Structural Characterization and Isomer Differentiation

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2003; 38: 555–572Published online 25 April 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.472

Structural characterization and isomer differentiationof chalcones by electrospray ionization tandem massspectrometry

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 tocharacterize the structures, and losses commonly observed include H2, H2O, CO and CO2, in additionto 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 withseveral diagnostic fragment ions appearing upon activation of the metal complexes. MSn experimentswere performed to support identification of some fragment ions and to verify the proposed fragmentationpathways. In several cases, MSn indicated that specific neutral losses occurred by stepwise pathways, suchas the neutral loss of 44 u as CH3

ž and HCOž, or CH4 and CO, in addition to CO2. Copyright 2003 JohnWiley & Sons, Ltd.

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

INTRODUCTION

Owing to their antioxidant abilities and emerging chemo-preventive properties against heart disease, aging andcancer,1 – 3 flavonoids have become the focus of increas-ing numbers of research studies. Flavonoids are a largegroup of phytochemicals that have the general structure of a15-carbon skeleton, which consists of two phenyl rings con-nected by a three-carbon bridge.4,5 More than 4000 knownflavonoids are classified into subgroups, including flavone,flavanone, flavonol, isoflavonoid, anthocyanidin and chal-cone. Chalcones, differing from all other flavonoids by theabsence of the C ring, are still considered a subclass offlavonoids.6 Chalcones, along with retrochalcones and dihy-drochalcones, in fact have an open structure and a carbonskeleton numbered in a way different from other flavonoids(Fig. 1). Chalcones have shown good physicochemical andbiological 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 akey role in flavonoid biosynthesis5,15,16 because they are the

ŁCorrespondence to: Jennifer S. Brodbelt, Department of Chemistryand Biochemistry, University of Texas, Austin, Texas 78712, USA.E-mail: [email protected]/grant sponsor: National Institutes of Health;Contract/grant number: NIH RO1 GM63512.Contract/grant sponsor: Welch Foundation; Contract/grantnumber: F-1155.

precursors of other flavonoids. Chalcones are first synthe-sized in plants by chalcone synthase (CHS) and then cyclizedto other flavonoids by chalcone isomerase (CHI). Since thechemopreventive properties of flavonoids (including chal-cones) depend on both the different functional groups andtheir relative positions, it is important to be able to charac-terize the structures and differentiate the isomers.

Chalcones are commonly found in licorice (liquorice) andapple seeds.6 Native chalcone glycosides tend to transformto 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. Thisisomerization has been evaluated in solution,17 – 19 and alsoby theoretical calculations.20

Although mass spectrometry has been involved inchalcone structural characterization since the early 1960s,most studies have focused on electron ionization21 – 29 withone report of chemical ionization,30 field desorption31 and afew on fast atom bombardment studies.32,33 Special attentionhas been given to 20-hydroxychalcones21,29 because oftheir different fragmentation patterns from other chalcones.The cyclization of chalcones to flavanones also has beenobserved in the gas phase.21 – 23 It was noticed that themass spectra of chalcones bearing a 20-hydroxyl functionalgroup were nearly identical with those of the isomericflavanones. Isomerization was first proposed and latersupported by experimental data.21 Based on experimentsusing the relatively energetic electron ionization method,

Copyright 2003 John Wiley & Sons, Ltd.

556 J. Zhang and J. S. Brodbelt

O

2′

3′

4′

5′

6′2

3

4

5

6

A B

1 1′

Chalcone Mw -OH -OCH3 -CH3

1 224 2′2 240 4,2′3 240 2′, 4′4 240 2, 2′5 240 3, 2′6 240 2′, 5′7 254 2′ 4′8 254 2′ 49 256 4, 2′,5′

10 268 2′ 4 5′11 270 2′, 6′ 4′12 270 2, 2′ 313 270 2′, 4′ 414 284 2′ 3, 415 284 2′ 3′, 4′16 284 2′ 4, 4′17 284 2′ 2, 4′18 288 3,4, 2′, 4′, 6′19 298 2′ 3, 4 5′20 298 4, 2′,5′

O

O

H3CO

OH

OHO

OOH

OH

OH

O

1

2

345

6

7

8

2′

3′

4′

5′

6′A

B

C

O

Chalcones Flavanones

FA (Mw 270)

FB (Mw 288)

Figure 1. Chalcone and flavanone structures studied.

it was concluded that an intramolecular equilibrium existsbetween a chalcone type and a flavanone type molecular ion.There have been no reports on the structural characterizationof 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 thenobtain their fragmentation patterns upon collision-activateddecomposition (CAD). Metal complexation, a method thathas proven to be an effective ionization mode for acidicflavonoids,34,35 is used as an alternative to differentiate someisomers.

EXPERIMENTAL

Chemicals and reagentsAll the chalcones and flavanones listed in Fig. 1 werepurchased 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 withoutfurther purification.

Each chalcone or flavanone stock solution was preparedin methanol at a concentration of ¾1 ð 10�3 M and keptprotected from light at 4 °C until use. 2,20-Bipyridine, lithium

chloride and cobalt(II), nickel(II), and copper(II) bromidewere dissolved in methanol at a concentration of ¾5 ð 10�3 M

and stored at room temperature.

Mass spectrometric conditionsAll the experiments were performed on a Thermo FinniganLCQ Duo quadrupole ion trap mass spectrometer. The sprayvoltage was kept at 4.5 kV (š). The temperature of theheated capillary was set at 200 °C. Nitrogen was used asthe sheath gas (20 arbitrary units). Auxiliary gas (nitrogen,30 arbitrary units) was added as needed. The injection timewas set at 10 ms except for the tandem mass spectrometric(MSn) experiments (50–100 ms). Each chalcone workingsolution was delivered to the ESI source at 5 µl min�1.The other parameters, including capillary voltage, octapoleoffsets and tube lens offset, were optimized for maximumabundance of the ions of interest. In solutions of chalconeswithout metal addition, the ions optimized were [L � H]�

(deprotonation mode) and [L C H]C or [L C Na]C (positiveion mode). When lithium chloride was added, the ions ofinterest were [L C Li]C. Upon addition of the auxiliary ligand2,20-bipyridine (bipy) and a transition metal, the [MII (L � H)bipy]C complex was optimized. The product ion spectra ofthe ions of interest were obtained by CAD, using helium asthe collision gas. The collision energy was varied such that the

Copyright 2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 555–572

ESI-MS/MS structural characterization of chalcones 557

relative intensity of the surviving parent ions was ¾5–10%.For the MSn experiments, the fragment ions of interest fromMS/MS were subject to further isolation and CAD, so thatonly ¾5–10% of the selected precursors survived the secondstage of activation.

The working solutions of chalcones (¾1 ð 10�5 M) weremade in methanol from their stock solutions with/withoutmetal addition. When a metal was added, the final con-centration ratios were 1 : 10 chalcone : lithium or 1 : 1 : 1chalcone : 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 resultingions and assess the ability to differentiate isomers, and MSn

was used to assist in elucidating the fragmentation pathways.

Protonation and lithium cationizationMost of the chalcones did not form detectable ions in thepositive ESI mode. Only chalcone 20, which does not haveany 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 (spectranot shown). The CAD spectra of these alkali metal adductswere relatively simple. The major fragment (m/z 311) of[20 C Na]C (m/z 321) was the loss of CO with subsequentaddition of H2O, whereas [20 C Li]C mainly underwent amethyl radical loss resulting in a fragment at m/z 290. Allthe other chalcones have at least one hydroxyl group, whichmakes 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 thatwere abundant enough to be isolated. Therefore, CAD of

protonated or alkali metal cationized chalcones was notpursued further.

DeprotonationChalcones other than 20 were easily deprotonated due totheir acidic nature (Fig. 2(A)). The CAD data of the resulting[L � H]� ions are summarized in Table 1 for chalconeswithout methoxy groups and in Table 2 for chalcones withmethoxy groups, and typical CAD spectra are shown inFigs 3–5 for three sets of isomers. To help rationalize theproposed fragmentation mechanisms, MSn experiments wereperformed on all the deprotonated chalcones (Table 3).

A-vs B-type fragmentsAs shown in Tables 1 and 2 and Figs 3–5, many of thefragment ions can be classified as A or B type, meaningthat these products retain the A or B ring portion of thechalcone while the second ring is lost. The loss of B ring(which results in the A type of ions) has been observedearlier for some chalcones by electron ionization.21,29 BothA- and B-type fragments have also been reported for othersubclasses of flavonoids by ESI.36 Examples of these types offragment ions are illustrated in Scheme 1. In Scheme 1, thesite of deprotonation is shown to accommodate the production structures. The resulting fragment ions may isomerizeor intraconvert to other resonance structures, and thus thestructures shown in Scheme 1 are only given to help guidethe reader in matching up structures that reasonably fit themasses of the fragments listed in the tables and figures.

MSn provided support for some of the A vs B fragmentassignments (Table 3). For example, MS/MS/MS indicatedthat the product ion m/z 119 of 2 was an ‘A’ ring fragmenteven though the A and B rings have the same isomericcomposition (the A ring fragment would be C8H7O� andthe B ring fragment would be C7H3O2

�). The assignment ofthe fragment ion as an ‘A’ ring fragment was verified by

140 180 220 260 300 340 380 420 460 500 540

m/z

0

283 [L - H]-

498 [CoII(L - H) bipy]+

Deprotonation

Cobalt complexation

Rel

ativ

e A

bund

ance

100

0

100 7.7 × 105

1.0 × 107

B)

A)

Figure 2. ESI spectra of chalcone 16 in both deprotonation and cobalt complexation modes with the relative intensities shown.



Table 1. CAD of deprotonated chalcones without methoxy groups (m/z, %), where ‘A’ and ‘B’ denote fragments retaining the A or Bring 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 � H2O 205 (18) 221 (1) 221 (100) 221 (60) 269 (4)P � CO 195 (100) 211 (4) 211 (100)P � H2 � CO 193 (3)P � C2H2O 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 C2H2O. For the other chalcones, there is noevidence 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 Bring 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) XP � 2CH4 C H2O 269 (40)P � CH3

ž 238 (100) 238 (100) 252 (100) 254 (100) 254 (15) 254 (100) 268 (100) 268 (100) 268 (100) 268 (90) 282 (100)P � 16b 237 (15) 237 (10) 251 (7) 267 (12) 267 (14)P � H2O 251 (73) 265 (2)P � CO 225 (9) 225 (3) 239 (4) 255 (44)P � 2CH3

ž 237 (3) 253 (88) 253 (5) 253 (84) 253 (100) 267 (92)P � 2CH4 251 (78)P � CH3

ž � H2O 236 (16)P � C2H2O 227 (23)P � CH3

ž � CO 210 (1) 224 (4) 226 (13) 240 (5) 254 (2)P � 44 209 (7)c 225 (11)c 225 (7) 225 (2)P � H2O � CO 223 (5)P � CH3

ž � CO2 210 (8)P � 2CH4 � CO 223 (30)P � 2H2O � 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 ofCH3

ž and Hž.c MSn experiments indicate that the loss of 44 u for 7 may be due to three pathways: the loss of CO2, the loss of CH3

ž and HCOž, andthe loss of CH4 and CO. MSn experiments indicate that the loss of 44 u for 11 may be due to two pathways: the loss of CO2 and the lossof CH3

ž and HCOž. 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.



70 100 130 160 190 220 250

m/z

Rel

ativ

e A

bund

ance

Chalcone 2

Chalcone 6

D)

E)

Chalcone 5

C) Chalcone 4

Chalcone 3

∗239

∗239

∗239

∗239

,∗239

237 (-H2)

221 (-H2O)211 (-CO)

195 (-CO2)B

161 (-C6H6)

B135 (-C8H8)

197

221 (-H2O)

195 (-CO2)B145 (-C6H6O)

A119 (C8H7O)

93 (C6H5O)

197 (-C2H2O)

195A91

B109

B135 (-C8H8)

148153

195 (-CO2)

A119 (C8H7O)

0

100

0

100

0

100

0

100

0

100

A)

B)

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.

80 120 160 200 240 280

m/z

Rel

ativ

e A

bund

ance

C)

B)

Chalcone 13

Chalcone 12

A) Chalcone 11

∗269

∗269

∗269

227 (-C2H2O)

254 (-CH3·)

254 (-CH3·)

225 (-CO2)

210 (-CH3·- CO2)

A175 (-C6H6O)

A149 (C9H9O2)

A123 (C7H7O2)

B93 (C6H5O)

254 (-CH3·)

251 (-H2O)

225/226

236 (-CH3· –H2O)B

165 (-C8H8)

B191 (-C6H6)

B148 (C8H4O3)

0

100

0

100

0

100

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.



80 100 120 140 160 180 200 220 240 260 280 300

m/z

Rel

ativ

e A

bund

ance

∗283

268 (-CH3·)253 (-2CH3·)

253 (-2CH3·)

253 (-2CH3·)

268 (-CH3·)

268 (-CH3·)

∗283

∗283

Chalcone 14

Chalcone 15

Chalcone 16

Chalcone 17

∗283

269 (-2CH4 + H2O)

268 (-CH3·)253 (-2CH3·)251(-2CH4)

255(-CO)

223 (-2CH4-CO)219 (-CO–2H2O)

145

0

100

0

100

0

100

0

100

A)

B)

C)

D)

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(L � H�� (m/z)

Chalcone RouteFrag-ment

Neutralloss %a

1 223 ! 221 ! 193 28 100165 56 13145 76 14

223 ! 195 ! 193 2 100

2 239 ! 195 ! 167 28 100119 76 64

239 ! 119 ! 93 26 10091 28 16

3 239 ! 197 ! 169 28 100141 56 25

239 ! 195 ! 193 2 75169 26 100167 28 37

239 ! 148 ! 120 28 100239 ! 135 ! 91 44 100

4 239 ! 119 ! 117 2 10091 28 18

239 ! 93 ! 65 28 100

5 239 ! 221 ! 211 28, CH2Ob 32193 28 100165 56 11

6 239 ! 237 ! 209 28 98195 42 39193 44 100

239 ! 221 ! 211 28, CH2Ob 13

Table 3. (Continued)


Neutralloss %a

193 28 100239 ! 211 ! 183 28 100

169 42 31167 44 60157 54 14143 68 17

239 ! 161 ! 133 28 31117 44 100

7 253 ! 238 ! 237 1 100209 29 20

253 ! 237 ! 209 28 22193 44 100

253 ! 225 ! 210 15 100

8 253 ! 238 ! 237 1 100210 28 21

253 ! 237 ! 209 28 100193 44 20

253 ! 225 ! 210 15 100

9 255 ! 211 ! 183 28 100255 ! 135 ! 125 28, CH2Ob 30

107 28 100255 ! 119 ! 93 26 100

10 267 ! 252 ! 251 1 100237 15 22224 28 37





Neutralloss %a

11 269 ! 254 ! 253 1 25226 28 100225 29 86177 77 18163 91 18

269 ! 251 ! 236 15 100

12 269 ! 254 ! 253 1 100236 18 60

269 ! 149 ! 134 15 100269 ! 123 ! 108 15 100

13 269 ! 254 ! 148 106 100269 ! 227 ! 212 15 100269 ! 148 ! 120 28 100

14 283 ! 268 ! 267 1 3253 15 100

283 ! 253 ! 225 28 100209 44 56

15 283 ! 268 ! 253 15 100

16 283 ! 268 ! 267 1 2253 15 100

283 ! 253 ! 235 18 16225 28 100211 42 43209 44 81185 68 18

17 283 ! 268 ! 253 15 100283 ! 255 ! 240 15 100283 ! 253 ! 238 15 56

209 44 100283 ! 251 ! 236 15 17

223 28 100283 ! 223 ! 208 15 100283 ! 145 ! 117 28 100

18 287 ! 151 ! 107 44 100

19 297 ! 282 ! 267 15 100297 ! 267 ! 252 15 25

239 28 100223 44 51

a Only fragments with more than 10% relative abundance arelisted, except for 14 and 16.b The observed loss of 10 u is due to loss of CO followed byaddition of H2O.

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.The assignments of m/z 135 of 3 and 161 of 6 as ‘B’ ringfragments were supported by the subsequent loss of CO2

in the MS/MS/MS of 135� and 161�, respectively, whichwould not be possible for A ring species owing to the lack ofoxygen atoms on the A ring.

Fragment ions at m/z 149 and 123 in the CAD spectrum ofdeprotonated 12 were assigned as A ring fragments becauseof the exclusive loss of 15 u observed in the MS/MS/MS ofm/z 123 and 149. The sole methoxy group of 12 is located onthe A ring.

Small neutral lossesIn addition to the cleavages due to A or B ring losses(see Fig. 1 for the location of the A and B rings), smallneutral losses of H2O, CO and CO2 were commonly observedand sometimes dominant for chalcones with only hydroxylgroups (Table 1). In addition to the expected neutral lossesof CO and CO2 and the formation of A and B ring fragments,there was an unusual loss of H2 for 1 and 6, as well as aprominent loss of H2O for 5, 6 and 11. The pathways forthese losses of H2 or H2O are not readily explained, noris it evident why these unusual losses are observed onlyfor certain chalcones. Another small neutral loss of C2H2O(42 u) was observed in the CAD spectra of deprotonatedchalcones 3, 6 and 13, and was especially prominent for 3.Each of these three chalcones has two hydroxyl groups onthe B ring, either in the meta- or para-position. The sameloss has also been observed for some other deprotonatedflavonoids.36 Extensive isotopic labeling is impractical owingto the high cost of the chalcones (up to $200 per mg) andthe synthetic difficulty of selective deuterium, 18O or 13Clabeling, in addition to the possibility of solution or gas-phase isomerization of the chalcones, as shown in Scheme 2for one simple deprotonated chalcone. Neutral losses suchas CO, CO2, and C2H2O also have been observed for otherdeprotonated flavonoids,36 such as for flavonols, flavonesand flavanones. In fact, the loss of CO2 from deprotonatedflavones has been proposed to involve a carbonyl group andan oxygen of the C ring in a rather unusual rearrangementreaction.36

For chalcones with both hydroxyl and methoxy groups,the most dominant losses were methyl radicals (and/or

100 120 140 160 180 200 220 240 260 280 300

m/z

A) MS2

C) MS4

B) MS3

253 (-2CH3⋅)

∗253

268 (-CH3⋅)

∗283

∗268

209 (-CO2)

225 (-CO)

Rel

ativ

e A

bund

ance

253 (-CH3⋅)

0

100

0

100

0

100

Figure 6. MSn of deprotonated chalcone 14. Parent ions arelabeled with asterisks.



methane for 7, 8, 10, 14 and 16) in addition to other smallneutral losses, such as CO (Table 2). The consecutive lossof methyl radicals during CAD was confirmed by MSn

experiments (Fig. 6 and Table 3), which showed sequentialloss of 15 u. Even though it is unusual to have radical lossesfrom even-electron molecular ions, this was observed notonly for deprotonated chalcones but also previously forother flavonoids.36 – 39

As shown in Fig. 6 and Table 3, MS/MS/MS experimentsproved to be useful in confirming some of the suspectedfragmentation pathways, including support for sequentiallosses. For example, for the deprotonated chalcones withoutmethoxy 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 precursorchalcone. For the deprotonated chalcones with two methoxygroups (14, 15, 16, 17 and 19), the loss of 30 u was in part dueto sequential loss of CH3

ž based on the consecutive loss of15 u seen in MS/MS/MS (Fig. 6). Note that this MS/MS/MSsequence does not rule out other pathways for the loss of

30 u, such as the loss of CO with H2. Even though 10 hasonly one methoxy group and one methyl group, the lossof 30 u seen for deprotonated 10 was likewise confirmed tobe at least in part due to loss of two methyl radicals in theMS/MS/MS experiments.

MS/MS/MS experiments also showed that the loss of44 u from deprotonated 7 is actually in large part due toconsecutive losses of CH3

ž and HCOž or consecutive lossesof CH4 and CO (Table 3). The consecutive losses of CH4

and CO are also observed in MS/MS/MS experiments fordeprotonated 8 even though this [P � 44] fragment is notobserved upon CAD of this deprotonated chalcone (Table 2).Similarly, the loss of 44 u from deprotonated 11 was foundto be due in part to consecutive losses of CH3

ž and HCOž.Furthermore, for deprotonated 6, one route for the loss of44 was proven to be consecutive losses of H2 and C2H2O,as confirmed by MS/MS/MS experiments (Table 3). Forthe other deprotonated chalcones that showed a loss of44 u in their CAD spectra, these unusual consecutive losses(i.e. CH3

ž and HCOž, CH4 and CO, H2 and C2H2O) werenot indicated based on MS/MS/MS experiments, hence the

O O- O- O-O

1 93- (B) 145- (B)

A B

O OH

2-O

-O119- (A)

O OH

4O-

O-

O-

O-O

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

O OH

6

O-O

OH

O

O-O-

161- (B)135- (B)

CO

O O-

3OH

CH2-

O

O-

91- (A) 135- (B)

O-

OH109- (B)

CO

O O-

OH

HO

9 107- (B)

-O

119- (A)

O

CO

O-

135- (B)

CO

O-

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



O OH

O-

OCH3 93- (B)

O-

123- (A)

OCH3

O-

OCH3

O-

149- (A) 175- (A)

OCH3

O-

12

CC

O

O O-

OHH3CO O

148- (B)13.

O-O

O O-

OHHO

18OH

HOO

O-

HO

151- (B)

CO

O OH

OCH3-O

165- (B)

O

-O OCH3

191- (B)

O-O

OCH3HO11

CO

O O-

OCH3OCH3

17 123- (B)

O-

OCH3

175- (B)

O-O

OCH3O-

CC

O

145- (A)

Scheme 1. (Continued).

O-

23

4

56

3'

4'5'

6'

O

23

4

56

3'4'

5'

6'

O-

O

O

23

4

56

3'4'

5'

6'

- O

I II

III

Scheme 2. Proposed resonant structures of deprotonatedchalcones. Note that flavanones have a different numberingsystem other than the chalcones as shown in Fig. 1. However,the same numbering system according to the original chalconeis used for the whole scheme to emphasize the positionsinvolved in the ring closure.

most likely pathway is simple loss of CO2, which has beenconfirmed for other flavonoids.

Deprotonated 7, 8, 10, 14 and 16 all dissociate by theloss of CH3

ž and the loss of CH4 upon collisional activation.MS/MS/MS experiments confirm that the loss of CH4 is inpart due to consecutive losses of CH3

ž and Hž. MS/MS/MSexperiments show that sequential losses of CH3

ž and Hž

may also occur for chalcone 11 even though the fragment[P � 16] is not directly observed in the CAD spectrum of thisdeprotonated chalcone (Table 2).

As discussed above for the MSn experiments, some ofthe neutral losses observed upon CAD of the deprotonatedchalcones may actually involve several pathways. Forexample, MSn experiments indicate that the loss of 44 umay be loss of CO2, loss of H2 with C2H2O, loss of CH3

ž withHCOž, and loss of CH4 with CO, depending on the identityof the chalcone. Likewise, in some cases the loss of 16 u is dueto consecutive loss of Hž and CH3

ž, not exclusively the loss ofCH4. These alternative pathways are shown in the footnotesof Tables 1 and 2. In general, the MS/MS/MS data indicatedthat some of the fragment ions observed in the CAD spectraof the deprotonated chalcones stemmed from consecutivelosses of small neutrals, thus adding a layer of complexity tothe rational interpretation of the fragmentation patterns ofchalcones. In addition, MSn experiments proved to be usefulin verifying some of the A versus B ring assignments.

Scheme 2 indicates the possible isomerization pathwayof chalcones to flavanones in order to explain some ofthe A and B type fragments in Scheme 1, which is in



accordance with earlier studies.21 – 23 To verify further theexistence of flavanone isomeric forms of the chalcones, twoflavanones isomeric to chalcones 11 and 18 were chosenfor ESI-MS and CAD. 5-Hydroxyl-7-methoxy flavanone (FA,Fig. 1) is an isomer of chalcone 11. The fragments uponCAD of the deprotonated FA were the same as that ofdeprotonated chalcone 11 (Table 2) but the abundances werevery different, with m/z 191 (100%) becoming the basefragment ion followed by m/z 165 (84%). This could beexplained by the much easier loss of the ‘B’ ring in theflavanone. The other major fragments were P � CH3

ž (42%),P � H2O (21%) and P � CO (14%). The second flavanonetested was 5,7,30,40-tetrahydroxyflavanone (eriodictyol, FB,Fig. 1), an isomer of chalcone 18. The CAD spectra of thedeprotonated ions of these two compounds were identical(Table 1). The similarity of the CAD patterns of the abovetwo isomer pairs is further strong evidence for possibleisomerization between the chalcone and flavanone forms.

Fragmentation patterns vs structural featuresIt is interesting that the fragmentation patterns of the variouschalcones may change dramatically upon rather modestchanges of substituents. For examples, substitution of amethoxy group for a hydroxyl group, such as the case forchalcones 3 versus 7, significantly changes the observed CADpatterns. Deprotonated 3 produces diagnostic A and B ionsupon CAD, in addition to the characteristic loss of C2H2O,whereas deprotonated 7 dissociates predominantly by loss ofCH3

ž, CH4, or CO, and forms no detectable A or B ions. Thedifferent CAD patterns of 3 and 7 might be at least partiallydue to the different numbers of possible isomeric forms: 3 hastwo different hydroxyl groups that can be deprotonated uponESI which results in two possible isomeric forms, whereas 7only has one hydroxyl group available for deprotonation. Asimilar lack of parallel fragmentation patterns occurs for thepair 13 and 16. These chalcones each have three substituentsin the same three positions, except that one has two hydroxylgroups and one methoxy group whereas the other has twomethoxy groups and one hydroxyl group. Deprotonated 13dissociates via three pathways, including loss of CH3

ž, loss ofC2H2O and formation of one diagnostic B ion. Deprotonated16 only undergoes loss of one or two CH3

ž units and givesno A or B fragment ions. The addition of an extra substituentcan also promote changes in the fragmentation patterns, asillustrated by the comparison of deprotonated 3 and 13 (thelatter having a methoxy group at the 4-position). The CADspectrum of deprotonated 13 lacks the detail of that observedfor deprotonated 3. In fact, the methoxy group in 13 is keyfor the dominant fragmentation pathway (loss of CH3

ž), andthe observed B fragment is not an analog of any of thoseobserved for deprotonated 3.

The comparisons summarized above are examples ofhow the interpretation of the fragmentation patterns ofrelated chalcones is not always straightforward. However,there are cases in which the fragmentation patterns ofcomplementary pairs show logical similarities. For example,chalcones 4 and 12 both have two hydroxyl substituentsat the 20- and 2-positions, and 12 also has one methoxygroup at the 3-position. Each of these undergoes the loss

of CO2, 94 u (loss of C6H6O, the B ring), 120 u (loss ofC7H2O4, another B ring loss) and 146 u (loss of C9H6O2,another B ring loss) upon collisional activation, which clearlysimplifies the interpretation of the fragmentation patternsof these related chalcones. In short, the chalcones do notfollow consistent structure-based fragmentation rules, thussuggesting that creation of mass spectral libraries will becritical for identifying chalcones in mixtures.

Isomer differentiationFive chalcone isomer series were included in this study:chalcones 2, 3, 4, 5 and 6 (Series 1, each containingtwo hydroxyl groups); chalcones 7 and 8 (Series 2, eachcontaining one hydroxyl group and one methoxy group);chalcones 11, 12 and 13 (Series 3, each containing twohydroxyl groups and one methoxy group); chalcones 14,15, 16 and 17 (Series 4, each containing one hydroxylgroup and two methoxy groups), and chalcones 19 and20 (Series 5, containing a combination of hydroxyl, methoxyand methyl groups). Chalcones in each series differ only bypositions of the same functional groups except in Series 5,the latter compounds having the same chemical formulasbut different combinations of functional groups. It wasour goal to differentiate these isomers by ESI-tandem massspectrometry.

CAD of the deprotonated chalcones was sufficient todifferentiate the chalcones in Series 1 and 3, which havemore than one hydroxyl group (Figs 3 and 4). Althoughsome common fragments are observed for each isomerseries, the individual fragmentation patterns are unique.For example, in Series 1, chalcone 3 gives unique A- andB-type fragment ions at m/z 91, 109 and 135 (with structuresproposed in Scheme 1). Chalcone 4 produces a distinctiveA and B fragment ions at m/z 93 and 145 and chalcone 6gives a very rich, diagnostic CAD pattern with two B-typeions (see Scheme 1). Chalcones 2 and 5, which have onlyone or two fragment ions, can nonetheless be distinguishedfrom the others owing to their lack of key ions present inthe CAD spectra of the other three chalcones. For Series 3,deprotonated 11 and 12 each produce an array of diagnosticions upon CAD, including several A- and B-type ions shownin Scheme 1, whereas deprotonated 13 is the only one ofthis series that dissociates by loss of C2H2O, in addition to aunique B-type ion.

CAD spectra of the deprotonated chalcones in Series 2and 4 did not provide adequate differentiation (Table 2 andFig. 5). The deprotonated chalcones 7 and 8 predominantlydissociate by loss of CH3

ž, and the unique loss of 44 u thatis observed only for deprotonated 7 is a minor pathway.For the Series 4 chalcones, a comparison of CAD patternsof isomeric chalcones 14, 15, 16 and 17 showed that 15 onlyshowed a minor loss of 2CH3

ž, while 14, 16 and 17 showedsignificant losses of 2CH3

ž (Table 2 and Fig. 5). However,even more striking was the variety of fragments observedfor deprotonated 17, which not only had neutral losses suchas CH3

ž, CH4, CO, 2CH3ž, 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 17differ only in the position of one methoxy group, and the



greater array of dissociation pathways for 17 is not readilyexplained based on this minor structural difference of theisomers. Deprotonated 17 gives a rich CAD spectrum that isdiagnostic, but the other three isomers are not differentiatedwith confidence owing to the dominant or exclusive methylradical losses upon CAD. Therefore, metal complexationwas examined as an alternative ionization method in aneffort to produce complexes that would give distinctivefragmentation patterns.

Differentiation of the two chalcones in Series 5 was aneasy case because these two chalcones differed significantlyin their ionization efficiencies in both the protonation anddeprotonation modes. Chalcone 20 does not deprotonate and19 does not protonate.

Metal complexationSince flavonoids in general deprotonate easily and have atendency to coordinate with metals, metal complexation hasbeen used as an alternative for structural characterization.34,35

2,20-Bipyridine, which is neutral and has a similar metalbinding strength to that of the deprotonated flavonoids, isused as an auxiliary ligand to promote stable complexes andprevent the formation of neutral dimeric species between themetal and two deprotonated flavonoids.34,35 The addition ofa transition metal (CoII, NiII or CuII) and 2,20-bipyridineto the chalcone solutions produced [MII (L � H) bipy]C

complexes. The signal intensities of the metal complexeswere similar to or higher than that of the correspondingdeprotonated chalcones (Fig. 2). CAD was then performed onthe metal complexes. In many cases, the metal complexes givemuch richer fragmentation patterns than the correspondingdeprotonated chalcones. An example is shown in Fig. 7for chalcone 7. Upon collisional activation, deprotonated

70 90 110 130 150 170 190 210 230 250 270

m/z

140 180 220 260 300 340 380 420 460 500

m/z

Rel

ativ

e A

bund

ance

A) (L – H)-

B) [CoII (L – H) bipy]+

∗253

∗468

238 (-CH3⋅)

225 (-CO)209 (-44)

237 (-16)

453 (-CH3⋅)425 (-CH3⋅ – CO)

397 (-CH3⋅ - 2CO)

362

338 [CoII (C7H7O2) bipy]+

292 [CoII (C6H5) bipy]+

247

233

216

228

157 348

0

100

0

100

Figure 7. CAD spectra of chalcone 7 in both deprotonationand cobalt complexation modes. Parent ions are labeled withasterisks. The losses of 16 and 44 u are explained in Table 2.

7 dissociated predominantly by loss of a methyl radical, withminor losses of methane, CO or 44 u. In contrast, the metalcomplex [CoII (7 � H) bipy]C produced several diagnosticfragment ions with possible structures proposed in Scheme 3,in addition to characteristic losses of CH3

ž and CH3ž with

one or two CO units.An example of the comparative CAD spectra for the

complexes of chalcone 14 containing different metals (CoII,NiII, CuII) is shown in Fig. 8. Both the cobalt and nickelcomplexes, presumably owing to the similar metal bindingstrength of the deprotonated chalcone and the auxiliaryligand and the similar coordination structures, dissociated to

100 150 200 250 300 350 400 450 500 550

m/z

Rel

ativ

e A

bund

ance

A) Cobalt complexation

B) Nickel complexation

C) Copper complexation

∗502

∗497

∗498216

247232/233

307

454 (-CO2)

468 (-2CH3⋅)

482 (-CH4)465 (-CH3⋅ - H2O)

308 [CoII (C6H5O) bipy]+

307 [NiII (C6H5O) bipy]+

495 (-H2)

369

231/232

246

214

219

237

251

0

100

0

100

0

100

Figure 8. CAD mass spectra of [MII (14 � H) bipy]C, whereMII D Co2C, Ni2C or Cu2C. Parent ions are labeledwith asterisks.

140 180 220 260 300 340 380 420 460 500 540

m/z

Rel

ativ

e A

bund

ance

216

247232/233

307

454 (-CO2)

468 (-2CH3⋅)

482 (-CH4)

∗498

465 (-CH3⋅ – H2O)

A) Chalcone 14

308 [CoII (C6H5O) bipy]+

483 (-CH3⋅)

∗498

468 (-2CH3⋅)

352

B) Chalcone 15

466 (-2CH4)

483 (-CH3⋅)

232/233216 247 ∗498

468 (-2CH3⋅)455 (-CH3⋅ - CO)

427338

C) Chalcone 16

D) Chalcone 17

∗498

483 (-CH3⋅)337

232/233216 247

0

100

0

100

0

100

0

100

Figure 9. CAD spectra of chalcone 14, 15, 16 and 17complexes with cobalt and 2,20-bipyridine. Parent ions [CoII

(L � H) bipy]C are labeled with asterisks.



O O-

OCH3

N

Co2+

N N N

Co2+

O-

OCH3

N N

Co2+

338+ 292+

-

7

N N

Co2+

318+

-

O O-

N

Co2+

N

H3CO

O O-

N

Co2+

N

O

N

Co2+

N

CH2-O-

N

Co2+

N

8360+ 308+322+

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’ denotefragments 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 � H2O 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 � H2O � 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 (CH3OH) bipy]C (m/z 247), [CoII (CH3O) bipy]C (m/z 246), [CoI (H2O) 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.



similar fragment ions upon CAD. Note that the fragmentions seen in the range m/z 200–255 are non-specificproducts containing 2,20-bipyridine, the metal, and up toone solvent (water or methanol) molecule. For most of thechalcones, the [CoII (chalcone—H) bipy]C fragmentationpatterns give the most useful array of diagnostic ions(see Fig. 9(A) as an example). Since 2,20-bipyridine has amuch greater copper binding energy compared to that ofeach chalcone, the whole chalcone molecule is generallylost 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 complexationThe CAD data for the cobalt complexes are provided inTables 4 and 5, and a representative series of CAD spectra

are shown in Fig. 9. For the chalcones without methoxygroups, commonly observed small neutral losses includedH2, H2O, and CO (Table 4), similar to the losses seen for thedeprotonated chalcones. Dominant losses observed for thechalcones with methoxy groups were CH3

ž, CH4, H2, H2O,CO and CO2 (Table 5). In addition, several less informativeproducts were observed for all the chalcones, such as [CoI

(CH3OH) bipy]C (m/z 247); [CoII (CH3O) bipy]C (m/z 246);[CoI (H2O) 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 aregenerally cobalt/2,20-bipyridine species that are non-specific.Relative to the CAD patterns of the deprotonated chalcones,the losses of H2O, H2 and CO are generally more prominentfor the metal complexes, [CoII (chalcone—H) bipy]C, andthe relative loss of 44 u is significantly lower. Moreover, thetypes 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’ denotefragments retaining the A or B ring system

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

Pa 468 (10) 468 (8) 482 (11) 484 (10) 484 (9) 484 (9) 498 (11) 498 (12) 498 (11) 498 (12) 512 (9) XP � H2 466 (4) 466 (8) 482 (100) 482 (8) 496 (7) 496 (6) 496 (2) 510 (3)P � CH3

ž 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)P � H2 � CH3

ž 451 (10)P � H2O 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)P � 2CH3

ž 452 (21) 468 (46) 468 (58) 468 (11) 468 (11) 482 (71)P � CH3

ž � CH4 467 (10)P � 2CH4 466 (24) 466 (12)P � CH3

ž � H2O 465 (11) 465 (11)P � CH3

ž � 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 � H2O � CO 452 (10)P � 2CO 412 (5)P � CH3

ž � H2O � CO 437 (8) 437 (8)P � CH3

ž � 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 (CH3OH) bipy]C (m/z 247), [CoII (CH3O) bipy]C (m/z 246), [CoI (H2O) 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.



chalcones compared to the metal complexes, a reasonableresult considering the vastly different structures of these twotypes of molecular ions.

The CAD spectra of the metal complexes gave a greaterarray of fragment ions, including at least one diagnostic ionfor each isomer, more than observed for the deprotonatedchalcones. The chalcones in Series 2, which relied on minorfragment ions (m/z 209 via loss of 44 u (7%) for 7 vs m/z 210via loss of CH3

ž and CO (1%) for 8) for differentiation in thedeprotonation mode, had distinctive fragment ions resultingfrom the cobalt complexes: 7, m/z 425, 397, 376, 362, 338,318, 292; and 8, m/z 412, 360, 332, 322, 308 (Table 5) (m/z376 and 332 are fragment ions with less than 10% relativeabundance and are thus omitted in the ‘Others’ column inTable 5). Structures for some of these diagnostic ions aresuggested in Scheme 3. Likewise, all the isomers in Series 4,which were not distinguishable by CAD in the deprotonationmode due to the dominant loss of one or two methyl radicalsfor 14, 15 and 16, had both distinctive CAD patterns andunique fragments upon CAD of the metal complexes: 14,m/z 307/308; 15, m/z 352; 16, m/z 455, 427, 338; and 17, m/z337 (Fig. 9), which led to easy differentiation. Dissociationof the metal complex of 16 results in an even-electron ionat m/z 338, while the metal complex of 17 gives a radicalion at m/z 337 that could be rationalized by the availabilityof a hydrogen at the 2-position. For 16, the hydrogen at the2-position could migrate to the 10-position while the bondbetween the ketone and 10-carbon is broken, resulting in afragment ion at m/z 338 and a ring closure between theketone and the A-ring at the 2-position. 17 has a methoxygroup instead of hydrogen at the 2-position, which makesthe hydrogen donation impossible. The structures of themost unique diagnostic ions are suggested in Scheme 4.Differentiation of the other isomer series was also possiblebased on the CAD patterns of the metal complexes (Tables 4and 5). Therefore, the CAD spectra of the cobalt complexesserved as distinctive fingerprints of the chalcones.

Nickel complexationThe fragmentation patterns of the nickel complexes (Tables 6and 7) were similar to that of the cobalt complexes, butwith H2 losses enhanced. The CAD mass spectra of thesecomplexes could also serve as fingerprints of the chalcones.

Copper complexationThe copper complexes, in contrast to the cobalt andnickel complexes, did not give significant chalcone-relatedfragments upon CAD (Tables 8 and 9 and Fig. 8(C)). Themain products upon CAD were complexes of copper and theauxiliary bipyridine ligand. Even when compound-specificfragments existed in the spectra, their abundances were toolow to be analytically useful.

MSn experiments were also performed on some selectedmetal complexes (Table 10). MS/MS/MS experiments of thecobalt and nickel complexes were also found to be useful infurther differentiation of the isomers in Series 4. For example,15 and 16 shared a major common fragment at m/z 483, butthe MS/MS/MS patterns of m/z 483 were unique for bothchalcone complexes (Table 10). MSn experiments were also

O

OCH3

H3CO

N N

O-

Co2+N N

O-

Co2+

14 308+

O

N N

O-

Co2+

OCH3

OCH3

N N

O-

Co2+

OCH3

O.

H

H

15 352+

·

O

H3CO

N N

O-

Co2+

OCH3

N N

O-

Co2+

OCH3

O

N N

O-

Co2+

O.

16

338+

427+

N N

OCH3

O-

OCH3

O

Co2+

N N

Co2+

O-

OCH3

·

17 337+

Scheme 4. Proposed structures of the unique fragment ions ofthe cobalt complexes of the chalcones in Series 4.

performed on those fragments that allowed differentiationof 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 structuresof those unique diagnostic fragments proposed in Scheme 4were based on the supporting MSn data.

CONCLUSION

Different ionization modes were compared for their utilityfor formation of chalcone ions and the subsequent CAD massspectra. Owing to their slightly acidic nature, chalcones wereeasily deprotonated. CAD upon the resulting deprotonated



Table 6. CAD of nickel complexes of chalcones without methoxy groups: [NiII (L � H) bipy]C (m/z, %), where ‘A’ and ‘B’ denotefragments 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 � H2O 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 � H2O � 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 (CH3OH) bipy]C (m/z 246), [NiII (CH3O) bipy]C (m/z 245), [NiI (H2O) 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’ denotefragments 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) XP � H2 465 (76) 465 (82) 479 (85) 481 (100) 481 (3) 481 (77) 495 (72) 495 (12) 495 (81) 495 (36) 509 (85)P � CH3

ž 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 � H2O 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)P � 2CH3

ž 451 (47) 467 (8) 467 (19) 467 (31) 467 (12) 481 (23)P � CH3

ž � CO 440 (4) 440 (2) 454 (11)P � CO2 453 (4) 453 (8)P � H2O � 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)P � CH3

ž � 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 (CH3OH) bipy]C (m/z 246), [NiII (CH3O) bipy]C (m/z 245), [NiI (H2O) 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 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 � H2O 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 (CH3OH) bipy]C (m/z 251), [CoII (CH3O) bipy]C (m/z 250), [CuI (H2O) 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) XP � CH3

ž 471 (1) 487 (1) 487 (12) 501 (1)P � H2O 470 (1)P � CO 444 (1) 444 (1) 460 (1) 474 (1) 474 (2)P � CH3

ž 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 (CH3OH) bipy]C (m/z 251), [CoII (CH3O) bipy]C (m/z 250), [CuI (H2O) 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

(L � H) bipy]C (m/z)

Complex Chalcone RouteFrag-ment

Neutralloss %

[CoII 2 454 ! 436 ! 408 28 100(L � H) 247 189 33bipy]C 233 203 30

216 220 10

14 498 ! 482 ! 464 18 100454 28 19



Neutralloss %

452 30 72436 46 10424 58 12410 72 52362 120 21344 138 20





Neutralloss %

326 156 36247 235 14233 249 20232 250 21216 266 12

498 ! 308 ! 280 28 10247 61 69233 75 100

498 ! 307 ! 306 1 18289 18 12279 28 100

15 498 ! 483 ! 468 15 57465 18 18454 29 39437 46 100352 131 88281 202 14247 236 23233 250 29232 251 12

498 ! 352 ! 268 84 51246 106 15232 120 100

16 498 ! 483 ! 482 1 44468 15 19455 28 67454 29 51427 56 37390 93 32247 236 82232 251 100

498 ! 427 ! 399 28 27397 30 13247 80 85233 194 100

498 ! 338 ! 295 43 25247 91 85233 105 100

17 498 ! 337 ! 336 1 55309 28 27308 29 21280 57 55279 58 22249 88 17247 90 80

23 104 100

[NiII 14 497 ! 495 ! 480 15 15(L � H) 479 16 100bipy]C 451 44 21

232 263 12

497 ! 369 ! 231 138 100



Neutralloss %

497 ! 307 ! 246 61 71232 75 100

15 497 ! 352 ! 337 15 100281 71 25246 106 81232 120 98214 138 28

16 497 ! 337 ! 246 91 76232 105 100

17 497 ! 337 ! 246 91 74232 105 100

497 ! 336 ! 246 90 75232 104 100

species gave simple and compound-specific fragments,which were sufficient in differentiating most of the isomers.The use of metal complexation as an alternative ionizationmode resulted in equal or greater signal intensities andled to more specific fragmentation patterns so that all theisomeric series were distinguished. MSn experiments provedto be useful in elucidating proposed fragmentation pathwaysand confirmed that many of the neutral losses occurredthrough stepwise pathways. Finally, it is clear that thechalcones do not follow consistent structure-specific rulesfor fragmentation, thus complicating the identification ofnew chalcones in mixtures.

AcknowledgementsThis work was supported by the National Institutes of Health (NIHRO1 GM63512) and the Welch Foundation (F-1155).

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Structural Characterization and Isomer Differentiation