Chromato–Mass Spectrometric Identification of Unusual Products of 4-Isopropylphenol Oxidation in Aqueous Solutions

Article abstract of DOI:10.1134/S1070363218010024

4-Isopropylphenol has been chosen as the simplest object to model the processes of oxidation of organic compounds with air oxygen in aqueous media, since it contains a hydrogen atom at the tertiary carbon atom in the α-position with benzene ring and a hydroxyl group enabling mass-spectrometric detection of the products in the negative ions mode. It has been stated that oxidation of 4-isopropylphenol with air oxygen in aqueous media becomes noticeable as the solution pH approaches the рK_а value of the substrate (10.25). The major product [4-isopropyl-2-(4-isopropylphenoxy)phenol] is formed via nucleophilic addition of the starting 4-isopropylphenol at the intermediate product of its oxidation, quinone methide. Intensity of electrochemical oxidation can be tubed by changing the electrode potential. The highest conversion of 4-isopropylphenol has been observed at potential 1.5–3.0 V, the formed compounds being the products of transformation of the same quinone methide intermediate. The obtained data have explained the formation and diversity of dimeric and oligomeric products of oxidation of natural flavonoids.

Full text of DOI:10.1134/S1070363218010024

ISSN 1070-3632, Russian Journal of General Chemistry, 2018, Vol. 88, No. 1, pp. 7–14. © Pleiades Publishing, Ltd., 2018.
Original Russian Text © I.G. Zenkevich, T.I. Pushkareva, 2018, published in Zhurnal Obshchei Khimii, 2018, Vol. 88, No. 1, pp. 9–17.
Chromato–Mass Spectrometric Identification
of Unusual Products of 4-Isopropylphenol Oxidation
in Aqueous Solutions
I. G. Zenkevich* and T. I. Pushkareva
St. Petersburg State University, Universitetskii pr. 26, St. Petersburg, 198504 Russia
*
e-mail: izenkevich@yandex.ru
Received August 14, 2017
Abstract―4-Isopropylphenol has been chosen as the simplest object to model the processes of oxidation of
organic compounds with air oxygen in aqueous media, since it contains a hydrogen atom at the tertiary carbon
atom in the α-position with benzene ring and a hydroxyl group enabling mass-spectrometric detection of the
products in the negative ions mode. It has been stated that oxidation of 4-isopropylphenol with air oxygen in
aqueous media becomes noticeable as the solution pH approaches the рK
major product [4-isopropyl-2-(4-isopropylphenoxy)phenol] is formed via nucleophilic addition of the starting
-isopropylphenol at the intermediate product of its oxidation, quinone methide. Intensity of electrochemical
а
value of the substrate (10.25). The
4
oxidation can be tubed by changing the electrode potential. The highest conversion of 4-isopropylphenol has
been observed at potential 1.5–3.0 V, the formed compounds being the products of transformation of the same
quinone methide intermediate. The obtained data have explained the formation and diversity of dimeric and
oligomeric products of oxidation of natural flavonoids.
Keywords: 4-isopropylphenol, aqueous media, dissolved air oxygen, free-radical oxidation, electrochemical
oxidation, quinone methides
DOI: 10.1134/S1070363218010024
Oxidation of natural and technogenic organic
compounds with dissolved air oxygen in aqueous
media is among the principal processes of their
transformation [1–4]. Therefore, elucidation of the
regularities of such processes is important for the
characterization of decompositions of toxic com-
pounds in different environmental objects. Moreover,
this is essential for the understanding of many in vivo
oxidation processes. For example, identification of
products of oxidation of biologically active com-
pounds is a prerequisite of elucidation of their anti-
oxidant activity mechanisms. The structure of the
oxidation products of many organic flavonoids including
the most widely spread quercetin [5, 6] and, hence, the
nature of its antioxidant activity have been scarcely
studied so far.
α-position with respect to heteroatoms) yielding
·
peroxide radicals RO and products of their further
2
transformations (Scheme 1).
The suggested schemes of quercetin (Q) oxidation
[5, 6] include such process involving the hydrogen
atom in position 2 of the pyran fragment of its
tautomer (Q*) as the first stage (Scheme 2).
Systematization (as an independent stage of identifica-
tion) of the earlier known products of quercetin oxida-
tion aiming to assign their structures with the
chromatographic peaks [6] has shown that half of the
detected products (28 of 57) have not been identified,
and only 8 of the products have been identified un-
ambiguously. The identified oxidation products include
poorly stable peroxides and hydroperoxides, unstable
Oxidation of organic compounds with dissolved
3
–
g
oxygen (ground state: triplet S ) generally obeys the
Scheme 1.
regulations of free-radical reactions, their first stage
being elimination of the most reactive hydrogen atoms
+
O2
+O
2
[R·]
·
…
[RO ]
RH
2
–HO·
(
mainly these at tertiary carbon atoms or in benzyl and
2
7

8
ZENKEVICH, PUSHKAREVA
Scheme 2.
OH
OH
OH
H
HO
O
O
HO
O
O
OH
OH
O
OH
OH
Q
Q*
hemiketals, hydrate and enol forms; their preparative
isolation from aqueous solutions is practically im-
possible. Moreover, the mixtures of the products of
flavonoids oxidation often contain dimers and trimers
of debatable origin, even at low concentration [6]. In
this regard, characterization of products of oxidation of
simpler model compounds under the same conditions
seems reasonable.
Oxidation of 4-isopropylphenol with air oxygen
in alkaline medium. To model oxidation of 4-iso-
propylphenol with air oxygen, we prepared its solu-
tions in a mixture of aqueous solution of NH HCO
(25 mM, рН ≈ 8.5) and acetonitrile (80 : 20 v/v) with
concentration ranging from 0.77 to 1.0 mg/mL and
bubbled them with air (~2 L/min) during 4 h. Since no
oxidation products were detected under those condi-
tions, the solution рН was adjusted to ~10 with aqueous
ammonia, and bubbling with air was continued during
4
3
The chosen objects should contain hydrogen atoms
at the tertiary carbon atoms. The simplest compound of
this type is isopropylbenzene [solubility in water
S (mol/L) at 20°С, pS = –logS = 3.39±0.01, i. e. about
1
h.
Properties of the oxidation products were unknown
0
.1 g/L]. However, this hydrophobic hydrocarbon
before the experiment, therefore to minimize the in-
formation loss, both of negative and positive ions
detection modes were used for recording the
electrospray mass spectra. In the positive ions mode,
the collected chromatograms contained numerous peaks
most of which corresponded to background signals of
admixtures; those could be eliminated by parallel
analysis of the specimens without the characterized
component or containing different concentrations of it.
As a result, a small number of oxidation products was
left for further consideration, much less than in the
case of quercetin [6], showing better selectivity of
(
log P 3.66±0.20 [7]) is noticeably volatile (vapor
pressure at 20°С is 3.3 mmHg), and this leads to
significant losses of the substrate from its aqueous
solution in contact with air, especially during bubbling.
The second issue is the detection of the formed
products (including phenol, dimethylphenylmethanol,
benzoic acid, and cumene hydroperoxide [4]): UV
detection is advantageous for these species, since mass-
spectrometric (HPLC-MS) detection of the signals of
positive as well as negative ions of the compounds
containing no phenolic hydroxyls is inefficient.
4
-isopropylphenol oxidation.
In view of the above reasons, more hydrophilic and
less volatile 4-isopropylphenol (log P 2.90±0.02, S ~
Table 1 lists retention times (t ) of major products
R
1
.1 g/L) seems more suitable as the model compounds
of oxidation of 4-isopropylphenol in two regimes of
separation (I and II), values of linear-logarithmic
retention indices in regime II, and mass numbers of the
for the study of free-radical oxidation with air oxygen
in aqueous solutions. Moreover, this compound is
interesting as such, since it models the behavior of 4-
substituted alkylphenols (4-octyl- and 4-nonyl-),
products of hydrolysis of their polyethoxy derivatives
used as nonionic surfactants [8] and exhibiting
endocrine toxicity.
+
[М + Н] ions in the positive ions detection mode
–
(designated as “+”) and of the [М – Н] ions in the
negative ions detection mode (symbol “–”). If the mass
spectrum contained strong signals of fragmentation
ions, their mass numbers are indicated and assigned as
well.
Herein, we discuss the results of chromato–mass
spectrometric (HPLC-ES-MS) identification of products
of oxidation of 4-isopropylphenol as a model com-
pound to elucidate the oxidation of more complex
compounds with air oxygen.
Even the small number of the listed components
clearly revealed the prominently different informative
content of the negative and positive detection modes.
For example, the compounds with retention times
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CHROMATO–MASS SPECTROMETRIC IDENTIFICATION OF UNUSUAL PRODUCTS
9
Table 1. Components detected in the products of 4-isopropylphenol oxidation with air oxygen in an aqueous solution at
a
рН ~10
+
–
t
R
, min
t
R
, min
(II)
RI
(II)
m/z [M + H] or [M – H]
(m/z of fragmentation ions)
Compound
(
I)
.5
.7
1
1
–
–
(+) 151 (110 [М + Н – С
(+) 151 (110 [М + Н – С
(–) 135
3
Н
Н
5
5
])
])
C
9
9
H
H
12O – 2H + [O] (structure not determined)
12O – 2H + [O] (structure not determined)
3
C
1
6.6
12.1
929
4-Isopropylphenol (initial substrate)
(
+) 137 (95 [М + Н – С
3 6
Н ])
1
2
2
7.6
2.7
4.1
–
18.9
–
–
1266
–
(+) 222 (177, 149)
Diethyl phthalate
(–) 269 (225 [М – Н – С
3
Н
8
])
4-Isopropyl-3-(4-isopropylphenoxy)phenol
Dibutyl phthalate
(+) 279 (205, 149)
a
Retention times of major components of the reaction mixtures are indicated in italic.
1
7.6 and 24.1 min (regime I) were assigned to diethyl
aqueous ammonia solution, followed by air bubbling.
Hence, the oxidation with air oxygen became
noticeable only when the solution pH approached the
рK value of 4-isopropylphenol (pK 10.25).
and dibutyl phthalates, widely used plasticizers [9]
getting into the specimens from distilled water stored
in plastic tare. However, those components contained
no functional groups with labile hydrogen atoms and
were observed only in the positive ions detection
mode. Another example of such sort was given by two
hydrophilic components with retention times 1.5 and
а
a
The only component found in the mixture of
products of 4-isopropylphenol oxidation under mild
conditions is a hydrophobic compound with t 22.7
R
(
regime I) and 18.9 min (regime II), formally
1
.7 min (regime I); their molecular mass (М = 150)
corresponding to the [136(С Н О) – 1(Н)]·2 dimer
9
12
suggested that they could be assigned to isomeric
products of two-stage oxidation of 4-isopropylphenol:
1
Alternatively, the second stage of their formation
involved addition of water followed by oxidation,
with molecular mass 270 Da (detected only in the
negative ions mode). The formation of such dimer in
fairly dilute aqueous solutions (0.77–1 mg/mL = 5.7–
36(С Н О) – 2(2Н) + 16([О]) = 150(С Н О ).
9 12 9 10 2
7
.4 mmol/L) should be commented in more detail.
1
36(С Н О) – 2(2Н) + 18(Н О) – 2(2Н) = 150(С Н О ).
According to generally accepted views, the first
9 12 2 9 10 2
Theoretically, three possible structures could be
suggested for such products: ortho-quinone and two
quinone methide one, 2-hydroxy-4-(1-methylethenyl)-
cyclohexa-2,5-dien-1-one being the most favorable
stage of the interaction of the compounds containing
labile hydrogen atoms at tertiary carbon (RH) is the
·
formation of stable radicals (R ) further transformed
·
2
into peroxide radicals (RO
), hydroperoxides (RO
H),
2
[
2
ground state energy Е(ММ+) 13.1 kJ/mol, Е(АМ-1) –
221.2 kJ/mol]. However, the made suggestions could
and other products (Scheme 1). 4-Isopropylphenol
molecule contains such hydrogen atom, the α-hydrogen
of the isopropyl group. It could be suggested that the
primary radicals formed from 4-alkylphenols could be
stabilized through the formation of unstable quinone
methide intermediates [in the case of 4-isopropyl-
phenol, 4-(1-methylethenyl)cyclohexa-2,5-dien-1-one
(Q1)] (Scheme 3).
not be clarified using the available analytical data.
Since the content of those components strongly
depended (down to complete disappearance) on the
concentration of 4-isopropylphenol in the solution,
they could be assigned to readily oxidized products,
quinone methides.
Starting 4-isopropylphenol was clearly detected in
both regimes of chromatographic separation (I and II)
during detection of positive as well as negative ions.
After 4 h of bubbling air (~2 L/min) through 100 mL
of the solution, products of 4-isopropylphenol oxida-
tion were not detected. They appeared only after
adjustment of the solution pH to ~10 by addition
In the absence of substituents in the para position
with respect to hydroxyl group, phenols form para-
benzoquinones upon oxidation [10]. Quinone methides
intermediates are well known in lignin chemistry [11];
they are chemically similar to quinones [12, 13], being
prone to the addition of nucleophilic agent (so called
nucleophilic coupled addition). Quinone methides are
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 1 2018

1
0
ZENKEVICH, PUSHKAREVA
Scheme 3.
Scheme 4.
OH
OH
O
O
OH
OH
+O2
+O2
_HO2
Nu
NuH
+
Nu
Q1
sometimes recognized as cross-linking agents [14]
used a ROXY cell designed to be incorporated in an
HPLC–MS system and enabling the detection and
identification of the oxidation products during their
generation [17–20]. It was found that the potential up
to 1.5 V was not enough to provide the reasonable rate
of 4-isopropylphenol transformation, whereas its
increase to 3.0 V allowed detection of more than ten
oxidation products in the negative ions detection mode,
independently of the solution pH.
(
Scheme 4).
Aqueous solutions of 4-isopropylphenol contain
two potential nucleophilic agents: water and 4-
isopropylphenol. The addition of water to quinone
methide Q1 leads to dihydroxyisopropylbenzene with
М 152 (detected under conditions of electrochemical
oxidation), further oxidation of which explains the
formation of the above-mentioned components with
М = 150. In the case of 4-isopropylphenol as the
nucleophile, isomeric 4-isopropyl-3- (А1) and 4-iso-
propyl-2-(4-isopropylphenoxy)phenol (А2) with М =
Table 2 lists the retention times of the products of
electrochemical oxidation of 4-isopropylphenol (separa-
tion regime II), their retention indices ranging from
–
2
70 are formed, as it is illustrated by Scheme 5.
820 to 1340, mass numbers of the [М – Н] ions and
other strong signals, possible identifications, and the
calculated (ACD/Labs software) hydrophobicity
parameters log P used to analyze the elution order.
Experimental log P value is given for the starting
substrate, whereas the isomers are characterized by the
parameter range. Retention times of the minor products
are given in italic in the first column to show the
relative contents of the components.
If the oxidation products contain only one of these
isomers (as was observed in this study), its structure
elucidation by means of chromato–mass spectrometry
analysis is impossible. If both products were present,
they could be assigned from the elution order (in view
of hydrophobicity) or from the data on their relative
stability (for example, the correspondence between
their fractions in the mixture and the ground state
energies).
The components listed in Table 2 include all the
compounds from Table 1 detected in the negative ions
mode. On top of them, 4-isopropylpyrocatechol (the
product of water addition to the quinone methide, М =
Electrochemical oxidation of 4-isopropylphenol
in aqueous solutions. As exemplified by quercetin,
the composition of the products of free-radical (with
air oxygen dissolved in water, also known as auto-
oxidation) and electrochemical oxidation processes are
very similar [6], enabling the comparison of the
formed products. Therefore, it was reasonable to
consider electrochemical oxidation of 4-isopropyl-
phenol in this study. Supposing that the corresponding
quinone methide was the primary intermediate, the
process could be described by Scheme 6.
1
52) and two dimeric products А1 and А2 (major)
with М = 270 were detected. They were assigned using
the ground state energies determined using the ММ+
and АМ-1 methods.
Isomer
A1
A2
24.2
Е(ММ+), kJ/mol
Е(АМ-1), kJ/mol
log P
24.6
–4396.1
–4397.6
6.33±0.32
5.94±0.33
Phenols (and, therefore, natural flavonoids) are
electrochemically active compounds, and electro-
chemical detectors can be used for their detection in
HPLC [15, 16]. In contrast to the chemical processes,
the rate of oxidation can be tuned by changing the
potential U of the electrochemical cell (detector). We
The obtained data showed that the minor isomer А1
(tR 15.7 min, RI 1097) was eluted before the major
isomer А2 (t 18.9 min, RI 1266), since the Е(ММ+)
R
and Е(АМ-1) values were somewhat lower for the
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CHROMATO–MASS SPECTROMETRIC IDENTIFICATION OF UNUSUAL PRODUCTS
11
Scheme 5.
O
OH
OH
O
4
-IPP
+
O
A1
A2
latter one. The log P values were not informative, as
their difference (0.39) did not exceed the estimated
accuracy (0.65).
products. That allowed the estimation of the order of
the products formation (that is, determine the primary
and secondary components). For example, the
formation of the products with М = 270 was observed
at potential of at least U = 0.7 V, the compounds with
М = 286 and 420 were formed at U = 0.95 V, and the
compound with М = 152 appeared at U = 1.4 V. The
highest yield of the oxidation products was observed at
U of 1.5 to 3.0 V.
Besides the mentioned components, the mixture of
products of the electrochemical oxidation contained
three isomeric dihydroxyisopropyl-(4-isopropylphen-
oxy)benzene with М 286. Six such isomers were
theoretically possible, and the presence of only two of
them could be due to either different probabilities of
their formation or overlapping of their peaks in the
chromatogram. They could be formed via water
addition to the products of oxidation of dimers А1 and
А2 (or vice versa) and were characterized by the log P
values of 5.01–5.78. Finally, the products contained
seven compounds with М = 420, isomeric dihydroxy-
isopropylbis-(4-isopropylphenoxy)benzenes formed via
a series of oxidation–nucleophilic addition processes.
The last detected component with М = 404 cor-
responded to one of hydrophobic isomeric 4-isopropyl-
bis-(4-isopropylphenoxy)phenols. It should be noted
that the two compounds eluted the last could not be
characterized by the retention indices, since their
retention times fell outside the linear gradient of
acetonitrile in the eluent (20 min) and corresponded to
the final isocratic region. The detection of the
reference and characterized components in different
elution regimes is impossible due to systematic
inaccuracy in the retention indices.
To verify the identification of the oxidation pro-
ducts with unambiguously elucidated structure, it was
reasonable to use the independent criteria, the simplest
one being the correlation of the retention indices in the
reversed-phase HPLC with the hydrophobicity
parameters log P. Since there were only three such
compounds (Table 2) [4-isopropylpyrocatechol (RI
8
2
22, log P 2.22), initial 4-isopropylphenol (RI 929, log P
.90), and 4-isopropyl-2-(4-isopropylphenoxy)phenol
(
RI 1266, log P 5.94)], the parameters of the
corresponding linear regression equation could be
estimated using the three points: a 117±7, b 575±28,
r 0.998, S 20. Nevertheless, good linear relationship
0
RI = alog P + b was observed, thus confirming the
correct identification of those compounds.
In summary, we found that the free-radical as well
as electrochemical oxidation of alkyl-substituted
phenols was accompanied by the formation of dimeric
(
or, generally, oligomeric) products. The nature of the
Oxidation of 4-isopropylphenol under conditions of
scanning voltammetry gave the values of the lowest
potential corresponding to the formation of certain
products was fully in line with the suggested genera-
tion of quinone methide intermediates. Other ways of
oxidation of alkyl-substituted phenols, for example,
with iron (III) chloride [21] have led to the formation of
dimeric and oligomeric products, possibly via the same
mechanism. Moreover, it could be suggested that the
transformations of 4-alkyl-substituted phenols (including
Scheme 6.
OH
O
_
_
4
-nonylphenols [8]) in aqueous media are strongly
+
O2 + 2e
+ 2OH
affected by the reactions of quinone methide inter-
mediates with nucleophilic agents present in such
media (for example, humic acids in the case of natural
water reservoirs).
Q1
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1
2
ZENKEVICH, PUSHKAREVA
Table 2. Components detected in the products of electrochemical oxidation of 4-isopropylphenol in an aqueous solution at
a
рН ~ 7.4 (separation regime II)
–
m/z [M – H] ,
m/z of fragmentation ions)
t
R
, min RI
Compound
log P
2.22
(
b
9
.2
822 151
4-Isopropylpyrocatechol
b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2.1
2.3
2.8
3.4
5.7
7.0
7.5
8.0
8.9
9.4
0.0
0.2
1.9
3.6
929 135
4-Isopropylphenol (starting)
2.90
937 285 (267 [М – Н – Н
959 419 (203)
2
О])
Isomer of dihydroxyisopropyl-(4-isopropylphenoxy)benzene
5.01–5.78
Isomer of dihydroxyisopropylbis-(4-isopropylphenoxy)benzene 8.04–8.93
986 285 (242 [М – Н – С
3
Н
7
])
Isomer of dihydroxyisopropyl-(4-isopropylphenoxy)benzene
4-Isopropyl-3-(4-isopropylphenoxy)phenol
5.01–5.78
6.33
1097 269 (226)
1164 285 (267 [М – Н – Н
1191 419 (401 [М – Н – Н
1217 419 (383)
2
2
О])
О])
Isomer of dihydroxyisopropyl-(4-isopropylphenoxy)benzene
5.01–5.78
Isomer of dihydroxyisopropylbis-(4-isopropylphenoxy)benzene 8.04–8.93
Isomer of dihydroxyisopropylbis-(4-isopropylphenoxy)benzene 8.04–8.93
b
1266 269 (225 [М – Н – С
3
Н
8
])
4-Isopropyl-2-(4-isopropylphenoxy)phenol
5.94
1294 419 (376)
Isomer of dihydroxyisopropylbis-(4-isopropylphenoxy)benzene 8.04–8.93
Isomer of dihydroxyisopropylbis-(4-isopropylphenoxy)benzene 8.04–8.93
Isomer of dihydroxyisopropylbis-(4-isopropylphenoxy)benzene 8.04–8.93
Isomer of dihydroxyisopropylbis-(4-isopropylphenoxy)benzene 8.04–8.93
1327 419 (401 [М – Н – Н
2
О])
1339 419 (267)
c
–
–
419
c
403 (385 [М – Н – Н
2
О])
Isomer of 4-isopropylbis-(4-isopropylphenoxy)phenol
a
b
Retention times of major components of the reaction mixtures are indicated in italic. Characteristics of those compounds were used for
c
the calculation of parameters of regression equation RI = alog P + b. Retention times of two components eluted last were outside the
linear gradient (20 min); the change in the elution regime made impossible the calculation of their retention indices.
The obtained data could reasonably explain the
formation of numerous dimeric products with mass
2М – 2] (М being the mass of starting substrates)
process by measuring the current. The solution of the
oxidation products was acidified with HCO H (~0.1%
2
[
in the analyzed specimen) prior to the analysis.
upon oxidation of quercetin and other flavonoids in
aqueous media. The primary oxidation products included
quinones as well as quinone methides existing in
different tautomeric forms [5, 6], the major pathway of
their further transformations involving nucleophilic
addition of the starting flavonoids. That resulted in the
formation of dimers containing flavonoid moieties
linked by oxygen bridges.
Electrochemical oxidation of 4-isopropylphenol
with mass-spectrometric detection of the products was
performed in the scanning voltammetry mode at the
potential scanning rate 2 mV/s at U from 0 to 1.5 (a)
and from 1.0 to 4.4 V (b).
Chromatographic analysis of the aqueous solutions
was performed using an Agilent 1290 Infinity chro-
matograph equipped with a mass-spectrometric detector.
Separation regime I: column (150×2.1 mm) Zorbax
Eclipse XDB-C8 (sorbent particles size 3.5 µm), pre-
column filled with the same sorbent, gradient elution
with mobile phases A (water–acetonitrile–formic acid,
99 : 1 : 0.1) and B (water–acetonitrile–formic acid,
10 : 90 : 0.1). Content of phase B in the eluent was
linearly increased from 5 to 80% during 20 min, kept
constant during 2 min, and then decreased to 5%
during 1 min, and the column was conditioned during
2 min. Eluent flow rate (рН ~ 2–3) 0.2 mL/min,
EXPERIMENTAL
Electrochemical oxidation of a solution of 4-
isopropylphenol (50–100 µg/mL) was performed in
mixtures of an aqueous solution of ammonium acetate
(
рН ~7.4) and acetonitrile (3 : 1 v/v) in a ROXY
electrochemical cell (Antec, Netherlands) equipped
with a Magic Diamond boron-doped electrode at 37°С
and flow rate 10 µL/min. The electrode potential was
varied between 2.5 and 3.5 V monitoring the oxidation
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CHROMATO–MASS SPECTROMETRIC IDENTIFICATION OF UNUSUAL PRODUCTS
13
applied specimen volume 10 µL, column thermostat
temperature 30°С. Separation regime II: column
without stirring during 1 day; no oxidation products
were detected in the mixture. Aqueous solution of
ammonia (10%) was added to рН ~10, and air was
bubbled through the solution during 1 h. Prior to the
analysis, the specimens were acidified with formic acid
(about 0.1%) to рН ~3. To exclude the signals of the
admixtures as well as minor or unstable components,
the starting substrate concentration in the solution was
varied.
(
100×2.1 mm) Acquity UPLC HSS T3 (sorbent
particles size 1.8 µm), pre-column filled with the same
sorbent, gradient elution with mobile phases A (water–
acetonitrile–formic acid, 99 : 1 : 0.1) and B (water–
acetonitrile–formic acid, 10 : 90 : 0.1). Content of
phase B in the eluent was linearly increased from 20 to
9
0% during 20 min, kept constant during 4 min, and
then decreased to 20% during 1 min, and the column
was conditioned during 2 min. Eluent flow rate (рН ~
Samples preparation and oxidation conditions for
quercetin has been described elsewhere [6].
2
–3) 0.2 mL/min, applied specimen volume 10 µL,
column thermostat temperature 30°С.
ACKNOWLEDGMENTS
Mass-spectrometric detection of the products of
-isopropylphenol and quercetin oxidation was
4
Authors are grateful to the directorate of Research
Institute of Hygiene, Occupational Pathology and
Human Ecology, Federal Medico-Biological Agency
of Russia (St. Petersburg) for the opportunity to
perform the experiments.
performed using a Bruker amaZON ETD spectrometer
with ions trap (Germany) under electrospray ionization
conditions in the positive and negative ions detection
modes. Voltage on the capillary –4.8 kV, nitrogen as
drying gas, 250°С, flow rate 9 L/min, mass scan range
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