On the difference in decomposition of taxifolin and luteolin vs. fisetin and quercetin in aqueous media

Article abstract of DOI:10.1007/s00706-016-1737-3

Abstract: The decomposition of flavonols quercetin and fisetin, flavone luteolin and flavanone taxifolin was studied in slightly alkaline solution under ambient conditions. The study was based on spectrophotometry and high-pressure liquid chromatography. Products formed by atmospheric oxygen oxidation and hydrolysis were identified by HPLC–DAD and HPLC–ESI-MS/MS. Only small differences in the chemical structure of flavonoids resulted in extremely variable oxidation pathways and products. Oxidation of flavonols led to the formation of both a benzofuranone derivative and several open structures. On the contrary, the benzofuranone derivative was not found as a product of taxifolin and luteolin oxidative decomposition. These compounds were oxidized to their hydroxylated derivatives and typical open structures. Quercetin was not identified as a possible oxidation product of taxifolin. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-016-1737-3

Monatsh Chem
DOI 10.1007/s00706-016-1737-3
ORIGINAL PAPER
On the difference in decomposition of taxifolin and luteolin
vs. fisetin and quercetin in aqueous media
1
1
1
1
•
ˇ
´ ˇ ´ ´ ´ ˇ
Sarka Ramesova Jana Kocabova Viliam Kolivoska
•
•
•
´
Romana Sokolova
•
Ilaria Degano2 Emanuela Pitzalis3
Received: 31 January 2016 / Accepted: 9 March 2016
Ó Springer-Verlag Wien 2016
Abstract The decomposition of flavonols quercetin and
fisetin, flavone luteolin and flavanone taxifolin was studied in
slightly alkaline solution under ambient conditions. The study
was based on spectrophotometry and high-pressure liquid
chromatography. Products formed by atmospheric oxygen
oxidation and hydrolysis were identified by HPLC–DAD and
HPLC–ESI-MS/MS. Only small differences in the chemical
structure of flavonoids resulted in extremely variable oxidation
pathways and products. Oxidation of flavonols led to the for-
mation of both a benzofuranone derivative and several open
structures. On the contrary, the benzofuranone derivative was
not found as a product of taxifolin and luteolin oxidative
decomposition. These compounds were oxidized to their
hydroxylated derivatives and typical open structures. Quercetin
was not identified as a possible oxidation product of taxifolin.
Graphical Abstract
Keywords Oxidation mechanism Á Fisetin Á Luteolin Á
Taxifolin Á Quercetin
Introduction
The redox properties of bioactive compounds and drugs
can give information about their role in metabolic pro-
cesses or their pharmacological activity [1–3]. The
bioactivity is also connected to the iron chelation ability
and the affinity of catechols to compete with iron binding
proteins [4, 5]. Electrochemical methods are frequently
employed as a part of biomimetic modelling of reductive or
oxidative drug metabolism [6, 7].
Flavonoid compounds are widely distributed in nature, in
fruits, vegetables, and leaves [8, 9]. Presently, these com-
pounds are extensively studied in aqueous solution because
of their pharmacological importance. They have antioxida-
tive, anticarcinogenic, anti-inflammatory, and antiviral
properties [10–12]. The chemical class of flavonoids
includes flavonols, flavones, isoflavonoids, anthocyanins,
flavanones, etc. [13]. Taxifolin (1, 3,5,7,30,40-pentahydroxy-
flavanone; Fig. 1), which occurs in citrus fruits, belongs to
the family of flavanones [13]. It is characterized by a rela-
tively strong antioxidant activity due to the substitution with
two OH groups at the position 30 and 40 of the B-ring. Its
molecule does not contain double bond between carbon
atoms C2 and C3, and thus its antioxidant activity is half of
that of quercetin (2, 2,3,30,40,5,7-pentahydroxyflavone;
Fig. 1) [14]. Despite that, its protective effect against cere-
bral ischemic reperfusion injury and leukocyte activation
was reported [15, 16]. Luteolin (3, 30,40,5,7-tetrahydroxy-
flavone; Fig. 1) is widely present in plants, vegetables as
artichoke, basil, celery, and parsley [8]. It is a flavone, whose
´
& Romana Sokolova
sokolova@jh-inst.cas.cz
1
´
J. Heyrovsky Institute of Physical Chemistry of ASCR, v.v.i.,
Dolejskova 3, 18223 Prague, Czech Republic
ˇ
2
3
Department of Chemistry and Industrial Chemistry,
University of Pisa, Via Moruzzi 13, 56124 Pisa, Italy
Institute of Chemistry of Organometallic Compounds, CNR,
Via G. Moruzzi, 1, 56124 Pisa, Italy
123

´
R. Sokolova et al.
OH
OH
OH
OH
4'
confirmed as the only oxidation product of quercetin
formed under inert atmosphere in non-aqueous solution and
in the cavity of b-cyclodextrin [24, 25]. This benzofura-
none derivative was isolated as the oxidation product of
quercetin and identified it by 1H NMR and 13C NMR [26],
it was also generated from quercetin by the peroxyl radical
generator 2,20-azobisisobutyronitrile [27]. Consistently,
2,4-dihydroxy-2-(4-hydroxy-3-methoxybenzoyl)-6-methoxy-
benzofuran-3(2H)-one was identified as the only oxidation
product of the methoxylated flavonol rhamnazin under inert
atmosphere [23]. Finally, the corresponding benzofuranone
derivative was confirmed after the oxidation of fisetin (4)
both in solution exposed to the air and degassed with argon
[22].
B
3'
HO
O
HO
O
O
2
7
A
5
OH
C
4
3
OH
OH
O
OH
1
2
OH
OH
OH
OH
HO
O
O
HO
O
O
OH
OH
3
4
Interestingly, the electrochemical oxidation of taxifolin
[28] and luteolin [29] under inert atmosphere of argon did
not show the formation of a benzofuranone derivative as it
was in the case of quercetin and fisetin [21, 22, 30]. In the
case of taxifolin, the redox properties are affected by its
specific structure, which does not allow the electron delo-
calization through the whole structure [28]. Although this
effect of delocalization of electrons is allowed in the case
of luteolin, the absence of formation of the benzofuranone
derivative of luteolin suggests that the highly reactive C3–
OH hydroxyl group has a significant contribution to the
whole oxidative process [31, 32]. The electrochemical
oxidation of both, luteolin and taxifolin, yielded to the
formation of related quinone derivatives and its subsequent
hydroxylation to its hydroxyl derivatives [28, 29].
Fig. 1 Chemical structure of taxifolin (1), quercetin (2), luteolin (3),
and fisetin (4)
chemical structure differs from quercetin by the absence of
C3–OH hydroxyl group. It exhibits antioxidant, anti-in-
flammatory, and anti-allergic activity [17, 18]. Fisetin (4,
2-(3,4-dihydroxyphenyl)-3,7-dihydroxychromen-4-one;
Fig. 1), another bioactive flavonoid present in nature, has
promising antioxidant, anticarcinogenic, and antibacterial
properties [11, 19].
The oxidation mechanism of the flavonol structure was
extensively studied in literature for quercetin (2). The large
variety of oxidation products of quercetin described in the
literature by several authors is related to the instability of
the primary formed oxidation products [20]. The oxidation
of flavonols in buffered solutions under inert atmosphere of
argon showed that both the oxidation potential and the
number of electrons and protons involved in the oxidation
strongly depend on the relative amount of dissociation
forms of flavonol in solution [21–23]. In the case of
quercetin, subsequent hydroxylation and the formation of
2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3-
(2H)-one take place (Scheme 1). This product was also
Under ambient conditions, flavonols quercetin and
fisetin were found to decompose fast [21, 22]. This phe-
nomenon made difficulties in the analytical determination
of oxidation products formed by oxidative electrolysis
under inert atmosphere. The purpose of this work is to
investigate the mechanism of oxidative decomposition of
flavanone taxifolin and flavone luteolin in aqueous solution
under ambient conditions. Their behaviour will be
Scheme 1
OH
O
OH
OH
OH
HO
O
O
HO
O
O
-
- 2e - 2H+
HO
O
O
,
H
2O
OH
OH
O
O
OH
R
R
R
2
,
4
,
R = OH
R = H
OH
OH
O
HO
O
HO
OH O
O
OH
O
OH
O
R
R
OH
123

On the difference in decomposition of taxifolin and luteolin vs. fisetin and quercetin in…
compared with the decomposition of flavonols quercetin
and fisetin.
verified that none of the oxidation products of 1 corre-
sponded to compound 2 (compare Fig. 3b, c to d).
Oxidation products of compounds taxifolin and quer-
cetin are summarized in Table 1. They were identified by
comparison of their absorption spectra obtained from
HPLC–DAD and confirmed by means of HPLC–ESI-MS/
MS analysis. The oxidation products of quercetin decom-
position were additionally identified by GC–MS analysis
after derivatization with silylating agent. Table 1 includes
the m/z values obtained for oxidation products of all
studied compounds. Scheme 2 shows chemical structures
of taxifolin oxidation products.
Result and discussion
Flavanone taxifolin
The oxidative decomposition of taxifolin (1) was first
studied by means of UV–Vis spectrophotometry. Figure 2a
shows the changes of the absorption spectra of 1 depending
on the time of exposure to the air oxygen. The absorption
spectrum of 1 under argon atmosphere is characterized by
bands with absorption maxima at 213, 244, and 321 nm.
During exposure to atmospheric oxygen the intensity of
absorption bands at 244 and 321 nm decrease. At the same
time the absorption bands at 286 and 400 nm increase. In
20 min the absorbance at 400 nm reaches the limiting
value. It is obvious that a significant change of the
absorption spectrum of 1 occurs at much longer times than
in the case of luteolin (2) (Fig. 2b).
Open structures
The formation of so-called ‘open structures’ was expected
(the compound 2-(3,4-dihydroxybenzyloxy)-4,6-dihydrox-
ybenzoic acid is here called ‘open structure—acid’ for
clarity reasons and applies to all the analogous products
formed for all the studied compounds). Such products were
found for 2 by Osman et al. [34]. Other ‘open structures’
were identified as the oxidation products of flavonols fisetin
(4) [22] (compound 2-(3,4-dihydroxybenzoyloxy)-4-hy-
droxybenzaldehyde is here called ‘open structure—
aldehyde’) and rhamnazin [23] (compound 2-hydroxy-6-
(4-hydroxy-3-methoxybenzoyloxy)-4-methoxyphenyl-2-
oxoacetic acid is here called ‘open structure—oxoacetic
acid’). Figure 4 shows the chemical structures of all open
structures types found by oxidation of flavonols, flavones,
or flavanones in our studies. These compounds are not
stable, and readily undergo hydrolysis and low molecular
weight hydroxyl compounds are formed (see Table 1).
The solution of taxifolin prepared under inert atmosphere
of argon was injected in the HPLC system. The chro-
matogram is shown in Fig. 3a. Afterwards, the vial was
opened and the solution was exposed to the air and shaken.
Chromatograms registered at different times of exposure to
air oxygen are reported in Fig. 3b and c. Several peaks
labelled as T1–T9 due to the oxidation products of 1
occurred in the chromatograms. To compare the oxidation
products with the already known oxidation products of
quercetin (2) [21, 33], the chromatogram of quercetin stan-
dard 2 was measured under the same conditions. It was
Fig. 2 a Shows the absorption spectra of 2.4 9 10-4 M taxifolin in
3.6 9 10-3 M KOH during the exposure to the air oxygen for a 0,
b 600 s, c 900 s, d 1200 s, e 19 h, f 40 h, g 7 days. b Shows the
absorption spectra of 3.5 9 10-5 M luteolin in 3.6 9 10-3 M KOH
during exposure to atmospheric oxygen for a 0, b 740 s, c 1240 s,
d 1920 s, e 2700 s, f 24 h
123

´
R. Sokolova et al.
aldehyde) and 2-(3,4-dihydroxybenzoyloxy)-4-hydroxy-
benzoic acid (F3, open structure—acid) as shown in Fig. 5
and Scheme 4. The oxidation product F3 elutes at very close
retention time to benzofuranone derivative F2. The area of
peak F3 in the chromatograms increased with time when the
solution was exposed to oxygen (Fig. 5b). Other peaks
belonging to low molecular weight hydroxyl compounds
were identified, as summarized in Table 1. Amongst the
investigated flavonols, only in the oxidized solutions of
fisetin a benzofuranone derivative could be distinguished
from the ‘open structure—acid’.
Conclusion
The present complex study was focused on the relation-
ship between the oxidation properties of several flavonoid
compounds in air, and their chemical structure. The dif-
ference in their chemical structures is often very small, as
the absence of one C3–OH hydroxy group or the absence
of C2–C3 double bond; but a significant influence on
electron transfer properties was assessed. Our recent
works showed that flavonol compounds, containing both
C2–C3 double bond and C3–OH hydroxy group in their
chemical structure formed a benzofuranone derivative
during their oxidation [21, 22]. In this paper, we prove
that those compounds which do not fulfil both these
conditions do not yield a benzofuranone derivative during
their oxidation, even in the presence of air oxygen. This
feature was assessed for flavanone taxifolin (the absence
of double bond in comparison to flavonol quercetin) and
flavone luteolin (the absence of C3–OH hydroxy group).
For these compounds, two typical oxidation pathways
were determined. The first pathway led through a quinone
intermediate to a hydroxylated compound formed by
nucleophilic addition of water. The second process was
the formation of open structures and their subsequent
decomposition to low molecular weight hydroxy com-
pounds. Similar open structures were identified also in the
case of flavonols quercetin and fisetin as side oxidation
products.
Fig. 3 HPLC chromatogram of taxifolin in 0.1 M KCl and
5.4 9 10-3 M KOH recorded after exposure to atmospheric oxygen
for different times: a 0 min, b 60 min, c 28 h. d Shows chromatogram
of solution of 2 a before and b after oxidation by oxygen. Detector set
at 280 nm
Flavone luteolin
The absorption spectrum of luteolin (3) in 3.6 9 10-3
M
KOH is characterized by four bands with absorption
maxima at 212, 266, 324, and 397 nm (Fig. 2b). The
absorption bands at 266 and 397 nm decrease during the
exposure of solution to oxygen. A new absorption band at
480 nm occurs, which is due to the formation of hydrox-
ylated luteolin [29, 35]. Oxidation products found after the
oxidation of flavone luteolin are summarized in Table 1
and Scheme 3.
Experimental
Flavonol fisetin
Fisetin, quercetin, taxifolin, and luteolin were purchased
from Sigma Aldrich. Chemicals used for preparation of
Britton–Robinson buffers of pH range 4.4–11.2
(0.04 mol dm-3 stock solutions of H3PO4, CH3COOH,
H3BO3 and 0.2 mol dm-3 NaOH) were of reagent grade
(p. a.). Potassium chloride and KOH (reagent grade p. a.)
Oxidation of fisetin (4) under ambient conditions leads to the
formation of 2-(3,4-dihydroxybenzoyl)-2,6-dihydroxyben-
zofuran-3(2H)-one (F2). This benzofuranone structure was
found to be typical oxidation product of flavonols [21–23,
30]. Other oxidation pathways yield 2-(3,4-dihydroxyben-
zoyloxy)-4-hydroxybenzaldehyde (F1, open structure—
123

On the difference in decomposition of taxifolin and luteolin vs. fisetin and quercetin in…
Table 1 Values of m/z of obtained oxidation products of compounds 1–4 from HPLC–ESI-MS/MS
Taxifolin
153, 109
Quercetin
Luteolin
153, 109
Fisetin
3,4-DHBA
153, 109
153, 109
[370, 355, 193
(3-TMSderivative)]
3,4-Dihydroxybenzaldehyde
2,4-DHBA
137
153, 137, 109
181, 153, 109
3,4-Dihydroxyphenyl oxoacetic acid
181, 153, 109
[383, 281 (3-TMS derivative)]
2,4-Dihydroxyphenyl oxoacetic acid
4-Hydroxyphenyl oxoacetic acid
4-Hydroxybenzaldehyde
2,4,6-THBA
181, 165, 153, 137, 109
165
121
169, 151, 125
169, 151, 125
[443, 353 (4-TMS derivative)]
2,5,7-Trihydroxy-4H-1-benzopyran-4-one
2,4,6-Trihydroxy-benzaldehyde
193 [25]
153, 125, 56
2,4,6-Trihydroxyphenyl oxoacetic acid
197, 169, 151, 125 197, 169, 151, 125
[471, 443, 369, 73 (4-TMS
derivative)]
Open structure—acid
305, 167
305
305, 167
[666, 651, 355 (5-TMS)]
317, 289, 207, 151
[678, 369, 281 (5-TMS derivative)]
Benzofuranone derivative
301, 273, 191, 153, 135
Open structure—aldehyde
20-Hydroxytaxifolin
Myricetin
289
273, 243, 163, 135, 109
319, 181, 153, 125 N/A
N/A
N/A
N/A
N/A
317, 301
N/A
N/A
N/A
6-Hydroxyluteolin
Open structure—oxoacetic acid
Quinone
N/A
301, 167, 125
333, 305, 261, 151
301
Quinoid structure
N/A
283, 209, 187,
171, 136, 125
Quinone open structure—
oxobenzaldehyde
315
Open structure—oxobenzaldehyde
317, 269, 167, 166
3-(3,4-Dihydroxyphenyl)-2-hydroxy-1-
(2,4,6-trihydroxyphenyl)prop-2-en-1-
one
303
[721, 647, 575 (6-TMS derivative)]
Dimer
601, 302
569, 417, 285, 151
Values obtained (for derivatized products) of 2 by GC–MS are in brackets
N/A not applicable
Stability of compounds in solution: UV–Vis
measurements
were used as 0.1 M and 0.36 mM solutions, respectively.
The solutions were prepared with ultrapure water (Milli-
pore). Methanol and acetonitrile were HPLC grade (Carlo
Erba, Milan, Italy). 3,4,5-Trihydroxybenzoic acid (more
than 99 % purity) was from Alpha Aesar (Lancaster,
England); 3,4-dihydroxybenzoic acid ([97 % purity) and
2,4-dihydroxybenzoic acid (97 % purity) were from Sigma
Aldrich (Milan, Italy). Standard solutions of all flavonoids
and low molecular hydroxybenzoic acids for HPLC were
prepared in methanol. All reagents and chemicals were
used without any further purification.
The 1.0-cm quartz cuvettes were used for recording the
absorption spectra during testing the stability of com-
pounds when exposed to the air oxygen. Spectral changes
were registered using Agilent 8453 diode array UV–Vis
spectrometer. The procedure of measurement of taxifolin
stability in solution was as follows: the first absorption
spectrum of solution prepared under inert atmosphere was
recorded in a closed cuvette; then, absorption spectra were
123

´
R. Sokolova et al.
Scheme 2
OH
OH
O
O
OH
OH
HO
O
O
HO
O
HO
O
O
H
2O
-2e- -2H+
/
or
O2
,
OH
OH
OH
OH
OH
OH
O
OH
1
Quinone
2'-Hydroxytaxifolin
Other oxidation decomposition products:
O
O2
O
OH
O
OH
OH
HO
OH
HO
HO
HO
OH
O
O
OH
OH
HO
OH
HO
O
O
OH
2-(2',4',6'-Trihydroxy-
phenyl)-2-oxoacetic acid
3,4-Dihydroxy-
benzoic acid
2,4,6-Trihydroxy-
benzoic acid
OH
OH
Open structure - acid
O
OH
OH
OH
H
HO
O
O
OH
OH
3,4-Dihydroxy-
benzaldehyde
OH
Myricetin
measured after opening the cuvette and manually shaking
of solution in the presence of air. Oxygen was removed
from the solution by passing a stream of argon.
OH
OH
OH
OH
O
O
O
High-pressure liquid chromatography
with photodiode array detector (HPLC–DAD)
HO
HO
O
O
OH
H
A high-pressure liquid chromatography Agilent 1200 Ser-
ies HPLC Systems equipped with diode array detector
(Agilent Technologies) was used. The chromatographic
separation was performed on analytical reverse phase C-8
column (HyPurity C8, 150 9 3 mm, 5 lm, Thermo Sci-
entific, Dubuque, USA) connected to C-18 pre-column
(HyPurity C18, 10 9 3 mm, 5 lm, Thermo Scientific,
Dubuque, USA). Column temperature: 20 °C. The gradient
elution programme used eluents (A): aqueous solution of
0.1 % H3PO4 and (B): acetonitrile. The gradient used for
solutions of fisetin and luteolin was: 0–2 min, 95 % A;
2–30 min, linear gradient to 40 % A; 30–35 min, linear
gradient to 30 % A; 35–60 min, linear gradient to 5 % A
and 95 % B (setup 1). The gradient used for solutions of
taxifolin and quercetin was: 0–2 min, 95 % A; 2–40 min,
linear gradient to 40 % A; 40–45 min, linear gradient to
30 % A; 45–70 min, linear gradient to 5 % A and 95 % B
R
O
R
Open structure - acid
Open structure - aldehyde
OH
OH
OH
OH
O
O
HO
O
O
HO
O
O
O
OH
H
R
O
R
Open structure - oxoacetic acid
Open structure - oxoacetaldehyde
Fig. 4 Chemical structures of ‘open structures’ resulting from the
flavonols and flavones oxidation by oxygen
123

On the difference in decomposition of taxifolin and luteolin vs. fisetin and quercetin in…
Scheme 3
OH
OH
OH
OH
OH
OH
O
O
O
HO
O
O
HO
O
O
HO
O
O
O
OH
H
H
OH
OH
OH
Open structure -
oxobenzaldehyde
Open structure - acid
Open structure - aldehyde
OH
OH
O
HO
HO
O
HO
O
O
OH
O
OH
O
Quinone
6-Hydroxyluteolin
HO
HO
HO
O
O
HO
OH
O
OH
H
OH
O
OH
OH
2,5,7-Trihydroxy-4H-1-
benzopyran-4-one
2,4,6-Trihydroxy-
benzaldehyde
3,4-Dihydroxybenzoic acid
(setup 2). The flow rate was 0.2 cm3 min-1, the injection
volume was 40 mm3. Diode array detector acquisition
parameters were: acquisition range 190–800 nm, 2 nm
step.
10 mm3. The programme was: 90 % A and 10 % B for
2 min; then to 50 % A and 50 % B in 9 min; then linear
gradient to 30 % A to 70 % B in 3 min; then to 10 % A to
90 % B in 4 min, hold for 4 min. Conditioning takes
3 min.
High-pressure liquid chromatography with electro-
spray ionization tandem mass spectrometer (HPLC–
ESI-MS/MS)
The ESI operating conditions were: drying gas (N2,
purity [98 %) 350 °C and 10 dm3 min-1; capillary volt-
age 4.5 kV; nebulizer gas 35 psig; sheath gas (N2, purity
[98 %) 375 °C and 11 dm3 min-1. The collision gas was
nitrogen (purity 99.999 %). The fragmentor potential was
175 V and the collision energy for the MS/MS experiments
ranged between 20 and 50 V depending on the target
compound.
HPLC–ESI-MS/MS was carried out using a 1200 Infinity
HPLC (Agilent Technologies, USA), coupled to a Jet
Stream ESI interface (Agilent Technologies) with a
Quadrupole-Time of Flight tandem mass spectrometer
6530 Infinity Q-TOF (Agilent Technologies). The chro-
matographic separation took place at 30 °C and was
performed on an analytical reverse phase C-18 column
(C18-extended 1.8 lm, 50 9 2.1 mm, Agilent Technolo-
gies, USA) connected to a C-18 pre-column (TC-C18 (2)
5 lm, 12.5 9 2.1 mm, Agilent Technologies, USA).
The eluents were: (A): 90 % H2O with 0.1 % formic
acid, and (B): 10 % acetonitrile with 0.1 % formic acid,
and the flow rate was 0.2 cm3 min-1. Injection volume was
High-resolution MS and MS/MS spectra were achieved
in negative mode in the range 100–3200 m/z; the mass axis
was calibrated using the Agilent tuning mix HP0321
(Agilent Technologies) prepared in acetonitrile.
Gas chromatography
The products of quercetin oxidation were identified using a
Trace GC gas chromatograph (Thermo Electron
123

´
R. Sokolova et al.
Fig. 5 HPLC chromatogram of fisetin in 0.1 M KCl and 3.6 9 10-3 M KOH: a prepared freshly under ambient conditions, b recorded in
60 min. Detector 280 nm. As insets are shown absorption spectra of oxidation products F1–F3 after subtraction of column baseline
Scheme 4
OH
OH
4'
O
O
B
HO
O
O
3'
OH
OH
HO
O
2
HO
O
O
-2e- -2H+
,
/
O2
H
2O
or
OH
7
A
C
3
OH
OH
O
5
F2
O
4
Quinoid structure
O2
O2
OH
O
HO
OH
OH
OH
OH
OH
OH
O
O
O
O
HO
O
HO
O
O
H
OH
O
F1
, open structure - aldehyde
F3
, open structure - acid
Corporation, USA) equipped with a PTV injection port and
a mass spectrometric detector based on an ion trap analyzer
(Polaris Q, Thermo Electron Corporation, USA). The PTV
injector was in the CT ‘splitless with surge’ mode at
280 °C with a surge pressure of 100 kPa, and the mass
spectrometer parameters were: ion source temperature
123

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21. Sokolova R, Ramesova S, Degano I, Hromadova M, Gal M,
Zabka J (2012) Chem Commun 48:3433
22. Ramesova S, Sokolova R, Degano I (2015) Electrochim Acta
182:544
23. Ramesova S, Degano I, Sokolova R (2014) Electrochim Acta
133:359
24. Ramesova S, Sokolova R (2014) Chem Listy 108:507
25. Ramesova S, Sokolova R, Degano I, Hromadova M, Gal M,
Kolivoska V, Colombini MP (2011) Collect Czech Chem C
76:1651
230 °C, electronic impact ionization (70 eV), scan range
m/z 50–700 and interface temperature 280 °C. Chromato-
graphic separation was performed on
a DB-5MS
chemically bonded fused silica capillary column (J & W
Scientific, Agilent Technologies) with stationary phase
5 % phenyl–95 % methylpolysiloxane, and of dimensions
0.25 mm ID, 0.1 lm film thickness, 25 and 30 m length.
The gas chromatographic conditions were as follows: ini-
tial temperature 57 °C, 2 min isothermal, then ramped at
10 °C min-1 up to 200 °C, 3 min isothermal, then ramped
at 20 °C min-1 up to 300 °C and then isothermal for
20 min. The carrier gas was He (purity 99.9995 %), at a
constant flow rate of 1.2 cm3 min-1. The peak assignment
was based on comparison with analytical reference com-
pounds and materials, with library mass spectra (NIST 1.7)
and with mass spectra reported in the literature.
To perform GC–MS analysis, oxidation products were
derivatised with a silylating agent N,O-bis(trimethylsi-
lyl)trifluoroacetamide (BSTFA). Derivatisation conditions
were: 10 mm3 of 2,4-dihydroxybenophenone (solution in
isopropanol; internal standard IS1) was added to the sam-
ple; the solution was dried and 30 mm3 of the
derivatisation agent BSTFA in 50 mm3 of AcOEt was
added; the reaction took place 30 min at 60 °C in closed
glass vials. Just before injection, 10 mm3 of hexadecane
(solution in isooctane; internal standard IS2) and 150 mm3
of AcOEt were added; 2 mm3 of the final solution was
injected in the GC system.
Theoretical calculations
26. Jorgensen LV, Cornett C, Justesen U, Skibsted LH, Dragsted LO
(1998) Free Radic Res 29:339
27. Krishnamachari V, Levine LH, Pare PW (2002) J Agric Food
Chem 50:4357
28. Kocabova J, Fiedler J, Degano I, Sokolova R (2016) Electrochim
Acta 187:358
29. Ramesova S, Sokolova R, Tarabek J, Degano I (2013) Elec-
trochim Acta 110:646
Quantum chemical calculations of organic compounds to
obtain their UV–Vis spectrum were performed using the
density functional theory (DFT) calculations with EDF2/6-
31G* density functional model (Spartan’14, v.1.1.8). Their
structures were optimized to the minimum of geometry.
Calculations were done for molecules in vacuum.
30. Sokolova R, Degano I, Ramesova S, Bulickova J, Hromadova M,
Gal M, Fiedler J, Valasek M (2011) Electrochim Acta 56:7421
31. Pyszkova M, Biler M, Biedermann D, Valentova K, Kuzma M,
Vrba J, Ulrichova J, Sokolova R, Mojovic M, Popovic-Bijelic A,
Kubala M, Trouillas P, Kren V, Vacek J (2016) Free Radic Biol
Med 90:114
Acknowledgments This work is supported by the Grant Agency of
the Czech Republic (14-05180S).
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