Identification of α-Tocopherol and Its Oxidation Products by Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry

Article abstract of DOI:10.1021/acs.jafc.9b06544

This study aimed to determine α-tocopherol (α-T) and its thermal oxidation products simultaneously. A novel method based on an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed. This approach was achieved by means of a BEH C18 analytical column under gradient elution conditions with eluents of acetonitrile/isopropanol (1:9, v/v) and acetonitrile/water (4:6, v/v). Compounds were elucidated through exact molecular mass and fragmentation ions obtained from the Q-TOF-MS detector. Two oxidation products, α-tocopheryl quinone and 5-formyl-?3-tocopherol, were identified, and one new compound was determined. This approach offered a simple, precise, and reliable method to determine oxidation products of α-T, which may give a way to understand the mechanism of the thermal oxidative process of α-T.

Full text of DOI:10.1021/acs.jafc.9b06544

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New Analytical Methods
Identification of #-tocopherol and its oxidation products
by ultra- performance liquid chromatography coupled
with quadrupole time-of-flight mass spectrometry
Chuanhui Tang, Guanjun Tao, Yue Wang, Yuanfa Liu, and Jinwei Li
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b06544 • Publication Date (Web): 19 Dec 2019
Downloaded from pubs.acs.org on December 22, 2019
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Identification of α-tocopherol and its oxidation products by ultra-
performance liquid chromatography coupled with quadrupole
time-of-flight mass spectrometry
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Chuanhui Tang, Guanjun Tao, Yue Wang, Yuanfa liu, Jinwei Li*
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State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi
214122, People’s Republic of China
*Corresponding Author’s E-mail: jwli@jiangnan.edu.cn (Li, J.W.)
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ABSTRACT
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This study aimed to determine α-tocopherol and its thermal oxidation products
simultaneously. A novel method based on an ultra-performance liquid
chromatography coupled with quadrupole time-of-flight mass spectrometry
(UPLC-Q-TOF-MS) was developed. This approach was achieved by means of a BEH
C18 analytical column under gradient elution conditions with eluent of
acetonitrile/isopropanol (1:9, v/v) and acetonitrile/water (4:6, v/v). Compounds were
elucidated through exact molecular mass and fragmention ions obtained from
Q-TOF-MS detector. Two oxidation products, α-tocopheryl quinone and
5-formyl-γ-tocopherol were identified and one new compound was determined. This
approach offered a simple, precise and reliable method to determine oxidation
products of α-T, which may give a way to understand the mechanism of thermal
oxidative process of α-T.
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KEYWORDS: α-tocopherol, UPLC-Q-TOF-MS, oxidation products, α-tocopheryl
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quinone
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INTRODUCTION
Vitamin E is naturally present in different food matrices such as edible oils,
grains, nuts and seeds, followed by eggs, fish and liver (1). Generally, it is categorised
into eight tocopherol homologues, four kinds of tocopherols and four tocotrienols (2).
Tocopherols differ from tocotrienols merely by the saturation degree of the isoprenoid
side chain. Tocopherols are effective in protecting cell membranes and lipids from
oxidation and peroxidation due to the function of donating phenolic hydrogens atom
capable of scavenging lipoperoxyl radicals (3, 4). Hence, vitamin E is commonly used
to increase the stability of foods during storage (5). Among all eight isoforms,
α-tocopherol (α-T) is widely used and considered as the most biologically active
isoform. Low intake of α-T may result in deficiency-associated ataxia, making it the
only one essential for humans (6, 7). Consequently, knowledge of α-T content in
foods is vital to make sure the optimal daily intake as an essential factor in human
health (8). Moreover, α-T possesses strong hydrogen-donating ability and functions as
effective chain-breaking antioxidant, protecting cell membranes from oxidative
damage (9). Lately, many literatures have demonstrated health benefits of α-T, such
as prevention and treatment of several diseases, including cardiovascular disease,
cancer, Alzheimer’s and Parkinson’s diseases (10). Therefore, a growing interest in
qualitative and quantitative determination of α-T has appeared.
As focus of previous studies, many researchers aimed to develop approaches for
determination of α-T, the most frequently used methods were based on high-pressure
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liquid chromatography (HPLC) (8). Besides, GC (gas chromatography) and SFC
(supercritical fluid chromatography) techniques have also been applied (11-13).
Techniques mentioned above are inevitable of defects in spite of certain advantages.
In terms of HPLC, UV (ultraviolet detector) and DAD (photodiode array detector)
detectors can easily obtain the absorption spectra of eluted compounds but proven to
be less sensitive and selective (12). These drawbacks were partly overcame through
FD (fluorophotometric detector) with fewer interfering peaks, whereas FD shows less
sensitivity than electrochemical detectors such as DAD. Recently, ELSD (evaporative
light scattering detector) has been successfully applied in many research fields but
with the least sensitivity since other components may co-elute with the target analytes
(13). Besides, extraction procedure is necessary before HPLC detection, a
time-wasting step and consumes large amount of hazardous organic solvents with
high cost (14). On the other hand, derivatization process is always necessary prior to
GC analysis in order to increase the volatility, stability, sensitivity of tocochromanols.
Even though significant achievements have been reached, most methods were
developed exclusively for α-T.
It is known that α-T is thermo-unstable and prone to degrade into different
metabolites over time. Numerous researchers have reported that in model systems,
α-T ultimately turned into α-tocopheryl quinone (α-TQ), 5-formyl-γ-tocopherol
(5-F-γ-T), as well as spirodimers and spirotrimers derived from α-T through oxidation
(15-17). However, there are no researches thoroughly illuminated the transformation
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of intermediate products as the structures from which products originated could be
further transformed to the final products even during analysis. As important indicators
and characterization of oxidative degree, the determination of α-T oxidation products
along with discovery of new ones is quite necessary (18, 19). However, limited
information is available on the simultaneous analysis of α-T and the degradation
products up to now. The reasons may be the followings: (i) occurrence of factors
might synergize or antagonize the metabolic process of α-T, for instance, heating
conditions along with other compounds (antioxidants, proteins and lipids), leading to
complex analytical issues; (ii) analytes are hard to separate and identify due to
unavailability of standards; (iii) levels of target objects are low or non-detectable. It is
of great necessity to improve existing analytical methods.
Ultra performance liquid chromatography-quadrupole time-of-flight mass
spectrometry (UPLC-Q-TOF-MS) is a novel approach with characteristics of rapid
speed, superior sensitivity and high selectivity. Mass spectrometry is powerful in
obtaining accurate information of molecular mass, which makes it appropriate for
precise identification in comparison with conventional detectors. The objective of this
study is to evaluate the suitability of using UPLC-Q-TOF-MS for simultaneous
assessment of α-T and its oxidation products, with neither prior extraction nor
derivatization. Additionally, the proposed method was further applied to determine
oxidation products of α-T in thermal-oxidative camellia oil.
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MATERIALS AND METHODS
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Chemicals and Reagents. Standard of α-tocopherol (HPLC grade, >96%) was
obtained from Sigma-Aldrich (SL, USA) and were stored at 4-8°C before use.
Isopropanol, acetonitrile and n-hexane of HPLC grade were from Fisher
Scientific (MA, USA), used as analysis mobile phase without any further purification.
Ultrapure water was obtained through a Milli-Qultrapure water purification system
(Bedford, USA). Other reagents were purchased from Sinopharm Chemical Reagent
Co. Ltd (Shanghai, China). Refined camellia oil was supplied by Hangzhou Choisum
Bio-Tech Co. Ltd (Hangzhou, China).
Conditions of thermal degradation of pure α-T. The thermal process was
according to the method of Chung (20) and Verleyen (21) with some modification.
Eight clean test tubes each with 10 mg α-tocopherol placed at the bottom were heated
at 180°C for 20 h in an oil bath. No air bubbling was applied and the tubes were
exposed in the air. Heated samples containing the mixture of α-T and its thermal
oxidative products were sampled at time intervals of 0, 1, 2, 3, 4, 8, 16, 20 h.
After heating, each test tube was immediately treated with ice-water bath, then
dissolved in 10 mL n-hexane and stored in dark glass flasks at -20°C before use. Our
research is vital to develop an UPLC method as no influence from exogenous
environment was brought in, even though the oxidation products may differ from
those in other model systems such as lipid matrices. Moreover, none multifarious
sample pretreatment was performed in order to guarantee the simultaneous existence
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of all target compounds as well as avoid any unnecessary loss.
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Chromatographic separation. An ultra-performance liquid chromatography
(UPLC) system (Waters, Milford, Massachusetts, USA) was equipped with a BEH
C18 analytical column (i.d. 2.1 × 50 mm, 1.9 μm, Waters, USA) to achieve liquid
chromatographic separation. The column temperature was 45°C. The flow rate was
300 μL/min and the injection volume was 1.0 μL. The use of acetonitrile/isopropanol
(1:9, v/v) as mobile phase A, acetonitrile/water (4:6, v/v) as mobile phase B were the
most preferable. The binary linear gradient elution applied was initiated with 35%
eluent A for 0.1 min, linear from 35% to 90% in 20 min, then increased to 100%
within 5 min and maintained 1 min. Finally, returned to the initial 35% in 0.1 min and
equilibrated for 4 min. Before next injection, the column was flushed with 5% mobile
phase A for 3 min.
Photo-Diode Array Detection. A Waters Acquity PDA detector was applied to
UPLC system to obtain UV signals meanwhile. The UV-vis spectra were recorded in
the wavelength range of 200-600 nm, extracted and utilized for tentative identification
of α-T and oxidation products, in accordance with their prospective retention time.
Wavelength for tentative identification of α-T was set at 295 nm whilst 275 nm was
set for oxidative products of α-T (21).
Mass spectra acquisition and data analysis. Mass data was acquired by a
Q-TOF-MS instrument (Waters, USA), with an electrospray ionization source (ESI)
E
probe operated in positive ion mode. Two acquisition modes, MS (where E
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represents the collision energy) and fast DDA (data dependent acquisition), were
utilized for TOF data collection.
E
ESI source parameters were set for MS and fast DDA experiments as the
following conditions: the capillary voltage was kept at 3.5 kV while the sampling
cone voltage was 20 V. The source temperature and desolvation temperature were set
at 100°C and 400°C respectively. High purity nitrogen (N ) and argon (Ar) were
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chosen as desolvation gas and collision gas severally with 40 L/h cone gas flow and
700 L/h desolvation gas flow.
E
In MS methodology mode, the mass range was scanned from 100 to 1500 m/z in
a full-scan mode, an interleaved collision energies were set at 6 eV (low energy) and
20 eV (high energy) respectively to acquire intact precursor ions and product ions
from a single analysis. Data was collected into separating channels within 0.5 s
duration and 20 ms inter-channel delay.
In fast DDA mode, the mass analyzer scanned over a mass range of 100-1000 Da
under full scan mode, the collision cell was operated with an alternating low energy
(collision energy of 6 eV) and elevated-energy (collision cell energy gradient ranged
from 15 to 35 eV). Scan duration and inter-scan delay were set at 1 s and 20 ms
respectively.
Mass accuracy was previously calibrated with sodium formate,
leucine-enkephalin in positive ion mode. MassLynx 4.1 software (Waters) was used
for instrument control and data analysis.
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Heat treatment of oil samples. 30 g fresh camellia oil was transferred into a 50
mL beaker and immersed in a 180°C-preheated oil bath. The beaker was exposed in
the air to simulate frying temperature of 180°C. The oils were sampled at time
intervals of 4 h for 36 h. After the heat treatment, the samples were purged with
nitrogen and then transferred into small brown bottles, and stored under -20°C until
analysis. All results listed are the mean values of two experiments.
Statistical Analysis. All experiments were performed in duplicate. All data
analysis was carried out utilizing the software SPSS (version 19.0).
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RESULTS AND DICUSSION
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UV-vis spectra of oxidative α-tocopherol. It has been witnessed that α-vitamers
are the most prone to oxidation among all forms as a result of their chromanol rings
possess strongest methylation degree, which favors resonance stabilization of
chromanoxy radicals (22). In this research, the UV spectra were observed before and
after thermal oxidation. The detector wavelength was recorded at 295 nm and 275 nm
(Fig.1) respectively for tentative identification of α-T and its oxidative products,
which were determined by their ultraviolet maximal adsorption. In comparison with
unheated samples (Fig.1 B and Fig.1 D), several additional peaks appeared after
heating as Fig.1 A and Fig.1 C showed, indicating that α-T degraded upon oxidation.
The appearance of unidentified peaks (retention time around 9.43 min, 12.86 min and
18 min) in the chromatograms could be observed obviously during the PDA detection.
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These results were consistent with Verleyen et al. (3), they found several peaks arisen
in HPLC-UV chromatogram of α-T heated under high temperatures in model triolein
and tripalmitin mixtures, which were proven to be α-TQ and epoxy-α-tocopheryl
quinones. Besides, Drotleff et al. reported similar results, in normal-phase HPLC-UV
chromatograms of eight tocochromanols after thermal oxidation, all chromatograms
were found with additional peaks emerged (22).
In this research, the new peaks were identified as main oxidation products of
interest formed during the 180°C heating, owing to no interferential abundance of
environment were brought in. It was impossible to compare the retention time with
standards directly as no standards were available. Thus, MS detection was performed
for further identification, which could be useful for identification of unknown
compounds as fragmentation spectra could provide information pertaining to chemical
substructures (23).
The development of new methods for oxidation products analysis
Optimization of chromatographic conditions. Considering α-T oxidation
products might be isomers with minor structural difference, UPLC and MS condition
were optimized for the best separation and detection.
In order to achieve ideal separation, better peak shape and narrow peak width, a
gradient elution program using acetonitrile/isopropanol (1:9, v/v) as mobile phase A,
acetonitrile/water (4:6, v/v) as mobile phase B was finally the most suitable system.
Different gradients were performed and it came out that when the binary gradient was
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initiated with 35% instead of 22% eluent A, the analysis time of each peak is
2-minutes shorter, which much saving the analysis time. Ammonium acetate (10 mM)
was added into mobile phases as an ionizing agent for a considerable gain in the
ionization efficiency required for mass spectrometry signal. Moreover, in order to
obtain shorter analysis time, high column efficiency as well as good resolution, a 1.9
μm particle size C18 column was chosen. The ultimate flow rate was maintained at
300 μL/min in consideration of sensitivity and resolution and the column temperature
was kept at 45°C to avoid high column pressure.
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Mass spectrometer conditions. Many researchers reported the ionization of
tocopherols and tocotrienols by ESI in both positive and negative modes. The
researches of Mejean reported the peak areas of analytes was higher in positive ESI
than negative ESI (24). In our research, positive mode was used. The ionization
energy of α-T was about 6.7 eV (24), so collision energies in our research were set
equal or greater than 6 eV. Multidimensional acquisition modes, including MSE
(where E represents the collision energy) and fast DDA (data dependent acquisition),
were utilized for TOF data collection for more precursor ions and cleaner
precursor-product ions. Therefore, complementary fragments information could be
provided by combining both methods, thus characterizing target compounds
selectively and comprehensively (25).
Identification of α-T and oxidation products. The developed method was
applied to investigate α-T degradation and its oxidation products formation
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simultaneously. Multiple approaches were used for comprehensive confirmation,
including database searching on publications for information of known products of
α-T. The chemical formula of potential oxidation components was deduced based on
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the high-accuracy [M + H] / [M + Na] precursor ion, then further identified and
confirmed by comparing raw detected fragment ions data with corresponding data
from relevant literatures.
Noticeably, several new peaks showed up in total ion current chromatograms of
Fig.2, indicating that α-T degraded after 180°C thermal oxidation experiments. In this
work, we primarily focused on the compounds eluted among 9.43, 9.88, 11.40, 12.30
min, which were almost in accordance with peaks of UPLC chromatograms in Fig.1.
As a result, three components were identified as oxidation products of α-T.
α-tocopherol. The peak at 11.4 min displayed an accurate molecular weight of
m/z 430.385 with the molecular formula of C H O , exactly corresponding to the
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molecular weight of α-T (Fig.3). It merely produced a prominent molecular ion peak
with tiny percentage of fragments at low collision voltage (Fig.3 A). When the [M]+
(m/z=429.37) was selected as the parent ion for MS/MS, it further generated several
structurally relevant fragments at a pair of ions with m/z 137.104, m/z 165.098 and
m/z 205.128 (Fig.3 B), which was helpful to characterize α-T. The aromatic ring of
α-T showed more stability and stronger bond ability than aliphatic side chain as a
result of resonance of electrons (20), and non-aromatic parts of α-T had a tendency to
decompose into two parts through oxidation. Hence, the fragment at m/z 165.098 was
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generated by the cleavage of the chromanol ring structure and the loss of a CH C≡CH
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fragment. The ion at m/z 205.128 was due to the characteristic fragmentation of
side-chain at C-2 and the loss of saturated hydrophobic side chain (-C H ). In the
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study of Stöggl et al. (26) , a HPLC-APCI (atmospheric-pressure ionization)-MS/MS
method was developed to detect vitamin E in food and phytopharmaceutical
preparations, fragments at m/z 205.2 and m/z 165.2 were reported to belong to α-T
characteristically, which was in agreement with our results. Additional peaks found at
137.104 m/z were part of the chroman ring, as shown in the fragmentation pathway in
Fig.3 B. Furthermore, the maximum UV-vis absorption of this peak was observed at
292 nm (data not shown), which was exactly corresponded with the characteristic
absorption of α-T revealed in previous works.
α-tocopheryl quinone. Among all oxidation products of α-T, α-TQ is an
important divalent oxidation product. Numerous investigations proved α-T degraded
into α-TQ after heat process (27, 28). Some authors tried to explain the formation
mechanism of α-TQ, among which one propose is that it is formed by rearrangement
of a 9-substiturd intermediate arising from the reaction between a cationic mesomer
and a nucleophile (29). However, Grams et al. (30) held different opinions that α-TQ
was derived from several end peroxide intermediates which generated from
cycloaddition of oxygen.
The compound with the retention time of 9.88 min showed a maximum UV-vis
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absorption at 275 nm and exhibited a molecular ion peak [M+H] at m/z 447.386,
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which was in accordance with published data of Chung et al. (20), indicating the
compound was α-TQ. Through oxidation, the elimination of OH groups to form
quinones resulted in shorter retention time compared with α-T. Resemble the
fragmentation patterns of α-T, α-TQ mainly decomposed at non-aromatic rings as
proposed in the potential fragment pathway in Fig.4. One molecule of α-T combined
one molecule of oxygen in α-TQ, and the presence of daughter ion at m/z 429.384 of
α-TQ was ascribed to the loss of oxygen. Additionally, a peak at m/z 469.390
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represented [M+Na] adduct ion indicated a common precursor ion in positive ion
mode that could confirm the existence of α-TQ. Apart from the above ions, a
prominent fragment ion at m/z 165.098 showed low abundance at low collision
energy while increased significantly with high collision energy. This indicated that the
chroman structure was apt to cleave as fragments on account of higher energy. These
fragments ions turned into ions at m/z 137.108 by successive losses of oxygen. The
elimination of the saturated side chain tail (C H ) lead to the formation of ions at
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m/z 219.141 sharing the similar fragmentation pathway with α-T.
5-formyl-γ-tocopherol. Peaks eluted at 9.43 min (Fig. 5) exhibited a
characterized molecular ion peak [M+H] at m/z 445.377, as well as a [M+Na]+
adduct ion at m/z 467.364, which was consistent with 7-formyl-β-tocopherol
(7-F-β-T) and 5-formyl-γ-tocopherol (5-F-γ-T), a pair of isomers. Two important
peaks observed at m/z179.081 and m/z 151.084 illustrated the loss of chroman
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structure of parent ions and the cleavage of a CH C≡CH fragment (17). It is obvious
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that the intensity of m/z 179.081 increased significantly as the collision energy was
given higher. The fragment at m/z 123.082 indicated that the aldehyde group on the
benzene ring was eliminated at the basement of fragment m/z 151.084. In addition,
fragment ions at m/z 427.371 were generated by the loss of phenolic hydroxyl groups.
The high stability of the p-π conjugation effect between phenolic hydroxyl groups and
the benzene rings made the abundance of it rather low.
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Malaika et al. (31) investigated oxidative products formed in polyethylene and
polypropylene during a melt process and reported the formation of 5-F-γ-T and
7-F-β-T. Same result was found in α-tocotrienol, researchers found homologous
aldehydic oxidation products of α-tocotrienol after thermal oxidation, such as
5-formyl-γ-tocotrienol (5-F-γ-T3) and 7-formyl-β-tocotrienol (7-F-β-T3) (22, 32). The
carbonyl groups on the benzene ring of these compounds could easily combine with
the hydroxyl groups in the ortho position, thus forming intramolecular hydrogen
bonds, further leading to relatively insignificant antioxidant properties than
α-tocotrienol. What is interesting is that in different matrices, compared with
5-formyl-γ-tocochromanols, 7-formyl-β-tocochromanols were always less likely to
form. Rosenau et al. (33) reported that 5a position of α-tocochromanols had a more
tendency to oxidize than position 7a, which accounted for more formation of 5-F-γ-T3
than 7-F-β-T3.
Drotleff et al. (22) applied a model n-hexane system to develop a new HPLC
method to separate tocochromanols and identify oxidation products of α-tocotrienol.
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They found 5-F-γ-T3 and 7-F-β-T3 shared the same fragments but showed different
characteristic UV-vis absorptions. In particular, 5-F-γ-T3 absorbed UV light at 386
nm, 290 nm and 275 nm while 7-F-β-T3 at 395 nm, 290 nm and 283 nm. Numerous
researches confirmed that these results showed no difference from the spectroscopic
characters of aldehydic oxidation products of α-tocopherol. Interestingly, in
agreement with 5-F-γ-T3, the compound eluted at 9.43 min in our study showed UV
absorption at 275 nm. Accordingly, it was proved compound eluted at 9.43 min as
5-F-γ-T since 5a position was more active to form oxidation products.
New oxidation products. In addition to the oxidative products reported in early
researches, one peak showed up which arouse our much interest. The compound
+
eluted at 12.3 min (compound I) possessed an ion peak [M+H] at m/z 473.415. As
mentioned above, 5-F-γ-T showed a mass higher than the molecular ion of α-T by
m/z=14, since methyl group on 5a position was oxidized and then substituted by an
aldehyde group. By that analogy, I was considered as ‘derivatives’ of 5-F-γ-T,
presented masses 28 Da higher than 5-F-γ-T, which were exactly twice of m/z=14,
suggesting the existence of two aldehyde groups in its structure. That means α-T was
probably deeply oxidized and other two methyl groups on 7a position and 8b position
were further substituted by corresponding aldehyde groups. Putative structural
formula was given in Fig.6. Fragments received from MS spectrum confirmed the
above assumption as well. I possessed a vital fragment ion at m/z 207.106, which
indicated the cleavage of the chromanol ring structure as well as the loss of a
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CH C≡CH fragment (17), this fragmentation pattern was the same with that of α-T
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and 5-F-γ-T. In addition, I presented a fragment at m/z 165.098, which was formed by
an additional loss of an aldehyde group and with the loss of an oxygen, it exhibited
ions at m/z=149.030. Apart from these ions, ions at m/z 414.269 might due to the
successive loss of two aldehyde groups. To the best of our knowledge, previous
studies have never reported this compound. We acknowledged that the formation of
compound I in our research was so tiny that more work need to identify its structure
further, including NMR (nuclear magnetic resonance) or FTIR analysis.
Analysis of α-tocopherol degradation and oxidation products formation in
the heating of pure α-T. α-T is influenced by many environmental elements,
especially easy to degrade under high temperatures. Several studies considered the
degradation of α-tocopherol in heated oils as first-order kinetics (34, 35). We
compared the peak area of each compound and focused on how each component
increased and decreased during the entire heating process. As shown in Fig.7, the
content of α-T followed a rapid loss and approximately half of the initial α-T
degraded during the first 1h, then it kept steady linear decreasing from 1h to 8 h.
Under the heating conditions, α-T was almost no more detectable in the heated
samples after 8 h. Along with steady decrease of α-T, α-TQ generated and its levels
increased in the first two hours, and then decreased almost 25% during the third hour.
It decreased sharply after heating 8 hours and the same tendency was found in
5-F-γ-T. Apparently, at the beginning of heating, α-T mainly degraded into both α-TQ
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and 5-F-γ-T, afterwards, they were not stable upon prolonged heating. Researches
proved that α-TQ formed and decomposed along several pathways (36), the
subsequent reactions such as the degradation of oxidative intermediates were also
assumed to follow first-order kinetics, and the formation and degradation of oxidation
products were considered as non-reversible processes (37). These researches indicated
α-TQ might further degraded into other oxidation products. Unknown compound with
molecular weight at 472.319 (compound I) made up low proportion and its amounts
remained steady with little fluctuation in the first 8 hours. At the end of heating, it
generally increased while the other compounds decreased during heating. The
potential reaction pathway leading to the formation of α-T oxidation products was
shown in Fig.8. It is proposed that main oxidation products were α-TQ and 5-F-γ-T at
the first heating period, along with remarkable decomposition of α-T. With the
prolonging of heating, both α-TQ and 5-F-γ-T degraded into compound I over time
until vanished.
Analysis of α-Tocopherol degradation and oxidation products formation in
the heating process of camellia oil. The proposed method was applied to determine
oxidation products of α-T in thermal-oxidative oil matrices. In order to maintain the
original state of refined oil, camellia oil was used in our research, which only
contained α-tocopherol, excluding interference from other tocopherols. It turned out
that the sole thermal oxidative product of α-T in camellia oil was α-TQ. The results
are in agreement with other researches (27, 28), which confirmed that α-T finally
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degraded during heating of oils to form α-TQ. Moreover, numerous studies showed
that tocopheryl quinone is one of the final oxidation products of tocopherols. Kreps et
al. proved that high temperature microwave heating, simulating pan-frying and
deep-fat frying process merely conducive to the formation of tocopheryl quinones in
both sunflower oil and rapeseed oil (16, 37). Previous studies accounted for this result
that the oxidation products existed in rather low concentrations and made
measurement difficult (27). Camellia oil used in our research only contained 120
mg/kg α-T, the concentration might be too low to form much oxidation products, and
the concentration of other oxidation products might under the limit of detection,
except for α-TQ. Moreover, it was partly caused by problems associated with
complexity of oil matrices, the abundance of some oil components could interfere
with oxidation products (3). As mentioned above, oxidative intermediates of α-T
easily degraded during heating, the formation and degradation of oxidation products
were non-reversible, which also made the intermediate processes fast and difficult to
capture.
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The contents of α-TQ were expressed as percentages relative to the initial peak
area of α-TQ at 1 h and the contents of α-T were expressed as percentages relative to
the initial peak area of α-T at 0 h. As shown in Fig.9 A, α-T degraded slowly during
the first 3 hours and only lost 10% of the initial content. This differed from the
heating of pure α-T. Lipid matrices were complex, containing many factors that might
synergize or antagonize the metabolic process of α-T. For instance, the differences in
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degree of saturation of the oils (38), and natural antioxidant content of other sterols
and phenolic compounds, leading to complex analytical issues (3). After heating for 4
h, α-T decreased sharply, which exactly corresponded to the quick increase of the
content of α-TQ (Fig.9 B). α-T remained decreasing till undetectable after 24 h. On
the contrary, α-TQ appeared a rising tendency in the primary 8 hours and then
decreased with longer heating time. One reason might be that only 50% α-T was
remained after 8 h, the generation of α-TQ was rather low in comparison with its
decomposition. The other reason for the decrease of α-TQ was that it was unstable at
high temperatures (28) and might degrade into other unknown oxidation products
(21).
In this work, a new method based on UPLC-Q-TOF-MS was developed for the
simultaneous determination of α-T and its oxidation products, which is more
convenient, time-saving and avoid the use of toxic organic solvents. In traditional
methods, isolation and extraction procedures were needed before HPLC analysis,
which cost large amount of organic solvents such as hexane and methanol (27, 28).
Furthermore, in comparision with the results of Murkovic (28), the elution time of α-T
was about 8 minutes shorter while α-TQ was about 12 minutes shorter, which was
more time-saving. The method showed a good separation and high accuracy and
successfully identified three oxidation products, among which two compounds were
α-TQ and 5-F-γ-T, one was a new compound with molecular weight at 472.4
(compound I). After heating, α-T firstly degraded into α-TQ and 5-F-γ-T, and then
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turned into compound I. This work may give a way to explain the mechanism of
thermal oxidative process of α-T. Further work is necessary to figure out the structure
of compound I, thus the identification of α-T oxidation product will be accomplished.
00
01
402
ABBREVIATIONS USED
403
404
405
406
407
408
409
410
UPLC-Q-TOF-MS, ultra performance liquid chromatography coupled with
quadrupole time-of-flight mass spectrometry; ESI, electrospray ionization source;
α-T, α-tocopherol; α-TQ, α-tocopheryl quinone; 5-F-γ-T, 5-formyl-γ-tocopherol;
7-F-β-T, 7-formyl-β-tocopherol; 5-F-γ-T3, 5-formyl-γ-tocotrienol; 7-F-β-T3,
E
7-formyl-β-tocotrienol; MS , DDA, two acquisition modes of mass spectrometry; FD,
DAD, ELSD, traditional detectors of GC and HPLC; HPLC, high-pressure liquid
chromatography; GC, gas chromatography; SFC, supercritical fluid chromatography;
FTIR, fourier transform-infrared spectroscopy; NMR, nuclear magnetic resonance.
411
AUTHOR INFORMATION
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413
414
415
416
417
418
Corresponding Author
*E-mail: jwli@jiangnan.edu.cn
Funding sources
This work was supported by the Natural Science Foundation of China (31871840) and
National Key R&D Program of China (2016YFD0401404).
Conflict of interest
The authors declare no competing financial interest.
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Figure 1. UPLC chromatograms of unheated and heated (4h at 180°C) samples with
PDA detector at 275 nm for α-T oxidation products and 295 nm for α-T. (A:
4h-heated sample, recorded at 275 nm; B: unheated sample, recorded at 275 nm; C:
4h-heated sample, recorded at 295 nm; D: unheated sample, recorded at 295 nm.).
Figure 2. Total ion current chromatograms of unheated and heated (4 h at 180°C)
samples by UPLC-Q-TOF-MS in positive mode. (A: 4h-heated sample; B: unheated
sample.).
Figure 3. The fragmentation pathways, extracted ion chromatogram and Q-TOF-MS
spectra of α-tocopherol in positive mode. (Retention time: 11.4 min; A, full-scan MS
spectrum; B, MS/MS spectrum of precursor ions at m/z 430.4.).
Figure 4. The fragmentation pathways, extracted ion chromatogram and Q-TOF-MS
spectra of α-tocopheryl quinone in positive mode. (Retention time: 9.88 min; A,
full-scan MS spectrum; B, MS/MS spectrum of precursor ions at m/z 447.4.).
Figure 5. The fragmentation pathways, extracted ion chromatograms and Q-TOF-MS
spectra of 5-formyl-γ-tocopherol in positive mode. (Retention time: 9.43 min. A,
full-scan MS spectrum; B, MS/MS spectrum of precursor ion at m/z 445.4.).
Figure 6. The proposed structural formula, fragmentation pathways, extracted ion
chromatograms and Q-TOF-MS spectra of compound eluted at 12.3 min in positive
mode. (Retention time: 12.3 min. A, full-scan MS spectrum; B, MS/MS spectrum of
precursor ions at m/z 473.4.).
Figure 7. Degradation of α-T and formation of oxidation products in pure α-T.
Figure 8. Proposed reaction pathway leading to the formation of α-T oxidation
products.
Figure 9. Degradation of α-T (A) and formation of α-tocopheryl quinone (α-TQ) (B)
in camellia oil. (Regarded initial peak area of α-T at 0h as 100%, the others expressed
as relative content in Fig.9 A; Regarded initial peak area of α-TQ at 1h as 100%, the
others expressed as relative content in Fig.9 B)
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