Photostability of alpha-tocopherol ester derivatives in solutions and liposomes. Spectroscopic and LC-MS studies

Article abstract of DOI:10.1016/j.jphotobiol.2016.03.032

α-Tocopherol (Toc) is known to degrade to the tocopheroxyl radicals (Toc?) by exposure to UV light irradiation. In the present study, the stability of Toc ester derivatives exposed to UV light was investigated and compared with Toc in organic solution and in phospholipid vesicles. To follow the depletion of Toc and its esters the absorbance and fluorescence methods were applied whereas degradation products were detected using LC-MS method. The irradiation with UVB light of air-equilibrated solutions of di-α-Tocopheryl malonate (DTMO), α-Tocopheryl malonate (TMO) and α-Tocopheryl succinate (TS) strongly modifies their absorption and fluorescence spectra. Upon UVB irradiation, absorption band at 279/285 nm becomes less pronounced indicating the photodegradation of esters. During irradiation, the fluorescence maximum of esters at 305 nm shifts to 326 nm, a maximum characteristic for Toc. Photorecovery of Toc from its esters derivatives was finally confirmed by LC-MS method. Among studied esters, only α-tocopheryl nicotinate (TN) did not undergo depletion and appeared resistant to UVB radiation. Kinetic studies indicated that photoinduced transformation occurs through the first order consecutive reaction chain mechanism. The photodissociation of Toc esters in the liposomes occurred with one order of magnitude slower than in organic solvents. Using MS/MS method it was found that final stable product of irradiation was α-tocopheryl quinone (TQ), an animal and plant metabolite of Toc.

Full text of DOI:10.1016/j.jphotobiol.2016.03.032

Photostability of alpha-tocopherol ester derivatives in solutions and liposomes.
Spectroscopic and LC–MS studies
Grazyna Neunert, Artur Szwengiel, Piotr Walejko, Stanislaw Witkowski,
Krzysztof Polewski
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DOI:
Reference:
S1011-1344(15)30006-3
doi: 10.1016/j.jphotobiol.2016.03.032
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17 August 2015
23 March 2016
23 March 2016
Please cite this article as: Grazyna Neunert, Artur Szwengiel, Piotr Walejko, Stanis-
law Witkowski, Krzysztof Polewski, Photostability of alpha-tocopherol ester deriva-
tives in solutions and liposomes. Spectroscopic and LC–MS studies, (2016), doi:
10.1016/j.jphotobiol.2016.03.032
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Photostability of alpha-tocopherol ester derivatives in solutions and liposomes.
Spectroscopic and LC-MS studies.
Grazyna Neunert1*, Artur Szwengiel2, Piotr Walejko3, Stanislaw Witkowski3, Krzysztof
Polewski1
1Department of Physics and Biophysics, Poznan University of Life Sciences, ul. Wojska Polskiego
38/42, 60-637 Poznan, Poland
2Institute of Food Technology, Poznan University of Life Sciences, ul. Wojska Polskiego 38/42,
60-637 Poznan, Poland
3Institute of Chemistry, University of Białystok, ul.Ciolkowskiego 1k, 15-245 Białystok, Poland
Abstract
-Tocopherol (Toc) is known to degrade to the tocopheroxyl radicals (Toc•) by
exposure to UV light irradiation. In the present study, the stability of Toc ester derivatives
exposed to UV light was investigated and compared with Toc in organic solution and in
phospholipid vesicles. To follow the depletion of Toc and its esters the absorbance and
fluorescence methods were applied whereas degradation products were detected using LC-MS
method.
The irradiation with UVB light of air-equilibrated solutions of di--Tocopheryl
malonate (DTMO), -Tocopheryl malonate (TMO) and -Tocopheryl succinate (TS) strongly
modifies their absorption and fluorescence spectra. Upon UVB irradiation, absorption band at
279/285 nm becomes less pronounced indicating the photodegradation of esters. During
irradiation, the fluorescence maximum of esters at 305 nm shifts to 326 nm, a maximum
characteristic to Toc. Photorecovery of Toc from its esters derivatives was finally confirmed
by LC-MS method. Among studied esters, only -tocopheryl nicotinate (TN) did not undergo
depletion and appeared resistant to UVB radiation. Kinetic studies indicated that
photoinduced transformation occurs through the first order consecutive reaction chain
mechanism. The photodissociation of Toc esters in the liposomes occurred with one order of
magnitude slower than in organic solvents. Using MS/MS method it was found that final
stable product of irradiation was -tocopheryl quinone (TQ), an animal and plant metabolite
of Toc.
Keywords: -tocopherol, UVB radiation, -tocopherol esters, absorption, fluorescence.

* Author for correspondence: e-mail: neunert@up.poznan.pl, Phone: +48618487496, Fax:
+48618487495
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1. Introduction
-Tocopherol (Toc), except its antioxidant function, fulfills a number of biochemical
and biological functions in the cell. However, Toc is unstable under the influences of light
and air. It has been shown that Toc under UV light irradiation is easily decomposed to the
tocopheroxyl radicals (Toc•) [1, 2]. Several studies have shown that photooxidation processes
lead to loss of antioxidant activity of vitamin E [3-5]. Such photochemical generation of free
Toc• by UV light is often a large problem for biological systems containing vitamin E
because Toc• is supposed to initiate and accelerate the lipid peroxidation (prooxidation) [6].
Reported studies have shown that during irradiation of Toc depending on the range of
light several products may be formed. Irradiation of Toc in methanol and hexane under 366
nm lead to its degradation with first order kinetics [7]. Such process was attributed to
formation of methoxy radicals, hydrogen peroxide formation and conversion of Toc to
phenoxy radicals. The another research has shown that during irradiation of tocopherol in
acetonitrile the formation of Toc• and its cationic form were observed whereas in methanol
only formation of Toc• occurred [8]. UVB (280-320 nm) irradiation of thin films of free Toc
on Petri dish yielded a mixture of dihydroxydimer, spirodimer, and spirotrimer [9]. Irradiation
under sunlight and UV light ( > 290 nm) of Toc in olive oil and hexane lead to its depletion
with a few products and two of them were identified by HPLC as 5-formyl-γ-tocopherol and
-tocopheryl quinone (TQ) [10]. Irradiation with UVB of γ-Toc in acetonitrile/H2O solution
and in soy phosphatidylcholine (PC) and dipalmitoyl phosphatidylcholine (DPPC) liposomes
lead to formation of photoproducts which include TQ, epoxyquinones, hydroperoxy-
tocopherones, Toc• and spirodimers [11]. TQ and its reduced form, -tocopheryl
hydroquinone (-TQH2), are found among oxidation products of Toc in both animal and
plant metabolisms [12, 13].
Commercially, vitamin E supplements contain mostly esterified forms e.g. acetate,
succinate or nicotinate. Toc succinate and acetate do not function as antioxidants, but can
serve as a source of a free Toc upon deesterification by the proper enzymes. Vitamin E and its
esterified forms can be exposed to sunlight or artificial UV light in several processes like skin
under sunlight [14, 15], preparation or refinement of foods [16] or in many processes in
pharmaceutical or cosmetic industry [17, 18]. So, the study on irradiation process of the
commercial forms of vitamin E may provide some knowledge about the role of exposure to
light influences the stability of the esterified form of vitamin E.
As mentioned above, several studies of photodegradation processes of Toc were
presented in the literature, but more detailed studies devoted to UV irradiation of tocopherol
esters are rather scarce. Published studies were mostly focused on irradiation of topically
applied esters, acetate or succinate, and its protection effect in photocarcinogenesis caused by
UV irradiation [14, 17, 19]. The results showed that the esters are not cleaved when applied to
dermis and epidermis. The process of bioconversion to free Toc when the acetate was
topically applied and irradiated with UVB was reported [19]. However, according to the
author's interpretation this process was not connected with irradiation. The photostability of
Toc acetate was studied by treating ethanol solution with 254 nm or 366 nm light [20].
Decrease of native fluorescence was observed during UVC (200-280 nm) irradiation whereas
applied UVA (320-400 nm) did not lead to degradation of Toc acetate.
To determine the photostability of Toc derivatives, we investigated the influence of
the UV light on the physicochemical properties of two novel esters: di--Tocopheryl
malonate (DTMO) and -Tocopheryl malonate (TMO), and synthesized -Tocopheryl
succinate (TS) as well as -tocopheryl nicotinate (TN) (Fig. 1). The effects of irradiation of
Toc esters were observed in organic solvents as well as in lipid membranes and compared
with native Toc. To follow the fate of Toc and its esters the absorbance and fluorescence
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methods were applied whereas detection of the degradation products was performed with LC-
MS method.
2. Materials and methods
2.1. Materials
Egg yolk phosphatidylcholine (PC), dipalmitoyl phosphatidylcholine (DPPC), 1,1-
diphenyl-2-picrylhydrazyl radical (DPPH), chloroform and methanol were purchased from
Sigma/Merck (Germany). Toc, TMO, DTMO, TN and TS were synthesized according to
procedures presented in [21, 22]. Ethanol, hexane, n-octane and acetonitrile were purchased
from POCh (Poland). All solvents were of spectroscopic grade. The ultra pure water from
MicroPure Water System manufactured by TKA (Germany) was used in all aqueous
solutions.
Fig. 1. Chemical structures of studied compounds.
2.2. Methods
2.2.1. Preparation of vesicles
Large unilamellar vesicles (LUV) with the incorporated Toc esters were prepared by
the extrusion method. The details of this procedure are given elsewhere [23]. Shortly, Toc and
its esters were dissolved in methanol to give a stock solution. For liposomes preparation of
dry DPPC or PC and esters were dissolved in chloroform and methanol, respectively, mixed
in different proportions and then the solvent was evaporated. The formed film was hydrated
with 0.1 M phosphate buffer (pH 7.4) and vortexed for 10 min. The resulting liposomal
suspension (0.08 mg/ml phospholipid final concentration) was extruded using a LiposoFast
Basic LF-1 extruder (Avestin Europe GmbH, Mannheim, Germany) with 100 nm pore
diameter.
The mean particle diameters of the LUV suspension was measured using method of
dynamic light scattering with Zetasizer Nano (Malvern Instruments, Worcestershire, UK)
[24]. The mean values of DPPC and PC liposome sizes: 120 and 105 nm, respectively, were
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determined from the liposomal size distribution histograms and were not changed
significantly in the present of studied compounds.
2.2.2. UVB irradiation
The sample was irradiated with a Xenon lamp (50 W). Irradiation treatment was
carried out at either 290 nm for Toc and at 280 nm for the esters (UVB) or at 320 nm for all
studied compounds (UVA). The intensity used in the experiment was 14 and 20 mW/cm2 of
UVB and UVA, respectively, as measured by a research radiometer (LM2, Carl Zeiss Jena,
Germany). During irradiation, the solution was gently mixed with a magnetic stirrer. All
samples were kept at 25 °C during exposure.
After various times of irradiation indicated in the text the cuvette was removed from
the UV beam to measure absorption and emission spectra. The sample was then brought back
to the irradiator, and the cycle was repeated until the spectrum of the sample did not change
noticeably between two successive irradiations.
2.2.3. Absorbance, fluorescence and fluorescence lifetimes measurements
Absorption spectra were measured with a Shimadzu UV 1202 spectrophotometer
(Shimadzu, Kyoto, Japan) with a spectral resolution of 1 nm. Steady-state fluorescence
emission spectra were recorded on a Shimadzu RF 5001PC spectrofluorometer (Shimadzu,
Kyoto, Japan) with a spectral resolution of 1 nm. Fluorescence lifetime measurements were
carried out with a TimeHarp 100 PC-board (PicoQuant, Berlin, Germany) for time-correlated
single photon counting (TCSPC) with 72 ps/channel resolution. The excitation source was a
sub-nanosecond pulsed UV LED 290 with maximum emission centered at 290 nm and a 0.7
ns wide pulse with full width at half maximum (FWHM), powered by a PDL 800-D driver.
The emission was detected by a PMA 182 photon sensor head (all the instruments from
PicoQuant, Berlin, Germany). The data were analyzed by an exponential reconvolution
method using a non-linear least square fitting program. Optimum fitting with minimisation of
the residuals was confirmed using a Chi-squared value χ2 < 1.4. All spectroscopic
measurements were performed using a 1 or 0.3 cm path length quartz cuvette at 22 ºC.
2.2.4. LC-MS analysis
Reversed-phase (C18 column) ultra high-performance liquid chromatography
electrospray ionization mass spectrometry (RP-UHPLC-ESI-MS) analysis was performed
using a Dionex UltiMate 3000 UHPLC (Thermo Fisher Scientific, Sunnyvale, CA) coupled to
a Bruker maXis impact ultrahigh resolution orthogonal quadrupole-time-of-flight accelerator
(qTOF) equipped with an ESI source and operated in positive-ion mode (Bruker Daltonik,
Bremen, Germany). The RP chromatographic separation was achieved with a Kinetex™ 1.7
µm C18 100 Å, LC column 100 × 2.1 mm (Phenomenex, Torrance, CA). The ESI-MS settings
were as follows: capillary voltage 4500 V, nebulizing gas 1.8 bar, and dry gas 9 L/min at 200
°C. The scan range was from mass-to-charge ratio (m/z) 80 to 1200. The mobile phase was
composed of methanol containing 0.5% of formic acid. The flow rate was 0.2 mL/min with a
isocratic elution. The sample injection volume was 2 µL. The column temperature was set at
40 °C. The ESI-MS system was calibrated using sodium formate clusters introduced by loop-
injection at the beginning of the LC-MS run. The LC-MS data were processed using Data
Analysis 4.1 software (Bruker Daltonik, Bremen, Germany). Molecular ions [M+H]+ were
extracted from full scan chromatograms and peak areas were integrated. The extraction
window of individual ion chromatograms was ± 0.05 m/z units. The compounds present in
each sample were identified by comparing their retention times with those of standards and
molecular mass and structural information from the MS detector.
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2.2.5. Data analysis
The Bruker Compass Profile Analysis 2.1 application (Germany) was used to analyze
LC-ESI experiments. The compounds were calculated by the Find Molecular Features (FMF)
algorithm, the fully defined compounds in a complex sample by combining retention time,
mass and quantitative information were generated as bucket table. The resulting compound
data were served as input data for statistical calculation. The bucket table was treated as
fingerprint for sample only the most abundant components were identified. Principal
component analysis (PCA) technique was used to dimensionality reduction of data to present
the samples in new coordinate system. The statistical analysis was performed using Statistica
software, Version 10, StatSoft Inc., OK-US. The most abundant compounds were identified in
MS/MS experiments. The SmartFormula3D (Bruker, Bremen, Germany) command was used
to calculate possible molecular formulae for a MS precursor ion by combining two spectra
MS and MS/MS. Proposed sum formulae of precursor ions were stored if at least one formula
for a fragment ion in combination with an explanation for the neutral loss exists.
2.3. Statistics
The results are presented as means ± standard deviation (SD) from three replicates.
Analyzed groups were compared using the analysis of variance (ANOVA) and determined by
the Tukey's post-hoc test at p<0.05 with the assistance of Statistica 10.0 (StatSoft, Tulsa, OK,
USA) software.
3. Results and Discussion
3.1. UV irradiation of Toc and its esters
It has been reported that tocopherols and its esterified forms absorb in the UVB range
[25, 26]. In methanol Toc has a main absorption band at 293 nm, while its esterified form
with TS showed an absorbance maxima at 279 nm and 285 nm (see Fig. 2 and Fig 3,
respectively). Our results indicate (data not shown) that the absorption spectra of the vitamin
E and its ester derivatives treated with UVA light did not show any change. Similar
observation were presented in the literature for Toc acetate [20, 27]. In contrast, the
spectroscopic properties of studied compounds changed considerably upon UVB illumination.
The absorption spectra of Toc and TS in methanol exposed to constant UVB taken at
different irradiation times are shown in Fig. 2 and Fig. 3, respectively. It could be seen, that
the characteristic absorption bands of Toc and TS decrease, indicating a degradation of
investigated compounds. Simultaneously, the absorbance increase is observed in the range
240-270 nm, which may be attributed to a one or a few products associated with Toc
oxidation which include TQ, epoxyquinones, hydroperoxy-tocopherones, Toc• [11] or
spirodimers [9]. As indicated by decreasing intensity of Toc characteristic peaks at 293 nm
the prolonged irradiation leads to total disappearance of Toc and increasing absorbance with
maximum at around 250 nm. Additionally, in the case of TS, the absorbance increase is
observed in the range 290-310 nm, suggesting formation of Toc since the other compounds
participating in this experiment do not absorb beyond 295 nm. The experiments carried out
for TS in a quartz cuvette of 0.3 cm path length, with absorption spectra taken every 10 min
of irradiation shown in Fig. 3B, revealed the shift of absorption maximum from 285 nm to
287 nm and absorption spectrum extending up to 320 nm. Beyond, the formation of maxima
at 255 and 266 nm are observed indicating on the presence of TQ arising from previously
formed Toc, what was finally confirmed by LC-MS method (see section below). Observed in
Fig. 3B absorbance progressing with irradiation reflects simultaneous processes of
photodegradation of TS, formation of Toc with its subsequent degradation and formation of
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TQ. Similar qualitative and quantitative spectral changes in series of experiments carried out
in other solvents and for DTMO and TMO were observed. Among investigated esters only
TN did not undergo decomposition under applied irradiation.
Fig. 2. Successively recorded absorption spectra of 25 M Toc in methanol irradiated at 0, 10,
21, 33, 45, 55, 67 and 82 min with 290 nm. Inset: The absorbance kinetics plotted as a
function of irradiation time, the positions of wavelengths are given in the legend.
Fig. 3. Absorption spectra of 25 M TS in methanol recorded at different irradiation
intervals with 280 nm. (A) Absorption spectra recorded in a 1 cm path length cuvette at 0, 20,
40, 60, 80, 100, 130, 150, 180, 210 and 230 min of irradiation. Inset: Absorbance kinetics of
TS plotted as a function of irradiation time, the positions of wavelengths are given in the
legend. (B) Absorption spectra recorded in a 0.3 cm path length cuvette at 0, 10, 20, 30, 40,
50, 70, 90 min of irradiation.
During irradiation, the isosbestic points at 230 and 274 nm for Toc, and at 234, 274
and 290 nm for TS were observed. These isosbestic points might indicate on complexity of
photoprocesses occurring during irradiation rather than direct conversion from TS to Toc and
TQ. Isosbestic point is a point on absorption spectrum where at least two chemical species
have identical molar absorption coefficients, which remain constant as reaction proceeds. This
formally evidences that reaction is proceeding without forming an intermediate or multiple
products. However, our findings of multiple isosbestic points during TS and Toc
phototransformation are not consistent with known evidences indicating that oxidation or
photooxidation of Toc occurs through consecutive reaction chain starting from chromanoxyl
radical to form different stable products depending on physical and chemical parameters of
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reaction environments as observed in Fig. 3B. Thus, in this case interpretation of isosbestic
point as an indicator of directly formed product does not hold.
3.2. Kinetic considerations
The progress of phototransformation of Toc or its esters was followed by measuring
the changes of absorbance maximum. Kinetic traces obtained during Toc irradiation plotted at
characteristic absorption maxima are given in inset Fig. 2. It shows that in irradiated solution
Toc disappears exponentially according to the first order kinetics yielding some
photoproducts. The plot of absorbance changes at 293 nm may be well fitted to a single
exponential function with photodissociation rate constant k = 3.83*10-4 s-1. Longtime lasting
experiments also showed exponential increase at 250 nm ascribed to TQ.
Kinetic traces of irradiated TS are given in inset Fig. 3A. It shows that disappearance
of TS occurs slowly with simultaneous absorbance increase at 250 and 300 nm ascribed to TQ
and Toc, respectively. For the first 120 min of irradiation, Toc and TQ kinetics show
exponential dependence indicating on the first order processes. In case of UVB generated Toc
kinetic after 120 min of irradiation, slow disappearance shows up. No interference from the
other compounds in this spectral range indicates that observed decrease is real. From the
presented kinetics, we may notice that formation of TQ is related with degradation of Toc.
The spectra taken every minute at beginning of irradiation (data not shown) indicate no lag
period for TQ formation what suggests that phototransformation from Toc to TQ occurs
instantly. Similarly, it shows that during irradiation of TS formation of Toc occurs
immediately with further consecutive reactions where undetectable transient products exhibit
fast transformation rates.
Differently from Toc, for TS absorption changes at 285 nm can be fitted to a linear
function. Similar linear decays of absorbance intensity were observed for other studied esters.
Such process very probably occurs due to overlapping with spectra of other species formed
during irradiation. Calculated rate constants of phototransformation for all tested esters in
different solvents are collected in Table 1.
Table 1. Photodissociation rate constants k [M/s]*10-6 of Toc esters calculated from absorption
data (means ± SD).
Solvent
TS
DTMO
TMO
Methanol (protic, polar)
Ethanol (protic, polar)
Hexane (aprotic, non-polar)
n-Octanol (protic, non-polar)
Acetonitrile (aprotic, polar)
2.26 ± 0.12
1.73 ± 0.10
3.93 ± 0.15
2.10 ± 0.13
4.66 ± 0.22
11.60 ± 0.11
11.10 ± 0.15
–
–
–
–
–
17.01 ± 0.24
–
–
It can be found that the fastest photodecomposition occurs for DTMO, followed by
TMO and TS. It is known, that the relationship between the structure and the function of the
terminal ester moiety of Toc play a significant role in the action of Toc esters [28 - 30]. Our
findings indicate that modification of individual functional moieties of Toc analogues (in this
case the length of the ester side group) also changes resistance to the UVB light. Interestingly
nicotinate derivative having similar absorption spectra does not undergo changes under
irradiation.
From Table 1 we may also notice that the rate constants of the photodissociation of
Toc esters are higher in aprotic solvents compared to protic ones. This may be important
regarding the fact that tocopherols are located in non-polar part of biological membranes. It
was shown that in polar protic solvents oxidation lead to different products and pathways
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compared to oxidation in inert polar aprotic solvents [31, 32]. Such difference arises from
increased electron mobility in protic solvents which finally leads to formation of quinone type
products [31].
From presented kinetics, it can be concluded that photodegradation of Toc and TS
occur via consecutive reaction mechanisms with TQ as a long term irradiation end-product.
Physical mechanism arises from fact that irradiation at absorption maximum with high-energy
radiation at 280/290 nm leads to efficient excitation of molecule and detachment of ester
moiety or in case of Toc formation as first intermediate of Toc• [11]. Calculated energy for
280/290 nm equals to 4.4 eV or 410 kJ/mol. This energy is comparable or higher to that of
heat formation as calculated for the acetate [33].
3.3. Fluorescence studies during irradiation
Intrinsic fluorescence of Toc and its esters was used possible to apply this technique to
follow progress of their decomposition. Fig. 4A presents changes of emission spectra of Toc
in methanol during irradiation. They show decreasing fluorescence intensity whereas shape
and maximum of the band located at 326 nm remains unchanged. After 80 min of irradiation,
the spectrum decreased to barely recorded level what indicates on disappearance of Toc in
this sample. The kinetics of Toc disappearance followed monoexponential decay with rate of
13.1 s-1 (inset Fig. 4A). The measured fluorescence lifetime for Toc sample without light
treatment was 1.2 ns [34] and did not change during UVB irradiation.
Fig. 4B presents TS fluorescence spectra in methanol during irradiation. It shows that
TS characteristic fluorescence maximum at 305 nm slowly increases with simultaneous shift
towards longer wavelength that after 40 min established at 326 nm and remained constant
during further irradiation. In this case, the isosbestic point at 308 nm indicates that
transformation from TS occurs via unknown precursor to Toc since no other fluorescent
species in the sample were detected. During up to 180 min of irradiation the fluorescence
intensity observed at 326 nm increases. Further irradiation resulted in decreasing fluorescence
intensity, inset Fig. 4B. Single fluorescence lifetime for TS in methanol of 3.4 ns was
obtained whereas after 180 min of irradiation the second shortest lifetime component was
noted, which value of 1.2 ns is identical with that obtained for original sample of Toc. Such
results revealed three processes occurring simultaneously, i.e. photodecomposition of TS
leading to formation of Toc with further Toc decomposition.
The spectral changes and kinetics of TS in the other investigated solvents, i.e. ethanol,
hexane, n-octanol, acetonitrile were similar to those obtained in methanol. The other
investigated esters shown results compatible with those obtained for TS.
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Fig. 4. Successively recorded emission spectra of 25 M Toc and TS in methanol during
irradiation. (A) Fluorescence spectra of Toc recoded after 0, 10, 21, 33, 45, 55, 67 and 82 min
of irradiation, excited at 290 nm. Inset: kinetic of peak fluorescence intensity. (B)
Fluorescence spectra of TS recorded at 0, 20, 40, 60, 80, 100, 130, 150, 180, 210 and 230 min
of irradiation. The excitation wavelength was 280 nm. Inset: kinetic recorded at spectra
maxima.
3.4. Photoproducts separation and determination with LC-MS
The LC-ESI-MS base peak chromatograms (BPCs) of Toc and TS before and after
UVB exposure were presented in Fig. 5. The main products of UVB radiation were
characterized during MS and MS/MS experiments, Fig. 6. TQ was formed after UVB
exposure of Toc. Additionally, it was observed that the Toc and TQ were also formed during
TS UVB radiation. The quality differences between peaks of Toc and TQ were computed on
basis of retention time (Fig. 5) and results of MS/MS experiments (Fig. 6).
Fig. 5. Typical LC-ESI-MS base peak chromatograms (BPCs) (A) Toc before (the continuous
line) and after (the dotted line) UVB exposure, (B) TS before (the continuous line) and after
(the dotted line) UVB exposure; the BPCs demonstrate the elution of Toc, TQ and TS peaks
at 181, 200, 160 s respectively.
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Fig. 6. Typical LC-ESI-MS mass spectrum of molecular ions for Toc: [M+H-H]+ (m/z 430),
[M+H-2H]+ (m/z 429) (A) and MS/MS spectra of [M+H-2H]+ ion (B, C – low and high m/z
range); mass spectrum of molecular ions for TQ [M+H-H2O]+ (m/z 429) (D) and MS/MS
spectra of [M+H-H2O]+ ion (E, F – low and high m/z range).
The precursor ion at 429 m/z from MS runs in both cases were selected (Fig. 6A, D).
The protonated fragments of Toc precursor ion (Fig. 6C) are presented in Fig. 6B (C13H17O2
at 205 m/z and C10H13O2 at 165 m/z). It was observed that ion at 430 m/z is not isotopic ion of
429 m/z – when the ion at 429 m/z was typed as the precursor for MS/MS experiment, the
signal of ion at 430 m/z was constant (comparing of Fig. 6A and 6C). This proves that Toc
generates two of molecular ions, namely [M+H-H]+ at 430 m/z and [M+H-2H]+at 429 m/z.
The formula C29H50O2 have been identified by SmartFormula3D algorithm command for Toc
during procedure of calculating possible molecular formulae for a MS precursor ion by
combining two spectra MS and MS/MS. The fragments C13H17O2 at 205.12 m/z, C10H13O2 at
165.09 m/z and C11H15O1 at 163.11 m/z (Fig. 6E) during MS/MS experiment for peak at 200 s
(Fig. 5) were generated. The formula C29H49O2 have been identified by SmartFormula3D
algorithm command for this peak, thus indicated that [M+H-H2O]+ is the molecular ion
formula for this peak and it is a TQ [35].
3.5. UVB irradiation of Toc and its esters in PC and DPPC liposomes
In biological cells Toc is located in cell membrane thus we carried out the UVB
irradiation experiments with Toc and its esters embedded into PC and DPPC liposomes as a
model of biological membranes. The absorption and fluorescence spectra of TS in PC and
DPPC liposomes resembled that obtained for TS in methanol solution, see Fig. 4b. The rise of
the absorption band around 290-310 nm and simultaneously decreasing peaks at 279 nm and
285 nm with irradiation time were observed (data not shown). Decreasing of TS characteristic
peaks is much slower and calculated rate constants for photodissociation of TS in PC/DPPC
equals to k = 0.010*10-4 M/s that is a half of that value in methanol. This same was observed
for Toc in PC with k = 0.47*10-4 s-1 compared to 3.83*10-4 s-1 in methanol. During irradiation
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of TS the maximum emission at 307 nm shifted towards longer wavelength of 325 nm as
observed in methanol. However, this increase was smaller compared with that reported in the
homogeneous solutions.
According to Ohara decreasing yield of the Toc and TS photodecomposition in the
liposome system might be explained by lower transmittance due to light-scattering of the
clouded solutions [36]. Toc and investigated esters are located in liposomal matrix which
could acts as shield and trap for the part of radiation. The measured transmittance of our
liposomal solution at 280 nm and 290 nm was 70% and 76%, respectively. Thus, we may
assume that light-scattering is one of main cause of decreased photodecomposition. We have
also noticed that calculated rate of decomposition in liposome solutions do not depend on type
of ester moiety attached to the chromanol ring. This suggests that location of the esters in
bilayer does not influence the rate of photodecomposition. Based on the presented results we
conclude that the photodissociation process of all Toc esters investigated in the liposomes is
similar to those observed in the homogeneous solution.
4. Conclusions
Direct absorption of UVB radiation by Toc ester leads to its decomposition and further
formation of free Toc, which finally transforms into biologically active quinone, a non-toxic
product. It was also shown that the yield of esters decomposition depends on the ester moiety
and homogeneity of the environment. To our best knowledge, this study presents the first time
the efficiency and mechanism of photodecomposition of vitamin E and its homologues
modulated by the UVB light in a single study. The results obtained during studies on
photodegradation of Toc and its newly synthesized esters may contribute to better
understanding the role of the UV light and improve synthesis of new Toc ester derivatives
more resistant to the UVB light.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgement
The authors acknowledge financial support by Poznan University of Life Sciences, Grant
number 508/782.00.
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Highlights




The stability of -tocopherol esters exposed to UV light was investigated.
The degradation of the esters were observed in organic solvents and in liposomes.
The yield of esters decomposition depends on the ester moiety and the environment.
The final stable product of esters irradiation was alpha-tocopheryl quinine.
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