Chemical cleavage of fucoxanthin from Undaria pinnatifida and formation of apo-fucoxanthinones and apo-fucoxanthinals identified using LC-DAD-APCI-MS/MS

Article abstract of DOI:10.1016/j.foodchem.2016.05.064

As the most abundant carotenoid in nature, fucoxanthin is susceptible to oxidation under some conditions, forming cleavage products that possibly exhibit both positive and negative health effects in vitro and in vivo. Thus, to produce relatively high amounts of cleavage products, chemical oxidation of fucoxanthin was performed. Kinetic models for oxidation were probed and reaction products were identified. The results indicated that both potassium permanganate (KMnO₄) and hypochlorous acid/hypochlorite (HClO/ClO^?) treatment fitted a first-order kinetic model, while oxidation promoted by hydroxyl radical (OH) followed second-order kinetics. With the help of liquid chromatography–tandem mass spectrometry, a total of 14 apo-fucoxanthins were detected as predominant cleavage products, with structural and geometric isomers identified among them. Three apo-fucoxanthinones and eleven apo-fucoxanthinals, of which five were cis-apo-fucoxanthinals, were detected upon oxidation by the three oxidizing agents (KMnO₄, HClO/ClO^?, and OH).

Full text of DOI:10.1016/j.foodchem.2016.05.064

Accepted Manuscript
Chemical Cleavage of Fucoxanthin from Undaria pinnatifida and Formation of
Apo-fucoxanthinones and Apo-fucoxanthinals Identified Using LC-DAD-AP-
CI-MS/MS
Junxiang Zhu, Xiaowen Sun, Xiaoli Chen, Shuhui Wang, Dongfeng Wang
PII:
S0308-8146(16)30744-0
DOI:
http://dx.doi.org/10.1016/j.foodchem.2016.05.064
FOCH 19213
Reference:
Food Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 September 2015
5 May 2016
11 May 2016
Please cite this article as: Zhu, J., Sun, X., Chen, X., Wang, S., Wang, D., Chemical Cleavage of Fucoxanthin from
Undaria pinnatifida and Formation of Apo-fucoxanthinones and Apo-fucoxanthinals Identified Using LC-DAD-
APCI-MS/MS, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.05.064
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Chemical Cleavage of Fucoxanthin from Undaria pinnatifida and
Formation of Apo-fucoxanthinones and Apo-fucoxanthinals
Identified Using LC-DAD-APCI-MS/MS
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Junxiang Zhu1, Xiaowen Sun1, Xiaoli Chen1, Shuhui Wang2, Dongfeng Wang1*
1 College of Food Science and Engineering, Ocean University of China, Qingdao
266003, the People’s Republic of China
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2 Qingdao Municipal Center for Disease Control & Prevention, Qingdao 266033, the
People’s Republic of China
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* Corresponding author. Tel.: +86 0532 82031575; fax: +86 0532 82032468; E-mail:
wangdf@ouc.edu.cn
Acknowledgments
This work was supported by the contribution of the Specialized Research Fund for the
Doctoral Program of Higher Education (Grant No. 20110132110007); the National
Natural Science Foundation of China (Grant No. 31371731); and the Independent
Innovation Major Project of Huangdao District Qingdao City (Grant No. 2014-3-11).
We acknowledge the Drs. Xu, Fan, and Cong for their technical assistance.

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Abstract: As the most abundant carotenoid in nature, fucoxanthin is susceptible to
oxidation under some conditions, forming cleavage products that possibly exhibit
both positive and negative health effects in vitro and in vivo. Thus, to produce
relatively high amounts of cleavage products, chemical oxidation of fucoxanthin was
performed. Kinetic models for oxidation were probed and reaction products were
identified. The results indicated that both potassium permanganate (KMnO4) and
hypochlorous acid/hypochlorite (HClO/ClO–) treatment fitted a first-order kinetic
model, while oxidation promoted by hydroxyl radical (OH·) followed second-order
kinetics. With the help of liquid chromatography-tandem mass spectrometry, a total of
14 apo-fucoxanthins were detected as predominant cleavage products, with structural
and geometric isomers identified among them. Three apo-fucoxanthinones and eleven
apo-fucoxanthinals, of which five were cis-apo-fucoxanthinals, were detected upon
oxidation by the three oxidizing agents (KMnO4, HClO/ClO–, and OH·).
Keywords: fucoxanthin, apo-fucoxanthins, chemical cleavage, reaction kinetics,
LC-DAD-APCI-MS/MS
Chemical compounds studied in this article
Fucoxanthin (PubChem CID: 5281239)

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1. Introduction
Fucoxanthin is the principal carotenoid present in the chloroplasts of brown seaweed.
It is the most abundant of all carotenoids, accounting for more than 10% of their
estimated total natural production (Miyashita et al., 2011). Numerous beneficial
nutritional and medicinal properties of fucoxanthin have been reported, including
excellent antioxidant (Fung, Hamid, & Lu, 2013; Takashima, Shichiri, Hagihara,
Yoshida, & Niki, 2012), anti-cancer (Satomi, 2012; Ye et al., 2014), anti-obesity
(Maeda, Hosokawa, Sashima, Funayama, & Miyashita, 2005; Kang et al., 2012),
anti-inflammatory (Heo et al., 2010) and anti-angiogenic (Sugawara, Matsubara,
Akagi, Mori, & Hirata, 2006) activity. Furthermore, fucoxanthin can help prevent
bone diseases, such as osteoporosis and rheumatoid arthritis (Das, Ren, Hashimoto, &
Kanazawa, 2010). These findings indicate that fucoxanthin can be used as a
nutritional supplement in diet or functional food, with a potential to lower the
incidence of obesity, cardiovascular disease, diabetes and cancer.
The distinct structure of fucoxanthin includes an unusual allenic bond, epoxy group,
and conjugated double bond in the polyene chain (Fig. 1). This structure is highly
unstable under some external conditions. In the previous study (Zhao, Kim, Pan, &
Chung, 2014), the decrease of total fucoxanthin and increase of cis-fucoxanthins,
owing to thermal processing, air exposure, and illumination, can be observed in
canola oil. Besides the isomerization, carotenoids are also susceptible to oxidation,
generating a great diversity of short-chain carbonyl compounds and, in some cases,
some volatile compounds (de Jesus Benevides, da Cunha Veloso, de Paula Pereira, &

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de Andrade, 2011). However, these compounds are not investigated in the relatively
few fucoxanthin stability studies (Kawee-ai, Kuntiya, & Kim, 2013; Sugimura et al.,
2012; Zhao, Kim, Pan, & Chung, 2014).
Besides the color fading, the products of carotenoid degradation are also supposed to
exhibit both positive and negative effects in vitro and in vivo. Taking lutein as an
example, its breakdown products have been reported to induce cytotoxic and
genotoxic effects in human retinal pigment epithelial cells (Kalariya, Ramana,
Srivastava, & van Kuijk, 2008; Kalariya, Ramana, Srivastava, & van Kuijk, 2009).
However, Nidhi, Sharavana, Ramaprasad and Vallikannan (2015) appraised the
anti-inflammatory efficacy of lutein UV-mediated cleavage products in counteracting
inflammation caused by lipopolysaccharide in rats. The results suggested that lutein
oxidation/photolysis produced fragments that exhibited higher antioxidant and
anti-inflammatory effects than the parent lutein molecule. Thus, an in-depth
understanding of carotenoid oxidative cleavage is significant not only for avoiding the
deterioration of fucoxanthin-enriched food quality during processing and storage, but
also for predicting possible effects on human health.
Normally, a trace amount of degradation products is formed in a complex food matrix,
which makes their detection and identification difficult (Rodriguez,
&
Rodriguez-Amaya, 2009). So in some previous studies, carotenoid cleavage was
performed with chemical methods to produce relatively high amounts of derivatives
with explicit structure (Caris-Veyrat, Schmid, Carail, & Böhm, 2003; Pennathur et al.,
2010; Sy, Dangles, Borel, & Caris-Veyrat, 2013). In addition, chemical oxidation can

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be used to investigate the stability of carotenoids under simulated food processing
conditions. For instance, de Jesus Benevides, da Cunha Veloso, de Paula Pereira and
de Andrade (2011) studied the ozonolysis of β-carotene, where solutions of this
compound were exposed to ozone concentrations similar to those used in the food
sanitization process.
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Therefore, this work is dedicated to the investigation of chemical (KMnO4,
HClO/ClO– and OH·) oxidation of fucoxanthin, probing the corresponding kinetic
models and identifying cleavage products, which could possibly underpin a further
study on bioactivity and safety evaluation of these products in vivo.
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2. Materials and methods
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2.1. Materials and Chemicals
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Dried seaweed (Undaria pinnatifida) was purchased from Rongcheng Jiayi Aquatic
Food Co., Ltd., Weihai, China. All dried samples were pulverized using a micro plant
grinder and sieved (200 µm sieves) to obtain a fine powder. The dried seaweed
powders were stored in the dark at room temperature until used.
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The standard of all-trans fucoxanthin was acquired from Sigma Chemical Company
(St. Louis, MO, USA). Solvents for HPLC including acetonitrile and methyl tert-butyl
ether (MTBE) were provided by Merck (Darmstadt, Germany). KMnO4, iron(II)
chloride tetrahydrate (FeCl2·4H2O), hydrogen peroxide (H2O2), sodium hypochlorite
(NaClO) and hydrochloric acid (HCl) were supplied by Sinopharm Chemical Reagent
Co., Ltd. (Shanghai, China). All other reagents used were of analytical grade.
2.2. Isolation of fucoxanthin from Undaria pinnatifida
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The extraction process was conducted using the methodology described by Xia et al.
(2013) with some modifications. All extractions were carried out in the dark to reduce
the possibility of photo-oxidation. Appropriate amounts of seaweed powders were
mixed with 80% (v/v) ethanol containing 0.01% (w/v) butylated hydroxytoluene
(BHT) to obtain a concentration of 0.25 g/mL. The mixtures were initially stirred by
an overhead stirrer for 0.5 h at room temperature, followed by static extraction, which
was conducted at 40 °C for 11.5 h and repeated three times. The extracts were
centrifuged at 5000 × g for 15 min and concentrated in vacuo. 95% (v/v)
methanol/hexane (1:1, v/v) was added to the ethanolic extract to isolate fucoxanthin.
After stirring for 1 min, mixtures separated into two distinct layers in a separation
funnel. The upper phase was discarded, and the lower one was concentrated for use in
subsequent experiments.
According to the modified methodology of fucoxanthin purification described by
Maeda, Hosokawa, Sashima, Funayama and Miyashita (2005), methanolic extracts
were subjected to silica gel adsorption chromatography. Silica gel (200-300 meshes,
Qingdao, China) was slurry-packed into a glass column and equilibrated with
petroleum ether/ethyl acetate (1:1, v/v). Extracts were adsorbed by portions of silica
gel of the same weight, loaded on the top of the column, and eluted in the dark using a
linear gradient of petroleum ether/ethyl acetate from 1:1 to 1:9 (v/v) for 60 min.
Fractions with different colors, i.e., green, reddish-orange, chartreuse, emerald and
yellow (from the top down), were collected in test tubes for HPLC analysis.
2.3. HPLC-VWD analysis

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To determine whether the fractions collected during the purification process contained
fucoxanthin, each portion eluted from the column was analyzed by an HPLC system
(1100, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an
autosampler, a column oven, and a variable wavelength detector (VWD). A
ZORBAX SB-C18 column (4.6 × 250 mm, 5 µm, Agilent Technologies Inc., Palo
Alto, CA, USA) was used at 25 °C, with the detection wavelength was set to 450 nm.
A gradient system based on a slight modification of a previous study (Zhao, Kim, Pan,
& Chung, 2014) was used. Elution was performed at a flow rate of 0.8 mL/min with
linear gradients of solvents A and B (A: acetonitrile/water 80/20, v/v; B: MTBE). The
solvent gradient used was 100% A at the start, with B linearly increased from 0 to 10%
in 10 min and held constant for 10 min before returning to initial conditions in 5 min.
At the end of the run, the system was equilibrated with 100% solvent A for 7 min.
After HPLC analysis, the fractions containing fucoxanthin were collected and
concentrated to a small volume, dried under nitrogen, and then stored in ethanol at
–20 °C until used.
2.4. Chemical oxidation procedure
Modified oxidation methodologies from previous studies (Gurak, Mercadante,
González-Miret, Heredia, & Meléndez-Martínez, 2014; Sommerburg et al., 2003;
Woodall, Lee, Weesie, Jackson, & Britton, 1997) utilizing KMnO4, HClO/ClO–, and
OH· were used, and the optical spectra were recorded on a Unico UV2102-PC
UV-visible spectrophotometer (Shanghai, China). The prepared fucoxanthin diluted
with dichloromethane (1.5 mL, 0.13 mM) was treated with 1 mL of 0.8 mM KMnO4,

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1 mL of 2.3 M NaClO containing 0.1 mL HCl (1 M), or 1 mL of 9.9 M H2O2
containing 0.2 mL FeCl2 solution (10 mM). The reaction mixtures were continuously
vortexed (4-32 min) in the dark, then washed five times using distilled water. After
centrifuging at 7000 × g for 2 min, the organic layer was separated under a steam of
nitrogen and re-dissolved in 1.5 mL dichloromethane for spectral analysis. The
UV-vis spectra were recorded from 200 to 700 nm, with the absorbance at 449 nm
used as a measure of fucoxanthin concentration in the following rate equation:
dA
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= −kAn
(1)
dt
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Here A, t, k and n represent the absorbance at 449 nm, the reaction time (min), the
reaction rate constant (min–1) and the kinetic order of the reaction, respectively.
Equation 1 can be integrated to obtain concentration as a function of time. For a
degradation reaction, integration of Equation 1 for n = 0, 1 and 2 (for zero-, first- and
second-order kinetics) leads to Equations 2, 3 and 4, which have been widely used to
model quality changes in carotenoid-rich foods owing to its simplicity and
satisfactory fit to experimental data (Van Boekel, 2008):
A = A − kt
0
(2)
(3)
A = A0e−kt
A0
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A =
(4)
1+ kA t
0
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Where, A0 is the initial absorbance of fucoxanthin at 449 nm. The best fit was
determined based on the respective coefficients of determination (R2), and the kinetic
parameters were estimated by non-linear regression using Origin 8 (Microcal Origin,

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USA).
2.5. LC-DAD-APCI-MS/MS analysis
The oxidation and control groups were analyzed using an HPLC system (1260,
Agilent Technologies Inc., USA) equipped with an autosampler, a column oven, a
diode array detector (DAD) and a triple quadrupole mass spectrometer with
atmospheric pressure chemical ionization (APCI) interface operated in positive
ionization mode. Samples were injected onto a ZORBAX Eclipse XDB-C18 column
(4.6 × 250 mm, 5 µm, Agilent Technologies Inc., USA). The mobile phase used a
gradient of solvent A (acetonitrile/water 80/20, v/v) and B (acetonitrile/water/MTBE
68/17/15, v/v/v). The flow was maintained at 0.8 mL/min, and the sample injection
volume was 10 µL. Detection wavelengths for UV-vis were set at 300, 350, and 400
nm. The following gradient flow program was used: mobile phase B increased from 0
to 60% in 10 min and kept constant for 10 min, then changed back to 0% in 7 min.
The MS operation parameters were adapted from Pennathur et al. (2010): capillary
voltage 2500 V, drying gas flow 5 L/min, drying gas temperature 350 °C, vaporizer
temperature 450 °C, and nebulizer pressure 20 psi. The mass range between m/z 200
and 800 was used for full scan mass spectra. The optimal fragmentor voltage for
fucoxanthin was 10 V, with a mass range of m/z 100 to 700 for MS/MS scan modes
containing product and precursor ion scans. The Agilent MassHunter (version 3.1)
software package was used for data acquisition and analysis (Agilent Technologies
Inc., USA).
3. Results and discussion

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3.1. Preparation of unoxidized fucoxanthin from Undaria pinnatifida
The fucoxanthin used in the chemical oxidation procedure was initially isolated from
Undaria pinnatifida by conventional solvent extraction, and the various purified
fractions were collected after silica gel column chromatography. The results of HPLC
analysis indicated the presence of fucoxanthin in both the crude extracts and the
reddish-orange pigment fraction. As shown in Fig. S1, peak I with a retention time of
11.48 min corresponded to the all-trans fucoxanthin as compared with the standard.
Similarly, previous research on color profiles of carotenoids separated by thin-layer
chromatography illustrated that fucoxanthin was a characteristic orange pigment
present in Undaria pinnatifida and Ectocarpus siliculosus (Mikami, & Hosokawa,
2013). In the LC-DAD-APCI-MS spectrum of the purified sample (Fig. S2), peak I
with a retention time of 12.49 min and a characteristic spectral signal of all-trans
fucoxanthin (λmax: 449 and 466 nm) was detected, with a relative content of up to
84.39%. Moreover, peaks II, III and IV with respective relative contents of 0.60%,
2.83% and 12.18% were identified as three cis-fucoxanthin isomers based on their
UV-vis spectra (Fig. S2B) and mass-spectrometric behavior (Fig. S2C). In the latter
analysis, the base peak in the full MS of all three isomers corresponded to dehydrated
protonated analyte with m/z 641. Specifically, compounds eluted at 13.16, 16.57, and
18.27 min were identified as 13,13’-cis-, 9’-cis-, and 13’-cis-fucoxanthin respectively,
in line with their hypsochromic shift peculiarity and the intensity of the cis peak
(DB/DII: 23.8, 10.2, 47.5%) according to prior research of Haugan, Englert, Glinz
and Liaacn-Jensen (1992). Since almost no other peaks were present in the

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chromatogram of the isolates from Undaria pinnatifida, the purified seaweed extracts
could be used as an unoxidized fucoxanthin sample for subsequent experiments.
3.2. Spectrum and kinetic analysis of fucoxanthin oxidation
Chemical oxidants, including KMnO4, HClO/ClO– and OH· are versatile reagents that
can react with carbon-carbon double bonds in carotenoids via diverse mechanisms to
produce different types of products. As a result of chemical oxidation, the pigment in
the model system faded, while its absorption peak flattened, shifted and finally
disappeared (Figs. 2A–2C). The spectral changes observed for the three reactions
indicated that the generated products were similar, based on the shift of λmax values
from 400–500 nm to 300–400 nm after oxidation for 30 min. The structure of
fucoxanthin features an unsaturated chain with seven conjugated double bonds, and
its characteristic UV-visible spectra exhibit a hyperconjugative effect (Fig. 1). The
disappearance of the fucoxanthin signal could be attributed to hyperconjugation loss
after oxidation (Pennathur et al., 2010). Furthermore, the kinetics of fucoxanthin color
loss in the reaction with chemical oxidants was investigated. After 30 min of
oxidation using KMnO4 and HClO/ClO–, a 60 and 87% decrease of fucoxanthin
concentration was observed (Fig. 2D), respectively. Similarly, when fucoxanthin was
oxidized by OH·, increasing the reaction time from 0 to 32 min caused a continued
reduction (0 to 77%) of absorption at 449 nm. The respective R2 of 0.976 and 0.990
indicated that both KMnO4 and HClO/ClO– showed a good fit to a first-order kinetic
model (n = 1), with the rate constants of 3.12 × 10–2 and 7.65 × 10–2 min–1,
respectively. Nevertheless, the reaction with OH· followed the second-order kinetics

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(R2 = 0.966, n = 2), with k = 1.69 × 10–1 min–1. The observed difference illustrated
that the mechanism of fucoxanthin with KMnO4 and HClO/ClO– was different to that
with OH·. Variation of kinetic order may be attributed to the complexity of carotenoid
behavior in radical-scavenging reactions, which includes the formation of
carotenoid/hydroxyl adducts and carotenoids radicals (Car·). In the field of
non-thermal food processing, the second-order kinetic model was used to describe
ultrasonic degradation of β-carotene in dichloromethane (Sun, Ma, Ye, Kakuda, &
Meng, 2010), which was also related to the reaction between carotenoids and free
radicals produced by ultrasound (Riesz, Berdahl, & Christman, 1985).
3.3. MS scan of fucoxanthin cleavage products
LC-MS technology has exerted a huge effect on the analysis of carotenoids, including
structure elucidation premised on molecular mass and fragmentation pattern. APCI
has become the most commonly used ionization technique for carotenoids, showing
high analytical sensitivity and being suitable for the ionization of carotenoids with
different polarities (Rivera, & Canela-Garayoa, 2012). Thus, LC-APCI-MS was
utilized to determine whether the observed reaction products were indeed due to the
oxidation and fragmentation of the parent fucoxanthin molecule.
The maximum absorbance of carotenoid cleavage products is dependent on the
number of conjugated double bonds present (Weesepoel, Gruppen, de Bruijn, &
Vincken, 2014). To detect the widest possible array of oxidation products using DAD,
detection wavelengths were set at 300, 350 and 400 nm based on the results of
spectral analysis (Figs. 2A–2C). Using these three wavelengths, a plethora of peaks

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generated by KMnO4-accelerated oxidation were detected, of which 17 could be
tentatively assigned (Fig. S3A). Some previous studies on carotenoid oxidation with
potassium permanganate (Caris-Veyrat, Schmid, Carail, & Böhm, 2003; Rodriguez,
& Rodriguez-Amaya, 2009; Gurak, Mercadante, González-Miret, Heredia, &
Meléndez-Martínez, 2014) indicated that oxygenated products found in the reaction
mixtures were apo-carotenoids. For example, potassium permanganate used by
Rodriguez and Rodriguez-Amaya (2009) could cleave the double bonds of lycopene
and generate apo-lycopenals without further oxidation to carboxylic acid. In this
reaction, a [3+2] electrocyclic addition of permanganate ion to the π-bond may be a
part of the initial step, leading to the formation of a cyclic Mn(V) ester.
Intramolecular electron transfer subsequently occurs to give the oxidized cyclic
Mn(VI) ester, which, in turn, produces final products bearing aldehyde groups by
rearrangement and fragmentation (Fatiadi, 1987).
Compared with the KMnO4-accelerated oxidation, the reaction with HClO/ClO–
produced similar products based on the resemblance of two chromatogram profiles.
As shown in Fig. S3B, 19 peaks found using different detection wavelengths and
eluted approximately between 2 and 8 min could be tentatively assigned. These
oxidation products were likewise identified as apo-carotenoids in a previous study on
astaxanthin (Weesepoel, Gruppen, de Bruijn, & Vincken, 2014). In this reaction, the
electron-rich olefin moiety in fucoxanthin initially acts as a nucleophile, with the
chloride atom of HClO/ClO– acting as an electrophile. After the formation of a
transformable carbonium ion, followed by the opening of the chloronium ion ring and

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an intramolecular SN2-type reaction of the formed chlorohydrin, fucoxanthin epoxides
are formed. When the epoxides encounter a second HClO molecule, the cleavage of
the carbon-carbon bond proceeds to generate two aldehydes (Pennathur et al., 2010).
When compared with the reactions of KMnO4 and HClO/ClO–, the retention time of
various peaks observed in the OH·-promoted oxidation was also in the range from 2 to
8 min, as shown in Fig. S3C. Scavenging of OH· generated in the Fenton reaction had
been investigated in the studies of carotenoids in vitro antioxidant activity.
Fucoxanthin and its stereoisomers were chosen as targets for the evaluation of their
OH· scavenging activity using chemiluminescence and electron spin-resonance
spectroscopy (D’Orazio et al., 2012; Zhang et al., 2014). When it comes to
carotenoid-radical interaction, the primary products of hydrogen abstraction from
carotenoids by OH· are the corresponding radicals (Car·), with the final degradation
products frequently being carbonyls and epoxides (Krinsky, & Yeum, 2003). One of
the reasons for their formation is the generation of carotenoids oxy-radicals (Car–O·),
comparable to the alkoxyl radicals (LO·) produced during lipid peroxidation, leading
to the cleavage of the polyene chain. A conceivable reaction sequence to generate the
Car–O· is as follows: Car + OH· → Car· + H2O; Car· + O2 → Car–OO·; Car–OO· +
Car → Car–OOH + Car·; Car–OOH + Fe2+ → Car–O· + Fe3+ + OH–. The
Car–O· radical is more reactive than either Car· or carotenoid peroxy radicals
(Car–OO·) and is able to abstract an electron from adjacent carbon-carbon bonds to
form two products. The latter reaction is known as β-scission. Additionally,
Car–O· can also lose an electron to become a ketone, or bond to an adjacent carbon to

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form an epoxide (Damodaran, Parkin, & Fennema, 2007).
In view of the above-mentioned studies, oxidation products obtained with the three
chemicals listed above were normally identified as apo- and epoxy-carotenoids.
Therefore, ion chromatograms of various degradation compounds with different
predicted m/z, including apo- and epoxy-fucoxanthins, were extracted from the total
ion chromatograms (TICs) of the mass spectra. The retention time in respective
extracted ion chromatograms (EICs) was compared with the values shown in Fig. S3.
Similarly to preceding research, the results showed the presence of
apo-fucoxanthinals and apo-fucoxanthinones, whereas apo-fucoxanthinoic acids and
epoxy-fucoxanthins were not detected in the reactions of fucoxanthin with KMnO4,
HClO/ClO– and OH· in this study. Some predominant fucoxanthin cleavage products
with m/z 267, 293, 307, 333, 359, 399, and 425 had different retention time in the
APCI positive mode. Although only oxidative modification was applied, the
compounds with m/z 267, 333, 359, and 425 were difficult to identify, since they can
exhibit structural or configurational isomerism, which was the main distinction
between fucoxanthin and other common carotenoids upon oxidative cleavage of the
polyene chain.
3.4. MS/MS fragments of fucoxanthin cleavage products
Since most carotenoids showed little fragmentation during APCI mass spectrometry,
collision-induced dissociation with product ion tandem mass spectrometry could be
helpful for structural characterization and elucidation of whether the observed
reaction products are indeed attributable to oxidation (van Breemen, Dong, &

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Pajkovic, 2012). Then identification was further verified by analyzing MS/MS
fragmentation of all [M+H]+ parent ions assigned. To optimize the MS/MS
parameters the prepared fucoxanthin was first analyzed in APCI positive ionization
mode. Protonated fucoxanthin with m/z 659, [M+H]+, was detected as the base peak
corresponded to the loss of water at m/z 641, [M+H−18]+, which is characteristic for a
compound with a hydroxyl group. The molecular ion peak with second-highest
relative abundance was observed at m/z 581 corresponding to the elimination of water
and acetic acid, [M+H−18−60]+, from protonated fucoxanthin (Fig. S2C). The
three molecular ions, [M+H]+, [M+H−18]+ and [M+H−18−60]+, were fragmented at
various voltages. The optimal voltage to obtain maximal structural information in the
MS/MS scan mode was determined to be 10 V.
Fig. S4 showed the EICs for m/z 293 (apo-11’-fucoxanthinal), which was located with
the full scan MS data accompanying a chromatographic peak in Fig. S3. As shown in
Fig. 3A, this compound fragmented to form a daughter ion with m/z 233,
corresponding to the loss of acetic acid, [M+H−60]+, which further ascertained the
structure of apo-11’-fucoxanthinal. Another fragment ion of m/z 125,
[M+H−18−56−94]+, was produced from the protonated molecule by cleavage the
C6’−C7’ bond of the polyene chain, with a loss of 56 amu previously observed in
APCI mass spectra of carotenoids containing an ε-ring (Rivera, Christou, &
Canela-Garayoa, 2014).
A product with m/z 307 was detected in the oxidation reactions with HClO/ClO– and
OH·, but not with KMnO4. Similarly to apo-11’-fucoxanthinal, it was situated with the

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full scan MS data accompanying only a chromatographic peak labeled as 2 in Figs.
S3B and S3C. As shown in Fig. 3B, an ion with m/z 289, [M+H−18]+, and its
derivative at m/z 207, [M+H–18–82]+, were observed, which indicated that the
dehydrated molecule underwent further bond cleavage between C9 and C10. Another
ion with m/z 109 was detected, attributable to bond cleavage between carbons 6, 7 and
12, 13.
As mentioned earlier, two structures were plausible for the compound at m/z 267:
apo-11-fucoxanthinal and apo-9’-fucoxanthinone. However, when comparing its EIC
(Fig. S4) with the chromatograms (Fig. S3), it located with the full scan MS data was
accompanied by only one chromatographic peak, denoted as 16, 18 and 19 in Figs.
S3A, S3B and S3C, respectively. MS/MS spectra were helpful for the structural
characterization. As shown in Fig. 3C, the fragmentation of peak 16 in Fig. S3A was
explained by consecutive cleavage of the end ring and the double bond between C7’
and C8’, yielding two fragment ions with m/z 211 ([M+H−56]+) and m/z 155
([M+H−56−56]+), which suggested that the structure of the compound with m/z 267
corresponded to apo-9’-fucoxanthinone rather than to apo-11-fucoxanthinal.
Analogous to apo-9’-fucoxanthinone (m/z 267), fragmentation patterns of the two
possible structural isomers of m/z 333 were studied using tandem mass spectrometry
to facilitate the distinction. To be exact, isomers with m/z 333 could have either
apo-15-fucoxanthinal or apo-13’-fucoxanthinone structures. When scrutinizing the
chromatograms and EICs in the MS scan mode, the compounds with m/z 333 were
found to take up two chromatographic peaks (Fig. S3). When fucoxanthin was

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subjected to KMnO4-promoted oxidation, one isomer of m/z 333 with a retention time
of 3.307 min was identified as apo-13’-fucoxanthinone based on the presence of two
fragment ions of m/z 135 and 161, as presented in Fig. 4A. The former was generated
from the protonated molecule by bond cleavage between C6’ and C7’ carbons of the
polyene chain, and the latter was produced by cleaving the double bond between C11’
and C12’, combined with the elimination of acetic acid and fragmentation of the end
ring. Another isomer of m/z 333 eluted at 3.460 min was identified as
apo-15-fucoxanthinal, showing a characteristic ion with m/z 287, produced by loss of
the terminal hydroxyl and carbonyl (Fig. 4B). In addition, both isomers shared a
common ion with m/z 109, ascribed to fragmentation patterns related to the various
splitting sites between conjugated double bonds.
Peak 4 and 6 in Fig. S3A belonging to the compounds with m/z 359 were identified as
apo-14’-fucoxanthinal and apo-15’-fucoxanthinal, respectively, based on their tandem
mass spectra. As shown in Fig. 4D, an iconic ion produced from ionized compound of
peak 4 was detected at m/z 205 and corresponded to the demethylated and dehydrated
terminal ring with cleavage of the double bond between C10 and C11, which verified
the structure of apo-14’-fucoxanthinal. Besides, the presence of the representative ion
with m/z 227 (Fig. 4C), [M+H−60−30−42]+, corresponding to the elimination of
acetic acid and two methyl groups combined with bond rupture between C13’ and
C14’, demonstrated that the compound of peak 6 was apo-15’-fucoxanthinal.
Similarly to the mass spectrometric behavior of isomers with m/z 333, a common ion
peak for apo-14’-fucoxanthinal and apo-15’-fucoxanthinal had m/z 109. Another

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common ion of the two isomers was m/z 161, corresponding to carbon-carbon bond
cleavage at positions 7, 8 and 14’, 15’ for the former, and at the 6’, 7’ double bond for
the latter.
When matching the Fig. S4 with the Fig. S3, it could be observed that compounds
with m/z 425 were ascribed to multiple chromatographic peaks, i.e., 10, 14 and 15 in
Fig. S3A. Although two constitutional isomers with a relative molecular mass of 424
existed (i.e., apo-12-fucoxanthinal and apo-10’-fucoxanthinal), the MS/MS result
suggested that the structure corresponded to apo-12-fucoxanthinal, due to the
presence of characteristic ions with m/z 425 in all cases. As depicted in Figs. 5A–5C,
the loss of toluene observed for most of the carotenoids was involved in the
production of fragment ions with m/z 255. This fragmentation indicated the presence
of extensive conjugation within the molecule (Rivera, Christou, & Canela-Garayoa,
2014). Additional ions were generated by cleavage of various carbon-carbon single or
double bonds as discussed above. Probably, the three peaks corresponded to the
different geometrical isomers of apo-12-fucoxanthinal. Since cis-isomers, including
13,13’-cis-, 9’-cis- and 13’-cis-fucoxanthin, were also formed from
all-trans-fucoxanthin in our unoxidized samples isolated from Undaria pinnatifida,
this conclusion seemed plausible. Based on the hypsochromic shift in λmax, the cis
absorption peak (Fig. 5D) and the elution order of cis-fucoxanthins, peaks 10, 14 and
15 in Fig. S3A were tentatively identified as all-trans-apo-12-fucoxanthinal,
9’-cis-apo-12-fucoxanthinal, and 13’-cis-apo-12-fucoxanthinal, respectively.
By the same token, cis-trans isomers were possible for m/z 399, which had only one

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possible chemical skeleton (apo-12’-fucoxanthinal). However, they were ascribed to a
number of chromatographic peaks, i.e., 9, 10, 11 and 12 in Fig. S3A. When
fucoxanthin underwent KMnO4-promoted oxidation, the generated peak 10 was
associated with both major and minor compounds with m/z 425 and m/z 399,
respectively. This bore a similarity to a previous study, which showed that the
chromatographic peak representing apo-12-fucoxanthinal was contaminated with
apo-11’-fucoxanthinal and apo-9’-fucoxanthinone, identified using mass spectrometry
and UV absorption spectroscopy, when fucoxanthin was allowed to react with the
zinc permanganate (Bonnett et al., 1969). As shown in Figs. S5A-5D, common ions
including m/z 109, 127, 173, 201, and 381 could be detected, in accordance with the
structure of apo-12’-fucoxanthin. Identification of cis-isomers with m/z 399 was
conducted similarly to above method for apo-12-fucoxanthinal isomers, although the
UV-vis absorbance spectrum of peak 10 was not available. Peak 9, 11 and 12 in Fig.
S3A with m/z 399 were tentatively identified as all-trans-apo-12’-fucoxanthinal,
13,13’-cis-apo-12’-fucoxanthinal, and 13’-cis-apo-12’-fucoxanthinal, respectively.
Finally, all apo-fucoxanthins formed during the chemically accelerated oxidation of
fucoxanthin were listed in Table 1.
4. Conclusion
Successful
identification
of
fucoxanthin
oxidation
products
by
LC-DAD-APCI-MS/MS demonstrates that a number of apo-fucoxanthinones and
apo-fucoxanthinals are formed in a relatively high amount when the fucoxanthin
isolated from Undaria pinnatifida is subjected to chemical cleavage using KMnO4,

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HClO/ClO–, and OH·. These compounds may be produced and further transformed
under some food processing and stored conditions. In addition to the
quantitative analysis of these products in fucoxanthin-enriched food during storage,
future studies should fully investigate the nutritional health and toxicity effects of
diets or functional foods with sufficient fucoxanthin content.

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Supplementary data
Additional HPLC-VWD analysis of crude extracts and purified reddish-orange
fraction of Undaria pinnatifida in Fig. S1, LC-DAD-APCI-MS analysis of purified
fraction of Undaria pinnatifida in Fig. S2, liquid chromatograms of fucoxanthin after
chemical degradation in Fig. S3, EICs of apo-fucoxanthins produced by reactions of
fucoxanthin with KMnO4, HClO/ClO–, and OH· in Fig. S4, MS/MS spectra of Peak 9,
10, 11, 12 and UV-vis spectra of Peak 9, 11, 12 from KMnO4-accelerated oxidation in
Fig. S5.

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Figure Captions
Fig. 1. Chemical structure of all-trans fucoxanthin.
Fig. 2. Spectral changes observed for reactions of fucoxanthin with (A) KMnO4, (B)
OH·, (C) HClO/ClO– and their kinetic analysis (D). n, kinetic order of reaction; R2,
coefficient of determination.
Fig. 3. MS/MS spectra of (A) apo-11’-fucoxanthinal with m/z 293 (Peak 6) and (C)
apo-9’-fucoxanthinone with m/z 267 (Peak 16) produced by KMnO4-accelerated
oxidation; MS/MS spectra of (B) apo-13-fucoxanthinone with m/z 307 (Peak 2)
produced by HCl/ClO–-accelerated oxidation.
Fig. 4. MS/MS spectra of (A) apo-13’-fucoxanthinone with m/z 333 (Peak 2), (B)
apo-15-fucoxanthinal with m/z 333 (Peak 3), (C) apo-15’-fucoxanthinal with m/z 359
(Peak 6) and (D) apo-14’-fucoxanthinal with m/z 359 (Peak 4) produced by
KMnO4-accelerated oxidation.
Fig. 5. MS/MS spectra of (A) all-trans-apo-12-fucoxanthinal with m/z 425 (Peak 10),
(B) 9’-cis-apo-12-fucoxanthinal with m/z 425 (Peak 14), (C)
13’-cis-apo-12-fucoxanthinal with m/z 425 (Peak 15) produced by
KMnO4-accelerated oxidation and their UV-vis spectra (D).
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Figures
Fig. 1
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Fig. 2
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