flavanone structures studied.

it was concluded that an intramolecular equilibrium exists
between a chalcone type and a flavanone type molecular ion.
There have been no reports on the structural characterization
of chalcones by electrospray ionization (ESI) or matrix-
assisted laser desorption/ionization (MALDI). In this work,
we used ESI to form parent ions of interest and then
obtain their fragmentation patterns upon collision-activated
decomposition (CAD). Metal complexation, a method that
has proven to be an effective ionization mode for acidic
flavonoids,34,35 is used as an alternative to differentiate some
isomers.
EXPERIMENTAL
Chemicals and reagents
All the chalcones and flavanones listed in Fig. 1 were
purchased from Indofine (Somerville, NJ). 2,20 -Bipyridine,
lithium chloride and cobalt(II), nickel(II) and copper(II)
bromide were purchased from Aldrich (Milwaukee, WI,
USA). HPLC-grade methanol was purchased from Fisher
(Fair Lawn, NJ, USA). All materials were used without
further purification.
Each chalcone or flavanone stock solution was prepared
in methanol at a concentration of ¾1 ð 10
3 M and kept
protected from light at 4 ° C until use. 2,20 -Bipyridine, lithium
chloride and cobalt(II), nickel(II), and copper(II) bromide
were dissolved in methanol at a concentration of ¾5 ð 10
3 M
and stored at room temperature.
Mass spectrometric conditions
All the experiments were performed on a Thermo Finnigan
LCQ Duo quadrupole ion trap mass spectrometer. The spray
voltage was kept at 4.5 kV (š). The temperature of the
heated capillary was set at 200 ° C. Nitrogen was used as
the sheath gas (20 arbitrary units). Auxiliary gas (nitrogen,
30 arbitrary units) was added as needed. The injection time
was set at 10 ms except for the tandem mass spectrometric
(MSn ) experiments (50–100 ms). Each chalcone working
solution was delivered to the ESI source at 5 µl min
1 .
The other parameters, including capillary voltage, octapole
offsets and tube lens offset, were optimized for maximum
abundance of the ions of interest. In solutions of chalcones
without metal addition, the ions optimized were [L
H]

(deprotonation mode) and [L C H]C or [L C Na]C (positive
ion mode). When lithium chloride was added, the ions of
interest were [L C Li]C . Upon addition of the auxiliary ligand
2,20 -bipyridine (bipy) and a transition metal, the [MII (L
H)
bipy]C complex was optimized. The product ion spectra of
the ions of interest were obtained by CAD, using helium as
the collision gas. The collision energy was varied such that the

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572

relative intensity of the surviving parent ions was ¾5–10%.
For the MSn experiments, the fragment ions of interest from
MS/MS were subject to further isolation and CAD, so that
only ¾5–10% of the selected precursors survived the second
stage of activation.
The working solutions of chalcones (¾1 ð 10
5 M) were
made in methanol from their stock solutions with/without
metal addition. When a metal was added, the final con-
centration ratios were 1 : 10 chalcone : lithium or 1 : 1 : 1
chalcone : transition metal : 2,20 -bipyridine.
RESULTS AND DISCUSSION
Four ionization modes were evaluated: protonation, depro-
tonation, lithium cationization and transition metal complex-
ation. CAD was then performed to characterize the resulting
ions and assess the ability to differentiate isomers, and MSn
was used to assist in elucidating the fragmentation pathways.
Protonation and lithium cationization
Most of the chalcones did not form detectable ions in the
positive ESI mode. Only chalcone 20, which does not have
any acidic hydrogens, formed sodium adducts [L C Na]C
(m/z 321) and not protonated species in the positive mode
(even with addition of acetic acid), or lithium adducts
[L C Li]C (m/z 305) with addition of lithium chloride (spectra
not shown). The CAD spectra of these alkali metal adducts
were relatively simple. The major fragment (m/z 311) of
[20 C Na]C (m/z 321) was the loss of CO with subsequent
addition of H2 O, whereas [20 C Li]C mainly underwent a
methyl radical loss resulting in a fragment at m/z 290. All
the other chalcones have at least one hydroxyl group, which
makes them slightly acidic. They did not form [L C H]C
(even with acid addition), [L C Na]C or [L C Li]C ions that
were abundant enough to be isolated. Therefore, CAD of
A) Deprotonation
100 7.7 × 105
Relative Abundance
B) Cobalt complexation
100 1.0 × 107
0
140 180
Figure 2. ESI spectra of chalcone 16 in both deprotonation and cobalt complexation modes with the relative intensities shown.
Copyright  2003 John Wiley & Sons, Ltd.
ESI-MS/MS structural characterization of chalcones 557
protonated or alkali metal cationized chalcones was not
pursued further.
Deprotonation
Chalcones other than 20 were easily deprotonated due to
their acidic nature (Fig. 2(A)). The CAD data of the resulting
[L
H]
ions are summarized in Table 1 for chalcones
without methoxy groups and in Table 2 for chalcones with
methoxy groups, and typical CAD spectra are shown in
Figs 3–5 for three sets of isomers. To help rationalize the
proposed fragmentation mechanisms, MSn experiments were
performed on all the deprotonated chalcones (Table 3).
A-vs B-type fragments
As shown in Tables 1 and 2 and Figs 3–5, many of the
fragment ions can be classified as A or B type, meaning
that these products retain the A or B ring portion of the
chalcone while the second ring is lost. The loss of B ring
(which results in the A type of ions) has been observed
earlier for some chalcones by electron ionization.21,29 Both
A- and B-type fragments have also been reported for other
subclasses of flavonoids by ESI.36 Examples of these types of
fragment ions are illustrated in Scheme 1. In Scheme 1, the
site of deprotonation is shown to accommodate the product
ion structures. The resulting fragment ions may isomerize
or intraconvert to other resonance structures, and thus the
structures shown in Scheme 1 are only given to help guide
the reader in matching up structures that reasonably fit the
masses of the fragments listed in the tables and figures.
MSn provided support for some of the A vs B fragment
assignments (Table 3). For example, MS/MS/MS indicated
that the product ion m/z 119 of 2 was an ‘A’ ring fragment
even though the A and B rings have the same isomeric
composition (the A ring fragment would be C8 H7 O
and
the B ring fragment would be C7 H3 O2
). The assignment of
the fragment ion as an ‘A’ ring fragment was verified by
283 [L - H]-
498 [CoII(L - H) bipy]+
220 260 300 340 380 420 460 500 540
m/z
J. Mass Spectrom. 2003; 38: 555–572
558 J. Zhang and J. S. Brodbelt

Table 1. CAD of deprotonated chalcones without methoxy groups (m/z, %), where ‘A’ and ‘B’ denote fragments retaining the A or B
ring system

1 2 3 4 5 6 9 18

Pa 223 (6) 239 (7) 239 (11) 239 (8) 239 (6) 239 (12) 255 (7) 287 (5)
P
H2 221 (41) 237 (93)
P
H2 O 205 (18) 221 (1) 221 (100) 221 (60) 269 (4)
P
CO 195 (100) 211 (4) 211 (100)
P
H2
CO 193 (3)
P
C2 H2 O 197 (100) 197 (17)
P
44 195 (55) 195 (15) 195 (22) 195 (74)b 211 (7)
‘A’ 119 (100) 91 (4) 93 (100)d 119 (11)
119 (50)
‘B’ 93 (3) 135 (44) 93 (100)d 161 (95) 135 (100) 151 (100)
145 (2) 109 (3) 145 (9) 135 (27) 125 (3)
107 (10)
Othersc 148 (21) 133 (4) 224 (25)
131 (10)
a
P D
L
H
, surviving parent ions.
b
MSn experiments indicate that the loss of 44 u may also be due to the loss of H2 and C2 H2 O. For the other chalcones, there is no
evidence for the loss of 44 u being anything other than the direct loss of CO2 .
c
Only fragment ions with more than 10% relative abundance are listed.
d
m/z 93 can be either A- or B-type ions owing to the same structure of A and B rings.

Table 2. CAD of deprotonated chalcones with methoxy groups (m/z, %), where ‘A’ and ‘B’ denote fragments retaining the A or B
ring system

7 8 10 11 12 13 14 15 16 17 19 20

Pa 253 (7) 253 (8) 267 (8) 269 (6) 269 (5) 269 (7) 283 (8) 283 (7) 283 (8)283 (13) 297 (10) X
P
2CH4 C H2 O 269 (40)
ž
P
CH3 238 (100) 238 (100) 252 (100) 254 (100) 254 (15) 254 (100) 268 (100) 268 (100) 268 (100) 268 (90) 282 (100)
b
P
16 237 (15) 237 (10) 251 (7) 267 (12) 267 (14)
P
H2 O 251 (73) 265 (2)
P
CO 225 (9) 225 (3) 239 (4) 255 (44)
ž
P
2CH3 237 (3) 253 (88) 253 (5) 253 (84) 253 (100) 267 (92)
P
2CH4 251 (78)
ž
P
CH3
H2 O 236 (16)
P
C2 H2 O 227 (23)
ž
P
CH3
CO 210 (1) 224 (4) 226 (13) 240 (5) 254 (2)
P
44 209 (7)c 225 (11)c 225 (7) 225 (2)
P
H2 O
CO 223 (5)
ž
P
CH3
CO2 210 (8)
P
2CH4
CO 223 (30)
P
2H2 O
CO 219 (17)
‘A’ 175 (9) 145 (26)
149 (100)
123 (19)
‘B’ 191 (5) 93 (33) 148 (30) 175 (4)
165 (23) 123 (5)
Othersd 236 (10)
a
P D
L
H
, surviving parent ions.
b
MSn experiments indicate that the loss of 16 u for 7, 8, 10, 14 and 16 may be due to two pathways: the loss of CH4 and the loss of
ž ž
CH3 and H .
c n ž ž
MS experiments indicate that the loss of 44 u for 7 may be due to three pathways: the loss of CO2 , the loss of CH3 and HCO , and
n
the loss of CH4 and CO. MS experiments indicate that the loss of 44 u for 11 may be due to two pathways: the loss of CO2 and the loss
ž ž
of CH3 and HCO . For the other chalcones, there is no evidence for the loss of 44 u being anything other than the direct loss of CO2 .
d
Only fragment ions with more than 10% relative abundance are listed.

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
 ESI-MS/MS structural characterization of chalcones 559

A) Chalcone 2
100 A
119 (C8H7O)
195 (-CO2)

∗239
0

B) Chalcone 3
100 197 (-C2H2O)
B
135 (-C8H8)
A B 148
91 109 153
195 ∗239
0
Relative Abundance

C) Chalcone 4
100 93 (C6H5O)
A
119 (C8H7O)
B 195 (-CO2)
145 (-C6H6O) ∗239
0

D) Chalcone 5
221 (-H2O)
100

∗239
0

E) Chalcone 6 221 (-H2O)

B 211 (-CO)
100 161 (-C6H6) 195 (-CO2) 237 (-H2)
B
135 (-C8H8)
197 ,∗239
0
70 100 130 160 190 220 250
m/z

Figure 3. CAD spectra of deprotonated chalcone isomers 2, 3, 4, 5 and 6. Parent ions [L
H]
are labeled with asterisks. ‘A’ and
‘B’ denote fragments retaining the A or B ring system.

A) Chalcone 11
100 254 (-CH3·)
251 (-H2O)
B
191 (-C6H6) 236 (-CH · –H O)
3 2
B
165 (-C8H8) 225/226
∗269
0

B) Chalcone 12
Relative Abundance

100 A
149 (C9H9O2)

210 (-CH3·- CO2)

B
93 (C6H5O) A 225 (-CO2)
123 (C7H7O2) A
254 (-CH3·)
175 (-C6H6O) ∗269
0

C) Chalcone 13
100 254 (-CH3·)

B
148 (C8H4O3) 227 (-C2H2O)
∗269
0
80 120 160 200 240 280
m/z

Figure 4. CAD spectra of deprotonated chalcone isomers 11, 12 and 13. Parent ions [L
H]
are labeled with asterisks. ‘A’ and ‘B’
denote fragments retaining the A or B ring system.

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
560 J. Zhang and J. S. Brodbelt

A) Chalcone 14
100 268 (-CH3·)
253 (-2CH3·)

∗283
0

B) Chalcone 15
100 268 (-CH3·)
Relative Abundance

253 (-2CH3·) ∗283

0

C) Chalcone 16
100 268 (-CH3·)
253 (-2CH3·)

∗283
0

D) Chalcone 17 255(-CO)
100 253 (-2CH3·)
268 (-CH3·)
251(-2CH4)
269 (-2CH4 + H2O)
145 223 (-2CH4-CO)
219 (-CO–2H2O) ∗283
0
80 100 120 140 160 180 200 220 240 260 280 300
m/z
Figure 5. CAD spectra of deprotonated chalcone isomers 14, 15, 16 and 17. Parent ions [L
H]
are labeled with asterisks.

Table 3. MSn data of deprotonated chalcone ions Table 3. (Continued)

(L
H
(m/z)
Frag- Neutral
Frag- Neutral Chalcone Route ment loss %a
Chalcone Route ment loss %a 193 28 100
1 223 ! 221 ! 193 28 100 239 ! 211 ! 183 28 100
165 56 13 169 42 31
145 76 14 167 44 60
223 ! 195 ! 193 2 100 157 54 14
143 68 17
2 239 ! 195 ! 167 28 100
239 ! 161 ! 133 28 31
119 76 64
117 44 100
239 ! 119 ! 93 26 100
91 28 16 7 253 ! 238 ! 237 1 100
209 29 20
3 239 ! 197 ! 169 28 100
253 ! 237 ! 209 28 22
141 56 25
193 44 100
239 ! 195 ! 193 2 75
253 ! 225 ! 210 15 100
169 26 100
167 28 37 8 253 ! 238 ! 237 1 100
239 ! 148 ! 120 28 100 210 28 21
239 ! 135 ! 91 44 100 253 ! 237 ! 209 28 100
4 239 ! 119 ! 117 2 100 193 44 20
91 28 18 253 ! 225 ! 210 15 100
239 ! 93 ! 65 28 100 9 255 ! 211 ! 183 28 100
5 239 ! 221 ! 211 28, CH2 Ob 32 255 ! 135 ! 125 28, CH2 Ob 30
193 28 100 107 28 100
165 56 11 255 ! 119 ! 93 26 100
6 239 ! 237 ! 209 28 98 10 267 ! 252 ! 251 1 100
195 42 39 237 15 22
193 44 100 224 28 37
239 ! 221 ! 211 28, CH2 Ob 13

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
 ESI-MS/MS structural characterization of chalcones 561

Table 3. (Continued) Fragment ions at m/z 149 and 123 in the CAD spectrum of
deprotonated 12 were assigned as A ring fragments because
Frag- Neutral
of the exclusive loss of 15 u observed in the MS/MS/MS of
Chalcone Route ment loss %a
m/z 123 and 149. The sole methoxy group of 12 is located on
11 269 ! 254 ! 253 1 25 the A ring.
226 28 100
225 29 86 Small neutral losses
177 77 18 In addition to the cleavages due to A or B ring losses
163 91 18 (see Fig. 1 for the location of the A and B rings), small
269 ! 251 ! 236 15 100 neutral losses of H2 O, CO and CO2 were commonly observed
and sometimes dominant for chalcones with only hydroxyl
12 269 ! 254 ! 253 1 100
groups (Table 1). In addition to the expected neutral losses
236 18 60
of CO and CO2 and the formation of A and B ring fragments,
269 ! 149 ! 134 15 100
there was an unusual loss of H2 for 1 and 6, as well as a
269 ! 123 ! 108 15 100
prominent loss of H2 O for 5, 6 and 11. The pathways for
13 269 ! 254 ! 148 106 100 these losses of H2 or H2 O are not readily explained, nor
269 ! 227 ! 212 15 100 is it evident why these unusual losses are observed only
269 ! 148 ! 120 28 100 for certain chalcones. Another small neutral loss of C2 H2 O
(42 u) was observed in the CAD spectra of deprotonated
14 283 ! 268 ! 267 1 3
chalcones 3, 6 and 13, and was especially prominent for 3.
253 15 100
Each of these three chalcones has two hydroxyl groups on
283 ! 253 ! 225 28 100
the B ring, either in the meta- or para-position. The same
209 44 56
loss has also been observed for some other deprotonated
15 283 ! 268 ! 253 15 100 flavonoids.36 Extensive isotopic labeling is impractical owing
16 283 ! 268 ! 267 1 2 to the high cost of the chalcones (up to $200 per mg) and
253 15 100 the synthetic difficulty of selective deuterium, 18 O or 13 C
283 ! 253 ! 235 18 16 labeling, in addition to the possibility of solution or gas-
225 28 100 phase isomerization of the chalcones, as shown in Scheme 2
211 42 43 for one simple deprotonated chalcone. Neutral losses such
209 44 81 as CO, CO2 , and C2 H2 O also have been observed for other
185 68 18 deprotonated flavonoids,36 such as for flavonols, flavones
and flavanones. In fact, the loss of CO2 from deprotonated
17 283 ! 268 ! 253 15 100 flavones has been proposed to involve a carbonyl group and
283 ! 255 ! 240 15 100 an oxygen of the C ring in a rather unusual rearrangement
283 ! 253 ! 238 15 56 reaction.36
209 44 100 For chalcones with both hydroxyl and methoxy groups,
283 ! 251 ! 236 15 17 the most dominant losses were methyl radicals (and/or
223 28 100
283 ! 223 ! 208 15 100
283 ! 145 ! 117 28 100 A) MS2 268 (-CH3⋅)
100
253 (-2CH3⋅)
18 287 ! 151 ! 107 44 100
19 297 ! 282 ! 267 15 100
297 ! 267 ! 252 15 25 ∗283
0
239 28 100
Relative Abundance

223 44 51 B) MS3
100 253 (-CH3⋅)
a
Only fragments with more than 10% relative abundance are
listed, except for 14 and 16.
b
The observed loss of 10 u is due to loss of CO followed by ∗268
0
addition of H2 O.
C) MS4
100 225 (-CO)
209 (-CO2)
the loss of 26 u upon CAD of m/z 119, and this loss of 26 u
(HC CH) must involve the carbons adjacent to the A ring. ∗253
The assignments of m/z 135 of 3 and 161 of 6 as ‘B’ ring 0
100 120 140 160 180 200 220 240 260 280 300
fragments were supported by the subsequent loss of CO2
m/z
in the MS/MS/MS of 135
and 161
, respectively, which
would not be possible for A ring species owing to the lack of Figure 6. MSn
of deprotonated chalcone 14. Parent ions are
oxygen atoms on the A ring. labeled with asterisks.

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
562 J. Zhang and J. S. Brodbelt

methane for 7, 8, 10, 14 and 16) in addition to other small
neutral losses, such as CO (Table 2). The consecutive loss
of methyl radicals during CAD was confirmed by MSn
experiments (Fig. 6 and Table 3), which showed sequential
loss of 15 u. Even though it is unusual to have radical losses
from even-electron molecular ions, this was observed not
only for deprotonated chalcones but also previously for
other flavonoids.36 – 39
As shown in Fig. 6 and Table 3, MS/MS/MS experiments
proved to be useful in confirming some of the suspected
fragmentation pathways, including support for sequential
losses. For example, for the deprotonated chalcones without
methoxy groups, the loss of 30 u in MS/MS/MS was
(H2 C CO). This could be seen by examining the sequence:
parent ion P !
P
2 !
P
30 or P !
P
28 !

P
30, where P represents the deprotonated precursor
chalcone. For the deprotonated chalcones with two methoxy
groups (14, 15, 16, 17 and 19), the loss of 30 u was in part due
ž
to sequential loss of CH3 based on the consecutive loss of
15 u seen in MS/MS/MS (Fig. 6). Note that this MS/MS/MS
sequence does not rule out other pathways for the loss of
30 u, such as the loss of CO with H2 . Even though 10 has
only one methoxy group and one methyl group, the loss
of 30 u seen for deprotonated 10 was likewise confirmed to
be at least in part due to loss of two methyl radicals in the
MS/MS/MS experiments.
MS/MS/MS experiments also showed that the loss of
44 u from deprotonated 7 is actually in large part due to
ž ž
consecutive losses of CH3 and HCO or consecutive losses
of CH4 and CO (Table 3). The consecutive losses of CH4
and CO are also observed in MS/MS/MS experiments for
deprotonated 8 even though this [P
44] fragment is not
observed upon CAD of this deprotonated chalcone (Table 2).
Similarly, the loss of 44 u from deprotonated 11 was found
ž ž
to be due in part to consecutive losses of CH3 and HCO .
Furthermore, for deprotonated 6, one route for the loss of
44 was proven to be consecutive losses of H2 and C2 H2 O,
as confirmed by MS/MS/MS experiments (Table 3). For
the other deprotonated chalcones that showed a loss of
44 u in their CAD spectra, these unusual consecutive losses
ž ž
(i.e. CH3 and HCO , CH4 and CO, H2 and C2 H2 O) were
not indicated based on MS/MS/MS experiments, hence the

O O- O- O O-

A B

1 93- (B) 145- (B)

O OH

-O
-O
2 119- (A)

O- O-
O O- O
-
CH2 C

OH OH O
3 91- (A) 109- (B) 135- (B)

O OH O- O O-

O- O-
4 93- (Aor B) 119- (A) 145- (B)

O O-
O OH O
O
C

O- OH
O-
6 135- (B) 161- (B)

O O
O O- C O
C

-O
HO
O- O-
OH
9 107- (B) 119- (A) 135- (B)

Scheme 1. Structures of the A- and B-type fragment ions of some deprotonated chalcones.

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
 ESI-MS/MS structural characterization of chalcones 563

O O-
O OH O
O
C

-O OCH3 -O OCH3 HO OCH3

11 165- (B) 191- (B)

O OH O-
C
C
O
O- O- O-
O- OCH3 OCH3 OCH3
OCH3
12 93- (B) 123- (A) 149- (A) 175- (A)

O O- O
-O

H3CO OH O
.
13 148- (B)

O O- O- O O-

C
C
O
OCH3 OCH3 OCH3 O- OCH3
- (B) - (A)
17 123 145 175- (B)

O O-
O-
O
C
HO HO OH HO O
OH
18 151- (B)

Scheme 1. (Continued).

4' Deprotonated 7, 8, 10, 14 and 16 all dissociate by the

5' ž
3' loss of CH3 and the loss of CH4 upon collisional activation.
6 O O-
6' MS/MS/MS experiments confirm that the loss of CH4 is in
5 O ž ž
3' 6 part due to consecutive losses of CH3 and H . MS/MS/MS
4 2 5 O ž ž
3
6' 4' - experiments show that sequential losses of CH3 and H
5' 4 2
may also occur for chalcone 11 even though the fragment
3
I II [P
16] is not directly observed in the CAD spectrum of this
deprotonated chalcone (Table 2).
As discussed above for the MSn experiments, some of
the neutral losses observed upon CAD of the deprotonated
chalcones may actually involve several pathways. For
4'
3' 5' example, MSn experiments indicate that the loss of 44 u
ž
6' may be loss of CO2 , loss of H2 with C2 H2 O, loss of CH3 with
O ž
6 HCO , and loss of CH4 with CO, depending on the identity
5 O- of the chalcone. Likewise, in some cases the loss of 16 u is due
ž ž
4 2 to consecutive loss of H and CH3 , not exclusively the loss of
3 CH4 . These alternative pathways are shown in the footnotes
III
of Tables 1 and 2. In general, the MS/MS/MS data indicated
Scheme 2. Proposed resonant structures of deprotonated that some of the fragment ions observed in the CAD spectra
chalcones. Note that flavanones have a different numbering of the deprotonated chalcones stemmed from consecutive
system other than the chalcones as shown in Fig. 1. However, losses of small neutrals, thus adding a layer of complexity to
the same numbering system according to the original chalcone the rational interpretation of the fragmentation patterns of
is used for the whole scheme to emphasize the positions chalcones. In addition, MSn experiments proved to be useful
involved in the ring closure. in verifying some of the A versus B ring assignments.
Scheme 2 indicates the possible isomerization pathway
most likely pathway is simple loss of CO2 , which has been of chalcones to flavanones in order to explain some of
confirmed for other flavonoids. the A and B type fragments in Scheme 1, which is in

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
564 J. Zhang and J. S. Brodbelt
accordance with earlier studies.21 – 23 To verify further the
existence of flavanone isomeric forms of the chalcones, two
flavanones isomeric to chalcones 11 and 18 were chosen
for ESI-MS and CAD. 5-Hydroxyl-7-methoxy flavanone (FA,
Fig. 1) is an isomer of chalcone 11. The fragments upon
CAD of the deprotonated FA were the same as that of
deprotonated chalcone 11 (Table 2) but the abundances were
very different, with m/z 191 (100%) becoming the base
fragment ion followed by m/z 165 (84%). This could be
explained by the much easier loss of the ‘B’ ring in the
ž
flavanone. The other major fragments were P
CH3 (42%),
P
H2 O (21%) and P
CO (14%). The second flavanone
tested was 5,7,30 ,40 -tetrahydroxyflavanone (eriodictyol, FB,
Fig. 1), an isomer of chalcone 18. The CAD spectra of the
deprotonated ions of these two compounds were identical
(Table 1). The similarity of the CAD patterns of the above
two isomer pairs is further strong evidence for possible
isomerization between the chalcone and flavanone forms.
Fragmentation patterns vs structural features
It is interesting that the fragmentation patterns of the various
chalcones may change dramatically upon rather modest
changes of substituents. For examples, substitution of a
methoxy group for a hydroxyl group, such as the case for
chalcones 3 versus 7, significantly changes the observed CAD
patterns. Deprotonated 3 produces diagnostic A and B ions
upon CAD, in addition to the characteristic loss of C2 H2 O,
whereas deprotonated 7 dissociates predominantly by loss of
ž
CH3 , CH4 , or CO, and forms no detectable A or B ions. The
different CAD patterns of 3 and 7 might be at least partially
due to the different numbers of possible isomeric forms: 3 has
two different hydroxyl groups that can be deprotonated upon
ESI which results in two possible isomeric forms, whereas 7
only has one hydroxyl group available for deprotonation. A
similar lack of parallel fragmentation patterns occurs for the
pair 13 and 16. These chalcones each have three substituents
in the same three positions, except that one has two hydroxyl
groups and one methoxy group whereas the other has two
methoxy groups and one hydroxyl group. Deprotonated 13
ž
dissociates via three pathways, including loss of CH3 , loss of
C2 H2 O and formation of one diagnostic B ion. Deprotonated
ž
16 only undergoes loss of one or two CH3 units and gives
no A or B fragment ions. The addition of an extra substituent
can also promote changes in the fragmentation patterns, as
illustrated by the comparison of deprotonated 3 and 13 (the
latter having a methoxy group at the 4-position). The CAD
spectrum of deprotonated 13 lacks the detail of that observed
for deprotonated 3. In fact, the methoxy group in 13 is key
ž
for the dominant fragmentation pathway (loss of CH3 ), and
the observed B fragment is not an analog of any of those
observed for deprotonated 3.
The comparisons summarized above are examples of
how the interpretation of the fragmentation patterns of
related chalcones is not always straightforward. However,
there are cases in which the fragmentation patterns of
complementary pairs show logical similarities. For example,
chalcones 4 and 12 both have two hydroxyl substituents
at the 20 - and 2-positions, and 12 also has one methoxy
group at the 3-position. Each of these undergoes the loss
Copyright  2003 John Wiley & Sons, Ltd.
of CO2 , 94 u (loss of C6 H6 O, the B ring), 120 u (loss of
C7 H2 O4 , another B ring loss) and 146 u (loss of C9 H6 O2 ,
another B ring loss) upon collisional activation, which clearly
simplifies the interpretation of the fragmentation patterns
of these related chalcones. In short, the chalcones do not
follow consistent structure-based fragmentation rules, thus
suggesting that creation of mass spectral libraries will be
critical for identifying chalcones in mixtures.
Isomer differentiation
Five chalcone isomer series were included in this study:
chalcones 2, 3, 4, 5 and 6 (Series 1, each containing
two hydroxyl groups); chalcones 7 and 8 (Series 2, each
containing one hydroxyl group and one methoxy group);
chalcones 11, 12 and 13 (Series 3, each containing two
hydroxyl groups and one methoxy group); chalcones 14,
15, 16 and 17 (Series 4, each containing one hydroxyl
group and two methoxy groups), and chalcones 19 and
20 (Series 5, containing a combination of hydroxyl, methoxy
and methyl groups). Chalcones in each series differ only by
positions of the same functional groups except in Series 5,
the latter compounds having the same chemical formulas
but different combinations of functional groups. It was
our goal to differentiate these isomers by ESI-tandem mass
spectrometry.
CAD of the deprotonated chalcones was sufficient to
differentiate the chalcones in Series 1 and 3, which have
more than one hydroxyl group (Figs 3 and 4). Although
some common fragments are observed for each isomer
series, the individual fragmentation patterns are unique.
For example, in Series 1, chalcone 3 gives unique A- and
B-type fragment ions at m/z 91, 109 and 135 (with structures
proposed in Scheme 1). Chalcone 4 produces a distinctive
A and B fragment ions at m/z 93 and 145 and chalcone 6
gives a very rich, diagnostic CAD pattern with two B-type
ions (see Scheme 1). Chalcones 2 and 5, which have only
one or two fragment ions, can nonetheless be distinguished
from the others owing to their lack of key ions present in
the CAD spectra of the other three chalcones. For Series 3,
deprotonated 11 and 12 each produce an array of diagnostic
ions upon CAD, including several A- and B-type ions shown
in Scheme 1, whereas deprotonated 13 is the only one of
this series that dissociates by loss of C2 H2 O, in addition to a
unique B-type ion.
CAD spectra of the deprotonated chalcones in Series 2
and 4 did not provide adequate differentiation (Table 2 and
Fig. 5). The deprotonated chalcones 7 and 8 predominantly
ž
dissociate by loss of CH3 , and the unique loss of 44 u that
is observed only for deprotonated 7 is a minor pathway.
For the Series 4 chalcones, a comparison of CAD patterns
of isomeric chalcones 14, 15, 16 and 17 showed that 15 only
ž
showed a minor loss of 2CH3 , while 14, 16 and 17 showed
ž
significant losses of 2CH3 (Table 2 and Fig. 5). However,
even more striking was the variety of fragments observed
for deprotonated 17, which not only had neutral losses such
ž ž
as CH3 , CH4 , CO, 2CH3 , and 2CH4 , but also gave one
diagnostic fragment ion at lower mass range (i.e. m/z 145),
an A ring species (Table 2 and Fig. 5). Chalcones 16 and 17
differ only in the position of one methoxy group, and the
J. Mass Spectrom. 2003; 38: 555–572
 ESI-MS/MS structural characterization of chalcones 565

greater array of dissociation pathways for 17 is not readily 7 dissociated predominantly by loss of a methyl radical, with
explained based on this minor structural difference of the minor losses of methane, CO or 44 u. In contrast, the metal
isomers. Deprotonated 17 gives a rich CAD spectrum that is complex [CoII (7
H) bipy]C produced several diagnostic
diagnostic, but the other three isomers are not differentiated fragment ions with possible structures proposed in Scheme 3,
ž ž
with confidence owing to the dominant or exclusive methyl in addition to characteristic losses of CH3 and CH3 with
radical losses upon CAD. Therefore, metal complexation one or two CO units.
was examined as an alternative ionization method in an An example of the comparative CAD spectra for the
effort to produce complexes that would give distinctive complexes of chalcone 14 containing different metals (CoII ,
fragmentation patterns. NiII , CuII ) is shown in Fig. 8. Both the cobalt and nickel
Differentiation of the two chalcones in Series 5 was an complexes, presumably owing to the similar metal binding
easy case because these two chalcones differed significantly strength of the deprotonated chalcone and the auxiliary
in their ionization efficiencies in both the protonation and ligand and the similar coordination structures, dissociated to
deprotonation modes. Chalcone 20 does not deprotonate and
19 does not protonate.
A) Cobalt complexation 468 (-2CH3⋅)
232/233 465 (-CH3⋅ - H2O)
100 482 (-CH4)
Metal complexation 247 454 (-CO2)
II +
Since flavonoids in general deprotonate easily and have a 308 [Co (C6H5O) bipy]
216 307
tendency to coordinate with metals, metal complexation has ∗498
0
been used as an alternative for structural characterization.34,35

Relative Abundance
2,20 -Bipyridine, which is neutral and has a similar metal B) Nickel complexation
231/232 307 [NiII (C H O) bipy]+
binding strength to that of the deprotonated flavonoids, is 100 6 5
246 495 (-H2)
used as an auxiliary ligand to promote stable complexes and
369
prevent the formation of neutral dimeric species between the 214
∗497
0
metal and two deprotonated flavonoids.34,35 The addition of
a transition metal (CoII , NiII or CuII ) and 2,20 -bipyridine C) Copper complexation
237
to the chalcone solutions produced [MII (L
H) bipy]C 100
complexes. The signal intensities of the metal complexes 219
were similar to or higher than that of the corresponding 251 ∗502
deprotonated chalcones (Fig. 2). CAD was then performed on 0
100 150 200 250 300 350 400 450 500 550
the metal complexes. In many cases, the metal complexes give m/z
much richer fragmentation patterns than the corresponding
deprotonated chalcones. An example is shown in Fig. 7 Figure 8. CAD mass spectra of [MII (14
H) bipy]C , where
for chalcone 7. Upon collisional activation, deprotonated MII D Co2C , Ni2C or Cu2C . Parent ions are labeled
with asterisks.

A) (L – H)-
100 238 (-CH3⋅) A) Chalcone 14 468 (-2CH3⋅)
100 232/233 465 (-CH3⋅ – H2O) 482 (-CH4)
247 454 (-CO2)
308 [CoII (C6H5O) bipy]+
216
307 ∗498
0
237 (-16)
B) Chalcone 15
225 (-CO)
100 483 (-CH3⋅)
209 (-44) ∗253 468 (-2CH3⋅)
Relative Abundance
Relative Abundance

0 466 (-2CH4)
70 90 110 130 150 170 190 210 230 250 270 352 ∗498
m/z 0

C) Chalcone 16
B) [CoII (L – H) bipy]+ 468 (-2CH3⋅)
100 483 (-CH3⋅)
100 233 455 (-CH3⋅ - CO)
453 (-CH3⋅) 232/233
228
247 425 (-CH3⋅ – CO)
216 247 338 427 ∗498
216 397 (-CH3⋅ - 2CO) 0
338 [CoII (C7H7O2) bipy]+
292 [CoII (C6H5) bipy]+ D) Chalcone 17
100 483 (-CH3⋅)
337
362 232/233
157 348 ∗468 216 247
∗498
0 0
140 180 220 260 300 340 380 420 460 500 140 180 220 260 300 340 380 420 460 500 540
m/z m/z

Figure 7. CAD spectra of chalcone 7 in both deprotonation Figure 9. CAD spectra of chalcone 14, 15, 16 and 17
and cobalt complexation modes. Parent ions are labeled with complexes with cobalt and 2,20 -bipyridine. Parent ions [CoII
asterisks. The losses of 16 and 44 u are explained in Table 2. (L
H) bipy]C are labeled with asterisks.

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
566 J. Zhang and J. S. Brodbelt

N N N N N N
N N
Co2+ Co2+ Co2+ Co 2+

O O- O-
- -

OCH3 OCH3
7
338+ 318+ 292+

N N
N N N N N
N
Co2+
Co2+ Co 2+
Co2+
O O- CH2-
O O- O-
O

H3CO
8
360+ 322+ 308+

Scheme 3. Proposed structures of the unique fragment ions of the cobalt complexes of the chalcones in Series 2.

Table 4. CAD of cobalt complexes of chalcones without methoxy groups: [CoII (L
H) bipy]C (m/z, %), where ‘A’ and ‘B’ denote
fragments retaining the A or B ring system

1 2 3 4 5 6 9 18

Pa 438 (9) 454 (10) 454 (10) 454 (5) 454 (10) 454 (8) 470 (9) 502 (7)
P
H2 436 (10) 452 (18) 452 (8) 452 (1) 452 (11) 468 (13) 500 (28)
P
H2 O 420 (2) 436 (18) 436 (20) 436 (100) 436 (100) 436 (51) 452 (61) 484 (36)
P
CO 410 (10) 426 (9) 426 (6) 426 (16) 442 (3)
P
H2
CO 408 (6) 424 (2) 440 (2)
P
CO2 410 (2)
P
H2 O
CO 408 (19) 408 (8) 408 (14) 424 (6)
P
2CO 382 (13)
‘A’ 318 (16) 334 (15) 292 (23) 334 (34) 308 (9)
292 (26)
‘B’ 308 (62) 308 (91) 324 (31) 308 (36) 324 (28) 324 (29) 376 (35)
Common ionsb 248 (24) 248 (26) 248 (22) 248 (1) 248 (9) 248 (12)
247 (32) 247 (43) 247 (33) 247 (1) 247 (24) 247 (25) 247 (2)
246 (10) 246 (10) 246 (10) 246 (13) 246 (8) 246 (4) 246 (2)
233 (38) 233 (52) 233 (40) 233 (2) 233 (33) 233 (29) 233 (6)
232 (47) 232 (37) 232 (40) 232 (77) 232 (2) 232 (30) 232 (20) 232 (23)
228 (14) 228 (17) 228 (50) 228 (9) 228 (4) 228 (6)
216 (100) 216 (100) 216 (100) 216 (5) 216 (14) 216 (55) 216 (11)
157 (12) 157 (15) 157 (12) 157 (1) 157 (5) 157 (6) 157 (2)
Othersc 332 (24) 332 (27) 348 (29) 437 (100) 466 (36)
309 (14) 334 (12) 370 (10) 417 (11)
380 (24)
350 (100)
346 (17)
337 (24)

a
P D [CoII (L
H) bipy]C , surviving parent ions.
b
Common product ions identified are [CoI (CH3 OH) bipy]C (m/z 247), [CoII (CH3 O) bipy]C (m/z 246), [CoI (H2 O) bipy]C (m/z 233),
[CoII (OH) bipy]C (m/z 232), [bipy C H]C (m/z 157).
c
Only fragment ions with more than 10% relative abundance are listed.

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
 ESI-MS/MS structural characterization of chalcones 567

similar fragment ions upon CAD. Note that the fragment
ions seen in the range m/z 200–255 are non-specific
products containing 2,20 -bipyridine, the metal, and up to
one solvent (water or methanol) molecule. For most of the
chalcones, the [CoII (chalcone—H) bipy]C fragmentation
patterns give the most useful array of diagnostic ions
(see Fig. 9(A) as an example). Since 2,20 -bipyridine has a
much greater copper binding energy compared to that of
each chalcone, the whole chalcone molecule is generally
lost upon collisional activation of the copper complexes,
resulting in non-diagnostic fragmentation patterns for the
[CuII (chalcone—H) bipy]C complexes (Fig. 9(C)).
Cobalt complexation
The CAD data for the cobalt complexes are provided in
Tables 4 and 5, and a representative series of CAD spectra
are shown in Fig. 9. For the chalcones without methoxy
groups, commonly observed small neutral losses included
H2 , H2 O, and CO (Table 4), similar to the losses seen for the
deprotonated chalcones. Dominant losses observed for the
ž
chalcones with methoxy groups were CH3 , CH4 , H2 , H2 O,
CO and CO2 (Table 5). In addition, several less informative
products were observed for all the chalcones, such as [CoI
(CH3 OH) bipy]C (m/z 247); [CoII (CH3 O) bipy]C (m/z 246);
[CoI (H2 O) bipy]C (m/z 233); [CoII (OH) bipy]C (m/z 232);
[bipy C H]C (m/z 157). In fact, ions below m/z 250 are
generally cobalt/2,20 -bipyridine species that are non-specific.
Relative to the CAD patterns of the deprotonated chalcones,
the losses of H2 O, H2 and CO are generally more prominent
for the metal complexes, [CoII (chalcone—H) bipy]C , and
the relative loss of 44 u is significantly lower. Moreover, the
types of A and B ions are different for the deprotonated

Table 5. CAD of cobalt complexes of chalcones with methoxy groups: [CoII (L
H) bipy]C (m/z, %), where ‘A’ and ‘B’ denote
fragments retaining the A or B ring system

7 8 10 11 12 13 14 15 16 17 19 20
a
P 468 (10) 468 (8) 482 (11) 484 (10) 484 (9) 484 (9) 498 (11) 498 (12) 498 (11) 498 (12) 512 (9) X
P
H2 466 (4) 466 (8) 482 (100) 482 (8) 496 (7) 496 (6) 496 (2) 510 (3)
ž
P
CH3 453 (92) 453 (100) 467 (100) 469 (1) 469 (1) 469 (100) 483 (19) 483 (100) 483 (100) 483 (100) 497 (100)
P
CH4 452 (17) 452 (21) 468 (17) 482 (100) 482 (12)
ž
P
H2
CH3 451 (10)
P
H2 O 450 (5) 450 (3) 466 (5) 466 (52) 466 (11)
P
CO 440 (6) 440 (5) 456 (2) 456 (2)
P
H2
CO 438 (8) 438 (4) 454 (4)
ž
P
2CH3 452 (21) 468 (46) 468 (58) 468 (11) 468 (11) 482 (71)
ž
P
CH3
CH4 467 (10)
P
2CH4 466 (24) 466 (12)
ž
P
CH3
H2 O 465 (11) 465 (11)
ž
P
CH3
CO 425 (57) 425 (4) 441 (3) 441 (13) 455 (16) 455 (9) 468 (8)
P
CO2 424 (6) 424 (3) 440 (4) 454 (27) 454 (6)
P
H2 O
CO 452 (10)
P
2CO 412 (5)
ž
P
CH3
H2 O
CO 437 (8) 437 (8)
ž
P
CH3
2CO 397 (36) 427 (11) 427 (3)
‘A’ 318 (5) 322 (6) 318 (4) 364 (100)
292 (13) 292 (9)
‘B’ 338 (34) 360 (9) 322 (6) 308 (34) 376 (5) 308 (15) 338 (12) 390 (3) 322 (8)
308 (30) 337 (69)
Common ionsb 248 (16) 248 (7) 248 (1) 248 (11) 248 (8) 248 (4) 248 (6) 248 (7) 248 (1)
247 (77) 247 (20) 247 (3) 247 (11) 247 (17) 247 (84) 247 (5) 247 (25) 247 (41) 247 (21)
246 (7) 246 (3) 246 (1) 246 (2) 246 (2) 246 (3) 246 (6) 246 (1) 246 (3) 246 (6) 246 (2)
233 (100) 233 (27) 233 (3) 233 (15) 233 (27) 233 (95) 233 (5) 233 (28) 233 (53) 233 (29)
232 (30) 232 (17) 232 (2) 232 (15) 232 (30) 232 (19) 232 (29) 232 (5) 232 (11) 232 (19) 232 (10)
228 (31) 228 (4) 228 (18) 228 (15) 228 (9)
216 (72) 216 (35) 216 (5) 216 (50) 216 (43) 216 (24) 216 (5) 216 (30) 216 (37) 216 (7)
157 (8) 157 (4) 157 (5) 157 (4) 157 (3) 157 (4) 157 (4) 157 (1)
Othersc 362 (23) 393 (18) 348 (15) 465 (11) 352 (11)
378 (12) 324 (18) 307 (14)
354 (21)

a
P D [CoII (L
H) bipy]C , surviving parent ions.
b
Common product ions identified are [CoI (CH3 OH) bipy]C (m/z 247), [CoII (CH3 O) bipy]C (m/z 246), [CoI (H2 O) bipy]C (m/z 233),
[CoII (OH) bipy]C (m/z 232), [bipy C H]C (m/z 157).
c
Only fragment ions with more than 10% relative abundance are listed.

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
568 J. Zhang and J. S. Brodbelt

chalcones compared to the metal complexes, a reasonable

result considering the vastly different structures of these two N N
N N
types of molecular ions. Co2+
Co2+
The CAD spectra of the metal complexes gave a greater O O-
O-
array of fragment ions, including at least one diagnostic ion
for each isomer, more than observed for the deprotonated
chalcones. The chalcones in Series 2, which relied on minor H3CO
OCH3
fragment ions (m/z 209 via loss of 44 u (7%) for 7 vs m/z 210 14 308+
ž
via loss of CH3 and CO (1%) for 8) for differentiation in the
deprotonation mode, had distinctive fragment ions resulting
from the cobalt complexes: 7, m/z 425, 397, 376, 362, 338, N N
N N
318, 292; and 8, m/z 412, 360, 332, 322, 308 (Table 5) (m/z Co2+
Co2+
376 and 332 are fragment ions with less than 10% relative O-
O O-
abundance and are thus omitted in the ‘Others’ column in OCH3 · OCH3
Table 5). Structures for some of these diagnostic ions are
H O.
suggested in Scheme 3. Likewise, all the isomers in Series 4, OCH3
H
which were not distinguishable by CAD in the deprotonation 15 352+
mode due to the dominant loss of one or two methyl radicals
for 14, 15 and 16, had both distinctive CAD patterns and
unique fragments upon CAD of the metal complexes: 14, N N
m/z 307/308; 15, m/z 352; 16, m/z 455, 427, 338; and 17, m/z Co2+
337 (Fig. 9), which led to easy differentiation. Dissociation O-
of the metal complex of 16 results in an even-electron ion
at m/z 338, while the metal complex of 17 gives a radical
N N
ion at m/z 337 that could be rationalized by the availability OCH3
Co2+
of a hydrogen at the 2-position. For 16, the hydrogen at the 338+
O O-
2-position could migrate to the 10 -position while the bond
between the ketone and 10 -carbon is broken, resulting in a
N N
fragment ion at m/z 338 and a ring closure between the H3CO OCH3
ketone and the A-ring at the 2-position. 17 has a methoxy Co2+

group instead of hydrogen at the 2-position, which makes 16 O O-

the hydrogen donation impossible. The structures of the

most unique diagnostic ions are suggested in Scheme 4.
O.
Differentiation of the other isomer series was also possible 427+
based on the CAD patterns of the metal complexes (Tables 4
and 5). Therefore, the CAD spectra of the cobalt complexes
N N N N
served as distinctive fingerprints of the chalcones.
Co2+ Co2+

Nickel complexation O O- O-
The fragmentation patterns of the nickel complexes (Tables 6 ·
and 7) were similar to that of the cobalt complexes, but
OCH3 OCH3 OCH3
with H2 losses enhanced. The CAD mass spectra of these
complexes could also serve as fingerprints of the chalcones.
17 337+

Scheme 4. Proposed structures of the unique fragment ions of

Copper complexation
The copper complexes, in contrast to the cobalt and
nickel complexes, did not give significant chalcone-related
fragments upon CAD (Tables 8 and 9 and Fig. 8(C)). The
main products upon CAD were complexes of copper and the
auxiliary bipyridine ligand. Even when compound-specific
fragments existed in the spectra, their abundances were too
low to be analytically useful.
MSn experiments were also performed on some selected
metal complexes (Table 10). MS/MS/MS experiments of the
cobalt and nickel complexes were also found to be useful in
further differentiation of the isomers in Series 4. For example,
15 and 16 shared a major common fragment at m/z 483, but
the MS/MS/MS patterns of m/z 483 were unique for both
chalcone complexes (Table 10). MSn experiments were also
the cobalt complexes of the chalcones in Series 4.
performed on those fragments that allowed differentiation
of the isomers (m/z 307 and 308 for 14, m/z 352 for 15,
m/z 338 and 427 for 16 and m/z 337 for 17). The structures
of those unique diagnostic fragments proposed in Scheme 4
were based on the supporting MSn data.
CONCLUSION
Different ionization modes were compared for their utility
for formation of chalcone ions and the subsequent CAD mass
spectra. Owing to their slightly acidic nature, chalcones were
easily deprotonated. CAD upon the resulting deprotonated

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
 ESI-MS/MS structural characterization of chalcones 569

Table 6. CAD of nickel complexes of chalcones without methoxy groups: [NiII (L
H) bipy]C (m/z, %), where ‘A’ and ‘B’ denote
fragments retaining the A or B ring system

1 2 3 4 5 6 9 18

Pa 437 (8) 453 (14) 453 (10) 453 (6) 453 (9) 453 (5) 469 (8) 501 (6)
P
H2 435 (75) 451 (56) 451 (66) 451 (45) 451 (42) 467 (100) 499 (100)
P
H2 O 435 (9) 435 (20) 483 (13)
P
CO 409 (6) 425 (7) 425 (6) 425 (3) 425 (4) 441 (15)
P
H2
CO 407 (28) 423 (24) 423 (44) 423 (30) 423 (17) 439 (43) 471 (8)
P
H2 O
CO 407 (2) 407 (9) 407 (1) 423 (20) 455 (6)
P
2CO 381 (8) 397 (4) 397 (3) 397 (5) 397 (2) 413 (3)
P
H2
2CO 379 (1) 395 (2) 395 (3) 395 (1)
‘A’ 317 (4) 291 (33) 291 (13) 307 (72) 349 (10)
291 (32) 323 (21)
‘B’ 307 (81) 307 (100) 323 (86) 307 (100) 307 (100) 323 (32) 323 (83) 375 (11)
Common ionsb 246 (36) 246 (24) 246 (41) 246 (1) 246 (40) 246 (14) 246 (66) 246 (9)
245 (14) 245 (5) 245 (16) 245 (6) 245 (3) 245 (5) 245 (8)
232 (49) 232 (32) 232(56) 232 (51) 232 (18) 232 (88) 232 (36)
231 (100) 231 (32) 231 (100) 231 (43) 231 (36) 231 (36) 231 (74) 231 (10)
214 (4) 214 (3) 214 (5) 214 (3) 214 (2) 214 (8) 214 (13)
Othersc 297 (29) 297 (42) 297 (100) 243 (12) 339 (18)
a
P D [NiII (L
H) bipy]C , surviving parent ions.
b
Common product ions identified are [NiI (CH3 OH) bipy]C (m/z 246), [NiII (CH3 O) bipy]C (m/z 245), [NiI (H2 O) bipy]C (m/z 232),
[NiII (OH) bipy]C (m/z 231), [NiI bipy]C (m/z 214).
c
Only fragment ions with more than 10% relative abundance are listed.

Table 7. CAD of nickel complexes of chalcones with methoxy groups: [NiII (L
H) bipy]C (m/z, %), where ‘A’ and ‘B’ denote
fragments retaining the A or B ring system

7 8 10 11 12 13 14 15 16 17 19 20

Pa 467 (11) 467 (11) 481 (11) 483 (11) 483 (9) 483 (8) 497 (6) 497 (11) 497 (10) 497 (12) 511 (10) X
P
H2 465 (76) 465 (82) 479 (85) 481 (100) 481 (3) 481 (77) 495 (72) 495 (12) 495 (81) 495 (36) 509 (85)
ž
P
CH3 452 (6) 452 (7) 466 (41) 468 (2) 468 (6) 468 (11) 482 (10) 482 (100) 482 (14) 482 (18) 496 (40)
P
CH4 481 (14) 495 (12)
P
H2 O 465 (2) 465 (12) 479 (6)
P
CO 439 (9) 439 (12) 453 (18) 455 (1) 455 (1) 455 (11) 469 (9) 469 (11) 469 (4) 483 (13)
P
H2
CO 437 (27) 437 (38) 453 (3) 453 (1) 453 (44)
ž
P
2CH3 451 (47) 467 (8) 467 (19) 467 (31) 467 (12) 481 (23)
ž
P
CH3
CO 440 (4) 440 (2) 454 (11)
P
CO2 453 (4) 453 (8)
P
H2 O
CO 437 (2) 437 (3) 451 (4) 451 (3) 451 (1) 465 (1)
P
2CO 411 (5) 411 (8) 425 (6) 427 (1) 427 (4) 441 (8) 441 (6) 455 (4)
P
H2
2CO 409 (2) 409 (3) 423 (1) 425 (1) 439 (2) 439 (2)
ž
P
CH3
CO2 438 (6) 452 (8)
‘A’ 291 (39) 321 (12) 291 (8) 363 (9) 321 (14) 321 (22)
291 (10) 291 (11) 291 (30)
‘B’ 337 (79) 307 (81) 321 (100) 307 (100) 307 (69) 337 (80) 337 (100) 321 (84)
Common ionsb 246 (37) 246 (44) 246 (37) 246 (7) 246 (7) 246 (54) 246 (73) 246 (20) 246 (54) 246 (72) 246 (73)
245 (15) 245 (13) 245 (13) 245 (5) 245 (11) 245 (20) 245 (17) 245 (11) 245 (18)
232 (50) 232 (62) 232 (51) 232(14) 232 (10) 232 (77) 232 (100) 232 (28) 232 (74) 232 (100) 232 (100)
231 (100) 231 (100) 231 (84) 231 (17) 231 (51) 231 (100) 231 (83) 231 (8) 231 (100) 231 (75) 231 (85)
214 (5) 214 (4) 214 (2) 214 (11) 214 (3) 214 (8) 214 (13) 214 (11)
Othersc 353 (12) 348 (12) 323 (87) 369 (30) 352 (13) 336 (65) 369 (38)
321 (13) 243 (13) 243 (13)
291 (10)

a
P D [NiII
L
H bipy]C , surviving parent ions.
b
Common product ions identified are [NiI (CH3 OH) bipy]C (m/z 246), [NiII (CH3 O) bipy]C (m/z 245), [NiI (H2 O) bipy]C (m/z 232),
[NiII (OH) bipy]C (m/z 231), [NiI bipy]C (m/z 214).
c
Only fragment ions with more than 10% relative abundance are listed.

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
570 J. Zhang and J. S. Brodbelt

Table 8. CAD of copper complexes of chalcones without methoxy groups: [CuII (L
H) bipy]C (m/z, %)

1 2 3 4 5 6 9 18

Pa 442 (5) 458 (5) 458 (5) 458 (8) 458 (5) 458 (8) 474 (6) 506 (3)
P
H2 O 440 (1)
P
CO 478 (40)
Common ionsb 251 (10) 251 (12) 251 (2) 251 (11) 251 (12) 251 (11) 251 (9)
250 (10)
237 (100) 237 (100) 237 (100) 237 (16) 237 (100) 237 (100) 237 (100) 237 (100)
236 (100)
219 (30) 219 (29) 219 (30) 219 (6) 219 (33) 219 (32) 219 (37) 219 (86)
Othersc 365 (5) 365 (2) 381 (5) 337 (3) 365 (5) 381 (5) 381 (2) 247 (6)
320 (2) 312 (8) 312 (2) 302 (2) 318 (2)
247 (2)

a
P D [CuII (L
H) bipy]C , surviving parent ions.
b
Common product ions identified are: [CuI (CH3 OH) bipy]C (m/z 251), [CoII (CH3 O) bipy]C (m/z 250), [CuI (H2 O) bipy]C (m/z 237),
[CuII (OH) bipy]C (m/z 236), [CuI bipy]C (m/z 219).
c
Only fragment ions with more than 2% relative abundance are listed

Table 9. CAD of copper complexes of chalcones with methoxy groups: [CuII (L
H) bipy]C (m/z, %)

7 8 10 11 12 13 14 15 16 17 19 20

Pa 472 (7) 472 (7) 486 (4) 488 (10) 488 (5) 488 (7) 502 (12) 502 (12) 502 (12) 502 (6) 516 (8) X
ž
P
CH3 471 (1) 487 (1) 487 (12) 501 (1)
P
H2 O 470 (1)
P
CO 444 (1) 444 (1) 460 (1) 474 (1) 474 (2)
ž
P
CH3 445 (1) 459 (2) 459 (2) 459 (1)

CO
Common 251 (10) 251 (11) 251 (14) 251 (5)
251 (8) 251 (11) 251 (10)251 (10) 251 (10) 251 (11) 251 (12)
ionsb 250 (10) 250 (9)
237 (100) 237 (100) 237 (100) 237 (100) 237 (74) 237 (100) 237 (100) 237 (100) 237 (100) 237 (100) 237 (100)
236 (100) 236 (97)
219 (25) 219 (36) 219 (32) 219 (74) 219 (27) 219 (37) 219 (37) 219 (36) 219 (37) 219 (37) 219 (34)
Othersc 395 (7) 365 (2) 379 (3) 350 (3) 368 (11) 381 (2) 381 (2) 425 (3) 473 (2) 395 (5) 379 (3)
337 (3) 351 (2) 348 (2) 332 (2) 367 (1) 351 (2) 365 (2) 364 (6) 395 (3) 364 (5) 378 (3)
337 (5) 330 (2) 272 (2) 339 (2) 350 (2) 364 (6) 346 (2) 364 (5) 346 (16) 360 (3)
326 (2) 326 (2) 247 (5) 312 (10) 356 (3) 217 (3) 351 (3) 356 (3)
247 (2) 241 (2) 346 (3)
326 (2)

a
P D [CuII (L
H) bipy]C , surviving parent ions.
b
Common product ions identified are [CuI (CH3 OH) bipy]C (m/z 251), [CoII (CH3 O) bipy]C (m/z 250), [CuI (H2 O) bipy]C (m/z 237),
[CuII (OH) bipy]C (m/z 236), [CuI bipy]C (m/z 219).
c
Only fragment ions with more than 2% relative abundance are listed.

Table 10. MSn data for selected chalcone complex ions [MII Table 10. (Continued)
(L
H) bipy]C (m/z)
Frag- Neutral
Frag- Neutral Complex Chalcone Route ment loss %
Complex Chalcone Route ment loss %
452 30 72
[CoII 2 454 ! 436 ! 408 28 100 436 46 10
(L
H) 247 189 33 424 58 12
bipy]C 233 203 30 410 72 52
216 220 10 362 120 21
344 138 20
14 498 ! 482 ! 464 18 100
454 28 19

Copyright  2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572
 ESI-MS/MS structural characterization of chalcones 571

Table 10. (Continued) Table 10. (Continued)

Frag- Neutral Frag- Neutral

Complex Chalcone Route ment loss % Complex Chalcone Route ment loss %

326 156 36 497 ! 307 ! 246 61 71

247 235 14 232 75 100
233 249 20 15 497 ! 352 ! 337 15 100
232 250 21 281 71 25
216 266 12 246 106 81
498 ! 308 ! 280 28 10 232 120 98
247 61 69 214 138 28
233 75 100 16 497 ! 337 ! 246 91 76
498 ! 307 ! 306 1 18 232 105 100
289 18 12 17 497 ! 337 ! 246 91 74
279 28 100 232 105 100
15 498 ! 483 ! 468 15 57 497 ! 336 ! 246 90 75
465 18 18 232 104 100
454 29 39
437 46 100
352 131 88 species gave simple and compound-specific fragments,
281 202 14 which were sufficient in differentiating most of the isomers.
247 236 23 The use of metal complexation as an alternative ionization
233 250 29 mode resulted in equal or greater signal intensities and
232 251 12 led to more specific fragmentation patterns so that all the
isomeric series were distinguished. MSn experiments proved
498 ! 352 ! 268 84 51
to be useful in elucidating proposed fragmentation pathways
246 106 15
and confirmed that many of the neutral losses occurred
232 120 100
through stepwise pathways. Finally, it is clear that the
16 498 ! 483 ! 482 1 44 chalcones do not follow consistent structure-specific rules
468 15 19 for fragmentation, thus complicating the identification of
455 28 67 new chalcones in mixtures.
454 29 51
427 56 37 Acknowledgements
390 93 32 This work was supported by the National Institutes of Health (NIH
247 236 82 RO1 GM63512) and the Welch Foundation (F-1155).
232 251 100
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