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Glycidyl Fatty Acid Esters in Refined Edible Oils: A
Review on Formation, Occurrence, Analysis, and
Elimination Methods
Wei-wei Cheng, Guo-qin Liu, Li-qing Wang, and Zeng-she Liu
Abstract: Glycidyl fatty acid esters (GEs), one of the main contaminants in processed oils, are mainly formed during the
deodorization step in the refining process of edible oils and therefore occur in almost all refined edible oils. GEs are potential
carcinogens, due to the fact that they readily hydrolyze into the free form glycidol in the gastrointestinal tract, which has
been found to induce tumors in various rat tissues. Furthermore, glycidol has already been identified as a “possible human
carcinogen’’ (group 2A) by the Intl. Agency for Research on Cancer (IARC). Therefore, significant effort has been
devoted to inhibit and eliminate the formation of GEs. The aim of this review is to provide a comprehensive summary
on the following topics: (i) GE occurrence data for different edible oils and oil-based food products, (ii) precursors of
GEs, (iii) factors influencing the formation of GEs, (iv) potential reaction mechanisms involving the leaving group and
reaction intermediates, and (v) analytical methods, including the indirect and direct methods. More importantly, the
various elimination methods for GEs in refined edible oils are being reviewed with focus on 3 aspects: (i) inhibition and
removal of reactants, (ii) modification of reactive conditions, and (iii) elimination of GE products.
Keywords: elimination methods, formation mechanisms, glycidyl fatty acid esters, heat-induced food toxicants, refined
edible oils
Introduction
Glycidyl fatty acid esters (GEs) have been identified as a new
class of food-processing contaminant. These substances contain
a common terminal epoxide group but exhibit different fatty
acid compositions (Figure 1). For the 1st time, this class of com-
pounds has been reported in edible oils after overestimation of
3-monochloropropane-1,2-diol (3-MCPD) fatty acid esters an-
alyzed by an indirect method (Weisshaar and Perz 2010). Since
1980, 3-MCPD esters have been studied as food-processing con-
taminants and are found in various food types and food ingredi-
ents, particularly in refined edible oils (Velisek and others 1980;
Küsters and others 2011; Razak and others 2012; Becalski and
others 2015). It has already been shown that, similar to 3-MCPD
esters, GEs may also be detected in significant concentrations in
refined edible oils (Zelinkova and others 2006; Masukawa and oth-
ers 2010; Weisshaar and Perz 2010). Although harmful effects on
CRF3-2016-1424 Submitted 9/1/2016, Accepted 12/1/2016. Authors Cheng
and G. Liu are with School of Food Science and Engineering, South China Univ. of
Technology, Guangzhou 510640, China. Author Wang is with Guangdong Testing
Inst. for Product Quality Supervision and China Natl. Quality Supervision and
Testing Center for Foods (Guangdong), Foshan 528300, China. Author Z. Liu
is with Bio-Oils Research Unit, Natl. Center for Agricultural Utilization Research,
Agricultural Research Service, U.S. Dept. of Agriculture, 1815 N. Univ. St., Peoria,
IL 61604, U.S.A. Author G. Liu is also with Guangdong Province Key Laboratory
for Green Processing of Natural Products and Product Safety, South China Univ. of
Technology, Guangzhou, 510640, China. Direct inquiries to author G. Liu (E-mail:
guoqin@scut.edu.cn).
humans and animals have not been demonstrated, the correspond-
ing hydrolysates, 3-MCPD and glycidol, have been identified as
rodent genotoxic carcinogens, ultimately resulting in the forma-
tion of kidney tumors (3-MCPD) and tumors at other tissue sites
(glycidol). Therefore, 3-MCPD and glycidol have been catego-
rized as ‘‘possible human carcinogens’’ (group 2B) and “probably
carcinogenic to humans’’ (group 2A), respectively, by the Intl.
Agency for Research on Cancer (IARC) (IARC 2000; Grosse
and others 2011). In 2009, diacylglyceride (DAG)-based oils pro-
duced by Kao Corp. (Japan) were banned from the global market
due to “high levels” of GEs (AOCS 2009).
Several reports have also suggested that a bidirectional trans-
formation process may occur not only between glycidol and 3-
MCPD but also their esterified forms in the presence of chloride
ions (Figure 1) (Weisshaar and Perz 2010; Destaillats and others
2012a; Shimizu and others 2012a; Ermacora and Hrncirik 2014a).
The transformation rate of glycidol to 3-MCPD was higher than
that of 3-MCPD to glycidol under acidic conditions in the pres-
ence of chloride ion (Kaze and others 2011). Concurrently, the
corresponding esters may exhibit similar conversion behavior. Fur-
thermore, orally administered glycidol and the corresponding es-
ters are metabolized to 3-MCPD in the gastrointestinal tract of
F344 rats (Onami and others 2015). This finding indicates that
the biological availability of 3-MCPD may be reduced by the re-
moval of GEs. In recent years, increasing attention is therefore
being paid to the formation mechanisms of GEs, together with
C2017 Institute of Food Technologists®
doi: 10.1111/1541-4337.12251 Vol. 00, 2017 rComprehensive Reviews in Food Science and Food Safety 1
Glycidyl esters in refined edible oils . . .
Figure 1–Potential conversion mechanism of GEs to 3-MCPD esters or 2-MCPD esters. R1, fatty acyl group.
their determination and their elimination methods, specifically in
edible oils and oil-based/fat-based food products.
Precursors of GEs in refined oils have been identified as par-
tial acylglycerols, that is, DAGs and monoacylglycerides (MAGs);
however, whether they also originate from triacylglycerides
(TAGs) is still a topic of controversial debates. Several authors
noted that pure TAGs were stable during heat treatment (such as
235 °C) for 3 h and were therefore not involved in the formation
of GEs (Destaillats and others 2012a; Shimizu and others 2012a).
However, our experimental results have shown that small amounts
of GEs are present in a heat-treated oil model consisting of almost
100% TAGs. The results from Fourier-transform infrared (FTIR)
studies indicate that the formation of GEs from TAGs can be at-
tributed to the pyrolysis of TAGs to DAGs and MAGs (Cheng
and others 2016). In contrast, 3-MCPD esters in refined oils can
be obtained from TAG (Destaillats and others 2012b; Hori and
others 2016). Presently, the mechanism for the formation of GE
intermediates and the relationship between GEs and 3-MCPD es-
ters are still unknown. At present, 2 methods have been developed
for the determination of GEs in edible oils: a direct one involv-
ing converting GEs into the derivatives and analyzing with gas
chromatography-mass spectrometry (GC-MS) as well as an indi-
rect one quantifying each GE with liquid chromatography-mass
spectrometry (LC-MS). Crews and others (2013) reviewed analyt-
ical methods of GEs in vegetable oils, some other food products,
and various biological samples. Here, the advantages and disad-
vantages of the routine quantification methods were compared. In
recent studies, however, GC-MS and nuclear magnetic resonance
(NMR) have also been used for the direct analysis of GEs in edible
oils (Steenbergen and others 2013; Song and others 2015). There-
fore, we believe that an updated review about these determination
methods is urgently needed. More importantly, efficient methods
should be developed for the reduction of GE levels to ensure the
safety of oils, while not negatively affecting food quality (such as
taste, aroma, and color) as well as consumer acceptance. To the best
of our knowledge, the only reviews on GE elimination methods
available to date have been reported by Craft and others (2013)
and Stadler (2015). However, these studies are based on limited
research results and the methods used involve the modification of
the refining process, renovation of deodorization equipment, as
well as the inhibition of DAG and MAG formation by agricultural
practices. Moreover, further studies on the formation mechanisms
of GEs, together with additional elimination methods, need to be
developed.
Our review aims to deliver a detailed summary of studies on the
occurrence of GEs in different refined edible oils, in combination
with mechanisms and factors responsible for the formation of GEs
and proposed measures for reducing the GE levels in refined edible
oils. Moreover, the identified gaps and future research prospects
are also discussed in subsequent sections.
Occurrence of GEs
Available data on GE contents in food is mainly limited to
refined edible oils and oil-based food products. However, it has
already been established that GEs are formed during the deodor-
ization step of oil-refining processes. As shown in Table 1, GEs
can be detected in diverse refined oils and fats such as palm oil,
rice oil, soybean oil, and corn oil. Among these, rice oil and palm
oil prove to be most susceptible to the formation of GEs, both
exceeding 30 mg/kg of oil. The precursor (DAG) contents of GEs
are particularly high in oil, ranging from 4% to 12% with a mean
of approximately 6.5% in palm oil (Long and others 2005). This
fact also explains the relatively high concentrations of GEs in palm
oil, while the actual reason for the high GE contents in rice oil is
still unclear. Crude or unrefined oils and fats, such as extra virgin
olive oil, do not contain GEs or merely trace amounts of GEs
(MacMahon and others 2013a).
Generally, a high-temperature treatment, such as deodorization,
is the most important factor for the formation of GEs. Apart from
deodorization, frying, barbecuing, and baking at high tempera-
ture also seem to result in the potential formation of GEs in edible
oils and oil-based food products. As reported previously, GEs are
detected in edible meat patties cooked by both gas-fired and char-
grilling cooking methods at approximately 200 °Corhigherin
concentrations ranging between 0.07 and 0.17 mg/kg and 0.67
and 1.11 mg/kg in meat samples, respectively (Inagaki and Hi-
rai 2016). GEs are also detected in cookies containing different
types of fat (MacMahon and others 2013a), presumably caused by
high-temperature baking. In contrast, Dingel and Matissek (2015)
have reported the absence of GEs formation in samples of potato
crisps and the corresponding oils during deep frying of potato
crisps. Subsequent reports confirmed that frying for a long time
(>8 h) led to the degradation of GEs, instead of the formation and
a higher GE degradation was found in the oil used to fry snacks
(95%) than that used to fry potato chips (87%). The corresponding
degradation rate largely depends on the type of oil (Aniołowska
and Kita 2015, 2016b, 2016c). Similarly, 3-MCPD esters in com-
mercial deep-fried food products containing fat were also found in
2Comprehensive Reviews in Food Science and Food Safety rVol. 00, 2017 C2017 Institute of Food Technologists®
Glycidyl esters in refined edible oils . . .
Table 1–Occurrence of GEs in refined edible oils and oil-based food products.
Class Food products Number Method Range (mg/kg)
Average
(mg/kg) References
Oils and fats Palm oil 3 Two-step SPE/LC-MS 9.26 to 9.40 – (Blumhorst and others 2013)
Palm oil 3 Two-step SPE/LC-MS 25.60 to 28.00 – (Shiro and others 2011)
Palm oil 2 NPLC/GC-MS – 30.20 (Steenbergen and others 2013)
Palm oil 20 Alkaline/Br−GC-MS 0.30 to 18.00 3.70 (Kuhlmann 2011)
Palm oil 4 Two-step -SPE/LC-MS – 31.24 (Aniołowska and Kita 2015c)
Palm oil and palm
Oil-based fats
12 GPC/GC-MS 0.32 to 6.30 2.38 (Weisshaar and Perz 2010)
Palm kernel oil 2 Alkaline/Br−GC-MS 0.30 to 2.50 – (Kuhlmann 2011)
Palm olein 3 LC-TOF-MS – 15.60 (Haines and others 2011)
Soybean oil 3 NPLC/GC-MS – 1.56 (Steenbergen and others 2013)
Soybean oil 3 UPLC: ultra performance
liquid chromatography-
TOF-MS
– 0.12 (Hori and others 2012)
Rice oil 3 Two-step SPE/HPLC-MS 27.22 to 28.76 – (Shiro and others 2011)
Rice oil 3 LC-TOF-MS – 33.70 (Haines and others 2011)
Olive oil 2 NPLC/GC-MS – 4.31 (Steenbergen and others 2013)
Corn oil 3 UPLC-TOF-MS – 2.09 (Hori and others 2012)
Sesame oil 3 LC-TOF-MS 1.30 to 3.70 – (Haines and others 2011)
Rapeseed oil 5 GC-MS 0.01 to 1.10 0.51 (Kuhlmann 2016)
Peanut oil 4 Alkaline/Br−GC-MS 0.40 to 1.10 – (Kuhlmann 2011)
Peanut oil 3 LC-MS/MS 0.44 to 0.57 0.49 (MacMahon and others 2013a)
Sunflower oil 11 GC-MS-HS 0.02 to 0.90 0.36 (Kuhlmann 2016)
Walnut oil 5 Acidic/Br−GC-MS – 5.83 (Ermacora and Hrncirik 2013)
Walnut oil 5 Alkaline/Br−GC-MS 0.70 to 1.40 – (Kuhlmann 2011)
Coconut oil 2 Alkaline/Br−GC-MS 0.50 to 3.00 – (Kuhlmann 2011)
Almond oil 1 LC-MS/MS – 0.03 (MacMahon and others 2013a)
Grapeseed oil 3 LC-MS/MS 0.14 to 3.02 1.14 (MacMahon and others 2013a)
Infant and baby
food
Infant formula (fat
fraction)
70 Acidic/Br−GC-MS 0.16 to 0.87 0.36 (W¨
ohrlin and others 2015)
Meat sample Pork, beef, chicken
cooked by gas-fired
frying pan and
charcoal grill
6 Two-step SPE/LC-MS 0.07 to 0.17 for
gas-fired frying
pan, 0.67 to
1.11 for
charcoal grill
– (Inagaki and Hirai 2016)
Conventional
samples
Oxtail soup (powder) 3 Acidic/Br−GC-MS – 0.31 (Küsters and others 2011)
Gravy (powder) 3 Acidic/Br−GC-MS – 0.07 (Küsters and others 2011)
Vegetable soup
(powder)
3 Acidic/Br−GC-MS – 0.14 (Küsters and others 2011)
Margarine 3 Acidic/Br−GC-MS – 3.63 (Küsters and others 2011)
Cookies 3 Acidic/Br−GC-MS – 0.94 (Shiro and others 2011)
Sauce for chicken
(powder)
3 Acidic/Br−GC-MS – 0.07 (Küsters and others 2011)
Chocolate—hazelnut
bar
3 Acidic/Br−GC-MS – 0.07 (Küsters and others 2011)
“–” Represents not available/analyzed data.
small amounts, ranging from 0 to 0.99 mg/kg, expressed by the 3-
MCPD equivalent (Arisseto and others 2015). This finding most
likely indicates that the digestion of GEs and 3-MCPD esters from
fried food exhibits a lower risk to human health. Moreover, some
food products, prepared using contaminated vegetable oils and fats
as the main ingredients, such as infant formula, margarine, creams,
and mayonnaise, may contain certain amounts of GEs derived from
the raw oil, without high temperature exposure (Küsters and oth-
ers 2011; Ermacora and Hrncirik 2014b). In recent years, with
increasing social awareness of oil safety, several industrial standards
have been established for limiting the GE contents in oils and
fats: below 0.60 mg/kg of oil set by China’s health care and food
industry, below 0.50 mg/kg of oil set by Nestl´
e, and below 0.10
mg/kg of oil set by Yili Company. However, no universally and
globally accepted standard has been set so far.
A significant discrepancy has also been observed among the GE
levels determined by different methods in the same type of oils.
For example, 30.2 mg/kg of GE levels in palm oil were detected
by GC-MS with normal phase LC (NPLC) separation, but only
9.26 to 9.40 mg/kg were detected by LC-MS with 2 solid phase
extractions (SPEs) (Blumhorst and others 2013; Steenbergen
and others 2013). Several likely reasons have been proposed
for this phenomenon: First, the crude oils, such as palm oil,
were collected from different geographic locations with varying
climatic conditions. This may have caused a distinct hydrolysis of
TAGs by lipase during the matur ity and harvesting of oil plants for
the formation of DAGs and MAGs (Siew and Ng 1995). Second,
the difference in refining process parameters, particularly the
deodorization step, likely caused discrepancies in the found GE
levels. Third, no unified standard method is available for analysis
of GEs, and the GE levels in the same type of oil determined by
these methods with different sensitivity, specificity, and accuracy
cannot be compared. This general limitation will be further
discussed in detail below. Finally, the overall reliability of some
data in certain cases may also be questionable.
Except for refined edible oils and fats, the data of GE occurrence
available for oil-based food products can be found listed in Table
1. The GE levels in infant formula (n=70) purchased from local
market s a re re port ed by W ¨
ohrlin and others (2015). The highest
concentration of GEs found was 0.87 mg/kg, expressed on a raw
fat basis. Ideally, the report motivated government officials and
industry to improve the safety of infant formula as an artificial
C2017 Institute of Food Technologists®Vol. 00, 2017 rComprehensive Reviews in Food Science and Food Safety 3
Glycidyl esters in refined edible oils . . .
substitute for human breast milk, commonly used for feeding in-
fants in their 1st year of life. To ensure the accuracy of GE content
in infant formula, a more recent report proposed a microwave ex-
traction method of fat with the limit of detection (LOD) and limit
of quantitation (LOQ) equaling 0.0008 and 0.0028 mg/L, re-
spectively (Marc and others 2016). In conventional oil-based food
sources, relatively high contents of GEs can be found in margar ine
and cookies, with a mean of 3.63 and 0.94 mg/kg, respectively.
The amounts of GEs in oil-based food products are caused by the
addition of contaminated oils and fats as well as high temperatures
in the manufacturing processes.
Formation of GEs
The occurrence of GEs in refined edible oils has attracted signif-
icant attention with respect to the formation mechanism, includ-
ing the precursors of GEs and factors influencing the formation
of GEs. The deodorization step in the oil refining process sig-
nificantly affects the formation of GEs (Weisshaar and Perz 2010;
Destaillats and others 2012a). In initial studies, GEs were regarded
as a pathway of 3-MCPD ester formation or their degradation
(Hamlet and others 2002; Svejkovska and others 2006). Subse-
quently, it was proposed that GEs, as well as 3-MCPD esters,
form an acyloxonium ion intermediate 1st and then rearrange
through charge migration, finally forming GEs (Weisshaar and
Perz 2010; Rahn and Yaylayan 2011a). However, it was also con-
sidered that GEs and 3-MCPD esters could be formed follow-
ing a different pathway, depending on the applied temperature
and reaction time (Haines and others 2011; Hrncirik and Duijn
2011). In this review, we focus on 3 aspects of the formation
of GEs from macroscopic and microscopic perspectives, that is,
precursors and factors influencing the formation of GEs for the
macroscopic section and reactive mechanisms for the microscopic
section.
Precursors
DAGs and MAGs, the minor components in edible oils, may
be formed not only through lipase hydrolysis of TAGs during ma-
turing, harvesting, and transportation of oil fruits/seeds, but also
through pyrolysis of TAG at high temperatures, including con-
ventional heating and deodorization (Shimizu and others 2012a;
Lucas-Torres and others 2014). Thus, different distributions of
DAGs and MAGs may be observed in the different varieties of oils
or the same type of oil from different locations, with MAGs being
present in much smaller amounts than DAGs. For example, the
total DAG contents, including 1,2-DAGs and 1,3-DAGs, in corn
oil, palm kernel oil, soybean oil, coconut oil, and sunflower oil
are all different, that is, 4.1%, 2.8%, 2.6%, 2.3%, and 2.2%, respec-
tively (Hamlet and others 2011). Moreover, due to the specificity
of TAG lipase at Sn-1 or Sn-3 position, the 1,2-DAG contents are
found to be higher than the 1,3-DAG contents in freshly extracted
oils. However, 1,3-DAGs exhibit a higher stability, which explains
the relative decrease in 1,2-DAG levels compared to 1,3-DAGs
and total DAG levels dur ing storage (Hamlet and others 2011).
Because of a longer water exposure compared to seed oil, fruit oils
are more susceptible to potential hydrolysis reactions. DAG levels
are therefore particularly high in fruit oils, such as palm oil and
olive oils. For example, palm oil features the highest amount of
DAG, ranging from 4% to 12%, with a mean of 6.5%. Further-
more, high contents of GEs have been reported in refined palm
oil (Pudel and others 2011; Aniołowska and Kita 2016a). The GE
levels in DAG-rich oil are 12 to 43 times higher than those in the
normal edible oil consisting chiefly of TAGs (Masukawa and oth-
ers 2010). Haines and others (2011) reported that only 5 mg/kg
or less of GEs were detected in oil consisting of a DAG content
of <2%. However, high DAG contents (>6%) in oil were found
to correspond to the prominent GE levels. This notion seems to
indicate that DAGs are likely to be involved in the formation of
GEs.
Furthermore, Destaillats and others (2012a) as well as Shimizu
and others (2012a) conducted a series of model reactions simulat-
ing oil deodorization with pure TAG, DAG, and MAG. GEs were
detected in heated DAG and MAG, but only in trace amounts in
TAG. Concurrently, Freudenstein and others (2013) also demon-
strated the contribution of DAGs and MAGs to the formation
of GEs in refined oil. In another approach, increasing amounts
of pure DAG and MAG were added into the model oil without
polar fractions and the mixtures were heated at 240 °Cfor2h.
In both tests, the formation of GE increased in a linear fashion,
with the increasing amounts of DAGs and MAGs. However, other
minor components, such as the phospholipids, chloride, and free
fatty acids (FFAs), did not affect or merely slightly affected the
formation of GEs. Therefore, it can be concluded that DAGs and
MAGs are the precursors of GEs. Our experiments also prove fur-
ther that MAGs exhibit a higher formation capacity than DAGs.
This could be achieved by comparing real edible oils and chem-
ical models (Cheng and others 2016). However, due to the low
molecular weight of MAGs that are readily stripped into the de-
odorizer distillate or due to possible interesterification reactions
from 2 MAGs to DAG, MAGs can be found in refined edible oils
only in trace amounts (<0.5%) (Goh and Timms 1985; Shimizu
and others 2012a), accounting for its low contribution to the for-
mation of GEs. Previously, glycerol had been shown to function
as a precursor of GEs (Freudenstein and others 2013), but it could
be completely removed by distillation off due to its low molecular
weight.
Factors
As mentioned above, GEs, as well as 3-MCPD esters, are formed
predominantly during the deodorization step in the oil refining
process. Deodorization temperature and time have been shown
to represent the most crucial factors for the formation of GEs.
Several research groups have reported that GE levels increase
upon increasing the incubation temperature ranging from 140 to
280 °C (Hrncirik and Duijn 2011; Destaillats and others 2012a).
Thus, the finding explains why no correlation between the GE
levels and DAG contents were observed in in the production of
commercial oil samples by different suppliers using different de-
odorization temperatures (Craft and others 2012). However, topic
of controversial debates is the issue whether GEs can degrade via
thermal degradation reactions at certain temperatures and reac-
tion time. Pudel and others (2011) reported that the GE content
increased with time below 250 °C, favoring the formation of
GEs. However, at temperatures up to 290 °C,theGElevelsde-
creased within 2 h, which was considered as a consequence of GE
degradation or distillation. GEs have been detected in deordorizer
distillates (Craft and others 2012), and recently, it has been found
that GE levels decreased rapidly with time after 2 h at 200 °Cin
DAG-model reaction occurring in sealed ampoule bottle (Cheng
and others 2016). If the deodorization temperature was set at
230 °C, GE levels increased with time in the range of 1 to 5 h
(Hrncirik and Duijn 2011). Therefore, the accumulation of GEs
in refined oil depends on the formation rate of GEs from DAGs
and MAGs as well as their degradation and distillation rate during
deodorization in oil refining process. It seems likely that the higher
4Comprehensive Reviews in Food Science and Food Safety rVol. 00, 2017 C2017 Institute of Food Technologists®
Glycidyl esters in refined edible oils . . .
the temperature that is implemented, the higher the degradation
rate of GEs that is observed in the deodorization step. This finding
may further explain why the GE content in frying oil decreases
during frying for a long per iod of time (>24 h), regardless of the
applied frying temperature (Aniołowska and Kita 2016b).
In the oil deodorization step, apart from deodorization tem-
perature and time, the stripping steam rate can also influence the
formation of GEs. ¨
Ozdikicierler and others (2015) investigated
the effects of process parameters (temperature, pressure, and strip-
ping steam) on the formation of GEs during steam distillation
of olive oil and olive pomace oil by response surface method-
ology. It was found that the interaction between the stripping
steam rate and temperature was statistically significant for the
formation of GEs. When the water flow rate was higher than
2.0 mL/min, the increasing mass of distillation vapor from oil to
cooler, which may contain MAGs, DAGs, and even GEs, likely
explained why the GE contents decreased despite of the high
temperature.
Additionally, due to an uneven heat distribution in oil
fruits/seeds during the roasting or drying processes, at the edge of
the roaster the temperature may increase beyond 200 °C(S
´
anchez
Moral and Ruiz M´
endez 2006), high enough to form GEs. In
some cases, small amounts of GEs could also be found in crude
oils (Matth¨
aus and others 2011; MacMahon and others 2013a;
Qi and others 2015). Although GE levels remain constant dur-
ing the refining process prior to deodorization, DAG and MAG
may be reduced to a certain degree (Bailey 2005), which indi-
rectly influences the formation of GEs. Additionally, it is well
known that GEs are unstable substances due to the presence of
an epoxide group in the chemical structures. It has been pro-
posed that the epoxy structure of GEs may be destructed under
acidic conditions, removing different amounts of GEs that depend
on the concentration of acid in refined oil (Matth¨
aus and others
2011). However, Freudenstein and others (2013) have suggested
that the presence of FFAs could support the formation of GEs.
Although it is believed there is little effect on the formation of
GEs in comparison with DAGs, certain amounts of FFAs exist
in bleached oil during physical refining. This notion seems to
indicate that a relatively low level of acid favors the formation
of GEs, but higher concentration of acid seems also to reduce
the occurrence of GEs in refined oil. However, further studies
and follow-up work must be conducted in order to confirm this
hypothesis.
Reactive mechanisms
It has already been well documented that DAGs and MAGs
represent the most important reactants for the formation of GEs
formed during high temperature exposure and depending on both
temperature and temperature exposure time. Unlike the chlorine-
containing 3-MCPD esters, GEs are formed without chloride.
Presently, there are 4 proposed mechanisms of GE formation de-
rived from DAGs and MAGs (Figure 2A), all involving an in-
tramolecular rearrangement through charge migration and dif-
fering from each other depending on either the nature of the
intermediate or the leaving group. Two of the proposed mecha-
nisms involve a common reactive intermediate formed by either
deacidification of 1,2-DAGs (Figure 2A, pathway d) or dehydra-
tion of MAGs (either 1-MAGs or 2-MAGs) (Figure 2A, pathways
candc´). The other 2 pathways consider a direct intramolecu-
lar rearrangement followed by elimination of fatty acid for DAGs
(either 1,2-DAGs or 1,3-DAGs) (Figure 2A, pathways b and b´)
or of water for 1-MAGs (Figure 2A, pathway a). The next sub-
section will review each mechanism, providing evidence support-
ing these mechanisms in refined edible oils and oil-based food
products.
For a long time, the cyclic acyloxonium ion, a well-known re-
active intermediate in organic chemistry (Paulsen 1971), has been
proposed as a possible reactive intermediate in the formation of
3-MCPD esters and 2-MCPD esters (Collier and others 1991;
Hamlet and others 2002; Vel´
ıˇ
sek and others 2002). It may be
readily formed by the elimination of hydroxyl groups from either
DAGs or MAGs. Rahn and Yaylayan (2011a) confirmed the actual
formation of cyclic acyloxonium ions through real-time monitor-
ing of FTIR spectra of pure acylglycerols with a chloride species
treated at 100 °C. Considering the similarity in molecular struc-
ture, formation condition, and widespread distribution between
3-MCPD esters and GEs (MacMahon and others 2013a), it has
been concluded that cyclic acyloxonium ions may also represent
a reactive intermediate in the formation of GEs (Weisshaar and
Perz 2010). However, the hypothesis has not yet been experi-
mentally confirmed. In a recent study conducted by our group,
the cyclic acyloxonium ion could be identified through FTIR
spectra of pure 1,2-dipalmitin and 1-monopalmitin during high-
temperature treatment (Cheng and others 2016). Therefore, we
can confirm that a cyclic acyloxonium ion I is formed through the
transformation of an electron along with the elimination of fatty
acid for 1,2-DAGs (Figure 2A, pathway d) and water for MAG
(Figure 2A, pathways c and c´). The cyclic structure may then be
opened through an intramolecular rearrangement at high temper-
ature, ultimately generating GEs. Theoretically, due to the steric
hindrance at the Sn-2 position, the cyclic acyloxonium ion cannot
be formed by the deacidification of 1,3-DAGs. Furthermore, in
comparison with DAGs, the reactivity of MAGs is expected to
proceed more rapidly in an acidic medium, such as partially hy-
drolyzed oils, not only due to more similar molecular structures
of MAGs and GEs, but also due to the superior leaving group
water compared to a fatty acid chain (Hamlet and others 2004).
However, Hrncirik and Duijn (2011) as well as Shimizu and others
(2012a) have concluded that the formation reactions of 3-MCPD
esters in the presence of chloride are completed within a relatively
short period of time. However, steady levels may be reached and
GEs form continuously throughout the heating period, without
chloride influencing the formation of GEs. The nonsynchronous
and independent occurrence lead to a possible conclusion that
3-MCPD esters and GEs feature no common intermediates. The
authors also reported that the levels of GEs and 3-MCPD esters
reached an equilibrium after 1 to 2 h of heating and depending
on the temperature level (Shimizu and others 2013a). The biggest
difference between the 2 formation reactions is the absence of
chloride, which may be responsible for the different formation
rates. It has also been reported that, due to the stability of 3-
MCPD esters, the plateau levels of 3-MCPD esters in the model
test are the result of completed reactions resulting from the lack of
an available chloride source. Hence, the formation of 3-MCPD
esters is limited to not only the quantity, but also the availabil-
ity of chloride, such as the molecular species. Conversely, GEs
represent unstable substances at high temperatures. Therefore, the
GE formation and transformation both take place in a simultane-
ous fashion, which may partially explain why GE levels reach an
equilibrium in a longer period of time than 3-MCPD esters.
To the best of our knowledge, Destaillats and others (2012a)
were the 1st to investigate the formation mechanisms of GEs
in refined palm oil. The authors conducted a series of model
reactions mimicking palm oil deodorization with pure TAG,
C2017 Institute of Food Technologists®Vol. 00, 2017 rComprehensive Reviews in Food Science and Food Safety 5
Glycidyl esters in refined edible oils . . .
Figure 2–Summary of the proposed pathways of GE formation: (A) reaction mechanisms derived from DAGs and MAGs and (B) mechanisms of
formation of DAGs and MAGs derived from TAGs at high temperature (Rahn and Yaylayan 2011a, 2011b; Destaillats and others 2012a). R1,R
2, and
R3, fatty acyl groups (either “same” or “different”).
DAG, and MAG. The results revealed that thermally treated DAG
with high amounts of GEs induced 5 times more FFAs than
thermally treated TAG with trace amounts of GEs did. However,
FFAs could be detected at a lower amount in thermally treated
MAG, also with high amounts of GEs. Based on the above results
and a previous report (Vel´
ıˇ
sek and others 2002), the acyloxonium
ion intermediates have been suggested to be generated through
the initial elimination of FFAs by abstraction of the proton in the
hydroxyl group (at the Sn-2 position for 1.3-DAGs and at the
Sn-3 position for 1,2-DAGs) and the vicinal carboxyl group. The
rearrangement through charge migration eventually results in the
formation of GEs (Figure 2A, pathways b and b´). In comparison
with the cyclic acyloxonium ion I, acyloxonium ion intermediates
exhibit a higher potential energy, suggesting a lower stability due
to the fact that these species do not feature a resonance-stabilized
structure (Hamlet and others 2011). This notion seems to suggest
the cyclic acyloxonium ion I in pathways c and d features a greater
potential for the formation of GEs than acyloxonium ions in
6Comprehensive Reviews in Food Science and Food Safety rVol. 00, 2017 C2017 Institute of Food Technologists®
Glycidyl esters in refined edible oils . . .
pathways b and b´(Figure 2A) do. Moreover, the latter species are
difficult to be detected due to their short time occurrence. There-
fore, the species have never been identified in vitro.ForMAGs,
the elimination of water could be initiated by abstraction of the
proton in the hydroxyl group as well as the hydroxyl group in the
vicinal diol following the formation of GEs (Figure 2A, pathway
a). This mechanism has been proposed in a similar fashion before
in the literature (Hamlet and others 2011). The reaction does not
occur in 2-MAGs, unless it is transferred to 1(3)-MAGs. The latter
is most notably due to the steric hindrance at the Sn-2 position.
The trace amounts of GEs detected in pure TAG suggest that
TAGs do not represent the precursors of GEs. However, DAGs and
MAGs can be formed from the direct hydrolysis of TAGs through
the elimination of fatty acids under high-temperature conditions.
Unlike GEs, the formation of 3-MCPD esters has been reported
to be predominantly derived from TAGs that involves the cyclic
acyloxonium ion II reaction intermediate (Destaillats and oth-
ers 2012b). The cyclic acyloxonium ion II may be attacked by
the chloride ion to form 3-MCPD esters and may also decom-
pose to form DAGs through the hydrolysis reactions (Figure 2B)
(Rahn and Yaylayan 2011a). DAGs and MAGs formed from TAGs
through the 2 routes described above may contribute to the minor
formation of GEs in pure TAG.
Except for the 4 possible pathways mentioned above, GEs may
also be formed through the decomposition of MCPD monoesters
involving 3-MCPD monoesters, and 2-MCPD monoesters rather
than their diesters. Vel´
ıˇ
sek and others (2002) have discussed the
decomposition of 3-MCPD, 2-MCPD, and their enantiomers in
alkaline media from a glycidol intermediate, which was found to
then hydrolyze to form glycerol. The latter was also observed as
the decay pathway of 3-MCPD and 2-MCPD in model dough
systems, as well as a side reaction in the production of epichloro-
hydrin via dehydrochlorination of dichloropropanols (Hamlet and
others 2003; Milchert and others 2012). Based on a classical or-
ganic chemistry theory, the combination of a hydroxy group and
a chlorine atom on neighboring carbon atoms is responsible for
the most common elimination reaction of vicinal chlorohydrins,
such as by a dehydrochlorination to form substituted oxiranes
(epoxides) (Ketola and others 1978). It has also been shown that
3-MCPD is almost 10 times more stable than 2-MCPD that de-
composes readily under alkaline conditions (Dolezal and Velisek
1994). More importantly, their formation mechanisms involving
the SN2 reaction are susceptible to steric effects (Collier and others
1991). As expected, the levels of 3-MCPD esters in almost all oils
are significantly higher than the levels of 2-MCPD esters (Koyama
and others 2015; J ˛
edrkiewicz and others 2016). Therefore, it seems
that the potential of 2-MCPD to form glycidol is much greater
than 3-MCPD. Similarly, the basic reactions of an esterified form,
such as the formation and decomposition, are considered to be
analogous to the reactions of the free form in the presence of a
vicinal chlorohydrin structure, such as 2-MCPD monesters and
3-MCPD-1-esters. The possible formation pathways of GEs de-
rived from both MCPD monoesters are shown in Figure 3. In
slightly acidic as well as neutral environments, the dehydrochlo-
rination of vicinal chlorohydrin groups in MCPD esters involves
the elimination of a chloride anion, resulting in the formation of
a carbocation intermediate. Then, the hydroxyl group in MCPD
monoesters may act as a nucleophile, leading to the formation of a
protonated epoxide and the formation of GE epoxides via proton
elimination (Figure 3). Conversely, the decomposition reaction of
MCPD esters takes place rather rapidly in an alkaline environment.
As outlined in Figure 3, the exogenetic hydroxyl anions react with
hydrogen in the hydroxyl group of chlorohydrin to reach an equi-
librium as the corresponding alcoholate anion. The alcoholate
oxygen then attacks the carbon bearing the leaving chloride atom
to form GEs as the rate-determining step in this dehydrochlori-
nation reaction. Due to the steric hindrance at the Sn-2 position,
the 3-MCPD-2-ester, the isomer of 3-MCPD-1-ester, cannot di-
rectly form GEs through the 2 routes described above at pH ࣘ7
and pH >7 until transformation into 3-MCPD-1-ester bearing
a vicinal chlorohydrin structure takes place (Figure 3). Also, the
high intrinsic hydrolysis tendency of oils exposed to alkali media
restricts the presence of alkali conditions in edible oils, and leads to
reduce contribution for the total GE levels compared with those
exposed to acidic and neutral media. Although these predictions
seem to be feasible in theory, 3-MCPD monoesters or 2-MCPD
monoesters in refined oils are only found in trace amounts, and,
in part, are derived from the hydrolysis of their corresponding
diesters, the most general form of 3-MCPD esters (Zelinkova and
others 2006; Ermacora and Hrncirik 2014a). Furthermore, it is
possible that 3-MCPD esters may be formed through the dehy-
drochlorination of dichloropropanol esters, which can be either
found not at all or merely in small amounts in some oils, such as
sunflower and tea seed oil (Milchert and others 2012; Yang and
others 2015; Kuhlmann 2016). It is thus believed that the contri-
bution of 3-MCPD or 2-MCPD esters to the formation of GEs
in refined edible oils is most likely negligible.
As mentioned before, the GE concentrations in refined oils can
be obtained as a result of competition reactions between the for-
mation and transformation, due to the fact that the epoxy group is
not stable upon heating. In comparison, the transformation of GEs
can be observed at lower temperatures, suggesting the transforma-
tion reaction exhibits a lower activation energy than the formation
reaction (Shimizu and others 2013a). To date, the transformation
mechanism of GEs in refined edible oils under high tempera-
ture conditions still remains unclear. The ring-opening reaction
of GEs has been demonstrated to take place under acidic con-
ditions, leading to the formation of DAGs and MAGs (Shimizu
and others 2012b). Accordingly, it seems possible that GEs may
transform back to MAGs and DAGs during heating in the pres-
ence of FFAs. However, the hypothesis for this action still remains
unexplored. It had been proposed before that one of the forma-
tion pathways of 3-MCPD esters takes place via GEs, meaning
that GEs may transform into 3-MCPD esters in the presence of a
chlorine source (Figure 1). Rahn and Yaylayan (2011b) as well as
Shimizu and others (2013b) have confirmed that GEs may repre-
sent precursors for 3-MCPD monoesters which exhibit merely a
very low concentration in edible oils (Zelinkova and others 2006).
However, the transformation rate between these species has been
shown to be very low (Shimizu and others 2013b), most notably
due to the fact that no significant difference between GE levels in
model reactions with and without a chloride source (Shimizu and
others 2012a). The thermal degradation test of 3-MCPD diesters
carried out in a model system mimics the deodorization process
of vegetable oils and also demonstrates that the continuous for-
mation of 3-MCPD monoesters is not due to the degradation of
the corresponding diesters, but instead is likely due to the trans-
formation of GEs in the presence of chloride ion (Ermacora and
Hrncirik 2014a). Additionally, degradation reactions of the ester
are thought to be possible to take place at high temperature, for
example, thermal degradation of TAGs during heating (Lucas-
Torres and others 2014). Therefore, it is believed that free glycidol
detected in the deodorizer distillate may be formed from GEs.
However, in a sealed heating system, the species is not formed
C2017 Institute of Food Technologists®Vol. 00, 2017 rComprehensive Reviews in Food Science and Food Safety 7
Glycidyl esters in refined edible oils . . .
Figure 3–Proposed pathway of GE formation derived from 2-MCPD esters and 3-MCPD esters in neutral, acidic, and alkaline media (Hamlet and others
2011). R, R1, and R2, fatty acyl groups (either “same” or “different”).
in diolein, whereas GEs are formed at identical concentrations
(Shimizu and others 2012a), indicating that the deacidification of
GEs via thermal degradation reactions is rather impracticable.
Analysis of GEs
Because of structural similarities of GEs and 3-MCPD esters,
initial methods for the detection of GEs provide the analytical pro-
tocol based on that of 3-MCPD esters, involving the transesterifica-
tion of esters to release the free forms (Divinova and others 2004;
Seefelder and others 2008; Weisshaar and Perz 2010). It has already
been shown that some technological drawbacks exist, ultimately
limiting the efficient detection of GEs as well as 3-MCPD es-
ters. For example, the absence of a proper chromophore renders
high-performance liquid chromatography (HPLC) with ultravi-
olet (UV) or fluorescence detectors unsuitable. Furthermore, a
high boiling point limits the use and development of a direct GC
method. To the best of our knowledge, only 1 report describes
the GC analysis of GEs with silylation in silicon oil. However, this
method is not applicable for edible oils consisting of high amounts
of acylglycerols (Engbersen and Van Stijn 1976). Therefore, as
shown in Table 2 and 3, therefore, the present analysis methods to
determine GEs in edible oils may be grouped into 2 categories:
indirect and direct methods. In the following subsections, we will
provide a comprehensive summary of the 2 methods.
Indirect analysis
The indirect analysis of GEs is based on the transforma-
tion of glycidol into a halogenated derivative involving a
derivatization reaction with derivatizing agents such as phenyl-
boronic acid (PBA), heptafluorobutyrylimidazole (HFBI),
bis(trimethylsiyl)trifluoroacetamide (BSTFA), and heptafluorobu-
tyric anhydride (HFBA). In indirect analyses, intact 3-MCPD
esters and GEs are 1st hydrolyzed into the corresponding free
3-MCPD and glycidol under either acidic or alkaline conditions,
followed by a purification process (liquid-liquid extraction),
derivatization, and quantification by GC-MS. The latter has been
regarded an official method by the German Society for Fat Science
(DGF) (DGF Standard Methods C III 18 (09) 2009). This method
consists of 2 pretreatments (option A and option B) with different
analytical mechanisms (Figure 4A). In option A, the released
glycidol from GEs by sodium methoxide, together with 3-MCPD,
can be converted to a 3-MCPD-PBA derivative in the presence of
chloride and PBA, thus determining the sum of GEs and 3-MCPD
esters. Option B only determines the level of 3-MCPD esters re-
sulting from the elimination of GEs by acid treatment; the total GE
amounts are calculated as the difference between both measure-
ments with a modification by multiplication with a stoichiometric
factor of 0.67. The development of the method is based on the as-
sumption that glycidol can be completely converted to 3-MCPD
in option A, and completely eliminated by acid treatment in option
B, whereas no other substance will react with inorganic chloride
to form 3-MCPD. As a reference, a modified approach has been
approved as a standard method by the American Oil Chemists’
Society (AOCS) (Joint AOCS/JOCS Official Method Cd 29c-13
2013).
Weisshaar (2008) showed that the acidic transesterification
procedure incorrectly produced higher levels of 3-MCPD
esters than the transesterification procedure with sodium
methoxide\methanol. This finding is most likely due to the addi-
tional formation of 3-MCPD under acidic conditions. Neverthe-
less, the previously published findings determined that, when free
3-MCPD is released by the alkaline-catalyzed transesterification,
the presence of GEs and chloride can lead to an overestimation of
3-MCPD ester levels in the tested oil (Kuhlmann 2008; Hrncirik
and others 2011). Conversely, Hrncirik and others (2011) demon-
strated that, due to the irreversible degradation of GEs during
acidic transesterification, the subsequent conversion to 3-MCPD
proved to be impossible. The latter seemed to be the reason for
a better robustness and selectivity of this acid transesterification.
Meanwhile, NMR studies indicated that a bidirectional conver-
sion takes place between GEs and 3-MCPD esters in option A with
the alkaline transesterification as depicted above (Kaze and others
2011). Furthermore, it was found that 37% of the 3-MCPD mass
was converted to glycidol during the derivatization step in option
A, while >70% of the glycidol mass was converted to 3-MCPD.
Additionally, the incomplete epoxide ring-opening reaction of
glycidol and its esters (about 90%) could also be observed by acid
treatment in option B. Furthermore, 3-MCPD and its esters were
determined to be formed from partial acylglycerols and related
chloride-containing substances in the tested oil during acidic pre-
treatment, which interfered with the accurate determination of the
3-MCPD esters. The results obtained led to an underestimation
of the GE contents in edible oils when this indirect method was
used. This, in turn, explains why lower GE levels were obtained
through the indirect method, compared to the direct method, us-
ing the same sample (Shimizu and others 2010). Furthermore, acid
8Comprehensive Reviews in Food Science and Food Safety rVol. 00, 2017 C2017 Institute of Food Technologists®
Glycidyl esters in refined edible oils . . .
Figure 4–Reaction scheme of the indirect determination of GEs by GC-MS: (A) chlorination and (B) bromination of GEs followed by PBA derivation. R,
R1, and R2, fatty acyl groups (either “same” or “different”).
C2017 Institute of Food Technologists®Vol. 00, 2017 rComprehensive Reviews in Food Science and Food Safety 9
Glycidyl esters in refined edible oils . . .
Table 2–Indirect analysis methods for GEs in edible oils.
Method
denomination Analyte Internal standard
Transesterification
(time)
Derivatization
agent Comments References
Alkaline (DGF
standard
method C-III 18
(09))
GEs 3-MCPD-d5Sodium methoxide
/methanol (10 min)
PBA Bidirectional
conversion between
glycidol and
3-MCPD leads to
inaccuracy of
method.
(DGF Standard
Methods C III 18
(09) 2009; Kaze
and others 2011)
Alkaline GEs and
3-MCPD
esters
1,2-Dipalmitoyl-3-
chloropropane-d5
Methanol/sulfuric acid (1
min)
PBA It is the 1st to
determine
simultaneously GEs
and 3-MCPD esters
in different food
products
(Küsters and others
2011)
Alkaline or acidic,
mild
GEs and 3-,
2-MCPD
esters,
1,2-Dipalmitoyl-3-
chloropropane-d5,
3-MBPD-d5
2.5 mg/mL methanolic
sodium hydroxide
solution (16 h); 1.8%
by volume of sulfuric
acid–methanol solution
(16 h)
PBA GEs were quantified
based on the 3-
MBPD/3-MBPD-d5
ratio due to the
transformation of
glycidol to MBPD.
(Kuhlmann 2011;
Ermacora and
Hrncirik 2013,
2014b)
Enzymatic
(Candida
rugosa)
GEs and
3-MCPD
esters
3-MCPD-d5,
3-MBPD-d5,
Lipase (30 min) PBA On the basis of
(Kuhlmann 2011)
alkaline,
methanolysis is
displaced by lipase
hydrolysis to
shorten the testing
time, shortening
the detection time
(Miyazaki and others
2012; Koyama and
others 2015;
Miyazaki and
Koyama 2016)
transesterification generally required a much longer reaction time
(16 h), based on the developed determination methods of 3-
MCPD esters reported previously (Zelinkova and others 2006;
Ermacora and Hrncirik 2012). To improve the accuracy and time
requirements of the indirect GE determination, a further modi-
fication of sample pretreatment was made to avoid using the acid
cleavage of the esters in the determination of GEs. Several re-
searchers also implemented the lipase-catalyzed hydrolysis of GEs
using a lipase from Candida rugosa (30-min incubation at room
temperature), which greatly shortens detection time (Miyazaki
and others 2012; Koyama and others 2015).
Different from the dependence of GE determinations on 3-
MCPD esters in the initial indirect methods described above,
Kuhlmann (2011) developed a new type of indirect determina-
tion method for GEs which allowed for the direct detection of
glycidol derivatives as well as 3-MCPD derivatives, independently
of one another (Figure 4B). The analytical protocol involves a
series of distinct steps: transesterification under mildly alkaline or
lipase-catalyzed conditions, transformation of glycidol into mono-
bromopropanediol (MBPD), derivatization of MBPD, and GC-
MS analysis. Two of 3 published methods by AOCS are based
on this protocol (Joint AOCS/JOCS Official Method Cd 29a-
13 2013; Joint AOCS/JOCS Official Method Cd 29b-13 2013).
Although this approach enables GEs to be determined in a si-
multaneous fashion with 3- and 2-MCPD esters, and lipase has
been employed for the hydrolysis of esters, avoiding the forma-
tion of additional GEs from MCPD esters or partial acylglycerols
(Miyazaki and others 2012; Koyama and others 2015), the in-
complete bromination may still result in an underestimation of
GE levels in the oils tested. Therefore, it becomes obvious why a
direct determination method, requiring no transesterification and
derivatization, is needed.
Direct analysis
Compared to the indirect determination of GEs, a direct
method generally quantifies the levels of every GE species bear-
ing different fatty acyl chains by LC-MS without any chemical
transformation. Therefore, the method provides full information
on the composition of the sample without any side reactions. An
overview of the methods from the recent literature can be found
listed in Table 3. The development of a direct method, instead of
an indirect method, has become of great interest in the last few
years. A major challenge of the method is that the presence of
large amounts of acylglycerols, especially TAGs in tested edible
oil, will negatively influence the precision, accuracy, and suscep-
tibility of the direct method, and therefore they must be removed
before analysis. The distribution pattern of GEs is directly related
to the fatty acid profiles of the tested oil (Aniołowska and Kita
2016a). It has already been proposed that the GE contents in most
edible oils may be adequately represented by the analysis of 7 GEs,
that is, GEs of lauric, myristic, palmitic, stearic, oleic, linoleic, and
linolenic acids (Dubois and others 2011). Compared with the indi-
rect method, a further separation and purification, as well as a larger
initial investment in analytical standards, are required for the com-
position complexity of the corresponding analyte. Several purifi-
cation techniques, such as gel permeation chromatography (GPC)
(Dubois and others 2011) and 2-step SPE, have been designed to
remove a large amount of tri- and partial acyl glycerides prior to
LC-MS analysis. From an inspection of Table 3, the 2-step SPE
purification, the 1st reversed phase SPE to remove nonpolar frac-
tions (such as TAG) and the following normal phase SPE removal
of partial acylglycerols, seem to be the superior pretreatment pro-
cess and are applied in a variety of developed direct methods (Table
3). Furthermore, it has already been demonstrated that the imple-
mentation of the SPE procedure in the order listed above provides
better analytical results than a SPE procedure in reversed order
(Masukawa and others 2010). If the diluted oil is injected directly
into the LC-MS system, a fast deterioration of system perfor mance
can be observed. Haines and others (2011) described the determi-
nation of 7 GEs and 20 3-MCPD esters in edible oils using LC-
time-of-flight-mass spectrometry (LC-TOF-MS) in the positive-
ion mode with the simplest implementation of sample prepa-
ration. The oil samples were only diluted by methanol–sodium
acetate solution/methylene chloride/ acetonitrile (0.26 mM, 1:8:1
10 ComprehensiveReviews in Food Science and Food Safety rVol. 00, 2017 C2017 Institute of Food Technologists®
Glycidyl esters in refined edible oils . . .
Table 3–Direct analysis methods for GEs in edible oils.
Method
denomination Analyte Calibration Sample preparation Chromatographic column
Mobile phase
composition
Recovery and
LOD/LOQ References
LC-MS, SIM:
selected ion
monitoring,
APCI, in
positive-ion
mode
Five GEs External standard
method
Liquid/liquid partitioning
of sample in
acetonitrile; 2-step SPE
cleanup: reversed
phase SPE using
Sep-Pak Vac RC C18
cartridge 500 mg
(Waters) followed by
the normal phase SPE
using a Sep-Pak Vac RC
Silica cartridge 500 mg
(Waters); evaporation
of eluent and
reconstitution of
residue in
methanol/2-propanol
1:1 by volume
UPLC-MS: Acquity UPLC
BEH: ethylene bridged
hybrid C18 column 100
×2.1 mm i.d., 1.7-µm
particle size (Waters);
HPLC-MS: L-column
ODS:
octadecasilyl-silica 150
×4.6 mm i.d., 5-µm
particle size
A: acetonitrile
/methanol/water
17:17:6 by volume; B:
2-propanol
71.3% to 94.6%
(average 79.4%)
for TAG-rich oils;
90.8% to 105.1%
(average 97.2%)
for DAG-rich oil,
LOQ: 0.0045 to
0.012 mg/mL
(Masukawa and
others 2010;
Masukawa and
others 2011)
LC-MS, SIM, APCI:
atmospheric
pressure
chemical
ionization, in
positive-ion
mode
Five GEs Internal standard
method with
C17:0-GE
Liquid/liquid partitioning
of sample in tert-butyl
methyl ether
(MTBE)/ethyl acetate
4:1 by volume; 2-step
SPE cleanup as
depicted by Masukawa
and others (2010);
evaporation of eluent
and reconstitution of
residue in
methanol/2-propanol
1:1 by volume
L-column ODS 150 ×4.6
mm i.d., 5-µm particle
size
A: methanol; B:
2-propanol
Close to 100%, LOQ:
82 to 110 mg/kg
(Shiro and others
2011; Blumhorst
and others 2013)
LC-TOF-MS for
method
development,
SIM, ESI, in
positive-ion
mode
LC-MS/MS for
routine analysis,
SIM, ESI, in
positive-ion
mode
Seven GEs Standard addition Liquid/liquid partitioning
of sample in
cyclohexane/ethyl
acetate (1:1 by
volume); GPC
extraction on Bio-Beads
S-X3 column 280 ×25
mm i.d.; additional
cleanup for oils
containing high DAG
and MAG content by
SPE: 500 mg silica
(eluent
dichloromethane)
LC-TOF-MS: Acquity UPLC
50 ×2.1 mm i.d.,
1.8-µm particle size
LC-MS/MS: Luna C18
column 50 ×3 mm i.d.,
3-µm particle size
LC-TOF-MS: A:
methanol/water/formic
acid 75:25:0.1 by
volume; B: 2-propanol:
formic acid 99.9:0.1 by
volume LC-MS/MS: A:
methanol/water/formic
acid 75:25:0.5 by
volume; B: 2-propanol:
formic acid 99.5:0.5 by
volume
68% to 111%
(average 93%),
LOQ: 0.05 to 0.10
µg/kg
(Dubois and others
2011)
LC-TOF-MS, SIM of
sodiated
adducts, ESI, in
positive-ion
mode
Seven GEs and 20 3-MCPD
mono- and diesters
Internal standard
method with
MCPD-d5
dioleic acid
ester and
palmitic
acid-d31 GEs
Dilution of sample in
mobile phase B with
0.1% by volume of
internal standard stock
solution
Luna C18 column 50 ×3
mm, i.d., 3-µm particle
size
A: 0.26 mM
methanol-sodium
acetate solution
(MSA)/methanol/
acetonitrile 1:8:1 by
volume; B:
MSA/methylene
chloride/acetonitrile
1:8:1 by volume
LOD: 0.07 to 0.29
mg/kg for GEs,
0.21 to 1.69
mg/kg for
3-MCPD
monoesters, and
0.10 to 0.40
mg/kg for
3-MCPD diesters
(Haines and others
2011)
(Continued)
C2017 Institute of Food Technologists®Vol.00,2017 rComprehensiveReviewsin Food Science and Food Safety 11
Glycidyl esters in refined edible oils . . .
Table 3–Continued.
Method
denomination Analyte Calibration Sample preparation Chromatographic column
Mobile phase
composition
Recovery and
LOD/LOQ References
LC-MS/MS, MRM,
APCI, in
positive-ion
mode
Five GEs Stable isotope
dilution analysis
with
d31-glycidyl
palmitate and
d35-glycidyl
stearate
Liquid/liquid partitioning
of sample in acetone;
2-step SPE cleanup:
reversed phase SPE
using Sep-Pak C18
cartridge followed by
normal phase SPE using
silica cartridge (300 ×
10 mm i.d.);
evaporation of eluent
and reconstitution of
residue in diethyl ether
Gemini C18, 250 ×2.0
mm, i.d., 5-µm particle
size
100% methanol 84% to 108%, LOD:
0.07 to 0.15
mg/kg using 10
mg sample and
0.001 to 0.003
mg/kg using 0.5
gsampleofoil
(Becalski and others
2012)
LC-TOF-MS, SIM of
sodiated
adducts, ESI, in
positive-ion
mode
Five GEs, 9 3-MCPD mono-
and diesters
Internal standard
method with
1-palmitoyl-3-
MCPD-d5,
1,2-bis-
linoleoyl-3-
MCPD-d5,
1,2-bis-
palmitoyl-3-
MCPD-d5
Liquid/liquid partitioning
of sample in n-hexane;
2-step SPE cleanup:
normal phase SPE using
Sep-Pak Plus SI
cartridges, 500 mg
(Waters) followed by
reversed phase SPE
using SPE Sep-Pak Plus
C18 cartridge, 500 mg
(Waters); evaporation
of eluent and
reconstitution of
residue in acetonitrile
AQUITY UPLC BEH C18
column 50 ×2.1 mm
i.d., 1.7-µm particle size
A: methanol /water
15:85 by volume; B:
methanol/phase
97.5:2.5 by volume
62.6% to 108.8%,
LOD: 0.16 ng/mL
for GEs, 0.86
ng/mL for
3-MCPD
monoesters, and
0.22 ng/mL for
3-MCPD diesters
(Hori and others
2012)
LC-MS/MS, MRM,
ESI, in
positive-ion
mode
Six GEs, 12 sn-1 and sn-2
3-MCPD monoesters
Internal standard
method with 6
deuterated GEs
and 1-oleoyl-3-
MCPD-d5,
1-palmitoyl-3-
MCPD-d5
Dissolution of sample in
20% by volume ethyl
acetate/MTBE; 2-step
SPE cleanup:
reversed-phase SPE
using 1000 mg/6 mL
C18 cartridge followed
by normal phase SPE
using 500 mg/3 mL Si
cartridge; evaporation
of eluent and
reconstitution of
residue in isopropanol
Pursuit XRs C18 column
150 ×2.0 mm i.d.,
3.0-µm particle size
A: 2 mM ammonium
formate /0.05% formic
acid in 92:8 by volume
methanol/water; B: 2
mM ammonium
formate /0.05% formic
acid in 98:2 by volume
isopropanol/water
84% to 115% for
GEs, 95% to
113% for sn-1
3-MCPD
monoesters,
76.8% to 103%
for sn-2 3-MCPD
monoesters, LOD:
below 0.03, 0.06,
and 0.18 mg/kg,
respectively
(MacMahon and
others 2013b)
GC-MS, SIM, ESI, in
positive-ion
mode
Seven GEs Internal standard
method with
glycidyl
palmitate-d5
Liquid/liquid extraction
of sample in
acetonitrile and
heptane; 2-step SPE
cleanup as depicted by
Masukawa and others
(2010); NPLC
separation: Lichrosorb
5 Diol columns 250 mm
×4.6 mm i.d.
DB-5 ms column 15 m ×
0.25 mm i.d., 0.10-µm
particle size
Helium gas 85% to 115%, LOD:
0.01 mg/kg
(Steenbergen and
others 2013)
1H NMR Intact GEs Internal standard
method with
benzene
Dissolution of sample in
hexane; silica gel
column (particle size 75
to 150 µm)
partitioning;
evaporation of eluent;
reconstitution of
residue in deuterated
chloroform
– 93.20% for palm
olein, 100.65%
for DAG-rich oil,
LOD: 0.0511
mmol/L, LOQ:
0.170 mmol/L
(Song and others
2015)
12 ComprehensiveReviews in Food Science and Food Safety rVol. 00, 2017 C2017 Institute of Food Technologists®
Glycidyl esters in refined edible oils . . .
by volume) prior to analysis. Nonetheless, the LOD of GEs in
refined palm oil was estimated to be about 0.1 mg/kg, with 1
exception of 0.29 mg/kg for glycidyl myristate. The LODs were
approximately 3 times higher than that reported by Shiro and oth-
ers (2011). Unfortunately, the analyte recoveries were not available
and likely varied significantly as a consequence of matrix effects.
In addition, the developed method by Hori and others (2012),
involving the addition of sodium salts to the mobile phase for
the formation of sodiated adducts, caused significant negative ef-
fects on the MS instrument. The latter finding demonstrates that
frequent instrument vvcleaning is critical, thereby avoiding a pre-
mature corrosion of the electrospray ionization (ESI) components,
such as the nebulizer needle.
Considering the high cost and complex data analysis of LC-
MS, Steenbergen and others (2013) have suggested a novel direct
detection method for intact GEs in edible oils based on GC-MS.
To reduce any interferences of acylglycerols, the preparation of
oil samples involves the extraction of analytes with acetonitrile
and heptane. The purification employs a 2-step SPE, as depicted
above, together with a final isolation of the GEs by NPLC. The
authors also considered that a potential formation of GEs might
occur from the precursors in the injection port of the GC with
a high temperature. Therefore, a cold on-column injection was
adopted to avoid potential thermal degradation of GEs with the
formation of artifacts. The LODs of GEs were determined to be
about 0.01 mg/kg for the individual GE, which proves to be signif-
icantly lower than the developed LC-MS-based methods (Table 3).
The recovery values were in the range of 85% to 115%, largely
depending on the chain identity and level of GEs. However, both
direct approaches based on GC-MS and LC-MS require a variety
of expensive reference mater ials and a rather complicated sam-
ple preparation process. This ultimately limits their applicability
in routine analyses. A more recent report describes another direct
method for the determination of GEs based on 1H NMR spec-
troscopy, an important analytical technique now widely used in
the study of lipids, such as the determination of fatty acid composi-
tion, real-time monitoring of transesterification, and adulteration
identification of vegetable oils (Anderson and Franz 2012; Zhang
and others 2013a; Vicente and others 2015). In this method, a
quantification formula was deduced from the diagnostic signals of
epoxy methylene protons at chemical shifts of 2.56 and 2.76 ppm.
By introducing a weighted average factor, the individual GE was
calculated as the stoichiometric ratio of the glycidol-to-esterified
lipid component followed by conversion of molar to weight per-
centage. The recovery values in palm olein and DAG-rich oil were
determined to be 93.20% and 100.65%, respectively, and the LOD
and LOQ were found to be 0.0511 and 0.170 mmol/L, respec-
tively, which was far higher than LC-MS-based method. These
results confirm the potential of the analytical model to for GEs
determination. Unfortunately, no information was provided re-
garding GE levels of various oils, which might have been below
the LOD due to the absence of a further purification process. Ac-
cordingly, further work is still required to evaluate the availability
of NMR method used as an alternative routine analysis of GEs in
refined oil.
From the above description of direct and indirect methods,
the major advantages of the indirect method are the very sim-
ple sample pretreatment saving detection time, no need for in-
tact GEs standards, which compensates for the lack of LC-MS-
based and GC-MS-based direct methods. After further develop-
ment of enrichment techniques, it is expected that the direct
method will progress as an alternative for the routine analysis
of GEs.
Elimination Methods of GEs
As mentioned above, GEs represent potential carcinogens
widely found in refined edible oils and oil-based food products,
with possible adverse effects on the human body and health upon
ingestion. Due to the lack of limit standards yet worldwide, there-
fore, there is an urgent need to mitigate GE levels to ensure the
quality and safety of edible oils and oil-based food products. Some
elimination methods have been shown to be promising on a re-
search laboratory or pilot plant scales and have therefore also been
adopted in the oil production industry. Most studies on the elim-
ination of GEs to date have been performed using palm oil, most
notably due to its extraordinarily high amounts of GEs and a gen-
erally high consumption rate around the world. The following
subsections will summarize the currently developed elimination
methods, based on the formation mechanisms of GEs, including
inhibition and removal of precursors, modification of formation
conditions, and elimination of formed GEs (Figure 5).
Inhibition and removal of precursors
Unlike 3-MCPD esters, the precursors of GEs are comprised
of only DAGs and MAGs rather than chloride ions which prove
to be water soluble. Therefore, we believe that the levels of 3-
MCPD esters can be reduced through the removal of chloride
ions by water wash even though this process does not change
the GE concentrations (Matth¨
aus and others 2011). It has already
been established that initial DAG and MAG contents in crude
oils exhibit a pronounced impact on the formation of GEs. The
removal of DAGs and MAGs seems to be the most straightforward
way to reduce the formation of GEs. As mentioned above, DAGs
and MAGs mainly form through hydrolysis of TAGs, resulting
from the activity of endogenous lipase after maturation of oil
plants before inactivation. It has also been reported that bruised
oil seeds/fruits display more lipolytic activity than undamaged oil
seeds/fruits. Moreover, postmature fruits, processing delay, as well
as rough handling of oil plant bunches may all contribute to the
presence of high DAG and MAG concentrations in crude oils
(Kopas and Kopas 2009; Matth¨
aus and Pudel 2014). Chilling the
oil fruit can also enhance lipase hydrolysis, which has been found
to induce up to 70% of FFAs in palm fruits when subjected to 5 °C
chilling (Sambanthamurthi and others 1995; Cadena and others
2013). Taken in concert, these factors explain why the GE contents
are different in palm oils from different locations as reported by
Matth¨
aus and others (2011). Only 1.3 mg/kg of GEs was detected
in palm oil from Ghana, but up to 14 mg/kg was found in palm oil
from Malaysia. For oil producers, the elimination of GEs should
begin with the selection of plants from different locations and
cultivation conditions. Taking some agricultural practices between
harvest and processing of oil fruits/seeds are essential to diminish
the activity of lipase. These practices include the modification
of harvesting conditions, minimizing cracking of oil fruits when
transforming to factories, avoiding bruised fruit, reducing the time
between harvest to milling, and so on. All these practices taken in
concert aim to inhibit the formation of DAGs and MAGs (Figure 5,
stage A: 1 to 7). For this reason, the sterilization step during milling
of oil seeds or fruits, intended to inactivate enzymes such as lipases
and to soften the fruit for speedy removal from the bunches, should
be kept at or below 120 °C (Figure 5, stage B: 8) (Craft and Nagy
2012; Stadler 2015).
C2017 Institute of Food Technologists®Vol.00,2017 rComprehensiveReviewsin Food Science and Food Safety 13
Glycidyl esters in refined edible oils . . .
Figure 5–Summary of the elimination methods of GEs in the oil seeds/fruits collection (Stage A), preparation (Stage B), refining (Stage C), and
application (Stage D) of edible oils. The provided information was obtained from different literature sources.
As mentioned above, the refining steps prior to deodorization,
that is, degumming, neutralization, and bleaching, may remove
some of the DAGs and MAGs. In part, this explains the concen-
tration reduction of 3-MCPD esters and GEs at different refining
stages (Pudel and others 2011). DAGs and MAGs can also be re-
moved by addition of adsorption materials (Figure 5, stage C: 12),
a process that has been well established for the removal of polar
components from frying oil (Yates and Caldwell 1993; Lin and
others 2001). Strijowski and others (2011) reported that amor-
phous magnesium silicate and calcined zeolite may reduce polar
components as well as DAGs and MAGs by approximately 25%.
Therefore, an improved bleaching procedure with additional re-
moval of polar components is needed in the future to ensure a
reduced formation of GEs as well as 3-MCPD esters during oil
deodorization. Based on a report by Craft and others (2012), it
seems feasible that the levels of DAGs and FFAs in crude palm oil
below 4% and 2.5%, respectively, should be kept to reduce the
formation of GEs during deodorization (Figure 5, stage C: 13).
Modification of formation conditions
From a chemistry point of view, it is well known that high
temperature represents the most important reaction condition for
the formation of GEs from DAGs and MAGs from a chem-
istry point of view. In the production of edible oil, the gen-
eral deodorization process of bleached oils involves steam dis-
tillation at temperatures between 250 and 260 °Cfor0.5to
3.0 h. This process represents the last, but indispensable step for
the removal of FFAs and any toxic polycyclic aromatic hydro-
carbons. Furthermore, this step is crucial for the decomposition
of pesticide residues and the inactivation of pigments. However,
the deodorization step during oil refining has also been demon-
strated to contribute to the widespread production of GEs in
refined edible oils (Weisshaar and Perz 2010; Destaillats and oth-
ers 2012a). Proper modification of deodorization temperature
and time are crucial parameters for inhibiting the formation of
GEs. Pudel and others (2011) have reported that the forma-
tion of GEs during deodorization at a temperature of <240 °C
is negligible (ࣘ5 mg/kg). However, at a temperature of 250 °C,
the GE concentrations increased significantly with time. To fur-
ther understand this phenomenon, Craft and others (2012) carr ied
out a laboratory-scale deodorization exper iment with pure DAG,
thereby confirming the significant formation of GEs at tempera-
tures above 230 to 240 °C. Hence, it may be feasible to reduce
the formation of GEs by keeping the deodorization temperature
below 240 °C (Figure 5, stage C: 14a). Instead of conventional de-
odorization, a short-path distillation (Figure 5, stage C: 14b) with
a condenser temperature of 60 °C, an evaporator temperature of
170 °C, a stirrer speed of 100 r/min, and a pump frequency of 20
Hz were used by Pudel and others (2016). The authors found that
the refined palm oil produced by this mild deodorization process
contained a low amount of GEs and 3-MCPD esters, whereas the
sensory quality, in terms of taste and odor could be improved. Ad-
ditionally, several studies involving a 2-stage deodorization (Figure
5, stage C: 15c), that is, 1 short step at high temperature (250 to
270 °C), and a 2nd longer step at a lower temperature (200 °C),
have been shown to significantly reduce the GE concentrations,
irrespective of the processing sequence (Pudel and others 2012;
Stadler 2015).
In addition, the roasting step during oil pressing also involves
a heat treatment, with a local temperature of over 200 °C, which
seems to be the proper temperature range for the formation of
GEs. It has already been reported that the roasting process leads
to the formation of 3-MCPD ester, as well as benzo(a)pyrene
(Cheng and others 2015; Li and others 2016). However, it is still
questionable whether GEs are formed in this process. Recently,
some findings, obtained through studies carried out in our
laboratory, have demonstrated that the levels of GE in hot-pressed
crude oils are significantly higher than the levels in cold-pressed
and solvent-extracted crude oils. Furthermore, owing to the
pyrolysis of TAG during roasting, DAG and MAG contents were
found to be slightly higher in hot-pressed crude oil compared
with the crude oils from the other 2 methods. The latter finding
may actually lead to an increased difference of GE levels among
the 3 refined oils. Therefore, further studies on the optimization
14 ComprehensiveReviews in Food Science and Food Safety rVol. 00, 2017 C2017 Institute of Food Technologists®
Glycidyl esters in refined edible oils . . .
of roasting parameters, such as roasting temperature, time, and
stirring speed, should be carried out in an effort to lower the GE,
DAG, and MAG contents in crude oils (Figure 5, stage B: 9, 10).
Together with the formation of 3-MCPD esters, it has been
reported that the formation of GEs is strongly dependent on the
pH value in oils (ˇ
Smidrkal and others 2011). Acidity has been
shown to have a strong influence on the formation of 3-MCPD
esters in crude oils heated at high temperature (Ramli and oth-
ers 2015). ˇ
Smidrkal and others (2011, 2016) reported that the
addition of alkali potassium or sodium bicarbonate to neutralize
FFAs in sunflower oil can significantly prevent the formation of
3-MCPD esters. A decrease in pH value may also induce a higher
potential to form GEs. Therefore, the neutralization of FFAs by
the addition of alkaline substances such as Na2CO3and NaHCO3
can be expected to reduce the formation of GEs during oil de-
odorization (Figure 5, stage C: 15). This, in turn, means that
the GE levels in chemically refined oils with preremoval of FFAs
in the neutralization step should be lower than the GE levels in
physically refined oils. Furthermore, compared with the latter pro-
cess, chemical refining requires the deodorization temperature at a
lower level (230 to 240 °C). Conversely, an increase in pH has also
been shown to reduce the GE levels, as well as 3-MCPD and its
esters, in refined oils (Sim and others 2004; Freudenstein and oth-
ers 2013). However, the underlying mechanisms of this reduction
in glycidol/3-MDPD (+esters) are still the topic of controversial
debates. In order to answer the question whether a high pH value
inhibits GE formation reactions or promotes their degradation,
further experimental data are required. More recently, it has also
been confirmed that the formation of 3-MCPD esters is related
to oil oxidation due to the participation of the free radicals in
the process of their formation (Zhang and others 2013b; Zhang
and others 2015). Indeed, the addition of antioxidants, intended
to inhibit oxidation or free radicals, to model systems involving
real oil models and chemical models, reduces the formation of
3-MCPD esters in both systems treated at 230 °C for 30 min,
compared with the corresponding control samples without ad-
dition of antioxidants (Li and others 2015). Zhang and others
(2016) also demonstrated that the formation of 3-MCPD esters
was mitigated by scavenging free radicals using antioxidants at the
molecular level. Unfortunately, whether GE levels are also reduced
through this treatment has never been the topic of investigations.
Therefore, further studies are needed to provide definite answers
to these questions.
Elimination of formed GEs
As discussed above, GEs have been proven to exist in almost
all refined oils. For all formed GEs, the elimination methods are
classified as physical adsorption and chemical degradation in the
literature. Physical adsorption to eliminate GEs involves a process
that does not destroy the molecular structure of GEs but adsorbs
the compounds in adsorption materials, such as activated car-
bon, magnesium silicate, zeolite, activated bleaching earth, and so
on (Figure 5, method 17). Previously, these adsorption materials
were applied for the removal of polar components (Lin and others
2001), polycyclic aromatic hydrocarbons (PAHs) (Leon-Camacho
and others 2003), and pesticide residues (Mendez and others 2005)
during oil refining. Recently, Str ijowski and others (2011) have
investigated the possibilities of removing 3-MCPD esters and GEs
from palm oil using different adsorption materials, such as amor-
phous magnesium silicate, zeolite, silicon oxide, sodium aluminum
silicate, calcium silicate, and magnesium silicate. The results ob-
tained show that calcined zeolite and synthetic magnesium silicate
could remove, by up to 40%, the 3-MCPD esters and GEs. The
treatment did not affect the oxidative stability and sensory prop-
erties of palm oil. Other studies have shown that mainly GEs
could be eliminated by addition of adsorption materials. How-
ever, the actual elimination mechanisms for the both compound
types still remain unclear. A study carried out thereafter not only
demonstrated the elimination of GEs by activated bleaching earth
in both TAG- and DAG-rich oils, but also investigated the elim-
ination process in a model system (Shimizu and others 2012b).
The authors also found that the elimination of GEs was not due
to adsorption of activated bleaching earth but rather through the
chemical transformation of GEs involving a ring-opening reac-
tion. However, it seems likely that GEs are 1st adsorbed, and then
a ring-opening reaction occurs in the adsorbed GEs on activated
bleaching earth pretreated by acid. Therefore, follow-up studies
should also focus on the evaluation of suitable adsorption materials
for effectively reducing the GEs contents in edible oils.
Due to the relatively low molecular weight of GEs and MAGs,
the compounds may be distilled off, to accumulate in distillates
during the deodorization/steam distillation process. For the 1st
time, Craft and others (2012) reported high amounts of GEs
(>100 mg/kg) in distillate samples, and proposed that GE lev-
els in refined palm oil can also be determined by the removal rate
of GEs into deodorization distillates. Therefore, the modification
of a steam distillation process may likely decrease the GE contents
in refined oils (Figure 5, stage C: 16a). ¨
Ozdikicierler and others
(2015) investigated the effects of steam distillation process parame-
ters (stripping steam rate, temperature, and pressure) on 3-MCPD
esters and GE formation in olive oil and olive pomace oil. The re-
sults obtained showed that the interaction between stripping steam
temperature and rate was statistically significant for the formation
of GEs. However, when the steam rate was sufficiently high, the
promoting effect of steam temperature on the formation of GE
was not significant. Under optimum conditions, determined by
response surface methodology, that is, steam distillation tempera-
ture of 230 °C, water flow rate of 1.2 mL/min, and pressure of
4 mbar for olive oil, as well as steam distillation temperature of
230 °C, water flow rate of 1.0 mL/min, and pressure of 2 mbar
for olive pomace oil, both ester contents in the oils could be re-
duced. This proves to be particularly true for GEs which could
be reduced to below 0.1 mg/kg. This elimination method may
be further examined for other edible oils exhibiting high levels of
GEs, such as palm oil and rice oil.
Additionally, as pointed out above, the formed GEs are not
stable under acidic conditions. Therefore, in an effort to reduce
the contents of GEs and 3-MCPD esters, Matth¨
aus and others
(2011) implemented the replacement of water by formic acid in
the formation of the strip steam during oil deodorization. A higher
concentration of formic acid led to lower amounts of the esters,
particularly GEs, indicating that the use of acid solutions instead
of water for the generation of strip steam during deodorization
seemed to be a feasible method for the reduction of GE levels
(about 35%) in refined edible oils (Figure 5, stage C: 16b). Differ-
ent from the physical adsorption discussed above, the elimination
mechanisms of this method may involve a structural degradation
of GEs involving some chemical reactions. From this study, we
assume that the addition of certain nontoxic substances to refined
oils enables GEs to degrade or to transform into harmless com-
pounds, such as glycerol, DAGs, MAGs, and so on (Figure 5,
method 18). It was shown previously that both glutathione and
cysteine could significantly lower the concentrations of formed
3-MCPD in the model system (Velisek and others 2003).
C2017 Institute of Food Technologists®Vol.00,2017 rComprehensiveReviewsin Food Science and Food Safety 15
Glycidyl esters in refined edible oils . . .
However, further investigations on the use of these specific sub-
stances should be required in order to decrease GE levels in refined
edible oils even more efficiently.
Oil storage plays an important part in the production and dis-
tribution of edible oil. In 2008, during an academic conference,
Matth¨
aus and others (2008) reported for the 1st time that the
concentrations of GEs and 3-MCPD esters decreased during oil
storage at low temperature. More recently, Matth¨
aus and oth-
ers (2015) further verified the notion that GEs in refined palm
oil stored at a temperature ranging between 5 and 15 °Cde-
graded significantly. However, at room temperature (20 °C) and at
–20 °C no degradation of GEs occurred. Interestingly, the levels of
3-MCPD esters remained constant at all tested temperature ranges.
Presumably, upon transportation or use, edible oils should be stored
or placed in the refrigerator (5 to 15 °C) to reduce the concen-
trations of GEs (Figure 5, stage D: 20). Another high-temperature
application, such as oil frying, has recently been shown to de-
grade GEs in frying oils due to the high temperatures (<190 °C)
and the long time exposure (Figure 5, stage D: 21). Here, the
degradation rate largely depends on oil types, fried raw materials,
and frying parameters (Aniołowska and Kita 2015, 2016a, 2016c).
Furthermore, when potato crisps are deep fried using high-oleic
sunflower oil on an industrial scale, no endogenous formation of
both GEs and 3-MCPD esters in frying oil can be observed and
the concentrations, particularly for GEs, are very low (about 0.09
mg/kg) (Dingel and Matissek 2015). Taken in concert, it can be
determined that only little attention needs to be paid to GEs in
frying oils, whereas the initial GE levels in refined oils require
more attention.
Apart from the general elimination methods described above,
the enzymatic removal of 3-MCPD and its esters has been reported
by Bornscheuer and Hesseler (2010). Herein, 3 kinds of enzymes,
halohydrin dehalogenase from Arthrobacter sp. AD2, epoxide hy-
drolase from Agrobacterium radiobacter AD1, and lipase A from
Candida antarctica, were used in this experiment. These enzymes
ultimately led to the conversion of 3-MCPD and its esters to the
harmless product glycerol. One of the enzymes, epoxide hydrolase,
may efficiently catalyze the hydrolysis of epoxides into the corre-
sponding vicinal diol. Therefore, the enzyme can also be applied
for the removal of GEs in refined edible oils (Figure 5, method 20).
Here, GEs may be hydrolyzed into nontoxic MAGs. Moreover,
the method does not require addition of organic solvents. Unfor-
tunately, no reports can be found in the literature highlighting the
enzymatic removal of GEs as of yet.
Conclusions
Oil deodorization represents a final and essential step in the
refining process of edible oil for the removal of odoriferous ma-
terials and other undesirable components. These include pesti-
cide residues, pigments, FFAs, and so on. Nevertheless, har mful
heat-processing contaminants, such as GEs and 3-MCPD esters,
are formed through pyrolytic reactions during high-temperature
deodorization. The common occurrence of GEs in edible oils
and oil-based food products, particularly infant formulas, has at-
tracted increasing attention in the oil processing industry in the past
10 y. However, currently, no universal, global regulations restrict-
ing the maximum allowable GE concentrations in edible oils exist.
Every aspect of GE studies, including analysis methods, formation
mechanisms, and elimination methods, is evaluated in detail. For
the analytical methods of GEs, and due to the uncertainty of the
indirect analysis methods, the current research mainly focuses on
direct methods, not only based on LC-MS. The precursors of
GEs, DAGs, and MAGs, have been well identified in model sys-
tems, but the reactive pathways from DAGs or MAGs to GEs still
remain controversial due to a lack of experimental data in the lit-
erature supporting any proposed mechanisms. This phenomenon
ultimately limits the development of effective elimination meth-
ods, potentially resulting in the large amounts of GEs in commer-
cially available edible oils. From the published reports, perhaps the
most effective and most pragmatic method to extensively elimi-
nate GEs in refined edible oils that exists to date is an upstream
intervention in edible oil production. Oil manufacturers and oil
plant growers need to work closely together in the future to reduce
GE contents in edible oils as much as possible. Additionally, owing
to the strong connection of formation mechanisms including pre-
cursors, formation conditions, and reaction process between GEs
and 3-MCPD esters, increased research efforts should focus on the
structural relationship of both species in formation mechanisms.
We believe that both GEs and 3-MCPD esters may be eliminated
by using effective, simple, and inexpensive techniques.
Acknowledgments
The authors express thanks for the financial support from the
Natl. Natural Science Fund of China (Nos. 31130042, 31271885,
31271884, 31471677), Natl. Hi-tech Research and Development
Project (No. 2013AA102103), The Natl. Key Research and De-
velopment Program of China (No. 2016YFD0400401–5), and
Public Welfare (Agriculture) Research Project (No. 201303072).
Mention of trade names or commercial products in this publication
is solely for the purpose of providing specific information and does
not imply recommendation or endorsement by the U.S. Dept. of
Agriculture (USDA). USDA is an equal opportunity provider and
employer.
Author Contributions
W. Cheng was responsible for the study design, searching and
interpreting the literature, and preparing the manuscript; G. Liu
and L. Wang were responsible for the study design and drafting the
final manuscript. Z. Liu gave valuable assistance in the major revi-
sion of the manuscript. All authors reviewed the final manuscript
before submission.
Conflicts of Interest
The authors declare no conflicts of interest.
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C2017 Institute of Food Technologists®Vol.00,2017 rComprehensiveReviewsin Food Science and Food Safety 19
