Chemistry and Physics of Lipids 165 (2012) 608–614

Contents lists available at SciVerse ScienceDirect

Chemistry and Physics of Lipids

journal homepage: www.elsevier.com/locate/chemphyslip

Triacylglycerols composition, oxidation and oxidation compounds in camellia oil

using liquid chromatography–mass spectrometry
Alam Zeb ∗
Department of Biotechnology, University of Malakand, Chakdara, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Camellia seed oil is one of most important edible oil, rich in oleic acid and contains many natural antiox-
Received 31 October 2011 idants with various biological activities. During preparation of foods or storage camellia oil oxidizes by
Received in revised form 6 March 2012 the auto-oxidation and produce oxidized compounds. Traditional analytical techniques like FFA, POV are
Accepted 12 March 2012
used for the determination of oxidation and adulteration of oils and fats. These methods were rarely
Available online 24 March 2012
able to detect the oxidized compounds produced and extent of oxidation. This paper presents the uses
of liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC–ESI-MS) for the
Keywords:
analysis of triacylglycerols (TAGs) composition and evaluation of auto-oxidation and oxidation products
HPLC–ESI-MS
Camellia oil
of camellia seed oil. The camellia oil was auto-oxidized for 12 months at room temperature. The TAGs
Auto-oxidation were identified from their characteristics fragmentations such as protonated molecular ion, ammonium
Oxidation products and sodium adducts, diacylglycerols, epoxy-diacylglycerols fragments and mono-acylglycerol fragments
Oxidized triacylglycerols in ESI-MS mass spectra. HPLC–ESI-MS data revealed the separation and identification of 15 TAGs. The
Free radical reaction major TAGs separated and identified in camellia seed oil were POO, OOO, OLO, PLO/POL, OLL, SOO, ALO
Lipid oxidation and OLLn. The auto-oxidation studies revealed a total loss of LnLLn, LnOLn, LLLn and OLLn amounting
about 13.5% total oxidation. The auto-oxidation products were epoxy hydroperoxides, epoxy epidioxides,
and mono-epoxides. It was observed that these were characteristic compounds produced in high oleic
oils.
© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction variety of human diseases or conditions such as aging, atheroscle-

rosis, arthritis, inflammatory bowel disease, neurodegenerative
Triacylglycerols are the principal components of plant oils. The disease, and some eye diseases (Halliwell, 1994). The oxidized fats
properties of the oils are dependent on the fatty acids composition. were found significance to human health since some of these oxi-
The presence of polyunsaturated fatty acids increases the poten- dation products may be carcinogenic (Yang et al., 1998). Jurek et al.
tial beneficial properties of the oils. However, the polyunsaturated (2005) showed that dietary lipid hydroperoxides are a key factor in
oils are more prone to oxidation of different kinds (Kamal-Eldin colon carcinogenesis. Recently Rohr-Udilova et al. (2008) observed
et al., 2003). Triacylglycerols are one of the prime determinants that hydroperoxides from the dietary oxidized oils enhances the
in the study of oxidation of oils. During the oxidation (auto-, growth of hepato-carcinoma. The analysis of oxidation and oxida-
thermal, photo) triacylglycerols are oxidized to hydroperoxides. tion compounds in oils or fats is thus an important point of concern
These hydroperoxides are highly reactive species (Hamilton et al., in food industries to health sectors.
1997). Hydroperoxides are then further oxidized or degraded to Camellia oleifera is a member of the Theaceae family, widely
a wide variety of secondary oxidation products. These oxidized known as tea seed plant. It is native to China, and South-east Asia
compounds are usually absorbed by the foods and consequently (Qizhi et al., 2008). It is also grown as an ornamental plant in West-
enters human body. It was established that oxidized fats and oils ern countries. It has been used in Chinese traditional medicine and
are toxic (Frankel et al., 1984) and are major contributing factor in a in cosmetics (Chaicharoenpong and Petsom, 2011). Camellia oil is
rich in oleic acid (C18:1) and contain many natural antioxidants
with various biological activities (Shyu et al., 1990). The camellia oil
has been observed to suppress cholesterol content in the body and
Abbreviations: ESI-MS, electrospray ionization mass spectrometry; TIC, total ion provide resistance to oxidative stress (Fu and Zhou, 2003). Camel-
chromatogram; ACS, American Chemical Society; BHT, butylated hydroxyl toluene; lia oil contains 75–80% oleic acid (Chaicharoenpong and Petsom,
A, arachidic; G, gadoic; L, linoleic; Ln, linolenic; P, palmitic; O, oleic; S, stearic;
2011), similar to olive oil, while the saturated fats are present in
[LOep ]+ , epoxide of LO.
∗ Tel.: +92 945 763442x3019. low amounts. Because of the high oleic contents, camellia oil pro-
E-mail address: Alamzeb01@yahoo.com vides health-promoting effects include lowering blood pressure,

0009-3084/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2012.03.004
 A. Zeb / Chemistry and Physics of Lipids 165 (2012) 608–614 609

cholesterol and triglycerides, thus helpful in preventing chronic

Hydroperoxy
10
diseases like cardiovascular diseases and cancer (Chen et al., 1998).

Epoxy/

DAGs
The oil is helpful in protecting liver against carbon tetrachloride
toxicity (Lee et al., 2007). Camellia oil is now commonly used as
11
cooking oil because of its high quality and healthful properties.
7
During preparation of foods and storage camellia oil oxidizes
and produce oxidized compounds. Traditional analytical tech- f
6 13
b 5
niques like FFA, POV are used for the determination of oxidation and c 8
a
adulteration of oils and fats. Hai and Wang (2006) used electronic d e
14 (B)
12 15
nose for the adulteration in camellia oil. It was found that electronic
nose was not a suitable method for the detection of the percentage
of adulteration in camellia oil. Other techniques like FTIR (Weng 10

et al., 2006) can also be successfully used, but the techniques are
time consuming and are not error free, and do not provide complete 7 After Auto-oxidation

Hydroperoxy
structural assignment. Therefore HPLC with DAD and MS detec- 5

Epoxy/

DAGs
11
tion can be a better choice (Byrdwell and Neff, 2001; Leskinen
4 8
et al., 2007). Mass spectrometry is especially a powerful technology 6

for analyzing lipids (Kofronova et al., 2009). The use of HPLC–MS 3

13

for the determination of triacylglycerols composition and adulter- 9

ation of triacylglycerols and consequently the camellia oil has not
DAGs
1 2 12 14 15
(A)
been reported. This paper describes the auto-oxidation products
in camellia oil using HPLC–ESI-MS method that was reported pre-
0 5 10 15 20 25 30 35
viously (Zeb and Murkovic, 2010a). The possible structures of the Time (min)
oxidation products formed were discussed.
Fig. 1. ESI-MS total ion chromatograms of camellia seed oil. (A) Un-oxidized fresh
sample, and (B) auto-oxidized sample. The identification of each peak number
2. Experimental (1–15) is given Table 1, and peaks a–f can be found at Table 2.

2.1. Reagents and materials

2.5. Statistical analysis

Ammonium and sodium acetate were from Sigma–Aldrich

All samples were measured in triplicate or otherwise men-
(Germany). All other chemicals and reagents were of ACS grade
tioned. Data of the percent peak areas and POV were analyzed by
from Sigma–Aldrich (USA). The fresh camellia seed oil was kindly
one-way analysis of variance (ANOVA) and Tukey’s multiple com-
provided by the Prof. Haiyan Zhong, Faculty of Food Science and
parison method at ˛ = 0.01 or ˛ = 0.05 using SigmaPlot for windows
Engineering, Central South University of Forestry & Technology,
version 11.0 (Systat Software, Inc., 2008).
China.
3. Results and discussion
2.2. Auto-oxidation of camellia oil
3.1. Triacylglycerols composition
A sample of 200 mL was stored in glass bottle and stored under
dark at room temperature (18 ± 5 ◦ C) for 12 months. A control sam- Identification and quantification of TAG is not very easy, because
ple was kept in refrigerator at −20 ◦ C. It was observed that no of its dependence on many factors, such as number and posi-
significant change occur in the refrigerated sample. tion(s) of double bonds, type of fatty acid, chain lengths and their
positions on the glycerol backbone. Similarly ionization of TAG in
mass spectrometry is also affected by different ion sources and its
2.3. Liquid chromatography–mass spectrometry
related parameters. The unequivocally correct quantification can
be achieved with authentic standards of all analyzed compounds
A sample of 40 mg ± 0.5 mg of the auto-oxidized camellia oil
in the sample (Kofronova et al., 2009). In the present work, the
was mixed with 1 mL of acetone and 3 mL of HPLC solvent
composition of the TAG was based on the relative peak areas.
and transferred to HPLC vials. Liquid chromatography was car-
The fresh samples of the camellia seed oil was analyzed for TAGs
ried out using an Agilent HP 1100 system coupled to ESI-MS
composition using HPLC–ESI-MS. A total of 15 TAGs were separated
(Agilent, Waldbronn, Germany). The separation of triacylglyc-
and identified in 35 min as shown in Fig. 1. The TAGs were identi-
erols was carried out using a Phenomenex C18 (150 mm × 3 mm)
fied from their characteristics fragmentation in mass spectra, like
(Germany) column. The elution was based on isocratic solvent
protonated molecular ion, ammonium and sodium adducts, dia-
system consists of 18% isopropanol in methanol with 0.1% acetic
cylglycerols fragments and mono-acylglycerol fragments. Table 1
acid. Other conditions of LC and MS were the same as reported
shows the TAGs composition and order of elution of TAGs in camel-
in our previous work (Zeb and Murkovic, 2010a,b). The separa-
lia seed oil. The highest amount (32.54%) was observed for POO
tion of all TAGs was achieved in 35 min. The TAGs were identified
(M+H+ as m/z 859 ion, M+18+ as 877, M+23+ as 882 ions) and OOO
based on the characteristics mass spectra and authentic standard
(M+H+ as m/z 885 ion, M+18+ as 902, M+23+ as 907 ions). Both
(triolein).
TAGs have a co-eluting DAG ion of m/z 603, which was identified
as (OO+ ) ion. The mixed composition of POO and OOO is closely
2.4. Peroxide value similar in relative quantity (35.4%) to refined olive oil as reported
recently (Zeb and Murkovic, 2011a) and also rapeseed oil (Zeb and
The peroxide value of the fresh and auto-oxidized camellia oil Murkovic, 2010a). The presence of high amounts of oleic acid gives
was determined using AOCS official method # Cd 8b-90 (AOCS, the camellia oil a higher stability and properties and that is the rea-
1998) and expressed as mequiv. O2 /kg of fat. son it is also known as “Eastern Olive oil” (Wang et al., 2006). The
610 A. Zeb / Chemistry and Physics of Lipids 165 (2012) 608–614

Table 1
Triacylglycerols composition of camellia oil determined by HPLC–ESI-MS.

Peak # Rt (min) TAG Peak area (%)a M+H+ M+NH4 + M+Na+ DG Fragments (m/z)

1 8.2 LnLLn a 0.93 875 892 897 LnLn 595 LLn 597 –
2 9.2 LnOLn a 877 894 899 LnLn 595 OLn 599 –
3 9.6 LLLn 3.93 877 894 899 LLn 597 LL 599 –
4 11.0 OLLn 8.00 879 896 901 LLn 597 OLn 599 OL 601
5 12.8 OLL 15.5 881 898 903 LL 599 LO 601 –
6 14.5 OLO b 17.97 883 900 905 LO 601 SL 603 –
7 15.0 POL b 857 874 879 PL 575 PO 577 LO 601
8 15.5 PLO b 857 874 879 PL 575 PO 577 LO 601
9 16.1 GLL 3.36 909 926 931 LL 599 GL 629 –
10 18.0 POO c 32.54 859 877 882 PO 577 OO 603 –
11 18.7 OOO c 885 902 907 OO 603 – –
12 20.2 GLO 1.75 911 928 933 LO 601 GL 629 GO 631
13 22.6 SOO d 9.42 887 904 909 OO 603 OS 605 –
14 23.6 ALO d 913 931 936 LO 601 AL 631 AO 633
15 28.2 AOO 2.14 912 932 937 AO 633 OO 603 –

Similar letters (a–d) in the column represent co-eluting peaks.

a
Epoxy DAGs (2.37%) and normal DAGs (0.42%).

presence of other minor components is usually one of the major play a critical role in the oxidation of TAGs. For this purpose
parameters for evaluating stability and biological properties. model TAGs like trilinolein and trilinolenin have been oxidized
The second highest amount was of the co-eluting peak of the to know the oxidative stability of vegetable oil TAGs (Frankel
OLO, POL and PLO. The OLO has protonated molecular ion (M+H+ ) et al., 1990; Neff and Byrdwell, 1998). TAGs were found to be
m/z 883, M+18+ as m/z 900, and M+23+ as m/z 905. The POL and oxidized by auto-oxidation. The oxidation products which are
PLO are of similar m/z values (M+H+ as m/z 857, M+18+ as m/z 874, mono, bis and tris-hydroperoxides were formed by the sequen-
M+23+ as m/z 879). They also share similar DAG+ ions (m/z 575 for tial addition of oxygen to the trilinolein. It was found that the
PL+ , m/z 577 for PO+ and m/z 601 for LO+ ). The regional-isomers initial products of oxidation were mono-hydroperoxides, which
were identified based on the relative intensity of the DAGs ions. were further oxidized to form a mixture of 1,2-bis-hydroperoxides
Leskinen et al. (2007) identified the regional-isomers composition and 1,3-bis-hydroperoxides (Kamal-Eldin et al., 2003). These bis-
of OLO and LOO, however in the present work only OLO was identi- hydroperoxides may form tris-hydroperoxides or converted to
fied, but other isomers was not identified. The possible reason may other oxidized compounds. All these hydroperoxides were com-
be the difference in the methods, instruments and variety of the oils. posed of their respective Z-E and E-E isomers. It has been found that
The third highest amount (15.5%) position was occupied by during the oxidation of trilinolein no positional preference has been
OLL. This TAG has protonated molecular ion of m/z 881, ammo- found between each of the sn-positions (Neff and Byrdwell, 1998).
nium adduct of m/z 898 and sodium adduct of m/z 903. The DAGs Fig. 1 shows the auto-oxidation and loss of certain TAGs. Total loss
ions were m/z 599 for LL+ and m/z 601 for LO+ . The composition of linolenic acid TAGs were observed. These include LnLLn, LnOLn,
of this TAG is relatively higher than olive oil (Zeb and Murkovic, LLLn and OLLn. Due to the higher unsaturation these TAGs were
2011a) and Hazel nut seed oil (12.3%), relatively equal in amounts unable to survive and resist auto-oxidation. The percent oxidized
to Brazil nut seed oil (16.7%), and lower than poppy seed oil of 19.9% compounds were 2.37% initially and reached a value of 13.51%
(Holcapek et al., 2003). SOO and ALO was placed on fourth position after one year of auto-oxidation. Thus the total increase in oxi-
based on relative percent peak area with a value of 9.42%. These dized compound was 11.14%. Similarly the initial peroxide value
two TAGs were found co-eluting at the retention time of 22.6 and was 13.8 mequiv./kg and after one year of auto-oxidation it reached
23.6 min. The amounts of these TAGs were relatively equal to rape- a value of 142.4 mequiv./kg (Fig. 2). Thus the total change in perox-
seed oil (Zeb and Murkovic, 2010a) and higher than most of the ide values was about 10.31%, which is relatively less than the value
seed oils reported by Holcapek et al. (2003). of total oxidized compounds. This shows that studying hydroper-
The fifth position on the basis of composition was occupied by oxides contents using mass spectrometry is more sensitive and
OLLn. This TAG was identified from its M+H+ (m/z 879), M+18+ (m/z reliable than usual titrimetric analysis.
896), M+23+ (m/z 901), DAGs ions such as LLn+ (m/z 597), OLn+ (m/z
120
599) and OL+ (m/z 601). The percent composition was found to be
8.0%. The composition of this TAG was previously reported in rel-
atively similar quantity in rapeseed oil (Zeb and Murkovic, 2010a) 100
and higher than rapeseed, soybean, sunflower and linseed oils pre-
viously reported by Holcapek et al. (2003). Other TAGs with smaller 80
composition are AOO (M+H+ m/z 912, M+18+ m/z 932, M+23+ m/z
POV (meq/kg)

937), LLLn (M+H+ m/z 877, M+18+ m/z 894, M+23+ m/z 899), GLL
60
(M+H+ m/z 909, M+18+ m/z 926, M+23+ m/z 931), GLO (M+H+ m/z
911, M+18+ m/z 928, M+23+ m/z 933) and LnLLn (M+H+ m/z 875,
M+18+ m/z 892, M+23+ m/z 897). The percentage composition of 40
these TAGs was 2.14, 3.93, 3.36, 1.75 and 0.93% respectively.
20

3.2. Auto-oxidation of camellia oil triacylglycerols

0
0 12
The oxidation of triacylglycerols (TAGs) follows the free rad- Oxidation Time (h)
ical reaction mechanism. However, steric hindrance, the level of
unsaturation, presence of a pro-oxidant, and the exposure time Fig. 2. Peroxide value (mequiv. O2 /kg) of control and auto-oxidized camellia oil.
 A. Zeb / Chemistry and Physics of Lipids 165 (2012) 608–614 611

100
(A) 100 (C)

Relative Intensity (%)

Relative Intensity (%)

80 80

60 60

40 40

20 20

0 0
200 300 400 500 600 700 800 900 1000 200 300 400 500 600 700 800 900 1000
m/z m/z

100
(B) 100
(D)

Relative Intensity (%)

Relative Intensity (%)

80 80

60 60

40 40

20 20

0 0
200 300 400 500 600 700 800 900 1000 200 300 400 500 600 700 800 900 1000
m/z m/z

100
(E)
Relative Intensity (%)

80

60

40

20

0
200 300 400 500 600 700 800 900 1000
m/z

100
(F)
Relative Intensity (%)

80

60

40

20

0
200 300 400 500 600 700 800 900 1000
m/z

Fig. 3. ESI-MS spectra of the auto-oxidized compounds (epoxides) of the camellia oil, (A) OLL epoxy hydroperoxide, (B) OOO epoxy epidioxide, (C) OOS epoxy epidioxide,
(D) OLO epoxide, (E) OOO epoxide, and (F) OOS epoxide.
612 A. Zeb / Chemistry and Physics of Lipids 165 (2012) 608–614

Table 2
Possible identification of triacylglycerols auto-oxidation products of the camellia oil using HPLC–ESI-MS.

Peak no. Possible compound Peak area (%) Characteristics m/z

M+H+ M+NH4 + M+Na+ Positional Other DAGs+ or

fragments fragments oxidized
DAGs+

a OLL epoxy-hydroperoxides 1.12 913.7 930.8 935.8 463.4, [M+H- [LL]+ 599.5,
493.4 H2 O]+ [LO]+ 601.5,
895.8, [LLep ]+
[M+H- 613.5,
2H2 O]+ [LOep ]+
879.8 615.5

b OOO epoxy-epidioxides 2.18 915.7 932.8 937.8 493.4, [M+H- [LOep ]+

519.4 H2 O]+ 615.5,
897.8, [OOep ]+
[M+H- 617.5,
H2 O2 ]+ [OSep ]+
881.8 619.5,
[Oep Oep ]+
631.5,
[Oep Sep ]+
633.5

c OOS epoxy-epidioxides 3.32 917.8 934.8 939.8 465.4 [M+H- [LO]+ 601.5,
2H2 O]+ [OO]+
883.8 603.5,
[OOep ]+
617.5,
[OSep ]+
619.5,
[Sep Sep ]+
635.5

d OLO epoxides 1.46 897.8 914.8 919.8 465.4 – [LL]+ 599.5,

[LO]+ 601.5,
[OOep ]+
617.5

e OOO epoxides 1.47 899.8 916.8 921.8 467.4 [M+39]+ [LO]+ 601.5,
937.8 [OO]+
603.5,
[OOep ]+
617.5,
[OSep ]+
619.5

f OOS epoxides 3.96 901.8 918.8 923.8 477.4, [M+H- [LO]+ 601.5,
493.4 H2 O]+ [OO]+
883.8 603.5,
[OSep ]+
619.5

3.3. Auto-oxidation products of camellia oil triacylglycerols was formed by the loss of epoxy oxygen and formation of another
unsaturation from the fragment ion of m/z 615 (LOep + ). Similarly
Three main classes of auto-oxidation compounds were iden- the m/z 613 corresponds to the LLep + . These fragments and their
tified. These were epoxy hydroperoxides, epoxy epidioxides, and relative intensities confirm that epoxy group was present on oleic
mono-epoxides. Table 2 shows the possible identification of TAGs acid group and hydroperoxide group was attached to outer (sn-3)
auto-oxidation products of the camellia oil. The epoxy group position, while sn-2 position is free. The next important fragmenta-
was identified the oxidized DAGs fragments as well as positional tion is known as positional fragments, because these ions confirm
fragments as discussed previously in detail (Byrdwell and Neff, the position of the epoxide and hydroperoxide. These ions are m/z
1998,1999; Zeb and Murkovic, 2010b). The first eluting compounds 463 and 493. The fragment ion with m/z 463 corresponds to the
was OLL epoxy hydroperoxides (peak a, Fig. 3A) with percent peak loss of C9 H16 O group from the LOep + of m/z 615. This confirms the
area of 1.12%. The protonated molecular mass (M+H+ ) of this com- position of epoxide at C9 or C10. Similarly the m/z 493 corresponds
pounds was m/z 913, ammonium adduct (M+18+ ) was m/z 930 and to the loss of C9 H16 from the LLep + of m/z 613. This means that
sodium adduct (M+23+ ) was m/z 935. A fragment with m/z 895 epoxide as well as hydroperoxide was present originally at posi-
corresponds to the loss of one water molecule (M+H-H2 O+ ), while tion 9 or 10. This is in accordance with our previous work (Zeb
m/z 879 corresponds to the loss of two water molecule as (M+H- and Murkovic, 2010b) as well as of the Byrdwell and Neff (1998,
2H2 O+ ). The epoxide was identified from its epoxy diacylglycerols 1999). Epoxy hydroperoxides from the oxidation of synthetic TAGs
fragments ion of m/z of 615 which corresponds to LOep + . This frag- (SOS, OPP, SSL, and OOS) was identified by Sjovall et al. (2001) using
ment was formed from the loss of an outer OH group from the single ion monitoring (SIM) mode of ESI-MS. They showed that
hydroperoxide forming an epoxide as previously observed in our epoxy hydroperoxides are produced in small quantities (3–7%) as
work (Zeb and Murkovic, 2010b, 2011a,b) and highly correlated compared to the monohydroperoxide (20–40%) at 37 ◦ C for 30 h.
with the Byrdwell and Neff (1998,1999) for hydroperoxides and However, in the present study less epoxy hydroperoxides have
epoxides. The fragment ion of m/z 599 corresponds to LL+ which been detected. These compounds were also identified as products
 A. Zeb / Chemistry and Physics of Lipids 165 (2012) 608–614 613

of thermal oxidation in the Rancimat (Zeb and Murkovic, 2011b). these compounds were produced in other high oleic oils. Liquid
This means that auto-oxidation as well as thermal oxidation follows chromatography–mass spectrometry was a successful technique
the same oxidation mechanism and results in to similar oxida- for the characterization of auto-oxidation as compared to tradi-
tion compounds but in different quantity depending on different tional methodologies.
parameters.
Similar the peak b was identified as OOO epoxy epidioxides with
Acknowledgements
the percent composition of 2.18%. The m/z 915 in the ESI-MS spectra
corresponds to M+H+ , m/z 932 was M+18+ and m/z 937 corresponds
The author is grateful of Prof. Dr. Michael Murkovic of the Insti-
to M+23+ of this compound (Fig. 3B). A fragment with m/z 897 cor-
tute of Biochemistry, Graz University of Technology, Graz, Austria
responds to the loss of one water molecule (M+H-H2 O+ ), while m/z
for providing all facilities to conduct this work. The experimental
879 corresponds to the loss of two oxygen and two hydrogen atoms
part was carried out at the above mentioned lab with the kind finan-
as (M+H-H2 O2 + ). The epoxide was identified from its epoxy diacyl-
cial support of Higher Education Commission (HEC) of Pakistan.
glycerols fragments ion of m/z of 615 which corresponds to LOep + ,
Prof. Dr. Haiyan Zhong, Central South University of Forestry and
m/z 617 corresponds to OOep + , m/z 631 and 633 corresponds to the
Technology, China for providing fresh camellia seed oil.
Oep Oep + and Oep Sep + respectively. The fragment ions at m/z 493.4
and 519.4 are formed by the loss of (C9 H16 O+ ) and (C8 H16 + ) from
the diepoxide ion (m/z 631.5). This explains that the epoxide was References
between C9 and C10. If this epoxide was at C8 and C9, a fragment
of m/z 477.4 and 507.4 would have been formed, which was not AOCS, 1998. Official Methods and Recommended Practices of the American Oil
Chemists’ Society, 5th edn. AOCS, Champaign (Method Cd 8b-90).
the case. Similarly peak c was identified as OOS epoxy epidiox- Byrdwell, W.C., Neff, W.E., 1998. Analysis of hydroxy-containing seed oils using
ides with relative percent composition of 3.32% higher than OOO atmospheric pressure chemical ionization mass spectrometry. Journal of Liquid
epoxy epidioxides. The m/z 917 in the ESI-MS spectra corresponds Chromatography & Related Technologies 21, 1485–1501.
Byrdwell, W.C., Neff, W.E., 1999. Non-volatile products of triolein produced at fry-
to M+H+ , m/z 934 was M+18+ and m/z 939 corresponds to M+23+ ing temperatures characterized using liquid chromatography with online mass
of this compound (Fig. 3C). A fragment with m/z 883 corresponds spectrometric detection. Journal of Chromatography A 852, 417–432.
to the loss of two water molecules (M+H-2H2 O+ ). The epoxy group Byrdwell, W.C., Neff, W.E., 2001. Autoxidation products of normal and genetically
modified canola oil varieties determined using liquid chromatography with
was identified from its epoxy diacylglycerols fragments ion of m/z
mass spectrometric detection. Journal of Chromatography A 905, 85–102.
of 617 which corresponds to OOep + , m/z 619 corresponds to OSep + , Chaicharoenpong, C., Petsom, A., 2011. Use of tea (Camellia oleifera Abel.) seeds in
and m/z 635 corresponds to the Sep Sep + . The fragment ions at m/z human health. In: Preedy, V.R., Watson, R.R., Patel, V.B. (Eds.), Nuts & Seeds in
Health and Disease Prevention. , 1st edn. Academic Press, London, Burlington,
465.4 are formed from the parent compound. The overall fragmen-
San Diego, pp. 1115–1122.
tation mechanism is similar to compound b. Interestingly the epoxy Chen, L.F., Qiu, S.H., Peng, Z.H., 1998. Effects of sasanguasaponin on blood lipids
epidioxides were identified only in high oleic triacylglycerols mix- and subgroups of high density lipoprotein cholesterol in hyper lipoidemia rat
ture (Zeb and Murkovic, 2010b) and in olive oil (Zeb and Murkovic, models. Pharmacology and Clinics of Chinese Materia Medica 14, 13–16.
Frankel, E.N., Neff, W.E., Miyashita, K., 1990. Autoxidation of polyunsaturated tria-
2011a). This may suggests that oils containing triacylglycerols of cylglycerols. II. Trilinoleneoylglycerol. Lipids 25, 40–47.
relatively similar quantity of fatty acids produce somehow similar Frankel, E.N., Smith, L.M., Hamblin, C.L., Creveling, R.K., Clifford, A.J., 1984. Occur-
oxidation products, independent of the auto- or thermal oxidation. rence of cyclic fatty acid monomers in frying oils used for fast foods. Journal of
the American Oil Chemist’s Society 61, 87–90.
Peak d, e and f were identified as OLO epoxides (Fig. 3D), OOO Fu, C.G., Zhou, P., 2003. Camellia oil: a new special type of plant oil. Food Science
epoxides (Fig. 3E) and OOS epoxides (Fig. 3F) respectively as shown and Technology 2, 19–21.
in Fig. 3. These epoxy moieties were identified from epoxy DAGs Hai, Z., Wang, J., 2006. Detection of adulteration in camellia seed oil and sesame oil
using an electronic nose. European Journal of Lipid Science and Technology 108,
ions and their fragmentation. For example the loss of epoxy oxy- 116–124.
gen from LOep + of m/z 601 produced a DAG ion with one more Halliwell, B., 1994. Free radicals, antioxidants, and human disease: curiosity, cause,
unsaturation having m/z 599 corresponds to LOep + (Fig. 3D). The or consequence? Lancet 344, 721–724.
Hamilton, R.J., Kalu, C., Prisk, E., Padley, F.B., Pierce, H., 1997. Chemistry of free
OOS epoxides were produced in higher amounts as compared to
radicals in lipids. Food Chemistry 60, 193–199.
the first two epoxides. These epoxides were identified from their Holcapek, M., Jandera, P., Zderadicka, P., Hruba, L., 2003. Characterization of triacyl-
protonated molecular ion, ammonium and sodium adducts, dia- glycerol and diacylglycerol composition of plant oils using high-performance
liquid chromatography–atmospheric pressure chemical ionization mass spec-
cylglycerols and epoxy diacylglycerols ions as shown in Table 2.
trometry. Journal of Chromatography A 1010, 195–215.
Previous studies showed that epoxides are the common oxidized Jurek, D., Udilova, N., Jozkowicz, A., Nohl, H., Marian, B., Schulte-Hermann, R., 2005.
products of most of oleic acid containing TAGs or oils (Byrdwell and Dietary lipid hydroperoxides induce expression of vascular endothelial growth
Neff, 1999). factor (VEGF) in human colorectal tumor cells. FASEB Journal 19, 97–99.
Kamal-Eldin, A., Mäkinen, M., Lampi, A., 2003. The challenging contribution of
hydroperoxides to the lipid oxidation mechanism. In: Kamal-Eldin, A. (Ed.), Lipid
Oxidation Pathways. AOCS Press, Champaign, IL, pp. 1–35.
4. Conclusion Kofronova, E., Cvacka, J., Jiros, P., Sykora, D., Valterova, I., 2009. Analysis of insect
triacylglycerols using liquid chromatography–atmospheric pressure chemical
ionization-mass spectrometry. European Journal of Lipid Science and Technol-
Liquid chromatography coupled to electrospray ionization mass ogy 111, 519–525.
spectrometry was successfully used for the characterization of Lee, C.P., Shih, P.H., Hsu, C.L., Yen, G.C., 2007. Hepatoprotection of tea seed oil
(Camellia oleifera Abel.) against CCl4-induced oxidative damage in rats. Food
triacylglycerols composition and studies of auto-oxidation and and Chemical Toxicology 45, 888–895.
oxidation products of camellia seed oil. A total of 15 TAGs were sep- Leskinen, H., Suomela, J.-P., Kallio, H., 2007. Quantification of triacylglycerol
arated and identified in 35 min of elution. The TAGs were identified regioisomers in oils and fat using different mass spectrometric and liquid
chromatographic methods. Rapid Communication in Mass Spectrometry 21,
from their characteristics fragmentation in ESI mass spectra of the
2361–2373.
protonated molecular ion, ammonium and sodium adducts, diacyl- Neff, W.E., Byrdwell, W.C., 1998. Characterization of model triacylglycerol (triolein,
glycerols, epoxy-diacylglycerols fragments and mono-acylglycerol trilinolein and trilinolenin) autoxidation products via high-performance liquid
chromatography coupled with atmospheric pressure chemical ionization mass
fragments. The major TAGs were POO, OOO, OLO, PLO/POL, OLL,
spectrometry. Journal of Chromatography A 818, 169–186.
SOO, ALO and OLLn. Camellia oil was rich in oleic acid and Qizhi, L., Yonghui, H., Haiyan, Z., Bedgood Jr., D.R., Prenzler, P.D., Robards, K., 2008.
linoleic acid. The auto-oxidation studies revealed a total loss The quality and volatile-profile changes of camellia oil (Camellia oleifera Abel.)
of LnLLn, LnOLn, LLLn and OLLn amounting about 13.5% total following bleaching. European Journal of Lipid Science and Technology 110,
768–775.
oxidation. The auto-oxidation products were epoxy hydroperox- Rohr-Udilova, N.V., Stolze, K., Sagmeister, S., Nohl, H., Schulte-Hermann, R., Grasl-
ides, epoxy epidioxides, and mono-epoxides. It was observed that Kraupp, B., 2008. Lipid hydroperoxides from processed dietary oils enhance
614 A. Zeb / Chemistry and Physics of Lipids 165 (2012) 608–614

growth of hepato-carcinoma cells. Molecular Nutrition and Food Research 52, Yang, C.M., Kendall, C.W., Stamp, D., Medline, A., Archer, M.C., Bruce, W.R., 1998.
352–359. Thermally oxidized dietary fat and colon carcinogenesis in rodents. Nutrition
Shyu, S.L., Huang, J.J., Yen, G.C., Chang, R.L., 1990. Study on properties and oxidative and Cancer 30, 69–73.
stability of tea seed oil. Food Sciences 17, 114–122. Zeb, A., Murkovic, M., 2010a. Analysis of triacylglycerols in refined edible oils by
Sjovall, O., Kuksis, A., Kallio, H., 2001. Reversed-phase high-performance liquid isocratic HPLC–ESI-MS. European Journal of Lipid Science and Technology 112,
chromatographic separation of tert-butyl hydroperoxide oxidation products of 844–851.
unsaturated triacylglycerols. Journal of Chromatography A 905, 119–132. Zeb, A., Murkovic, M., 2010b. Characterization of the effects of ␤-carotene on the oxi-
Wang, L., Lee, F.S.C., Wang, X., He, Y., 2006. Feasibility study of quantifying and dation of triacylglycerols using HPLC–ESI-MS. European Journal of Lipid Science
discriminating soybean oil adulteration in camellia oils by attenuated total and Technology 112, 1218–1228.
reflectance MIR and fiber optic diffuse reflectance NIR. Food Chemistry 95, Zeb, A., Murkovic, M., 2011a. Carotenoids and triacylglycerols interactions during
529–536. thermal oxidation of refined olive oil. Food Chemistry 127, 1584–1593.
Weng, R.H., Weng, Y.M., Chen, W.L., 2006. Authentication of Camellia oleifera Abel. oil Zeb, A., Murkovic, M., 2011b. Determination of thermal oxidation and oxidation
by near infrared Fourier transform Raman spectroscopy. Journal of the Chinese products of ␤-carotene in corn oil triacylglycerols. Food Research International,
Chemical Society 53, 597–603. doi:10.1016/j.foodres.2011.02.039.