Structural characterization of nitrated 2′- hydroxychalcones by electrospray ionization tandem mass spectrometry

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605

ISSN: 1469-0667 © IM Publications LLP 2009doi: 10.1255/ejms.1020 All rights reserved

EuroPEAN JourNALofMASSSPEctroMEtry

chalcones (1,3-diaryl-2-propen-1-ones) are an important class of natural compounds belonging to the flavonoid family.1 these compounds have proved to possess an impressive array of pharmacological and agrochemical activities, namely anti-protozoal, anti-inflammatory, immunomodulatory, anti-oxidant, (inhibiting lipid peroxidation), anti-tumor, anti-malarial, anti-fungal, anti-microbial and anti-viral activities, which are currently under investigation.2–8 certain natural and synthetic derivatives bearing 2¢-hydroxy derivatives have also exhibited a wide spectrum of biological activities with potential applications as biocides and pharmacological drugs.1,2 Nitroflavonoids are selective and competitive ligands

for central benzodiazepine receptors and posses anxiolytic properties, having the presence of the nitro groups essential for these activities.9–11

Mass spectrometry has been widely employed for the structural characterization of chalcones, most studies have focused on electron ionization,12–16 chemical ionization,17 field desorption,18 fast atom bombardment,19,20 a few on ESI16,21,22 and one using atmospheric pressure chemical ionization (APcI).23 However, only a few studies reported the use of tandem mass spectrometry (MS/MS) for structural characterization of chalcones ionized by ESI21,22 and APcI conditions.23 Interestingly, chalcones behave peculiarly

Structural characterization of nitrated 2¢-hydroxychalcones by electrospray ionization tandem mass spectrometry

Ana I.R.N.A. Barros,a,* Fernando M. Nunes,a Cristina Barros,b Artur M.S. Silvab and M. Rosário M. Dominguesb

achemistry Department, university of trás-os-Montes e Alto Douro, 5001-911 Vila real, Portugal. E-mail: [email protected] Department, university of Aveiro, 3810-193 Aveiro, Portugal

Isomeric 2¢-hydroxychalcones bearing nitro and methoxy groups in different positions of their skeleton were analyzed by tandem mass spectrometry (MS/MS) with electrospray ionization (ESI), in positive mode. Collision-induced dissociation of the protonated molecules, [M + H]+, formed under electrospray conditions were studied and it was found that the product ion spectra of these chalcones presented different fragmentation patterns depending on the position of the substituents on the molecule. The product ion spectra (ESI-MS/MS) of the B ring ortho-nitro substituted 2¢-hydroxychalcone and of the 4¢-methoxychalcones showed loss of OH•, 2OH• and combined losses of OH• and H2O. These fragment ions were absent in the spectra of the respective meta- and para isomers. The observed differences in the product ion spectra of these nitrochalcones allowed identification of the o-nitro derivatives. Distinction between the meta- and para- derivatives was not achieved. Chalcones bearing 6¢-methoxy substituents showed distinct fragmentation from the one observed for their isomers, 4¢-methoxychalcones, since they present only one fragment ion, a typical (0,aA – H)+ and, therefore, do not allow detailed structural information to be obtained, nor to differentiate between the o-, m- or p-nitro isomers. Overall, it was found that small changes in the substitution pattern of chalcones change their fragmentation considerably in the ESI-MS/MS, and that these features permit the differentiation of specific isomers of these 2¢-hydroxynitrochalcones.

Keywords: chalcones, 2¢-hydroxynitrochalcones, electrospray, tandem mass spectrometry, isomers differentiation

Introduction

A.I.R.N.A. Barros et al., Eur. J. Mass Spectrom. 15, 605–616 (2009)received: 17 february 2009 n revised: 26 June 2009 n Accepted: 27 June 2009 n Publication: 5 August 2009

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606 Structural Characterization of Nitrated 2¢-Hydroxychalcones

under MS/MS conditions, since the fragmentation pattern of chalcones changes dramatically with rather small changes in substituents.21,22 thus, the fragmentation pattern of related chalcones is not always straightforward which shows that mass spectrometry characterization of differentially substituted chalcones is needed to provide background data essential for the analysis of chalcones in mixtures.21,22

Mass spectrometry is, nowadays, an invaluable tool for providing structural information, especially when low amounts and/or not very pure samples are available for analysis, as well as in the study of mixtures. these advantages are even more significant as it can be used for tracing modification and/or degradation of target bioactive compounds during in vivo cellular metabolism as, for example, chalcones.2

Mass spectrometry is now an indispensable tool needed in all phases of new drug discovery and development. It is first used for the structural characterization of all new compounds and then for their pharmaceutical profiling. the new compounds that show activity in a high throughput screening assay are then brought to a discovery drug metabolism group for further evaluation.24

So, this work presents the characteristic fragmentation of the [M + H]+ ions of isomeric 2 ¢-hydroxychalcones bearing nitro groups in different positions of the B ring (figure 1, structures 2–4), bearing nitro groups on the B

ring and methoxy groups in 4¢ and/or 6¢ positions of the A ring (figure 1, structures 6–8, 10–12, 14–16) and of their corresponding non nitrated derivatives (figure 1, structures 1, 5, 9, 13). the obtained fragmentation patterns were used for their structural characterization. furthermore, the results obtained were considered for the differentiation of positional isomers of the nitro derivatives in the B ring and of methoxy substituents in the A ring of studied chalcones (figure 1).

Experimental2¢-Hydroxychalcones were synthesized by the base-catalyzed aldol condensation of 2¢-hydroxyacetophenones and the appropriate benzaldehyde derivatives.25,26 the purity of all synthesized chalcones was assessed by 1H-NMr, 13c-NMr, bi-dimensional HSQc and HMBc and elemental analysis.25

Electrospray mass spectra and product ion mass spectra were obtained in positive mode and were acquired using a Q-tof 2 instrument (Micromass, Manchester, uK). the samples for electrospray analyses were prepared by diluting 1 µL of the chalcone solutions in chloroform (~ 10–5 M) in 200 µL of methanol. Nitrogen was used as nebulizer gas and argon was used as collision gas. Samples were introduced into the

11

R1’ R2’ R1 R2 R3

1 H H H H H

2 H H NO2 H H

3 H H H NO2 H

4 H H H H NO2

5 CH3O H H H H

6 CH3O H NO2 H H

7 CH3O H H NO2 H

8 CH3O H H H NO2

9 H CH3O H H H

10 H CH3O NO2 H H

11 H CH3O H NO2 H

12 H CH3O H H NO2

13 CH3O CH3O H H H

14 CH3O CH3O NO2 H H

15 CH3O CH3O H NO2 H

16 CH3O CH3O H H NO2

Figure 1

OOH

H

H R1

R2

R3R1' R2'

Figure 1. Structures of the studied 2¢-hydroxychalcone derivatives (1–16).

Page 3

A.I.R.N.A. Barros et al., Eur. J. Mass Spectrom. 15, 605–616 (2009) 607

mass spectrometer using a syringe pump with a flow rate of 10 µL min–1. the needle voltage was set at 3000 V, the ion source at 80ºc and desolvation temperature at 150°c. cone voltage was 35 V. collision-induced decomposition mass spectra (MS/MS) were acquired by selecting the desired ion with the quadrupole section of the mass spectrometer and using collision energy between 20–25 eV. the product ions were analyzed with the tof analyzer. Pseudo-MS3 experiments were performed increasing the cone voltage to 80 V. Data acquisitions were carried out with a Micromass MassLynx 4 data system. In the MS/MS spectra, the lock mass in each product ion spectrum of chalcones was the calculated monoisotopic mass/charge of the precursor ion. the methanol and chloroform (riedel-de-Haën) were high purity solvents (HPLc grade). Methanol-d1 (cH3oD) (riedel-de-Haën) was used in the solution preparation to obtain the deuterated molecular species.

Results and discussionthe ESI-MS spectra, obtained in positive mode for all the studied 2¢-hydroxychalcones 1–16, showed the presence of [M + H]+ ions and their corresponding m/z values are summarized in table 1. Each [M + H]+ ion was induced to fragment by collision with a gas and the obtained product ion spectra (MS/MS) were analyzed. chalcone characteristic product ions are assigned by an adaptation of the nomenclature proposed by Ma et al.27 for the fragmentation of flavonoids. thus, the i,jA+ and i,jB+ labels refer to the fragments containing intact A- and B-rings, respectively, in which the superscripts i and j indi-cate the propenone bonds that have been broken (Scheme 1). Identification of the product ions observed was confirmed by exact mass measurement and elemental composition deter-mination. the most probable elemental composition of all the fragment ions was obtained with a high degree of confidence,

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

[M + H]+ 225 270 270 270 255 300 300 300 255 300 300 300 285 330 330 330

[P – cH3]+• 210

(100)—

255 (5)

255 (20)

240 (25)

— — — — — — — — — — —

[P – oH]+•

—253 (13)

— — — — — — — — — — — — —

[P – H2o]+ 207 (20)

252 (8)

252 (13)

252 (11)

237 (18)

282 (13)

282 (11)

— —

[P – 2oH]+•

—236 (34)

— — —266 (8)

— — — — — — — — — —

[P – oH – H2o]+•

—235 (100)

— — —265 (16)

— — — — — — — — — —

[P – 46]+(a) 179 (60)

224 (22)

224 (91)

224 (100)

209 (50)

254 (6)

254 (50)

254 (47)

— —

[P – H2o – No2]+

—206 (15)

—236 (8)

— — —

[P – c6H2]+

—196 (45)

—226 (6)

— — —

[P – H2o – co – No2]+

—178 (12)

178 (11)

178 (13)

208 (11)

208 (8)

— — — — — — — —

[P – c7H2o]+ 168 (20)

198 (6)

[1,bA – H]+ 147 (20)

— — —177 (12)

— — — — — — — — — —

[0,1¢B – H]+ 131 (40)

176 (8)

176 (100)

176 (66)

131 (28)

176 (35)

176 (92)

176 (100)

—176 (7)

176 (9)

176 (9)

[0,aA – H]+ 121 (90)

121 (26)

121 (76)

121 (64)

151 (100)

151 (100)

151 (100)

151 (100)

151 (100)

151 (100)

151 (100)

151 (100)

181 (100)

181 (100)

181 (100)

181 (100)

[0,1¢B – H – No2]+

—130 (15)

130 (10)

130 (22)

130 (21)

130 (6)

130 (8)

— — — — — — — —

(a)this loss may be due to two pathways in nitrochalcones, loss of No2 or loss of co and H2o as observed for the simple chalcone

Table 1. Ions observed in the product ion spectra of the [M + H]+ ions of 2¢-hydroxychalcones 1–16 with indication of their m/z values and relative abundance (RA%).

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608 Structural Characterization of Nitrated 2¢-Hydroxychalcones

as presented in table 2, which shows, as an example, the results obtained for compounds 1–4. this table contains the empirical formula, observed and calculated mass/charge ratios, double bond equivalent and mass error for the frag-ment ions observed in MS/MS spectra of 2¢- hydroxychalcones 1–4. the errors between the observed masses and calculated ones range from 0 mDa to 5.1 mDa (0.2–21.1 ppm) indicating good mass accuracy and confirming the composition of the fragments.

Non substituted 2¢-hydroxychalcone (1)the product ion spectra of the protonated molecule of the non-substituted 2¢-hydroxychalcone (1), with an m/z 225, was studied in order to identify the product ions due to cleavage

11

Scheme 1

OOH

H

H

R' R1'

2'3'

4'5'

6'

32

1

65

4

0

α

β

A B

0,αA+

0,1'B+

1,βA+

Scheme 1. Ion nomenclature used for the fragmentation of chalcones.

Predicted Formula Calculated mass (Da)

Observed mass (Da)

Error (mDa)

Error (ppm)

DBEa

Compound 1 ([M + H]+ C15H13O2)

c14H10o2(–cH3 ) 210.0681 210.0663 –1.8 –8.5 10.0 c15H11o(–H2o) 207.0810 207.0774 –3.6 –17.3 10.5 c14H11(–H2o – co) 179.0861 179.0834 –2.7 –14.9 9.5

c9H7o2 (1,bA – H)+ 147.0446 147.0397 –4.9 –33.4 6.5

c9H7o (0,1¢B – H)+ 131.0497 131.0473 –2.4 –18.2 6.5

c7H5o2(0,aA – H)+ 121.0290 121.0264 –2.6 –21.1 5.5

Compound 2 ([M + H]+ C15H12NO4)

c15H11No3(–oH) 253.0739 253.0749 1.0 4.0 11.0c15H10No3(–H2o) 252.0661 252.0664 0.3 1.3 11.5c15H10No2(–2oH) 236.0712 236.0712 0 0.2 11.5c15H9No2(–H2o – oH) 235.0633 235.0684 5.1 2.6 12.0

c14H10No2(–H2o – co) 224.0712 224.0727 1.5 6.9 10.5

c15H12o2 (–No2) 224.0837 224.0727 –11.1 –49.2 10.0c14H8No 206.0606 206.0636 3.6 4.6 11.5c9H10No4 196.0610 196.0639 2.9 14.9 10.0c14H10(–H2o – co – No2) 178.0783 178.0764 –1.9 –10.4 10.0

c9H6No3 (0,1¢B – H)+ 176.0348 176.0363 1.5 8.7 7.5

c8H10No3 168.0661 168.0680 1.9 11.5 4.0

c7H5o2 (0,aA – H)+ 121.0290 121.0289 –0.1 –0.5 5.5

c9H6o (0,1¢B – H – No2)+ 130.0419 130.0410 –0.9 –6.7 7.0

Compound 3 ([M + H]+ C15H12NO4)

c14H9No4(–cH3) 255.0532 255.0611 –4.6 –18.2 10.5c15H10No3(–H2o) 252.0661 252.0694 3.3 13.2 11.5c14H10No2(–H2o – co) 224.0712 224.0750 3.8 17.2 10.5c15H12o2 (–No2) 224.0750 224.0750 –8.7 –39.0 10.0

c9H6No3 (0,1¢B – H)+ 176.0348 176.0363 1.5 8.7 7.5

c7H5o2 (0,aA – H)+ 121.0290 121.0300 1.0 8.6 5.5

c9H6o (0,1¢B – H – No2)+ 130.0419 130.0440 2.1 16.4 7.0

Table 2. Calculated and experimental elemental composition of the most abundant fragments for the compounds 1–4.

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A.I.R.N.A. Barros et al., Eur. J. Mass Spectrom. 15, 605–616 (2009) 609

of the hydroxyl-chalcone moiety [figure 2(a)] and the ions identified are shown in table 1. the major fragment ion of the 2¢-hydroxychalcone (1), corresponding to the base peak, was due to the loss of 15 Da yielding the fragment ion at m/z 210, attributed to the loss of a methyl radical. the loss of a methyl radical has been previously observed in the product ion spectra of protonated molecules of chalcones without a 2¢-hydroxy substituent, formed under APcI conditions.23 In this case, the elimination of cH3

• only occurred after loss of co.23

for the 2¢-hydroxychalcone 1, the elimination of a methyl radical is not easily rationalized and cannot be explained by simple bond cleavage. Probably, it involves an initial isomerization of chalcone into flavanone, a-cleavage between the c=o and the adjacent carbon and migration of a hydrogen (already observed in fragmentation of M+• of cyclic ketones)28 and the elimination of a methyl radical. It is well known from solution chemistry that this intramolecular cyclization of 2¢-hydroxychalcone into flavanone occurs easily.29 furthermore, the isomerization of chalcone into flavanone has already been proposed to occur in the gas phase, prior to the fragmentation pathways, in the analysis of 2¢-hydroxychalcones under ESI conditions in negative mode.21

tandem mass spectra of deuterated species were obtained in order to give some clarification on the mechanism of this fragmentation and understand the influence of protons on it [figure 2(b)]. It was found that after deuteration, the [M – H + 2D]+ ion was formed, which means that the hydroxyl group hydrogen was exchanged by a deuterium and a second D+ was added to the molecule. Analyzing the correspondent product ion spectra [figure 2(b)], it was possible to observe the loss of neutral species with 15 Da, 16 Da and 17 Da that should correspond to loss of cH3

•, cH2D• and cHD2

•, respectively. taking into consideration all these data, a mechanism is proposed (Scheme 2) for the elimination of cH3

• and cH2D

• / cHD2•.

other characteristic and structurally informative fragment ions27 observed in the spectrum of the 2¢-hydroxychalcone 1 were those resulting from c–c linkage cleavage of the

propenone unit yielding fragment ions at m/z 121 (0,aA – H)+, m/z 147 (b,1A – H)+ and at m/z 131 (0,1¢B – H)+. fragment ions at m/z 207, m/z 197 and m/z 179 were attributed to fragment ions [M + H – H2o]+, [M + H – co]+ and [M + H – H2o – co]+, respectively. Loss of co should be preceded by a 2¢-hydroxychalcone isomerization into flavanone as referred to above.21 In fact, loss of co is a typical elimination of cyclic ketones28 and was observed for other flavonoids21 and cyclic compounds with a carbonyl group.30

the product ion identification was confirmed by exact mass measurement and molecular formula determination as previously reported (table 2).

2¢-hydroxychalcones bearing one nitro group (2–4)the ESI-MS/MS spectra were obtained for the protonated molecules, [M + H]+ of 2¢-hydroxychalcones 2, 3 and 4, [figures 3(a)–(c)], bearing a nitro group in the ortho- 2 [figure 3(a)], meta- 3 [figure 3(b)] and para- 4 [figure 3(c)] positions of ring B, respectively.

Predicted FormulaCalculated mass

(Da)Observed mass

(Da)Error (mDa)

Error (ppm)

DBEa

Compound 4 ([M + H]+ C15H12NO4)

c14H9No4(–cH3) 255.0515 255.0532 –1.7 –6.5 11.0c15H10No3(–H2o) 252.0661 252.0640 –2.1 –2.1 11.5c14H10No2(–H2o–co) 224.0712 224.0719 0.7 3.3 10.5c15H12o2(–No2) 224.0837 224.0750 –11.8 –52.8 10.0

c9H6No3(0,1¢B – H)+ 176.0348 176.0326 –2.2 –12.3 7.5

c7H5o2(0,aA – H)+ 121.0290 121.0278 –1.2 –9.5 5.5

c9H6o(0,1¢B – H – No2)+ 130.0419 130.0422 0.3 2.6 7.0

aDBE: double bond equivalents, corresponding to the number of double bonds and rings. to convert the reported DBE value to the actual number of double bonds, add 0.5 owing to an even-electron state.

Table 2 (continued). Calculated and experimental elemental composition of the most abundant fragments for the compounds 1–4.

11

m/z 100 120 140 160 180 200 220

%

0

100

%

0

100 210.1

179.1 121.0 131.0 178.1 147.0 207.1

179.2 225.1

211.1

180.1 179.1

122.0 132.1 148.0 210.1 212.1

227.1

m/z 100 120 140 160 180 200 220

%

0

100

%

0

100

m/z 100 120 140 160 180 200 220

%

0

100

%

0

100 210.1

179.1 121.0 131.0 178.1 207.1

179.2 225.1

210.1

179.1 121.0 131.0 178.1 207.1

179.2 225.1

211.1

180.1 179.1

122.0 132.1 148.0 210.1 212.1

227.1

[M+H] +

[M - H+2D] +

-H2O-(H2O+CO)

-CO

-DHO

-(DHO+CO)

-COm/z

100 120 140 160 180 200 220

%

0

100

%

0

100 210.1

179.1 121.0 131.0 178.1 207.1

179.2 225.1

210.1

179.1 121.0 131.0 178.1 207.1

179.2 225.1

211.1

180.1 179.1

122.0 132.1 148.0 210.1 212.1

227.1

m/z 100 120 140 160 180 200 220

%

0

100

%

0

100

m/z 100 120 140 160 180 200 220

%

0

100

%

0

100 210.1

179.1 121.0 131.0 178.1 207.1

179.2 225.1

210.1

179.1 121.0 131.0 178.1 207.1

179.2 225.1

211.1

180.1 179.1

122.0 132.1 148.0 210.1 212.1

227.1

[M+H] +

[M - H+2D] +

-H2O-(H2O+CO)

-CO

-DHO

-(DHO+CO)

-CO

-CH3.

-CDH2.

(a)

(b)

( 0, α A - H) +

( 0, α A - D) +

( 0,1’B-H)+

( 0,1’ B-D)+

(1,βA-H)+

(1,βA-D)+

m/z 100 120 140 160 180 200 220

%

0

100

%

0

100 210.1

179.1 121.0 131.0 178.1 147.0 207.1

179.2 225.1

211.1

180.1 179.1

122.0 132.1 148.0 210.1 212.1

227.1

m/z 100 120 140 160 180 200 220

%

0

100

%

0

100

m/z 100 120 140 160 180 200 220

%

0

100

%

0

100 210.1

179.1 121.0 131.0 178.1 207.1

179.2 225.1

210.1

179.1 121.0 131.0 178.1 207.1

179.2 225.1

211.1

180.1 179.1

122.0 132.1 148.0 210.1 212.1

227.1

[M+H] +

[M - H+2D] +

-H2O-(H2O+CO)

-CO

-DHO

-(DHO+CO)

-COm/z

100 120 140 160 180 200 220

%

0

100

%

0

100

Figure 2

Figure 2. Product ion spectra of the [M + H]+ (a) and [M – H + 2D]+ (b) of 2¢-hydroxychalcone (1) formed under ESI conditions.

Page 6

610 Structural Characterization of Nitrated 2¢-Hydroxychalcones

11

OH (D) OH (D)

+

O

H (D)H

OH (D)

+

Loss of CD2H.

O

HC

O+

.

C+

O

H (D)H

H (D)

O

O

H (D)H

H (D)

O +

OCH

O+

- CH3.(-CHD2

.)

.

Loss of CH3. and CDH2

.

OH (D) OH (D)

+

O

H (D)H

OH (D)

+

C

O

H

H (D)

OH (D)

+

O

O(D) H

CH

H

H (D)H

+

O

O(D) H

C

(D) H

H (D)H

+

H

O

O(D) H

CH

C

H

. +

- CH3(-CH2D)

O

O(D) H

CH

(D) H

HH

+

O

O(D) H

C

(D) H

HH

+

H

O

O(D) H

CH

C

(D) H

. +

- CH3

or

. ..

Pathway A

Pathway B

O

O(D) H

C

(D) H

O

O(D) H

HC

(D) H

+

- CH3.

HH

H

.

+

HH

H

O

O(D) H

HC

(D) H

+

.

OH (D) OH (D)

+

O

H (D)H

OH (D)

+

Loss of CD2H.

O

HC

O+

.

C+

O

H (D)H

H (D)

O

O

H (D)H

H (D)

O +

OCH

O+

- CH3.(-CHD2

.)

.

OH (D) OH (D)

+

O

H (D)H

OH (D)

+

Loss of CD2H.

O

HC

O+

.

C+

O

H (D)H

H (D)

O

O

H (D)H

H (D)

O +

OCH

O+

- CH3.(-CHD2

.)

.

Loss of CH3. and CDH2

.

OH (D) OH (D)

+

O

H (D)H

OH (D)

+

C

O

H

H (D)

OH (D)

+

O

O(D) H

CH

H

H (D)H

+

O

O(D) H

C

(D) H

H (D)H

+

H

O

O(D) H

CH

C

H

. +

- CH3(-CH2D)

O

O(D) H

CH

(D) H

HH

+

O

O(D) H

C

(D) H

HH

+

H

O

O(D) H

CH

C

(D) H

. +

- CH3

or

. ..

Pathway A

Pathway B

O

O(D) H

C

(D) H

O

O(D) H

HC

(D) H

+

- CH3.

HH

H

.

+

HH

H

O

O(D) H

HC

(D) H

+

.

Loss of CH3. and CDH2

.

OH (D) OH (D)

+

O

H (D)H

OH (D)

+

C

O

H

H (D)

OH (D)

+

O

O(D) H

CH

H

H (D)H

+

O

O(D) H

C

(D) H

H (D)H

+

H

O

O(D) H

CH

C

H

. +

- CH3(-CH2D)

O

O(D) H

CH

(D) H

HH

+

O

O(D) H

C

(D) H

HH

+

H

O

O(D) H

CH

C

(D) H

. +

- CH3

or

. ..

Pathway A

Pathway B

O

O(D) H

C

(D) H

O

O(D) H

HC

(D) H

+

- CH3.

HH

H

.

+

HH

H

O

O(D) H

HC

(D) H

+

.

Loss of CH3. and CDH2

.

OH (D) OH (D)

+

O

H (D)H

OH (D)

+

C

O

H

H (D)

OH (D)

+

O

O(D) H

CH

H

H (D)H

+

O

O(D) H

C

(D) H

H (D)H

+

H

O

O(D) H

CH

C

H

. +

- CH3(-CH2D)

O

O(D) H

CH

(D) H

HH

+

O

O(D) H

C

(D) H

HH

+

H

O

O(D) H

CH

C

(D) H

. +

- CH3

or

. ..

Pathway A

Pathway B

O

O(D) H

C

(D) H

O

O(D) H

HC

(D) H

+

- CH3.

HH

H

.

+

HH

H

O

O(D) H

HC

(D) H

+

.

Scheme 2 Scheme 2. Proposed mechanism for the loss of CH3

•, CH2D• and CHD2• from [M + H]+ and/or [M – H + 2D]+ ions of 2¢-hydroxychalcone (1).

Page 7

A.I.R.N.A. Barros et al., Eur. J. Mass Spectrom. 15, 605–616 (2009) 611

Several common and structurally informative fragments of the chalcone structure were observed. these ions were the ones at m/z 121 (0,aA – H)+, resulting from c–c linkage cleavage

of the propenone unit, at m/z 176 corresponding to (0,1¢B – H)+

bearing the nitro moiety, at m/z 130 [(0,1¢B – H)+ – No2•] and

fragment ions and at m/z 252, m/z 224 and m/z 178 attributed to losses of H2o, combined losses of H2o and co, and loss of H2o + co + No2, as can be seen in figure 3 and table 1. Scheme 3 presents the structures of the ions identified, the molecular formulas of which were confirmed by elemental composition determination (table 2). the structure of the ion at m/z 130 was in accordance with Zhang et al.21 the ion at m/z 224 can be attributed to the loss of a nitro group (–46 Da), and/or to the combined loss of co and H2o, since it was observed as a product ion formed by loss of a neutral of 46 Da, with high relative abundance (60% RA) in the product ion spectra of the non-substituted 2¢-hydroxychalcone (1). Nevertheless, the elimination of No2

• is a characteristic loss of nitro-substituted and nitroaromatic compounds21,31 and is also observed for other nitro-substituted chalcones.23 to elucidate the fragmentation pathway yielding the fragment ion observed at m/z 224, pseudo-MS3 experiments were performed in the Q–tof instrument by increasing the cone voltage to 80 V and the MS/MS of fragment ions generated in the source were obtained. the pseudo-MS3 data of the dehydration fragment ion [M + H – H2o]+, with an m/z 252, was acquired confirming that it was indeed a precursor of the m/z 224 ion, as suggested in Scheme 2. Also, pseudo-MS3 experiments performed on ion m/z 224 yielded fragment ions formed by the loss of H2o and loss of co yielding ions with m/z 206 and m/z 196, confirming the contribution of the fragmentation pathway

11

Figure 3

[M+H]+

[M+H]+

[M+H]+-CH3.

-OH.

-H2O

m/z100 120 140 160 180 200 220 240 260

%

0

100

%

0

100

%

0

100 235.1

196.1121.0 130.0 224.1206.1

236.1

253.1 270.1

176.0

121.0

147.1

224.1

270.1252.1

224.1

121.0 176.0

130.0 178.1 270.1255.1

-(CO+NO2)

-(H2O+CO)

-(H2O+CO)

-(H2O+OH.)

(0,1’B-H)+-NO2

(0,1’B-H)+- HCO.

(0,1 B-H)+

a

b

c(0,1 B-H)+

-(OH.+OH.)

(0,αA-H)+

(0,αA-H)+

(0,αA-H)+

(0,1’B-H)+-NO2

[M+H]+

[M+H]+

[M+H]+-CH3.

-OH.

-H2O

m/z100 120 140 160 180 200 220 240 260

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100 235.1

196.1121.0 130.0 224.1206.1

236.1

253.1 270.1

176.0

121.0

147.1

224.1

270.1252.1

224.1

121.0 176.0

130.0 178.1 270.1255.1

-(CO+NO2)

-(H2O+CO)

-(H2O+CO)

-(H2O+OH.)

(0,1’B-H)+-NO2

(0,1’B-H)+- HCO.

(0,1 B-H)+

a

b

c(0,1 B-H)+

-(OH.+OH.)

(0,αA-H)+

(0,αA-H)+

(0,αA-H)+

(0,1’B-H)+-NO2

168.1 

Figure 3. Product ion spectra of the [M + H]+ ions of, respectively, ortho, meta and para -nitro-2¢-hydroxychalcones 2(a), 3(b) and 4(c).

22

Scheme 3

Scheme 3

OH O

N

O

OH+

O

N

O

OH+

m/z 252

OH O

NO2

A

B

H

+ OH O

A

+

m/z 121

O

NO2

m/z 176+

O

+ .

m/z 130

- NO2.

N OH

O

+

m/z 224

- CO

Scheme 3. Common fragmentation pathways of [M + H]+ of chalcones 2–4.

Page 8

612 Structural Characterization of Nitrated 2¢-Hydroxychalcones

by loss of the nitro group (No2•) to the formation of the ion

at m/z 224. Nevertheless, data from the exact mass analysis (table 2) confirm that this ion was mainly due to the combined loss of co and H2o since the error determined for this ion is lower (6.9 ppm) than the error determined for the product ion due to loss of No2 (error = –49.2 ppm), as exemplified in the case of chalcone 2. Analysis of the product ion spectra of the deuterated ions [M – H + 2D]+ (figure 4), when compared with the protonated ions (figure 3), revealed that the corresponding product ion shifted to a value one unit higher (m/z 225), revealing that it was due to the loss of co plus HDo. Still, a lower abundant ion at m/z 224 that was due to the loss of No2

• and/or loss of co plus H2o could be observed, as discussed earlier. the presence of the nitro substituent in these substituted chalcones may be confirmed by the presence of an ion at m/z 176 corresponding to the (0,1¢B – H)+ ion bearing a nitro moiety and the fragment ions at m/z 178 and m/z 130, corresponding to ions [(M + H) – H2o – co – No2]

+ and (0,1¢B – H)+, respectively. Both ions were absent in the product ion spectra of the non-substituted 2¢-hydroxychalcone 1, confirming its attribution and the location of the nitro group attached to the B-ring. Interestingly, the ions at m/z 224 and m/z 121 showed high relative abundances for compounds 3 and 4 and lower relative abundance for compound 2.

comparing the data obtained for the nitro chalcones with that obtained for the unsubstituted one (1), summarized in table 1, it can be observed that ions due to loss of cH3., the base peak of compound 1, are absent (in the case of 2) or showed low relative abundance (for 3 and 4). the presence of the nitro group notably changes the fragmentation pattern of this class of compounds. this finding is in accordance with the results reported by Zhang et al.21 that point out the influence of the

substituents of chalcones in the distinct fragmentation pattern observed. this different fragmentation pattern is probably due to a different protonation position in the nitro derivatives when compared with the non-nitro chalcones. the nitro group is a known electron-withdrawing group that is known, for example, to increase considerably the gas-phase bond dissociation enthalpy of the hydroxyl group in nitro substituted phenols.32

furthermore, noteworthy differences are observed by comparing the product ion spectra of the nitro-2¢-hydroxychalcones 2, 3 and 4 [figures 3(a)–(c)], which are very relevant bearing in mind that they are positional isomers of these nitro derivatives. Main differences occur in the product ion spectra of compound 2 with an ortho-nitro group, where new fragments were identified. these new ions are due to the loss of oH•, 2oH• and (oH• + H2o) with formation of the ions, respectively, at m/z 253, m/z 236 and m/z 235, ions at m/z 206 and m/z 207 due to combined loss of H2o + co + H2o and H2o + co + oH•, as well as the ions at m/z 196 and m/z 168. the structures proposed for these ions are presented in Scheme 4. these ions were absent in the spectra of the meta- and para-isomers. the combined elimination of 2oH• and H2o plus oH• was observed for nitro-substituted porphyrins in the b-pyrrolic position31 and loss of 2oH• was also observed for nitro-isoflavonoids.33 Similar to that suggested for porphyrins, this elimination of 2oH• may probably be triggered by interaction of the nitro group with hydrogen from the ca of the propilidene bridge, which proceeded via the formation of an intramolecular five-membered ring, as presented in Scheme 4. the loss of the second oH• should be favored since a conjugated system is formed after this loss (ion at m/z 236, Scheme 4). In both mechanisms, we propose that at least a fraction of the molecules are protonated on the nitro group. tandem mass spectra of deuterated species were obtained in order to give some clarification on the mechanism of this fragmentation and understand the influence of the proton on it. It was found that after the addition of cH3oD, the ion formed under ESI conditions was the [M – H + 2D]+ion, as observed previously. the results obtained by the analysis of the product ion spectra of the deuterated ions [M – H + 2D]+ of these 2¢-hydroxychalcones [figures 4(a)–(c)], which showed an increase of one mass unit in the corresponding fragments when compared with the non-deuterated ions, support the proposed mechanism.21

overall, the position of the nitro group in the B ring changes the ESI-MS/MS spectra of the corresponding 2¢-hydroxychalcones considerably. these fragment ions were not observed for the chalcones bearing the nitro substituent in the meta- or para position of the phenyl ring since, in these cases, the proposed rearrangement cannot possibly occur due to steric reasons. In fact, the location of the nitro group in the ortho position allows discriminatory migration of an hydroxyl group via intra-molecular six-membered cyclization during an intermediate transition state (shown in Scheme 4), which is not allowed when the nitro group is located in the meta- or para positions of the aromatic ring. this intra-molecular cyclization accomplishes selective unimolecular

11

m/z120 140 160 180 200 220 240 260 280

%

0

100

%

0

100

%

0

100 122.0

121.0

236.0

197.1 130.0 235.0 254.1 272.1

122.0

121.0

225.1 176.0

147.0 207.1 272.2 252.1

225.1

176.0 122.0

207.1 272.1 256.0

m/z120 140 160 180 200 220 240 260 280

%

0

100

m/z120 140 160 180 200 220 240 260 280

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100 122.0

121.0

236.0

197.1 130.0 235.0 254.1 272.1

122.0

121.0

236.0

197.1 130.0 235.0 254.1 272.1

122.0

121.0

225.1 176.0

147.0 207.1 272.2 252.1

122.0

121.0

225.1 176.0

147.0 207.1 272.2 252.1

225.1

176.0 122.0

207.1 272.1 256.0

[M - H+2D] +

[M - H+2D] +

[M - H+2D] +

-(HDO-OH?)

-(HDO+CO)

-(HDO+CO)

(OD+OH ? )

( 0, α A - D) +

(0,1’B-D)+

( 0, α A - D) +

(0,1’B-D)+

(a)

(b)

(c)

( 0, α A - D) +

m/z120 140 160 180 200 220 240 260 280

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100 122.0

121.0

236.0

197.1 130.0 235.0 254.1 272.1

122.0

121.0

225.1 176.0

147.0 207.1 272.2 252.1

225.1

176.0 122.0

207.1 272.1 256.0

m/z120 140 160 180 200 220 240 260 280

%

0

100

m/z120 140 160 180 200 220 240 260 280

%

0

100

%

0

100

%

%

0

100

%

0

100

%

0

100

%

0

100

0

100

Figure 4

Figure 4. ESI-MS/MS spectra of the [M – H + 2D]+ of of respec-tively ortho, meta and para-nitro-2¢-hydroxychalcones 2(a), 3(b) and 4(c)

Page 9

A.I.R.N.A. Barros et al., Eur. J. Mass Spectrom. 15, 605–616 (2009) 613

decomposition of the precursor ions, namely, allowing new and alternative fragmentation pathways which are energetically favorable and specific for the isomers bearing the nitro group in the ortho position. the observed differences in the product ion spectra of these nitrochalcones allowed the presence of the ortho derivative to be distinguished. Differentiation between the meta- and para isomers was not achieved.

Unsubstituted and nitro 2¢-hydroxy-4¢-methoxychalcones (5–8)to study the influence of the presence of a 4¢-methoxy group in the chalcone fragmentation, the corresponding non-nitrated chalcone (5) was studied by ESI-MS/MS. comparing the frag-ment ions observed in the product ion spectrum of chalcone 5 with that of chalcone 1 (table 1), it was found that the presence of a 4¢-methoxy group does not significantly change the frag-mentation pattern of the 2¢-hydroxychalcone. In fact, the same fragmentation pathways were observed for both chalcones 1 and 5, although yielding product ions with different relative

abundances. In the case of chalcone 5, the major fragment ions were the result of c–c bond cleavage of the propenone unit yielding fragment ions at m/z 151 corresponding to (0,aA – H)+. the presence of the methoxy group did not produce any frag-mentation specific for this functional group, as it was deduced by the analysis of the product ion spectrum of the [M + H]+ ion of the 2¢-hydroxy-4¢-methoxychalcone 5 and compared with the product ion spectrum of the [M + H]+ ion of the 2¢-hydroxy-chalcone (1) (table 1).

the nitro 2¢-hydroxy-4¢-methoxychalcones (5–8) showed, as can be seen in table 1, several common ions at m/z 151 and m/z 176, corresponding to the characteristic (0,aA – H)+• and the nitro (0,1¢B – H)+ ions, confirming the presence of the methoxy group linked to the A ring and the nitro moiety to the B ring. the other common fragmentations are due to loss of H2o (m/z 252), H2o + co (m/z 254) and combined elimination of co + H2o + No2

(m/z 208). Interestingly, the ortho-nitro derivative 6 showed exclusive fragmentation patterns when compared to the meta- 7 and para- 8 nitro-derivatives, which were equivalent to those

22

O

O

N

O-HO+

OH O

N

N

O

HO

HO O

+

N

HO

OHOm/z 168

m/z 196

OH O

N

O-HO+

O

+.N

OOHOH

+.

m/z 253

-OH . (OD . )

O

N +

m/z 252

O-HO

- H2O (-HDO)

M+H+

- OH . (-OD .)

- OH .

O

N

O

+.

m/z 235

OOH

N

CH +

m/z 236

- CO

m/z 224 - OH . (-OD .)m/z 207

m/z 206

N

O

+

- H2O(-HDO)

Scheme 4

Scheme 4. Fragmentation pathways observed only for chalcone 2, with the ortho-nitro group in its B ring.

Page 10

614 Structural Characterization of Nitrated 2¢-Hydroxychalcones

described for the ortho nitrochalcone 2, thus allowing the differentiation of the ortho-derivative. fragment ions at m/z 283, m/z 266 and m/z 265, due to the elimination of oH•, 2oH• and oH• plus H2o for the ortho-nitro derivative must be formed by the same fragmentation pathways as proposed above for the non-methoxylated nitro derivatives.

the presence of a methoxy substituent in the 4¢-position of the A ring did not significantly change the product ion spectra of the 2¢-hydroxynitrochalcones.

Unsubstituted and nitro-2¢-hydroxy-6¢-methoxychalcones (9–12)the 2¢-hydroxynitrochalcone derivatives with the methoxy substituent in the 6¢ position of the A ring (compounds 10–12) were also studied by MS/MS. the non nitrated 2¢-hydroxy-6¢-methoxychalcone 9 was studied in order to identify the typical fragmentation observed in the MS/MS spectra due to the 6¢-methoxy moiety. As can be seen in table 1, compound 9 showed a product ion spectrum with only one fragment ion at m/z 151 corresponding to the (0,aA – H)+ ion. the presence of a 6¢-methoxy group significantly changes the fragmentation pattern of the 2¢-hydroxychalcone, when compared with the fragmentation observed for its isomer, the 4¢-methoxy-2¢-hydroxychalcones 5 and also for the 2¢-hydroxychalcone 1. Interestingly, the product ion spectra of the corresponding 6¢-methoxynitrochalcones 10–12 (table 1), presented the same behavior as compound 9, since there was only one fragment ion at m/z 151 in their product ion spectra. Very limited structural information can be drawn from these spectra and differentiation between positional nitro isomers cannot be obtained. the influence of the 6¢-methoxy group on the fragmentation mechanism of the 2¢-hydroxychalcones can be rationalized as being due to steric effects of the 6¢-methoxy group which introduces a torsion on co–ca,26 which is probably the reason for this preferential cleavage site.

Unsubstituted and nitro-2¢-hydroxy-4¢,6¢-dimethoxychalcones (13–16)the influence of the introduction of two 4¢- and 6¢-methoxy groups in the A ring of 2¢-hydroxy-4¢,6¢-dimethoxychacone was studied. It was found that this modification of the substi-tuition pattern also changes the fragmentation of the MS/MS spectra when compared with the non-substituted 2¢-hydroxy-chalcone 1, or with the other methoxy 2¢-hydroxychalcone presented before. In fact, the ESI-MS/MS spectra of the 2¢-hydroxy-4¢,6¢-dimethoxynitrochalcone (13) only showed the ion at m/z 181 which resulted from the propenone cleavage 0,aA+, similar to what was observed for chalcones 9–12, with a 6¢-methoxy moiety, but distinct from the 4¢-methoxy substi-tuted ones 5–8. the corresponding nitro 2¢-hydroxychalcones 14–16, presented an additional ion at m/z 176, corresponding to the (0,1B – H)+ ion, with the B ring bearing the nitro group, so confirming the presence of the nitro moiety linked to these chalcones. this fragmentation pattern for the three isomers 14–16 was similar to the nitro group, respectively, in the o-,

m- or p positions of the B ring, so differentiation between the isomers cannot be seen.

Conclusionfragmentation induced by low-energy collision dissocia-tion, corresponding to the elimination of oH•, 2oH• and oH• plus H2o allowed distinguishing between the ortho-nitro-2¢-hydroxychalcone derivatives 2, 6 from the meta- 3, 7 and para- 4, 8 derivatives of 2¢-hydroxychalcones and 2¢-hydroxy-4¢-methoxychalcones, which did not show the losses. However, the presence of a methoxy moiety in the 6¢ position of ring A significantly changed the product ion spectra of these 2¢-hydroxychalcones, since only one fragment, the (0,aA – H)+ fragment ion at m/z 151, was observed. the presence of two methoxy moieties in the 4¢,6¢-dimethoxychalcones also resulted in limited fragmentation (ions at m/z 151 and m/z 176). these differences may be due to steric effects of the 6¢-methoxy groups, inducing torsion on co–ca. No loss of No2

• nor of co was observed from the precursor ion, although these eliminations occurred in combination with loss of water, ([M + H] – H2o – co+) and ([M + H] – H2o – co – No2

+)• or from the (0,1B – H)+ ion, in the case of nitro group. thus, small changes in substituents linked to the chalcone moieties changed the fragmentation pattern of chalcones considerably.

Acknowledgmentsthe authors acknowledge the financial support of fEDEr, fct-Portugal, research unit of chemistry in Vila real (PoctI-SfA-3-616) and research unit 62/94 “Química orgânica, Produtos Naturais e Agro-Alimentares” of university of Aveiro.

References1. D.N. Dhar, The Chemistry of Chalcones and Related

Compounds. John Wiley & Sons Inc., New york, uSA (1981).

2. Z. Nowakowska, “A review of anti-infective and anti-inflammatory chalcones”, Eur. J. Med. Chem. 42, 125 (2007). doi: 10.1016/j.ejmech.2006.09.019

3. M.L. Go, X. Wu and X.L. Liu, “chalcones: An update on cytotoxic and chemoprotective properties”, Curr. Med. Chem. 12, 483 (2005).

4. J.f. Stevens, c.L. Miranda, B. frei and D.r. Buhler, “Inhibition of peroxynitrite-mediated LDL oxidation by prenylated flavonoids: the alpha, beta-unsaturated keto functionality of 2¢-hydroxychalcones as a novel anti-oxidant pharmacophore”, Chem. Res. Toxicol. 16, 1277 (2003). doi: 10.1021/tx020100d

5. I.S. Arty, H. timmerman, M. Samhoedi, Sastrohamidjojo, Sugiyanto and H. Van der Goot, “Synthesis of

Page 11

A.I.R.N.A. Barros et al., Eur. J. Mass Spectrom. 15, 605–616 (2009) 615

benzylideneacetophenones and their inhibition of lipid peroxidation”, Eur. J. Med. Chem. 35, 449 (2000). doi: 10.1016/S0223-5234(00)00137-9

6. J.r. Dimmock, A. Jha, G.A. Zello, t.M. Allen, c.L. Santos, J. Balzarini, E. De clercq, E.K. Manavathu and J.P. Stables, “cytotoxic 4¢-aminochalcones and related com-pounds”, Pharmazie 58, 227 (2003).

7. M. cabrera, M. Simoens, G. falchi, M.L. Lavaggi, o.E. Piro, E.E. castellano, A. Vidal, A. Azqueta, A. Monge, A.L. de cerain, G. Sagrera, G. Scoane, H. ceretto and M. Gonzalez, “Synthetic chalcones, flavanones, and flavones as antitumoral agents: Biological evaluation and structure-activity relationships”, Bioorg. Med. Chem. 15, 3356 (2007). doi: 10.1016/j.bmc.2007.03.031

8. G.S.B. Viana, M.A.M. Bandeira and f.J.A. Matos, “Analgesic and antiinflammatory effects of chal-cones isolated from Myracrodruon urundeuva Allemao”, Phytomedicine 10, 189 (2003). doi: 10.1078/094471103321659924

9. H. Viola, M. Marder, c. Wolfman, c. Wasowski, J. Medina and A. Paladini, Bioorg. Med. Chem. 7, 373 (1997). doi: 10.1016/S0960-894X(97)00020-6

10. M. cárdenas, M. Marder, V.c. Blank and L.P. roguin, “Antitumor activity of some natural flavonoids and syn-thetic derivatives on various human and murine cancer cell lines”, Bioorg. Med. Chem. 14, 2966 (2006). doi: 10.1016/j.bmc.2005.12.021

11. c. Wolfman, H. Viola, M. Marder, c. Wasowski, P. Ardenghi, I. Izquierdo and A.c. Paladini, “Anxioselective properties of 6,3¢-dinitroflavone, a high-affinity benzodiazepine receptor ligand”, Eur. J. Pharmacol. 318, 23 (1996). doi: 10.1016/S0014-2999(96)00784-4

12. c. Van De Sande, J.W. Serum and M. Vandewalle, “Studies in organic mass spectrometry-XII: mass spec-tra of chalcones and flavanones. the isomerisation of 2¢-hydroxychalcone and flavanone”, Org. Mass Spectrom. 6, 1333 (1972). doi: 10.1002/oms.1210061208

13. c.E. Ardanaz, P. traldi, u. Vettori, J. Kavka and f. Guidugli, “the ion-trap mass-spectrometer in ion structure studies—the case of [M – H]+ ions from chalcone”, Rapid Commun. Mass Spectrom. 5, 5 (1991). doi: 10.1002/rcm.1290050103

14. V.S. Parmer, S.K. Sharma, A. Vardhan and r.K. Sharma, “New fragmentation in the electron impact mass spec-trometry of derivatized pyrano-1,3-diphenylprop-2-enones”, Org. Mass Spectrom. 21, 109 (1986).

15. Z. Nowakowska, “Structural assignment of chalcones and differentiation of their isomeric derivatives by elec-tron ionization induced fragmentation”, Rapid Commun. Mass Spectrom. 18, 2516 (2004). doi: 10.1002/rcm.1643

16. Z. Nowakowska, “Structural characterization and differentiation of isomeric w-bromoalkoxy derivatives of (E)-chalcone by means of mass spectrometry”, Eur. J. Mass Spectrom. 14, 37 (2008). doi: 10.1255/ejms.905

17. V.S. Bankova, N.N. Mollova and S.S. Popov, “chemical ionization mass-spectrometry with amines as reactant

gases. 1 Amine chemical ionization mass-spectrometry of flavonoid aglycones”, Org. Mass Spectrom. 21, 109 (1986). doi: 10.1002/oms.1210210304

18. B. Hoffmann and J. Holzl, “chalcone glucosides from Bidens pilosa”, Phytochemistry 28, 247 (1989). doi: 10.1016/0031-9422(89)85049-6

19. J.K. Prasain, t. yasuhiro, J.X. Li, K. tanaka, P. Basnet, H. Dong, t. Namba and S. Kadota, “Six novel diaryl-heptanoids bearing chalcone or flavanone moiety from the seeds of Alpinia blepharocalyx”, Tetrahedron 53, 7833 (1997). doi: 10.1016/S0040-4020(97)00474-2

20. K. redl, B. Davis and r. Bauer, “chalcone glycosides from Bidens campylotheca”, Phytochemistry 32, 218 (1993). doi: 10.1016/0031-9422(92)80140-A

21. J. Zhang and J. Brodlet, “Structural characterization and isomer differentiation of chalcones by electrospray ionization tandem mass spectrometry”, J. Mass Spectrom. 38, 555 (2003). doi: 10.1002/jms.472

22. L. Zhang, L. Xu, S.S. Xiao, Q.f. Liao, Q. Li, J. Liang, X.H. chen and K.S. Bi, “characterization of flavonoids in the extract of Sophora flavescens Ait by high-performance liquid chromatography coupled with diode-array detec-tor and electrospray ionization mass spectrometry”, J. Pharm. Biomed. Anal. 44, 1019 (2007). doi: 10.1016/j.jpba.2007.04.019

23. y. tai, S. Pei, J. Wan, X. cao and y. Pan, “fragmentation study of protonated chalcones by atmospheric pres-sure chemical ionization and tandem mass spectrom-etry, Rapid Commun. Mass Spectrom. 20, 994 (2006). doi: 10.1002/rcm.2404

24. W.A. Korfmacher, “use of mass spectrometry for drug metabolism”, Curr. Drug Metabol. 7, 455 (2006). doi: 10.2174/138920006777697945

25. A.I.r.N.A. Barros, A.M.S. Silva, I. Alkorta and J. Elguero, “Synthesis, experimental and theoretical NMr study of 2¢-hydroxychalcones bearing a nitro substituent on their B ring”, Tetrahedron 60, 6513 (2004). 10.1016/j.tet.2004.06.005

26. A.r. Alcantara, J.M. Marinas and J.V. Sinisterra, “Synthesis of 2¢-hydroxychalcones and related compounds in interfacial solid–liquid conditions”, Tetrahedron Lett. 28, 1515 (1987). doi: 10.1016/S0040-4039(01)81030-3

27. y.L. Ma, Q.M. Li, H. Van den Heuvel and M. claeys, “characterization of flavones and flavonol agycones by collision-induced dissociation tandem mass spectrom-etry”, Rapid Commun. Mass Spectrom. 11, 1357 (1997). doi: 10.1002/(SIcI)1097-0231(199708)11:12<1357::AID-rcM983>3.0.co;2-9

28. f.W. McLafferty and f. turecek, Interpretation of Mass Spectra. university Science Books, Mill Valley, uSA, p. 186 (1993).

29. t.A. Mabry and A. ulubelen, “flavonoids and related plant phenolics”, in Biochemical Applications of Mass Spectrometry, first supplementary volume, Ed by G.r. Waller and o.c. Dermer. John Wiley & Sons, New york, uSA, p. 1131 (1980).

Page 12

616 Structural Characterization of Nitrated 2¢-Hydroxychalcones

30. M.r.M. Domingues, P. Domingues, M.M. oliveira, L.H.M. carvalho, A.f. oliveira de campos and A.J. ferrer correia, “Electrospray tandem mass spectrometry of 2H-chromenes”, Rapid Commun. Mass Spectrom. 18, 2969 (2004). doi: 10.1002/rcm.1713

31. M.r.M. Domingues, M.G.o.S. Marques, P. Domingues, M.G.P.M.S. Neves, J.A.S. cavaleiro, A.J. ferrer correia, o.V. Nemirovskiy and M.L. Gross, “Differentiation of positional isomers of nitro meso-tetraphenyl-porphyrins by tandem mass spectrometry”, J. Am. Soc. Mass Spectrom. 12, 381. (2001). doi: 10.1016/S1044-0305(01)00207-0

32. J.S. Wright, E.r. Johnson and G.A. DiLabio, “Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants” J. Am. Chem. Soc. 123, 1173 (2001). doi: 10.1021/ja002455u

33. J.K. Prasain, r. Patel, M. Kirk, L. Wilson, N. Botting, V.M. Darley-usmar and S. Barnes, “Mass spectrometric methods for the analysis of chlorinated and nitrated isoflavonoids: a novel class of biological metabolites”, J. Mass Spectrom. 38, 764 (2003). doi: 10.1002/jms.492