Electrospray tandem mass spectrometry analysis of methylenedioxy chalcones, flavanones and flavones

Article abstract of DOI:10.1002/rcm.6577

RATIONALE Several methylenedioxy chalcones, flavanones and flavones substituted with mono-, di- and trimethoxy groups have been used in the treatment of proliferative conditions like cancer and inflammatory diseases. The application of these flavonoids in

Full text of DOI:10.1002/rcm.6577

Research Article
Received: 2 February 2013
Revised: 20 March 2013
Accepted: 21 March 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 1303–1310
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6577
Electrospray tandem mass spectrometry analysis of
methylenedioxy chalcones, flavanones and flavones
1
2
José C. J. M. D. S. Menezes1,2 , José A. S. Cavaleiro , Shrivallabh P. Kamat ,
*
Cristina M. R. F. Barros1 and M. Rosário M. Domingues1
*
1Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal
2Department of Chemistry, Goa University, Taleigao Plateau, Goa 403 206, India
RATIONALE: Several methylenedioxy chalcones, flavanones and flavones substituted with mono-, di- and trimethoxy
groups have been used in the treatment of proliferative conditions like cancer and inflammatory diseases. The
application of these flavonoids in biology requires an analytical method to ensure a detailed knowledge of their
structures after drug metabolism.
METHODS: Electrospray ionization mass (ESI-MS) and tandem mass (ESI-MS/MS) spectra were acquired using a Q-TOF 2
instrument. Fragmentation patterns and their pathways were analyzed by CID-MS2–3 spectra acquired in a LXQ linear ion
trap mass spectrometer using standard isolation and excitation procedures (activation q value of 0.25, activation time of
30 ms). ESI-MS and ESI-MSn conditions: spray voltage 5 kV, nitrogen 8.00 sheath gas flow rate (arb), heated capillary
temperature 275ꢀC, capillary voltage 10.99 V; tube lens voltage 75.01 V.
RESULTS: The ESI-MS/MS spectra of chalcones were nearly identical to their corresponding isomeric flavanones with
+
0,aA+/1,3A+ and 0,1’B+/1,4B cleavages. Other common losses are of CH3, H2O, HCHO and C2H2O. The characteristic loss
•
of C2H2O and absence of a 0,aB+/1,3B+ product ion allows to distinguish between the 2- or 4-methoxy-substituted
chalcones and flavanones. Common losses of CH3, CH3 and H, and C2H2O2 characteristic for the presence of
methylenedioxy groups were observed in flavones.
•
•
•
CONCLUSIONS: The substitution pattern on the B-ring leads to distinct base peak formation in the flavones. In addition,
differentiation of isomers with methoxy substituents in ortho and para positions of the B-ring was achieved using MS/MS
in chalcones and flavanones. This method will be helpful for identification of these compounds in biological mixtures.
Copyright © 2013 John Wiley & Sons, Ltd.
Flavonoids and their derivatives which form a large class of
food constituents are naturally present in fruits, vegetables
as well in beverages, such as red wine and tea. They form
an integral part of the human diet as many of them alter
metabolic processes and have a positive impact on health.[1]
Scientific interest in these substances has been stimulated by
the potential health benefits arising from the antioxidant
activities of these polyphenolic compounds.[2] As a dietary
component, flavonoids are thought to have health-promoting
properties due to their high antioxidant capacity in both
in vivo and in vitro systems.[3] Apart from antioxidant activities,
flavonoids also have scavenging effects on activated
carcinogens and mutagens, act on proteins that control cell
cycle progression and altered gene expression.[4]
3,4 positions have been tested for treatment of proliferative
condition like cancer and inflammatory conditions.[5] The
3,4-methylenedioxy-3’,4’,5’-trimethoxychalcone 1 is a potent
anticancer prodrug activated by the tumor-selective
catalytic activity of the cytochrome P450 enzyme CYP1B1
to give the 3,4-dihydroxy group which is a potent broad
spectrum tyrosine kinase inhibitor.[6] Synthetic flavones
like 7,8,3’,4’-tetrahydroxyflavone 2 and their analogues
are known to inhibit telomerase, and have been intensively
explored as anticancer agents.[7] The corresponding
tetramethoxychalcone
3 is also known to inhibit the
generation of an inflammatory mediator (Fig. 1).[8]
Several methoxy-substituted flavonoids like tangeretin, a
5,6,7,8,4’-pentamethoxyflavone, found in the peel of most citrus
fruits, can act as a prodrug, forming its primary metabolite 40-
hydroxy-5,6,7,8-tetramethoxyflavone, and both inhibit cell cycle
progression in primary hepatocytes.[9] Tangeretin also protects
against the development of experimentally induced Parkinson’s
disease in rats.[10] In addition, nobiletin (5,6,7,8,3’,4’-
hexamethoxyflavone), along with tangeretin detected and
isolated from orange juice, was found to inhibit P-glycoprotein
drug efflux transporter, but not the cytochrome P450 (CYP)
isozyme CYP3A4 and can be potential agents for reversing
multidrug resistance or for recovering the bioavailability of
certain drugs.[11]
In this regard newer synthetic flavonoid moieties based
on naturally occurring ones having specific groups have
been synthesized and tested for biological activity. For
example, chalcones having the methylenedioxy group at
* Correspondence to: M. R. M. Domingues or J. C. J. M. D. S.
Menezes, QOPNA, Department of Chemistry, University
of Aveiro, 3810-193 Aveiro, Portugal.
E-mail: mrd@ua.pt; josemenezes@ua.pt
Rapid Commun. Mass Spectrom. 2013, 27, 1303–1310
Copyright © 2013 John Wiley & Sons, Ltd.

J. C. J. M. D. S. Menezes et al.
Figure 1. Structures of chalcones and flavones with biological activity.
Because all these biochemical activities like antioxidant,
cardiovascular, anticancer, and other age-related disease
protective properties are dependent on the individual
flavonoid structure, each new compound needs to be studied
systematically to assess its structure before and after drug
metabolism. Mass spectroscopy is routinely used for the
structural elucidation of flavonoid metabolites in in vivo
studies. The main conjugations expected for in vivo metabolites
are O-methylation, O-glucuronidation and O-sulfation and
they can be easily identified by the mass they add to the original
molecule.[12] Mass spectrometric techniques are indispensible
tools for the identification and structural studies of flavonoid
glycosides using electrospray ionization (ESI) and liquid
chromatography/mass spectrometry (LC/MS) and have been
previously reviewed.[13]
Considering the importance of methylenedioxy- and methoxy-
substituted flavonoids, and in continuation of our previous
study,[14] it was desirable to synthesize new flavonoids with such
substitution patterns for biological evaluation. In this paper we
describe the detailed mass spectral characterization of synthetic
chalcones, flavanones and flavones derived from 2’-hydroxy-
3’,4’-methylenedioxyacetophenone (4) and mono (2- and 4-), di
(3,4-) and tri (3,4,5-) methoxybenzaldehyde using electrospray
ionization tandem mass spectrometry (ESI-MS/MS).
for the fragment ions observed in the MS/MS spectra of
the studied compounds.
MS3 spectra were acquired in a LXQ linear ion trap mass
spectrometer (ThermoFinnigan, San Jose, CA, USA) with
an ESI source. Samples were introduced into the mass
spectrometer at a flow rate of 8 mL min–1. ESI conditions used
to obtain ESI-MS and ESI-MSn spectra were: spray voltage
5 kV, nitrogen 8.00 sheath gas flow rate (arb), heated
capillary temperature 275ꢀC, capillary voltage 10.99 V, and
tube lens voltage 75.01 V. CID-MS2–3 experiments were
performed on mass-selected precursor ions using standard
isolation and excitation procedures (activation q value of
0.25, activation time of 30 ms). The collision energy used
was between 20–24 (arbitrary units). Data acquisition was
carried out with an Xcalibur data system (ThermoFinnigan,
San Jose, CA, USA).
Synthesis
The syntheses of chalcone, flavanone and flavones derivatives
were carried out using the reported procedure (Scheme 1).[14]
The experimental details of the flavonoids used in this study
are described in the Supporting Information.
EXPERIMENTAL
Mass spectrometry
Positive ion ESI mass spectra and tandem mass spectra
were acquired using a Q-TOF 2 instrument (Micromass,
Manchester, UK). The samples for ESI analyses were
prepared by diluting 1 mL of the flavonoid solutions in
chloroform (~10-5 M) in 200 mL of methanol. Nitrogen was
used as the nebulizer gas and argon as the collision gas.
Samples were introduced into the mass spectrometer at a
flow rate of 10 mL min–1, the needle voltage was set at
3000 V and the ion source temperature was 80ꢀC. The cone
voltage was 35 V. Collision-induced decomposition tandem
mass spectra were acquired by selecting the desired ion with
the quadrupole section of the mass spectrometer and using
a collision energy of 30–50 eV. The product ions were
analyzed with the time-of-flight (TOF) analyzer. In MS
and MS/MS experiments, the TOF resolution was set to
~9000. In MS/MS experiments, the Q1 resolution was set
to ~0.7 Da. Data acquisitions were carried out with a
Micromass Mass Lynx data system. The lock mass in each
product ion spectrum was the calculated monoisotopic
mass/charge of the precursor ion and the empirical
formula, observed and calculated mass/charge ratio,
double-bond equivalent and mass error were determined
Scheme 1. Synthesis of methylenedioxy-substituted mono-,
di- and trimethoxychalcones, flavanones and flavones; (I)
Benzaldehyde derivative, alcoholic KOH; (II) TFA, reflux,
2 h; (III) Cat. I2, DMSO, reflux, 45 min.
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Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 1303–1310

ESI-MS of methylenedioxy chalcones, flavanones and flavones
RESULTS AND DISCUSSION
Fragmentation of chalcones and flavanones 5–12
The main product ions observed in the ESI-MS/MS spectra of
the [M+H]+ ion of chalcones and flavanones (5–12), the
most probable identification and the corresponding relative
abundance (RA) are summarized in Table 1. The fragmentation
pathways of the studied chalcones and flavanones show
several common features but also show interesting differences.
The common fragmentation observed for chalcones or
flavanones involves 0,aA+/1,3A+ cleavage giving a product ion
at m/z 165 for all compounds 5–12 and 0,1’B+/1,4B+ product ions
at m/z 161 for 5, 6, 9, and 10; m/z 191 for 7, and 11; and m/z 221
for 8 and 12. These pairs of product ions (0,aA+/1,3A+ and
0,1’B+/1,4B+) indicate that the methylenedioxy substituent is
attached to the A-ring and is constant for all compounds
whereas the B-ring of each compound carries either the
mono-, di- or trimethoxy substituents.
The ESI-MS spectra of the chalcones, flavanones and flavones
5–16 showed the [M+H]+ ions and a minor [M+Na]+ ion. The
fragmentations of the [M+H]+ ions were induced by collision
with a gas under tandem mass spectrometry conditions (ESI-
MS/MS). The MS/MS spectra of the chalcones were similar
to the isomeric flavanones with similar substituents. The
cyclization of 2’-hydroxychalcones to the corresponding
isomeric flavanones has been observed in the gas phase
and justifies their similar behavior.[15] Hence, the discussion
of chalcones and flavanones (5–12) will be presented
together based on the similar fragmentation patterns
observed while those of flavones (13–16) will be discussed
separately.
The ion nomenclature for the product ions for chalcones,
flavanones and flavones has been adapted as proposed in
literature (Scheme 2). The i,jA and i,jB labels are used to
designate product ions containing intact A- and B-rings, and
the subscripts i and j refer to the C-ring bonds that have been
broken.[16] The fragmentations or proposed structures in
chalcones, flavanones and flavones were confirmed by MS3
experiments. The product ions observed in the MSn
experiments and not observed in the MS2 spectra were noted
by their m/z values and are presented in Supplementary
Tables S1 and S2 (see Supporting Information).
The 0,1’B+/1,4B+ product ions are the base peak in the spectra
of compounds 6–8 and 10–12, while, in the case of compounds
5 and 9, this ion was observed in 12% and 16% relative
abundance (RA) (Table 1). It is worth mentioning here that
differences in the position of substituents on the B-ring of
chalcones (or flavanones), or the ortho-position in the cases of
5 and 9, and para-position in the case of 6 and 10, lead to similar
fragmentation but with different RAs, thus allowing the
differentiation of these pairs of isomers. The presence of the
Scheme 2. Ion nomenclature used for the fragmentation of
flavonoids.[18]
Table 1. Identification of the [M+H]+ ions and summary of the main product ions observed in the ESI-MS2 spectra for the
chalcones 5–8 and flavanones 9–12 with the indication of their relative abundances (%RA)a
Compound
5
6
7
8
9
10
11
12
Formula
C17H15O5 C17H15O5 C18H17O6 C19H19O7 C17H15O5 C17H15O5 C18H17O6 C19H19O7
[M+H]+m/z
299.0
299.0
284 (3)
281 (4)
269 (2)
251 (3)
329.1
314 (2)
311 (2)
299 (1)
281 (<1)
287 (1)
-
359.1
299.0
299.0
284 (3)
281 (4)
269 (2)
251 (3)
329.0
314 (1)
311 (2)
299 (1)
281 (<1)
287 (<1)
-
359.1
[P-CH3]+
-
-
-
-
344 (<1)
-
-
-
-
344 (<1)
•
[P-H2O]+
341 (2)
341 (2)
[P-HCHO]+
-
-
-
-
[P-HCHO-H2O]+
[P-C2H2O]+
257.0 (100) 257 (<1)
317 (9)
257.0 (100) 257 (<1)
317 (8)
[P-CO-H2O]+
[P-C2H2O2]+
-
253 (1)
241 (2)
223 (1)
-
-
-
-
-
253 (1)
241 (2)
223 (1)
-
-
-
-
-
271 (<1)
253 (<1)
-
-
271 (<1)
253 (<1)
-
[P-CO-HCHO-H2O]+
-
-
+
+
•
0,1’B+/1,4B - CH3]
-
206 (2)
165 (13)
-
206 (2)
165 (15)
•
[
0,aA+/1,3A+
0,1’B+/1,4B+
[P-B]+
165 (38)
165 (36)
165 (20)
165 (86)
165 (39)
165 (18)
161 (12)
161 (100) 191 (100) 221 (100)
161 (16)
161 (100) 191 (100) 221 (100)
191 (3)
191 (2)
-
-
-
191 (3)
191 (3)
-
-
-
[
[
0,1’B+/1,4B+]-HCHO
-
-
-
191 (28)
190 (4)
193 (4)
151 (3)
-
-
-
191 (34)
190 (4)
193 (4)
151 (4)
0,1’B+/1,4B - OCH3]
-
133 (5)
-
-
163 (3)
-
-
133 (5)
-
-
163 (3)
-
+
+
•
•
0,aB+/1,3B+
-
-
[P-C2H2O-B]+
151 (76)
151 (88)
aP refers to the precursor ion, i.e. the ion undergoing MS2 [M+H]+ (m/z) and B indicates the B-ring of chalcone/flavanone.
Rapid Commun. Mass Spectrom. 2013, 27, 1303–1310
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J. C. J. M. D. S. Menezes et al.
257.1
[P-C H O]
100
•
observed are 15 Da ( CH3), 18 Da (H2O) which are typical of
chalcones and flavanones[17] along with losses of neutral
species with 30 Da (HCHO), 42 Da (C2H2O), 58 Da
(C2H2O2); combined losses of 46 Da (CO+H2O), 48 Da
(HCHO+H2O) and 76 Da (CO+HCHO+H2O). Losses of
15 Da, 18 Da, 30 Da and 48 Da were observed in the case of
chalcones 6, 7, 8; flavanones 10, 11, 12 and were curiously
found to be absent in chalcone 5 and flavanone 9 wherein the
methoxy group is in the ortho-position in the B-ring. Generally
methoxy-substituted chalcones showed product ions derived
by loss of 15 Da and 18 Da and combined losses of 46 Da
(CO+H2O), as observed in the present cases.[17] Loss of 30 Da
was proposed[17] as loss of CO followed by 2H in case of non-
methoxylated chalcones, but can also be considered as loss of
HCHO[19] from the methoxy substituents by addition of H to
the ring. Similarly, combined loss of 46 Da (CO+H2O) was only
seen for chalcone 6 and its isomeric flavanone 10. Loss of 76 Da
(CO+HCHO+H2O) was observed for chalcones 6, 7 and
flavanones 10 and 11. Loss of 58 Da (C2H2O2) is characteristic
for the presence of a methylenedioxy group[19] and was
observed with low RA for chalcones 6, 7 and flavanones 10
and 11 (see Table 1; Fig. 2 for chalcone 6; see Supplementary
Figs. S1, S4 and S5, Supporting Information, for 7, 10 and 11).
The loss of 42 Da (C2H2O) is known to be characteristic for
substituted chalcones and flavanones[17,20] and it was
observed as the base peak at m/z 257 for compounds 5 and
9; at m/z 257 in compounds 6, 10; and at m/z 287 in 7, 11 with
low RA (see Table 1; Scheme 3). This loss in compound 5 and
9 (100%RA) can be rationalized as arising due to the
proximity of the 2-methoxy substituent on the B-ring as
shown in 2-methoxy- or hydroxychalcones.[20] This cleavage
of 42 Da (C2H2O) is also proposed for flavones and
flavonols[16,21] and can also account for the formation of
these ions in the chalcones 5, 8 and flavanones 9, 12. In
compounds 8 and 12 the loss of 42 Da produced a product
ion at m/z 317 with 9% and 8% RA, respectively. This
fragmentation serves as a characteristic for distinguishing
between the ortho- and para-methoxychalcones (5 and 6)
and -flavanones (9 and 10) and also between the
3,4-dimethoxy- and 3,4,5-trimethoxychalcones (7 and 8)
and -flavanones (11 and 12).
5
[P-C H O-B]
151.0
0,
A
165.0
[M+H]
299.1
0,1’B
161.1
0
m/z
160
180
200
220
240
x4
260
280
300
161.1
100
0,1’B+
6
0,
A+
[P- CH3]+
[P-H2O]+
165.0
[P-CO-HCHO-H2O]+
[P-HCHO-H2O]+
[P-C2H2O2]+
251.1
[P-HCHO]+
[M+H]+
299.1
281.1
284.1
269.1
B+
0,
241.1
P-B+
191.0
223.1
133.1
0
100
m/z
120
140
160
180
200
220
240
260
280
300
Figure 2. ESI-MS2 spectra of the [M+H]+ ions at m/z 299 of
positional isomeric chalcones 5 and 6.
electron-donating methoxy substituent at the ortho-position in
chalcone 5 (or flavanone 9) changes the fragmentation pattern
compared to the para-position in 6 (or 10) and serves as a
diagnostic feature for differentiating between the positional
isomers [see Fig. 2 for chalcones 5 and 6; see Supplementary
Figs. S3 and S4 (Supporting Information) for flavanones 9
and 10]. Previous works have shown that substituted
chalcones (o- and p-methoxy-substituted) show different
fragmentation patterns for isomeric chalcones depending
on the position of substituents.[17] Similarly, specific
product ions were formed/observed in the case of o-nitro
substituents in the B-ring of chalcones and were absent in
the m- or p-nitro-substituted chalcones.[18]
The 0,aB+/1,3B+ product ions were also observed for the
studied compounds and were detected at m/z 133 for 6 and 10,
m/z 163 for 7 and 11, and m/z 193 for 8 and 12, while they were
absent in case of 5 and 9. [see Fig. 2 for 5 and 6; Supplementary
Figs. S1, S2, S3, S4, S5 and S6 (Supporting Information) for7, 8, 9,
10 and 11.] These 0,aB+/1,3B+ product ions can also assist as
diagnostic product ions to distinguish between the positional
isomers 5 versus 6 and 9 versus 10 (see Table 1).
MS3 experiments for the ions at m/z 161, 191 and 221
(
0,1’B+/1,4B+), in the case of compounds 6, 10; 7, 11; and 8,
12, gave the product ions at m/z 133, 163 and 193
(
0,aB+/1,3B+), respectively, by loss of CO (–28 Da). The MS3
experiment on the product ion observed at m/z 221 for
compound 8 and 12 gave the product ions observed in its
MS2 spectrum at m/z 190 (- OCH3) and 191 (-HCHO),
•
confirming the fragmentation pathway for these ions along
•
with the product ions at m/z 206 (- CH3) and 189 (CH3OH)
(see Supplementary Table S1, Supporting Information).
Apart from the i,jA+ and i,jB+ product ions chalcones and
flavanones also show losses of small molecules and/or
radicals from the [M+H]+ ion. The most common losses
Scheme 3. Proposed fragmentation pathway observed in the
MS2 spectra of chalcones 5, 8 and flavanones 9, 12.
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Rapid Commun. Mass Spectrom. 2013, 27, 1303–1310

ESI-MS of methylenedioxy chalcones, flavanones and flavones
Table 2. Identification of the [M+H]+ ions and summary of the main product ions observed in the ESI-MS2 spectra for the
flavones 13–16 with the indication of their relative abundances (%RA)a
Compound
13
14
15
16
Formula
C17H13O5
C17H13O5
C18H15O6
C19H17O7
[M+H]+m/z
297.0 (40)
297.0 (24)
327.0 (23)
357.0 (25)
[P-CH3]+
282 (66)
282 (26)
312 (11)
342 (2)
•
+
•
•
[P- CH3- H]
281 (18)
-
311 (100)
341 (25)
[P-HCHO]+
-
-
-
327 (44)
[P-CH3OH]+
265 (19)
-
-
-
+
•
•
[P- CH3- H-H2O]
-
-
-
323 (14)
[P-CO2]+
-
-
283 (28)
313 (11)
+
•
•
[P- CH3-HCHO]
-
-
282 (5)
312 (5)
[P-CO-H2O]+
-
-
-
-
311 (10)
+
•
•
[P- CH3-CO]
254 (13)
254 (100)
-
[P-C2H2O2]+
239 (5)
239 (14)
269 (2)
299 (6)
+
•
•
[P- CH3-H2O-CO]
-
-
266 (13)
296 (100)
[P-HCHO-CH3OH]+
-
-
-
295 (5)
+
•
•
[P- CH3-H2O-2CO]
-
-
-
268 (23)
+
•
•
[P- CH3-H2O-CO- HCO]
-
-
-
267 (28)
+
•
•
[P-2HCHO- OCH3]
-
-
-
266 (5)
+
•
•
[P- CH3-2CO]
-
226 (12)
224 (3)
211 (11)
196 (4)
186 (6)
183 (6)
165 (12)
-
-
-
+
•
•
[P-C2H2O2- CH3]
-
254 (2)
-
[P-C2H2O2-CO]+
-
241 (<1)
-
+
•
•
[P-C2H2O2-CO- CH3]
-
-
-
-
+
•
•
[P- CH3-CO-C3O2]
-
-
[P-C2H2O2-2CO]+
1,3A+
-
213 (<1)
165 (<1)
-
-
165 (2)
-
165 (6)
164 (100)
1,3A+
•
aP refers to the precursor ion, i.e. the ion undergoing MS2 [M+H]+ (m/z).
Fragmentation of flavones 13–16
respectively. This product ion (–16 Da) corresponds to the
base peak of the spectrum of flavone 15 (see Supplementary
Fig. S7, Supporting Information). It is worth mentioning that,
although the loss of 16 Da is observed for all flavones in
this series except for flavone 14, it was specifically observed
as base peak for the 3,4-dimethoxyflavone 15, indicating
that it can serve as diagnostic ion to distinguish it from
the other derivatives in this series. Similar sequential loss
of 16 Da was confirmed by MSn experiments for
methoxychalcones.[17]
Loss of 58 Da (C2H2O2) characteristic for the presence of a
methylenedioxy group[19] leads to the formation of the product
ion at m/z 239 for compound 13, 14; m/z 269 for 15; and at m/z
299 for 16(see Supplementary Fig. S8 Suporting Information).
Comparatively this product ion was observed with higher %
RA in the case of flavone 14 (see Table 2).
The main product ions [M+H]+ observed in the ESI-MS/MS
spectra of flavones (13–16) and the corresponding relative
abundance (RA) are summarized in Table 2. The common
fragmentation pathways for all these compounds will be
discussed followed by the interesting differences which
help in differentiating between the two monomethoxy
(13 and 14; see Fig. 3) and also the dimethoxy- and
trimethoxyflavones (15 and 16; see Supplementary Figs. S7
and S8, Supporting Information). The characteristic retro-
Diels-Alder (RDA) fragmentation for flavones or the 1,3A+
product ion at m/z 165 was observed for all flavones 13, 14,
15 and 16. In case of compound 13 the same RDA product
•
ion losses the H radical to give a radical ion as base peak at
m/z 164 (Fig. 3). Here the ketene type intermediate is
proposed since, under MS3 experiment, loss of 28 Da was
observed (see Supplementary Table S2 for MS3 product ions
in Supporting Information).[16] This product ion serves as a
diagnostic for distinguishing the o-methoxychalcone 13 from
its p-methoxy isomer and the di- and trimethoxy derivatives
in which this ion is absent.
The loss of small molecules and/or radicals from the
[M+H]+ ion was also observed. Loss of 44 Da (CO2)
was seen in flavones 15 and 16 to give product ions at
m/z 283 and 313, and this loss has been shown to occur by
the contraction of the C-ring of flavonoids using ESI-MS2 in
the negative mode.[21] Loss of 46 Da (CO+H2O) gave a product
ion at m/z 311 in flavone 16. Specific loss of 32 Da (CH3OH)
which proves the presence of a methoxy group was observed
only in the case of flavone 13 to give a product ion at m/z 265.
•
The most common loss of 15 Da ( CH3) was observed for all
flavones 13, 14, 15 and 16. This loss is easily detected and
proves the presence of the methoxy groups in flavonoids.[16]
Loss of 16 Da seen for flavones 13, 15 and 16 is considered
•
Combined loss of 43 Da (CO+ CH3) giving an ion at m/z 254
•
•
as a combined loss of CH3 and H. This fact was proved
by MS3 experiments which showed that ion at m/z 282 in 13,
m/z 312 in 15 and m/z 342 in 16 (all due to loss of 15 Da or
was seen for flavone 13 and forms the base peak for
14; thus, this product ion is able to differentiate between the
p-methoxyflavone 14 from its o-methoxy isomer 13 which has
•
•
CH3) leads to the product ions at m/z 281, 311 and 341,
a base peak arising from the RDA reaction and loss of H as
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Copyright © 2013 John Wiley & Sons, Ltd.
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J. C. J. M. D. S. Menezes et al.
MS/MS.[16] A further MS3 experiment on m/z 254 in the case
of 14 gave rise to m/z 226 (100% RA) which was also seen in
the MS2 spectrum in 12% RA and was deduced to be due
to further loss of CO. A product ion at m/z 186 observed
in the MS2 spectrum was also seen as second most
abundant ion in MS3 and as the base peak in the MS4 on
the ion of m/z 254 (see Supplementary Table S2, Supporting
Information). This loss was deduced to have arisen from
•
combined loss of 43 Da (CO+ CH3) followed by 68 Da
(C3O2) which has been observed in flavonoids and has been
ascribed to arise from the A-ring containing a 1,3-dihydroxy
group.[21] In the present case this loss of 68 Da (C3O2) can
be proposed as arising from the methylenedioxy group on
the A-ring. Some of the above proposed fragmentation
mechanisms in the MS2 and MS3 of flavone 14 are
represented in Scheme 4.
•
Loss of 61 Da ( CH3+H2O+CO) gives ions at m/z 266 for 15
and forms the base peak at m/z 296 for flavone 16 (see
Supplementary Fig. S8, Supporting Information). The
stepwise losses of these product ions were derived by MS3
experiments on the product ion at m/z 312 in 15 and m/z 342
in 16 which explained their formation and confirmed the
pathway by comparing their %RA in the MS2 as well as
MS3 spectra (see Table 2 and Supplementary Table S2,
Supporting Information). This stepwise loss is in complete
agreement with the fragmentation of flavones having higher
or lower degree of methoxylation which starts with loss of
the methyl radical, followed by H2O and a carbonyl
Figure 3. ESI-MS2 of the [M+H]+ ions at m/z 297 of positional
isomer flavones 13 and 14.
group.[22] Combined loss of CH3 and HCHO (45 Da) gives
•
discussed earlier (see Fig. 3). An MS3 experiment on the ion at
ions at m/z 282 for 15 and m/z 312 for 16. Characteristic loss
of a formaldehyde group was observed only in the case of
flavone 16 to give an ion at m/z 327; although unusual,
it has been observed for methoxy- and methylenedioxy-
substituted lignans.[19]
•
m/z 282 (- CH3) in 13 and 14 gave rise to m/z 254 due to
•
combined loss of CH3 followed by CO. This type of combined
loss has been specifically observed in flavones having the
methoxy group at the p-position on the B-ring using FAB CID
Scheme 4. Proposed fragmentations pathways observed in the MS2 and MS3 of
flavone 14.
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Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 1303–1310

ESI-MS of methylenedioxy chalcones, flavanones and flavones
Product ions at m/z 266 and 267 observed only in flavone 16
Acknowledgements
can be explained by combined loss of 91 Da (2 HCHO and
We gratefully acknowledge University of Aveiro and Goa
University for providing necessary laboratory facilities. Thanks
are due to Fundação para a Ciência e a Tecnologia (FCT,
Portugal), European Union, QREN, FEDER and COMPETE
for funding the QOPNA research unit (project PEst-C/QUI/
UI0062/2011); the Portuguese National NMR Network and
Portuguese Mass Spectrometry Network (RNEM) (REDE/
1504/REM/2005) also supported by funds from FCT. JCJMDS
Menezes thanks QOPNA for a research grant.
•
•
OCH3) and 90 Da (combined loss of CH3+H2O+CO
•
+HCO ). The fragmentation pathway for 90 Da was
confirmed as that proposed by carrying out MS3 experiments.
It was observed that the product ion at m/z 267 was derived
•
from product ions at m/z 296 (loss of CH3+H2O+CO)
indicating the stepwise formation. Proposed fragmentations
in the MS2 and MS3 of flavone 16 are represented in
Supplementary Scheme S1 (see Supporting Information).
The most probable elemental compositions of the product
ions were obtained with a high degree of confidence (see
Supplementary Table S3, Supporting Information). The errors
between the observed masses and calculated values are
around –35.7 to 17.6 mDa (–176.5 to 69.2 ppm), indicating
very good mass accuracy and confirming the compositions
of the observed product ions.
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The electrospray ionization tandem mass spectrometry of
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flavanones and flavones show several interesting features.
The ESI tandem mass spectra of all the studied 2’-
hydroxychalcones were identical to the corresponding
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most common fragmentation of chalcones or flavanones
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common ions for all studied compounds. The common losses
•
observed in chalcones and flavanones are 15 Da ( CH3),
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•
•
•
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•
base peak due to RDA cleavage followed by loss of a H
•
radical in the former while sequential loss of CH3
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of methoxy and formaldehyde groups, respectively. This
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SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
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Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 1303–1310