Formation of potentially toxic carbonyls during oxidation of triolein in the presence of alimentary antioxidants

Article abstract of DOI:10.1007/s00706-017-2036-3

Abstract: A relation between oil uptake and cancer as well as induction of hepatic inflammation was shown earlier. It is discussed that the main oil oxidation products—hydroperoxides and carbonyls—might be the reason for the mentioned diseases. In this manuscript quantitative determination of aldehydes which are formed during oxidation of triolein—as a model substance—using the Rancimat 679 is described. The oxidation of 11?g of triolein is carried out at 120?°C sparging air with a flow of 20?dm³/h for 10?h. A series of aliphatic aldehydes starting from hexanal to decanal as well as decenal was identified by LC–MS/MS and quantified as DNPH derivatives. In addition, the total amount of carbonyls was determined. Based on the calibration with hexanal, all other dominant substances were in the similar concentration range with maximum concentrations of 1.6?μmol/cm³ of hexanal, 2.3?μmol/cm³ of heptanal, 2.5?μmol/cm³ of octanal, 3.2?μmol/cm³ of nonanal, 4.0?μmol/cm³ of decanal after 6?h. The total amount of carbonyls reached a maximum after 6?h being 27?μmol/cm³ for triolein without antioxidant. The results of this investigation will be a basis for further toxicological studies on oxidized oils.

Full text of DOI:10.1007/s00706-017-2036-3

Monatsh Chem (2017) 148:2031–2035
DOI 10.1007/s00706-017-2036-3
ORIGINAL PAPER
Formation of potentially toxic carbonyls during oxidation
of triolein in the presence of alimentary antioxidants
Marini Damanik1
Michael Murkovic1
•
Received: 11 May 2017 / Accepted: 10 July 2017 / Published online: 19 October 2017
Ó The Author(s) 2017. This article is an open access publication
Abstract A relation between oil uptake and cancer as well
as induction of hepatic inflammation was shown earlier. It
is discussed that the main oil oxidation products—hy-
droperoxides and carbonyls—might be the reason for the
mentioned diseases. In this manuscript quantitative deter-
mination of aldehydes which are formed during oxidation
of triolein—as a model substance—using the Rancimat 679
is described. The oxidation of 11 g of triolein is carried out
at 120 °C sparging air with a flow of 20 dm3/h for 10 h. A
series of aliphatic aldehydes starting from hexanal to
decanal as well as decenal was identified by LC–MS/MS
and quantified as DNPH derivatives. In addition, the total
amount of carbonyls was determined. Based on the cali-
bration with hexanal, all other dominant substances were in
the similar concentration range with maximum concentra-
tions of 1.6 lmol/cm3 of hexanal, 2.3 lmol/cm3 of
heptanal, 2.5 lmol/cm3 of octanal, 3.2 lmol/cm3 of non-
anal, 4.0 lmol/cm3 of decanal after 6 h. The total amount
of carbonyls reached a maximum after 6 h being 27 lmol/
cm3 for triolein without antioxidant. The results of this
investigation will be a basis for further toxicological
studies on oxidized oils.
Graphical abstract
Hexanal (1.6 μmol/cm3
Heptanal (2.3 μmol/cm3
Octanal (2.5 μmol/cm3
Nonanal (3.2 μmol/cm3
Decanal (4.0 μmol/cm3
)
)
)
)
)
Keywords Triolein Á Lipid oxidation Á Aldehydes Á
DNPH Á Antioxidants Á b-Carotene Á a-Tocopherol
Introduction
Prolonged exposure to oxidized lipids can be detrimental to
human health. It has been reported that lipid hydroperox-
ides from processed dietary oils can enhance the growth of
hepatocarcinoma cells [1]. Another issue was published by
¨
Bohm and co-workers [2] who found that oxidized fat
might result in liver inflammation which was shown in
experimental rats. In the latter publication, no relation to a
specific substance or group of substances was established.
Therefore, a quantitative characterization of oxidized oils
is a prerequisite to start further mechanistic investigations
on the toxic principles of oxidized oils.
In earlier manuscripts the formation of different perox-
ides (hydroperoxides, epidioxides) and epoxides was
described in detail [3, 4]. A second group of reactive
products from oil oxidation are the carbonyls which are
formed during further reactions of the primary peroxides.
The formation of carbonyls during oil oxidation was
investigated earlier in rapeseed oil [5] when it was shown
that as a result of the degradation of the intermediate
& Marini Damanik
marini.damanik@student.tugraz.at
1
Institute of Biochemistry, Graz University of Technology,
Graz, Austria
123

2032
M. Damanik, M. Murkovic
110
100
90
80
70
60
50
40
30
20
10
0
hydroperoxides a great variety of aldehydes and ketones
are formed. It is well known that medium and short chain
aldehydes which are formed from higher unsaturated fatty
acids are the key aroma-active components which are
responsible for a rancid aroma. These are C7–C11
monounsaturated aldehydes, or C6–C9 dienals, or C5
branched aldehydes or some C8 ketones which are impor-
tant contributors to the oil aroma having negative attributes
(rancid, winey-vinegary, fusty, muddy sediment, musty)
[6].
6 hours oxidation
4 h oxidation
fresh triolein
These carbonyls and other compounds such as alde-
hydes, terminal alkenes, carboxylic acids, and aliphatics
are formed by a b-cleavage of lipid alkoxyl radicals [7].
The fragmentation reactions of allylic hydroperoxides by
the Hock rearrangement create two different carbonyls
from the same lipid peroxide, i.e. aldehydes or ketones [8].
Mechanistically, this is achieved by the insertion of one
oxygen atom into the lipid carbon backbone and delocal-
ization of a positive charge on a carbon or oxygen atom.
The main aim of this work was to establish an analytical
method for a quantitative determination of the carbonyls in
the lipid (oxidized oil) phase for determination of the ali-
mentary exposure. For the easier identification and
assignment of the reaction products, a defined matrix (tri-
olein) which did not contain any interfering compounds
was used. It was decided to use the derivatization with 2,4-
dinitrophenylhydrazine (DNPH)—a commonly known
derivatization reagent for carbonyls—that would give the
possibility to identify and quantify single components as
well as the total carbonyl content. These derivatives are
stable and can be analysed by HPLC with ESI-MS in the
negative mode or by its absorption at 400 nm [9–11].
With this background, we have started to characterize
the formation of carbonyls during oxidation of triolein in
the oil phase and not the volatile phase in which these
compounds are mostly related to sensory sensation. On
basis of reversed phase chromatography with UV and MS
detection, the non-evaporating carbonyl compounds
formed during oxidation of triolein were identified and
quantified. In this case, triolein was used as model oil. The
results of these experiments will be a basis for future work
on food oil oxidation. In addition, the interaction of
antioxidants (b-carotene, a-tocopherol) with the carbonyl
formation was analysed.
4
6
8
10
12
Retention time / min
Fig. 1 Chromatogram of DNPH derivatives of fresh triolein and after
4 h as well as 6 h of oxidation in the Rancimat
carbonyls from fatty acid oxidation are too volatile to be
analysed by LC/MS. Therefore, the DNPH adds molecular
weight to increase sensitivity and selectivity [12]. Negative
ionization using APCI was the most sensitive MS method
which could be used. The hydrazones formed were anal-
ysed using visible light absorption (400 nm) or by MS. The
advantage of detection at 400 nm is that practically all
peaks that appeared in the chromatogram were DNPH
derivatives. In the control chromatograms only the baseline
could be observed at 400 nm (Fig. 1). The semiquantitative
evaluation of the carbonyls was based on a calibration
using hexanal with the peak area relating to the molar
concentration of hexanal. The linear calibration of hexanal
was done in the range of 8–1000 lg/cm3 (0.08–10 mM).
The formation of carbonyls could be explained by a Hock
cleavage which was reviewed by [7]. This might explain
the formation of, e.g. decanal and 2-undecenal which are
produced by homolytic cleavages on either sides of the
8-hydroperoxide and nonanal from either the 9- or 10-hy-
droperoxide. The linearity of the hexanal analysis was
tested in the range of 5–1300 lg/cm3 according to Mandel
[13] with a limit of detection of 4.1 lg/cm3 and a limit of
quantification of 13 lg/cm3.
The formation of carbonyls began after 2 h of oxidation
at 120 °C while bubbling air through the oil. A strong
increase of carbonyl formation was observed between 2
and 4 h with a maximum after 6 h and a slight decrease or
constant concentrations afterwards. A series of linear
aldehydes (from 6 carbons to 10 carbons) could be iden-
tified positively by LC–MS. Based on the calibration with
hexanal all other dominant substances were in the similar
concentration range with maximum concentrations of
1.6 lmol/cm3 of hexanal, 2.3 lmol/cm3 of heptanal,
2.5 lmol/cm3 of octanal, 3.2 lmol/cm3 of nonanal,
4.0 lmol/cm3 of decanal after 6 h (Fig. 4). These identified
Results and discussion
Secondary oxidation products analysis
The derivatization of carbonyls is commonly used in
chromatographic analysis. The hydrazones formed are
stable enough for MS experiments. In some cases the
123

Formation of potentially toxic carbonyls during oxidation of triolein in presence of…
2033
28
1000000
800000
600000
400000
200000
0
27
26
25
24
23
22
21
20
19
2
0
6
7
8
9
10
11
12
13
14
0,00,1
2
4
6
8
10
Retention time / min
Oxidation time / h
Fig. 2 Selected ion traces of the DNPH conjugates of the saturated
and unsaturated aldehydes found in oxidized triolein
Fig. 3 Formation of the total amount of carbonyls during oxidation
of triolein at 120 °C with constant air sparging
aldehydes were volatile and undergo further degradation
reactions which may explain the reduced concentration.
The double peaks which could be seen after 4 h of
oxidation were attributed to Z/E isomer formation since no
difference in the mass spectrum and UV absorption could
be observed. After 6 h of oxidation the double peaks dis-
appear resulting in a shift in the relative concentrations
(Fig. 1).
stabilization was obtained by addition of 300 ppm of
ascorbyl palmitate to soybean oil (SO). In addition, a
combination of ascorbyl palmitate (300 ppm) and a-toco-
pherol (1000 ppm) was able to limit hydroperoxide and
hexanal formation in SO at 35 °C for 12 weeks [20].
Antioxidants inhibit lipid oxidation by quenching free
radicals and physically stabilizing the micelles at reaction
sites [21]. As described in [22] the oxidation starts after 5 h
in both, the refined and fortified oils (containing b-car-
otene), with the formation of hydroperoxides. In this case,
the addition of b-carotene resulted in a pro-oxidant effect.
b-Carotene was still measurable after 10 h [22]. During the
course of the oxidation in the experiments described here,
the levels of the formed carbonyls were lower in the
samples with added antioxidants (Fig. 4). In addition, the
pattern of the chromatograms did not change significantly
The identification of the peaks was done by LC–MS/MS
as is shown in Fig. 2. The earlier mentioned saturated
aldehydes (hexanal–decanal) including decenal could be
identified. The fragmentation of all of these compounds
was similar with a specific fragment of m/z = 182 origi-
nating from the DNPH moiety.
In this experiment, nonanal and octanal were the main
products. 2-Nonenal and 2,4-decadienal which are the
known degradation products of linoleic acid were not
detected. Additional compounds (heptane, heptanol,
octane, 1-undecene, and 2-undecenal) that were described
earlier are produced by a homolytic fission of the inter-
mediary formed R–O bond [14–19]. However, non-
carbonyls could not be detected with the method described
here.
4,0
3,5
3,0
2,5
2,0
1,5
1,0
The total amount of carbonyls formed is shown in
Fig. 3. Similar to the single aldehydes, a maximum con-
centration is reached after 6 h with a decrease afterwards.
The maximum concentration is 27 mM which is based on a
calibration with hexanal.
-tocopherol
triolein
-carotene
0,5
0,0
Influence of lipid-soluble antioxidants on carbonyl
formation
Oxidation time / h
Fig. 4 Formation kinetics of single compounds during oxidation of
triolein in presence of lipid-soluble antioxidants at 120 °C with
constant air sparging for 10 h (filled circle hexanal; filled square
heptanal; filled upward triangle octanal; filled downward triangle
nonanal; filled diamond decanal)
In a second series of experiments, two lipid-soluble
antioxidants were added, namely a-tocopherol and b-car-
otene. The earlier experiments showed that the best
123

2034
M. Damanik, M. Murkovic
Experimental
2,4-Dinitrophenylhydrazine (2,4-DNPH) was purchased
from Sigma-Aldrich (St. Louis, USA), hydrochloric acid
(HCl, 37%) was purchased from Merck (Darmstadt, Ger-
many), all solvents (e.g. methanol, ACN, acetone) used
were of HPLC grade and were purchased from Merck
(Darmstadt, Germany), acetic acid was purchased from
Roth (Karlsruhe, Germany), triolein was purchased from
FLUKA (Buchs, Switzerland), b-carotene and a-toco-
pherol were from Sigma-Aldrich (St. Louis, USA).
control
-tocopherol
-carotene
4
5
6
7
8
9
10
11
12
13
Retention time / min
Analysis of secondary oxidation products in oil
Fig. 5 Comparison of the carbonyl formation in presence of lipid-
soluble antioxidants (a-tocopherol, b-carotene) after 10 h of oxida-
tion at 120 °C. The carbonyls are measured as DNPH derivatives
detected at 400 nm
Oxidation of triolein with Rancimat
The triolein oil samples were subjected to oxidation in a
Rancimat (679, Metrohm, Herisau, Switzerland). Eleven
grammes of sample was used for the oxidation experi-
ments. The temperature was set to 120 °C and the air flow
to 20 dm3/h. Triolein was treated for up to 10 h. The
oxidized samples were cooled immediately after the oxi-
dation and stored under nitrogen below -18 °C.
indicating the same carbonyls are formed but at lower
amounts. The sample containing a-tocopherol showed a
higher formation of octanal after 10 h (Fig. 5).
For this, b-carotene and a-tocopherol were dissolved in
acetone and added to the triolein. The concentrations of the
antioxidants were 300 lg/g 0.5 lg. The fortified triolein
was stirred for 15 min and then flushed with nitrogen for
30 min to remove the acetone before sealing airtight in
glass bottles. For the oxidation in the Rancimat 20 g of
triolein was used. The concentrations which were applied
were in the same range as it can be expected in natural oils.
The a-tocopherol concentration was 300 lg/g and the level
of b-carotene was 500 lg/g. The concentrations were
selected according to the concentrations normally occur-
ring in red palm oil which are in the range of 600–1000 lg/
g for vitamin E and 400–3500 lg/g for a- and b-carotene
[23].
Derivatization with 2,4-dinitrophenylhydrazine (DNPH)
To 1 cm3 of the oxidized oil samples 4 cm3 of acetonitrile
was added and mixed with 3 cm3 of the reagent 2,4-DNPH
(3.48 mg/cm3). The reaction mixture was kept in the dark
for 1 h. After completion of the reaction, 2 cm3 ethyl
acetate was added for extraction and 1 g KCl for better
phase separation. This mixture was thoroughly shaken for
30 s and centrifuged for phase separation. The organic
layer was analysed without further treatment by HPLC.
Liquid chromatography–mass spectrometry condition
for aldehydes identification
The analyses of the DNPH derivatives of the carbonyls
formed during oxidation were done by HPLC (Agilent
1100, Waldbronn, Germany) using a reversed phase col-
umn (Kinetex, EVO C18, 150 9 3 mm, 5 lm,
Phenomenex, Aschaffenburg, Switzerland). For elution a
gradient was used starting with methanol (45%), water
(30%), and acetonitrile (25%) changing to methanol (6%),
water (4%), and acetonitrile (90%) linearly within 15 min.
The absorption of the eluent was measured at 400 nm for
the presence of the DNPH derivatives.
Conclusion
The formation of carbonyls was quantified during oxidation
of triolein by derivatization of the formed products with
DNPH which is a selective reagent for aldehydes and
ketones. Some of the oxidized fragments of triolein were
identified by LC–MS/MS—using APCI in negative
mode—being hexanal (m/z = 279), heptanal (m/z = 293),
octanal (m/z = 307), nonanal (m/z = 321), decenal (m/
z = 333), decanal (m/z = 335), and undecenal (m/
z = 347). Using a Rancimat for reproducible oxidation
experiments with a constant air flow at defined tempera-
tures (120 °C) the production of carbonyls showed a good
repeatability. The formation of carbonyls from triolein
showed a maximum after 6 h with a slight or no decrease
during prolonged oxidation.
For mass selective detection, a QTRAP 2000 (AB Sciex,
Framingham, MA, USA) was used. Ionization was done
using the APCI mode with a gas drying temperature of
250 °C, capillary voltage of 4000 V, and a fragmentor
potential of 150 V.
123

Formation of potentially toxic carbonyls during oxidation of triolein in presence of…
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Acknowledgements Open access funding provided by Graz
University of Technology. This work was supported by Ministry of
Research, Technology and Higher Education of the Republic of
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Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
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