Journal of Chromatography A, 1314 (2013) 208215

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Simultaneous determination of total fatty acid esters of

chloropropanols in edible oils by gas chromatographymass
spectrometry with solid-supported liquidliquid extraction
Qing Liu a,1 , Feng Han a,b,1 , Ke Xie a,b , Hong Miao a, , Yongning Wu a,b,
a
b

Key Laboratory of Food Safety Risk Assessment, Ministry of Health; China National Center of Food Safety Risk Assessment, Beijing 100021, China
Department of Food Science and Engineering, Wuhan Polytechnic University, Wuhan 430023, China

a r t i c l e

i n f o

Article history:
Received 5 March 2013
Received in revised form 1 August 2013
Accepted 20 August 2013
Available online 7 September 2013
Keywords:
Chloropropanols esters
Gas chromatographymass spectrometry
(GCMS)
Edible oils
Solid-supported liquidliquid extraction
(SLE)
Heptauorobutyrylim idazole (HFBI)

a b s t r a c t
This study aimed to establish a novel robust method for the simultaneous determination of total fatty
acid esters of 4 chloropropanols including fatty acid esters of 3-monochloropropane-1,2-diol (3-MCPD
esters), 2-monochloropropane-1,3-diol (2-MCPD esters), 1,3-dichloropropan-2-ol (1,3-DCP esters) and
2,3-dichloropropan-1-ol (2,3-DCP esters) in edible oils. In this method, sodium methylate in methanol
was used as the reagent for the ester cleavage reaction of chloropropanols esters. The reaction products were extracted by a sodium sulfate solution, and then puried by solid-supported liquidliquid
extraction (SLE) using diatomaceous earth (HydromatrixTM ) as the sorbent. Finally, the extracts were
derivatized with heptauorobutyrylim idazole (HFBI) and analyzed by gas chromatographymass spectrometry (GCMS). Quantication was achieved using deuterated chloropropanols as their respective
internal standards, including 3-MCPD-d5 , 2-MCPD-d5 , 1,3-DCP-d5 and 2,3-DCP-d5 . A good linear relationship between peak area and concentrations was obtained within the range of 0.0252.000 mg L1 with
a correlation coefcients not less than 0.999 for all the chloropropanols esters. The limits of detection
(LODs) of esters of 3-MCPD, 2-MCPD, 1,3-DCP and 2,3-DCP (calculated as corresponding chloropropanols)
were 30, 30, 100 and 30 g kg1 , respectively. The average recoveries of the 3-MCPD esters and the
4 chloropropanols spiked at 0.1, 0.5 and 2 mg kg1 into blank oil matrix were typically in a range from
70.7% to 113.3%. The robust method validation data including calibration, LOD/LOQ, accuracy and repeatability and prociency test results (Z-score: 0.5) of the ofcial Food Analysis Performance Assessment
Scheme (FAPAS) indicated that the present quantitative method could be successfully applied to the
determination of total chloropropanols esters in various edible oils.
Crown Copyright 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction
3-Monochloropropane-1,2-diol (3-MCPD) and other chloropropanols such as 2-monochloropropane-1,3-diol (2-MCPD),
1,3-dichloropropan-2-ol (1,3-DCP) and 2,3-dichloropropan-1-ol
(2,3-DCP) have been recognized as contaminants in various food.
They were rst detected in acid-hydrolyzed vegetable proteins
(acid-HVP) [1], and then were also detected in bakery products,
meat and sh products, and soups [2,3]. Recently, 3-MCPD fatty
acid esters have been identied and could be found in a variety
of processed foods and food ingredients. They are believed to be
formed at high temperatures during the deodorization step of the
oil rening process [4]. In rened oils, the concentrations of 3-MCPD
esters may reach a thousand times more than the levels of free

Corresponding authors at: No. 7 Panjiayuannanli, Chaoyang District, Beijing

100021, China. Tel.: +86 10 67779118; fax: +86 10 67779118.
E-mail addresses: miaoh@cfsa.net.cn, miaohong0827@163.com (H. Miao),
wuyongning@cfsa.net.cn (Y. Wu).
1
These authors contributed equally to this work.

3-MCPD [3,5]. Additionally, 3-MCPD esters have even been

detected in human breast milk and other foods, such as liquid
seasoning or bakery foods recently [3,6]. These fatty acid esters
of chloropropanols aforementioned represent the bound form of
3-MCPD, from which free 3-MCPD could be released by a lipasecatalysed hydrolysis reaction [7,8]. To date, the adverse effects of
3-MCPD have been concerned due to its kidney and reproduction
toxicity, as well as immune-suppression and potential carcinogenic
effects [5,913]. The Germany Federal Institute for Risk Assessment
(BfR) made the health-risk assessment of 3-MCPD esters, based on
available analytical method of 3-MCPD esters and by assuming that
100% of the 3-MCPD is released from the 3-MCPD esters exposure
[10]. It is estimated that dietary exposure of infants and adults to
3-MCPD esters (calculated as 3-MCPD) was 520 times as much as
TDI of 2 g kg1 body weight per day adopted by Joint FAO/WHO
Expert Group on Food Additives in 2001 [14,15].
In recent years, different analytical methods were reported
for the determination of 3-MCPD fatty acid easters. The methods
include either the conversion of the esters into a free forms (e.g.
free 3-MCPD) for analysis (indirect methods) [1618], or the

0021-9673/$ see front matter. Crown Copyright 2013 Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2013.08.074

Q. Liu et al. / J. Chromatogr. A 1314 (2013) 208215

direct determination of the individual esters separately (direct

methods) [1921]. The direct method, mainly with LCMS, can
only detect some of the individual chloropropanols esters since
it is not possible to commercially obtain the standard substance
of all chloropropanol esters. However, there are many kinds of
fatty acids in oil, which leading to different chloropropanols
esters including mono-esters and di-easters. Therefore, it is very
difcult for the direct method to detect all the chloropropanols
esters. The measurements of total chloropropanols esters are
critical when estimating the health-risk of these contaminants.
With the direct method, the health-risk of chloropropanols esters
may be underestimated because some chloropropanls esters may
not be analyzed. Compared to the direct methods, the indirect
analytical methods, which the present study applied, have obvious
advantages, such as higher sensitivity due to the detection of the
free forms of chloropropanols, the need of reference standards and
the internal standards of the 4 corresponding chloropropanols for
the quantication and comprehensive analysis of all the chloropropanols esters and so on. More signicantly, the results obtained
from the indirect methods can be rst-hand used for health-risk
assessment. Therefore, the indirect methods are more practical.
In most of the indirect methods, 3-MCPD esters were commonly
cleavaged into free 3-MCPD by transesterication in acid or alkaline medium [12,13,16,22,23], or hydrolyases [17], followed by
purication, derivatization and quantication of free 3-MCPD by
GCMS. The most prevailing indirect methods derive from the German Society for Fat Science (DGF) standard methods C-III 18 (09)
[6,13,17,2428], derivatizing 3-MCPD with phenylboronic acid
(PBA). However, the DGF-based methods for the determination of
chloropropanols esters may not be able to avoid the interference
from glycidyl esters. Besides, these methods need a programmedtemperature vaporization (PTV) inlet. Otherwise it could conduct
baseline shift seriously, which considerably inuence chromatographic measurement efciency. Furthermore, another
disadvantage of all these methods based on DGF C-III 18 (09) treatment is that the fatty acid esters of 1,3-DCP and 2,3-DCP cannot be
detected because their conversion products cannot react with PBA.
Although 3-MCPD was known as the most important food
contaminant among the four chloropropanols, the other chloropropanols (free and conjugated forms of 2-MCPD, 1,3-DCP and
2,3-DCP) should also be measured, especially as the conjugated
forms of these three chloropropanols may be present in foods. However, to our best knowledge, no well-validated method is available
for simultaneously detecting all the fatty acid esters of chloropropanols in foods.
The aim of the present study is to develop a novel analytical
procedure for the simultaneous determination and differentiation of fatty acid esters of four chloropropanols (their chemical
structures were shown in Fig. 1a) in edible oils. This method
transforms conjugated-chloropropanols into free chloropropanols
by sodium methoxide, and efciently puried by solid-supported
liquidliquid extraction (SLE). N-Heptauorobutyrylimidazole
(HFBI), as the derivatization reagent, reacts with these four
chloropropanols to form detectable compounds for simultaneous GCMS analysis. A scheme of the reaction is shown in
Fig. 1b using 3-MCPD esters as an example. Within the scope of
method development, critical pretreatment parameters such as
ester cleavage time and purication steps were also explored and
optimized.
2. Experimental
2.1. Chemicals and materials
Anhydrous sodium sulfate, sodium chloride and sodium
methoxide were obtained from the Jinke Institute of Fine

209

Fig. 1. The chemical strictures of fatty acid of 4 chloropropanols (a) and the scheme
of the reaction in this method (b).

Chemicals (Tianjin, China). Anhydrous sodium sulfate was heated

for 8 h at 200 C and then allowed to cool down and kept in a desiccator until use. Methyl-tert-butyl ether (MTBE) was purchased
from SigmaAldrich (St. Louis, MO). High performance liquid chromatography (HPLC) grade hexane, methanol, ethyl acetate and
glacial acetic acid were purchased from J. T. Baker (Phillipsburg,
NJ, USA). Ultrapure water obtained from a Milli-Q lter system
(Millipore, Bedford, MA, USA) was used throughout the analysis. N-Heptauorobutanoylimidazole (HFBI) was purchased from
Pierce Biotechnology (Rockford, IL, USA). Sodium methoxide was
dissolved in methanol (0.5 mol L1 ) for transesterication. The
aqueous solutions of sodium chloride (NaCl) and sodium sulfate
(Na2 SO4 ) were prepared with the concentration of 200 g L1 . Chem
ElutTM columns using diatomaceous earth (HydromatrixTM ) as the
sorbent were obtained from Agilent (Palo Alto, CA, USA) and were
used for the SLE cleanup.

2.2. Standards
3-Monochloro-1,2-propandiol
(3-MCPD,
98%),
1,3dichloropropan-2-ol (1,3-DCP, 97%) and 2,3-dichloropropan-1-ol
(2,3-DCP, 97%) were purchased from Fluka (Weinheim, Germany)
while 2-monochloro-1,3-propandiol (2-MCPD, 98%) and 3-MCPD
esters of rac-oleoyl, rac-linoleoyl, rac-linolenoyl, rac-1,2dilinolenoyl, rac-1,2-dilinoleoyl, rac-palmitoyl, rac-1,2-dioleoyl,
rac-1,2-distearoyl and glycidyl esters of oleate, linoleate, linolenate, stearate, palmitate (with chemical purities of 98%) were
purchased from Toronto Research Chemicals (Toronto, Canada).

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Q. Liu et al. / J. Chromatogr. A 1314 (2013) 208215

1,3-Dichloropropan-2-ol-d5 (1,3-DCP-d5 , 98%) was purchased

from Isotec (Weinheim, Germany). 3-Monochloro-1, 2-propandiold5 (3-MCPD-d5 , 99%) was purchased from Dr. Ehrenstorfer
(Augsburg, Germany). 2-Monochloro-1,3-propandiol-d5 (2-MCPDd5 ), 2,3-dichloropropan-1-ol-d5 (2,3-DCP-d5 ), 3-MCPD-d5 esters
for rac-1-oleoyl, rac-1,2-dilinolenoyl, rac-1,2-dilinoleoyl, rac1-linoleoyl, rac-1-linolenoyl, rac-1,2-dioleoyl, rac-1,2-distearoyl,
rac-1-palmitoyl, and glycidyl-d5 esters of oleate, linoleate, linolenate, all with chemical purities of 98%, were purchased from
Toronto Research Chemicals (Toronto, Canada). The deuterium
atoms of the above isotope standards are labeled in the skeleton of
glycerol or glycidyl other than in the corresponding carbon chain
of fatty acid. Individual stock solutions (1000 mg L1 ) were prepared by dissolving an appropriate amount of each standard in
ethyl acetate and stored at 20 C in amber glass vessels. Mixed
working solutions of standards (3-MCPD, 2-MCPD, 1,3-DCP and 2,3DCP) and deuterium-labeled standards (3-MCPD-d5 , 2-MCPD-d5 ,
1,3-DCP-d5 and 2,3-DCP-d5 ) at 10 mg L1 were prepared by merging all of the above stock solutions and diluting with hexane, and
then stored at 20 C.
2.3. Apparatus
Detection and quantication were performed with an Agilent Technologies 7890A gas chromatograph equipped with a
Series 5975C quadrupole mass selective detector (Agilent Technologies, Palo Alto, CA, USA). Data acquisition and processing were
performed using an Agilent workstation (GCMSD For 1701EA).
Separation was performed on a capillary column of DB-5MS
(30 m 0.25 mm I.D., 0.25 m lm thickness) from Agilent Technologies (Palo Alto, CA, USA) for all the analysis.

2.4. GCMS analysis

Splitless injection mode was used and the temperature of injector was set at 250 C. The injection volume was 1 L. The GC
temperature programming was 50 C for 1 min, followed by a
2 C min1 ramp to 90 C and a nal ramp of 40 C min1 to 250 C,
and hold at 250 C for 5 min. The total GC run time of analysis was
30 min per sample. The ultrapure grade helium was employed as
carrier gas at a ow rate of 1 mL min1 .
The tandem quadrupole instrument was operated in the
selected ion-monitoring mode (SIM). The analytes were ionized
by electron impact (EI), 70 eV of ion energy, with a solvent delay
time of 8 min. The temperatures of transfer line, MS source, and
MS quadrupole were 280 C, 230 C, and 150 C, respectively. The
electron multiplier voltage was 1000 V, and the dwell time for each
measured ion was set to 25 ms.
Table 1 lists the studied compounds and the internal standards
along with their retention times, selected ions, limits of detection
and quantication. The quantitation and qualier abundances were
determined by injection of standards under the same chromatographic conditions using full-scan with the mass-to-charge ratio
ranging from 60 to 400 m/z. The analytes were identied by their
retention times, the identication of quantitation ion and qualier
ions, and the ratios of the qualier ions to the quantitation ion.

2.5. Samples
The edible oil samples, including a crude palm oil and various rened vegetable oils such as camellia-seed oil, sunower oil,
peanut sesame blend oil, phytosterols oil, tea-seed oil and sesame
blend oil, were purchased from local markets. Samples of virgin
olive oils were used as matrix blanks and for the preparation of

spiked samples. They were analyzed in advance and fatty acid esters
levels of four chloropropanols were found to be below the LODs.
2.6. Sample pretreatment
About 0.1 g oil sample was accurately weighted into a 12 mLglass test tube with screwed cap, and 20 L working internal
standard solution as well as 0.5 mL MTBE/ethyl acetate mixed solution (8:2, v/v) were added. After vortex, 1 mL of NaOCH3 solution
was added into the tube with a full vortex, and followed by placing
for 4 min at room temperature. Subsequently, 3 mL hexane followed by 3 mL of glacial acetic acid in Na2 SO4 solution (1:29, v/v)
were added. The upper phase was discarded after full vortex and
liquidliquid phase separation.
The aqueous extracts were further puried by SLE with Chem
ElutTM column. After loading the extracts and holding for 3 min,
the column was washed with 20 mL hexane to remove the nonpolar components, and then, the target compounds were eluted
with 80 mL of diethyl ether. The eluents were collected into 150 mLasks lled with 15 g anhydrous Na2 SO4 , then evaporated to almost
dryness in a 35 C water bath by a vacuum rotary evaporator.
The residue was dissolved in 2 mL hexane and derivatized with
40 L HFBI at 70 C for 20 min. After cool down, 1 mL NaCl solution
was added into the derivatives, and the mixture was shaken for 30 s.
After dried with a little amount of anhydrous Na2 SO4 , the upper
layer was transferred into a sampler vial for GCMS determination.
2.7. Validation
The quantication of the chloropropanols esters was achieved
by using 4 deuterium isotope standards as internal standards.
The series of calibration standards, including the levels of 25, 50,
100, 200, 400, 800 and 2000 g L1 containing 4 deuterated internal standards at 100 g L1 , were prepared and derivatized, and
injected into GCMS for three times. The average peak areas of the
analytes (A) and of the internal standards (Ai) were recorded. All
the results were calculated using internal standard correction.
The limit of detection (LOD) and limit of quantication (LOQ),
following International Union of Pure and Applied Chemistry
(IUPAC) recommendation [29], were dened and determined as
the minimum detectable amount of analyses from blank oil spiked
extract with a signal-to-noise ratio (S/N) of 3:1 and 10:1, respectively.
The accuracy and repeatability of the method were assessed by
the quantication of ve replicates of analyte-free olive oils, and
spiked at three levels of concentration (0.1, 0.5 and 2 mg kg1 ) with
fatty acid esters of 3-MCPD and glycidol. Additionally, to check the
efciency of ester cleavage, four chloropropanols were individually
spiked into the blank matrix with the same levels as 3-MCPD esters.
3. Results and discussion
3.1. Selection of internal standards
In this study, the selection of internal standards is critical
for analyzing 3-MCPD esters in edible oils. The standards of
esters of 3-MCPD and glycidyl and their one-to-one corresponding deuterium-labeled 3-MCPD-d5 and glycidyl-d5 esters were
hydrolyzed, respectively. Meanwhile, the 3-MCPD esters or glycidyl
esters respectively spiked by 3-MCPD-d5 were also hydrolyzed. The
3-MCPD levels determined were expressed as recovery rates. The
results shown in Table 2 clearly indicated that the recoveries with
the spiking of internal standard 3-MCPD-d5 ranged from 43.3% to
114.0% (RSD, 0.326.4%), while the recoveries with the spiking of
corresponding esters of 3-MCPD-d5 or glycidyl-d5 as internal standards ranged from 69.2% to 310.1% (RSD, 0.715.6%).

Q. Liu et al. / J. Chromatogr. A 1314 (2013) 208215

211

Table 1
Method parameters including retention times, selected ions, regression equation, linear range, and limits of detection and quantication (LOD and LOQ, g kg1 ).
Chloropropanols derivatives

Retention times

Characteristic fragment ions (m/z)a

Regression equation

3-MCPD
3-MCPD-d5
2-MCPD
2-MCPD-d5
1,3-DCP
1,3-DCP-d5
2,3-DCP
2,3-DCP-d5

15.062
14.857
15.471
15.279
11.296
11.127
12.138
11.922

253, 289, 291, 275

257, 294, 296, 278
253, 111, 277, 275
257, 116, 280, 278
75, 77, 275, 277
79, 81, 278, 280
75, 77, 111, 253
79, 81, 116, 257

y = 1.91x 0.125

y = 1.10x 0.095

y = 1.52x 0.144

y = 2.52x 0.194

Linear range (mg L1 )

0.0252.000

0.0252.000

0.0252.000

0.0252.000

LOD

LOQ

30

30

100

30

100

100

200

100

Quantier ions are underlined.

The results in Table 2 showed that the recoveries with

3-MCPD-d5 as internal standards were generally better than
the esters of 3-MCPD-d5 or glycidyl-d5 , especially for rac 1linoleoyl-3-MCPD, rac 1-linolenoyl-3-MCPD, glycidyl-linoleate and
glycidyl-linolenate. In other words, the recoveries of unsaturated
mono-esters with the one-to-one corresponding deuteriumlabeled standards did not satisfy the recoveries requirements.
Therefore, 3-MCPD-d5 was adopted as the internal standard. Moreover, different oil having a different prole of fatty acid esters, it
was not appropriate to select a single deuterium labeled ester of
3-MCPD as the internal standard for the analysis of all the edible
oils.
Considering there are no commercial deuterium-labeled esters
of 2-MCPD, 1,3-DCP, and 2,3-DCP available, deuterium-labeled 2MCPD, 1,3-DCP, and 2,3-DCP can also be used as one-to-one internal
standards.
3.2. Sample preparation
Some investigations demonstrated that fatty acid esters of glycidol (oxirane-2-methanol, 2, 3-epoxy-1-propanol) may interfere
the determination and lead to the over-estimated values of 3MCPD esters, which were almost completely transformed into
cyclic 1,3,2-dioxaboralane derivative of 3-MCPD by derivatization
using phenylboronic acid in NaCl solution [30]. Consequently, the
Na2 SO4 solution was used to extract the target compounds instead
of NaCl solution. From Table 2, it is concluded that glycidyl esters
cannot be cleavaged into free 3-MCPD using Na2 SO4 solution as
extraction solution either in blank solvent or blank oil matrix. Additionally, seven oil samples were determined using NaCl solution

and Na2 SO4 solution, respectively. Lower values of chloropropanols

esters were obtained using Na2 SO4 solution instead of NaCl solution (Table 3). Based on the above facts, the Na2 SO4 solution was
selected as the solvent for extracting the target compounds to avoid
the interference for determination of chloropropanols esters by
glycidyl esters.
The cleavage time of esters, the diethyl ether amount, and types
of derivatization agents can inuence quantitatively the reaction.
In this study, the optimum cleavage time was investigated in detail.
One mL of sodium methoxide solution (0.5 mol L1 ) was added into
palm oil to react for 2, 4, 6, 8, 10 and 12 min, respectively. The ester
cleavage time of 2 min was shown to provide the highest quantity of 3-MCPD (see supplementary Table S1). With the increase of
ester cleavage time, the decrease of 3-MCPD level was observed.
However, the ratios of the area of 3-MCPD to 3-MCPD-d5 were
close between each other with different ester cleavage time treatments, the RSD of which was 6.4%. If internal standard method was
applied to the determination, the ester cleavage time only affect
the sensitivity rather than the accuracy. Considering the internal
standard quantication and operation feasibility for a large amount
of sample preparation in a batch, a cleavage time of 4 min was
selected.
Supplementary material related to this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.chroma.2013.08.074.
Fig. 2 shows the inuence of the diethyl ether amount used to
elute chloropropanols from the diatomaceous earth on the peak
areas, which affect the sensitivity. The results indicated that the
peak areas of 3-MCPD and 3-MCPD-d5 increased with the increasing volume of diethyl ether in the range of 4070 mL, and then the

Table 2
The comparison of average recoveriesa of 3-MCPD esters and glycidyl esters with different internal standards, and recoveries of the studied glycidyl esters compounds in
solvent and oil matrix using different extraction solvents.c
Compounds

Oleoyl-3-MCPD
Linoleoyl-3-MCPD
Linolenoyl-3-MCPD
1,2-Dilinolenoyl-3-MCPD
1,2-Dilinoleoyl-3-MCPD
Palmitoyl-3-MCPD
1,2-Dioleoyl-3-MCPD
1,2-Distearoyl-3-MCPD
Glycidyl-oleate
Glycidyl-linoleate
Glycidyl-linolenate
Glycidyl-stearate
Glycidyl-palmitate
a
b
c
d

Spiking in solvent

Spiking in blank oil

IS: 3-MCPD-d5

IS: Corresponding esters-d5 b

NaCl

NaCl

Na2 SO4

NaCl

Na2 SO4

114.0 (0.3)
70.4 (16.1)
93.3 (26.4)
97.5 (1.4)
96.5(2.7)
99.8 (4.1)
73.5 (14.2)
100.1 (4.3)
74.0 (4.1)
43.3 (13.5)
81.4 (13.7)
111.6 (13.7)
105.1 (3.5)

69.2 (6.7)
222.9 (15.6)
259.6 (0.7)
96.7 (5.8)
98.3 (3.0)
93.8 (5.3)
75.6 (4.5)
92.8 (2.2)
98.8 (2.1)
310.1 (2.7)
232.7 (0.9)

ND
ND
ND
ND
ND

13.7 (1.3)
10.8 (1.6)
15 (11.3)
14.4 (3.2)
18.7 (10.0)

ND
ND
ND
ND
ND

d
d

IS: 3-MCPD-d5

Results are the mean of ve replicates (%), and RSD % are given in parentheses.
The results are recoveries of 3-MCPD esters and glycidyl esters using corresponding 3-MCPD-d5 esters and glycidyl-d5 esters as internal standards.
The blank solvent and blank oil matrix were all spiked at 0.5 mg kg1 .
Deuterium-labeled glycidyl esters for stearate and palmitate are not available. ND: not detected.

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Q. Liu et al. / J. Chromatogr. A 1314 (2013) 208215

Table 3
Concentration of chloropropanols esters in oil samples using Na2 SO4 solution and NaCl solution as extraction solvent (mg kg1 ).
3-MCPD esters

Camellia-seed oil
Sunower oil
Peanut sesame blend oil
Phytosterols corn oil
Sunower oil
Tea-seed oil
Sesame blend oil

2-MCPD esters

1,3-DCP esters

2,3-DCP esters

Na2 SO4

NaCl

Na2 SO4

NaCl

Na2 SO4

NaCl

Na2 SO4

NaCl

1.452
0.957
0.262
0.428
1.014
0.277
0.320

1.540
1.018
0.273
0.489
1.023
0.456
0.434

1.008
0.675
0.215
0.338
0.726
0.232
0.281

1.060
0.696
0.211
0.366
0.765
0.228
0.265

ND
ND
ND
ND
ND
ND
ND

ND
ND
ND
ND
ND
ND
ND

ND
ND
ND
ND
ND
ND
ND

ND
ND
ND
ND
ND
ND
ND

ND, not detected.

moderate growth was observed from 70 to 90 mL (Fig. 2). Therefore,

80 mL diethyl ether was selected for the elution of chloropropanols.
According to DGF standard methods C-III 18 (09), PBA was
used as the derivatization agent in many previous studies
[14,16,22,3032]. The advantage is that 3-MCPD released from 3MCPD esters in NaCl solution can be derivatized directly, which
makes it to be a relatively easy and convenient method. The major
disadvantage, however, is that 1,3-DCP and 2,3-DCP released from
their corresponding easters cannot be derivatized with PBA and
could not be detected. In addition, if the split/splitless injector had
been used instead of the PTV injector, baseline noise and shift would
have seriously affected the response of the target compounds. After
eight consecutive injections of the same sample, a baseline drift
appeared as a result and therefore accurate quantication cannot
be achieved.
It is well known that instrumental detection capability depends
directly on the cleanliness of the extract. In order to overcome the
disadvantage of traditional methods of DGF [24], a novel method,
which based on the cleanup procedure of the solid-supported
liquidliquid extraction (SLE) by commercial column of diatomaceous earth were proposed in this work. In late 1997, SLE as an
innovative purication method was proposed for the purication
of combinatorial libraries [33]. The technique involves supporting
an aqueous buffer on a bed of coarse mesh, calcinated diatomateous
earth sold under the product name Hydromatrix. It has already been
used with success mainly in the biological uid analysis [3437].
The application of SLE to the analysis of phenolic compounds has
been described in balsamic vinegar, olives and wine [38,39]. One
of the advantages of SLE over extraction is fully compatible with a

wide range of solvents, regardless of density. Extractions are readily

performed in any solvent that is not miscible with water. In addition, because emulsions are not an issue with SLE, solvent mixtures
can be used regardless of density. Therefore, the technique could
be used for extracting chloropropanols from the aqueous solution.
The extracted chloropropanols were transferred from the aqueous
phase to the organic phase (ethyl ether) through this purication
step of absorbing water and removing impurities. Thereafter, the
four chloropropanols released from all the chloropropanols esters
can be derivatized with the HFBI and analyzed by GCMS. Fig. 3
shows the chromatograms and spectrums of standard sample analysis. The baseline noise and drift are inconspicuous.
3.3. Method validation
3.3.1. Calibration curves
The calibration data and regression equation of the calibration
curves are presented in Table 1. The ANOVA test for the linearity of the calibration curves was performed within the range of
0.0252.000 mg L1 , and the linearity was always satised with
correlation coefcients (r) not less than 0.999.
3.3.2. Limits of detection (LODs) and quantication (LOQs)
The limits of detection (LODs) and quantication (LOQs) were
considered as the minimum amount of target analyte that produces a chromatogram peak with a signal-to-noise ratio (S/N) of
three and ten times as the background chromatographic noise,
respectively. The S/N was measured at the spiked levels of 10, 20,
30, 40, 50, 100, 200, and 300 g kg1 , respectively. Results from

Fig. 2. Inuence of diethyl ether amount.

Q. Liu et al. / J. Chromatogr. A 1314 (2013) 208215

213

Fig. 3. The chromatograms and mass spectrums of the processed virgin olive oil sample with the optimized method at the fortied level of 0.5 mg kg1 . (1) 1,3-DCP-d5
derivative, (2) 1,3-DCP esters derivative, (3) 2,3-DCP-d5 derivative, (4) 2,3-DCP esters derivative, (5) 3-MCPD-d5 derivative, (6) 3-MCPD esters derivative, (7) 2-MCPD-d5
derivative, (8) 2-MCPD esters derivative.

Table 1 show that LODs of fatty acid esters of 3-MCPD, 2-MCPD,

1,3-DCP and 2,3-DCP (calculated as free forms) were 30, 30, 100
and 30 g kg1 , respectively.
3.3.3. Recovery experiments
The accuracy and precision of the method were examined by the
reproducibilities using spiked blank sample matrices. The recovery estimation of this method was performed on spiked blank oil
samples at concentration levels of 0.1, 0.5 and 2 mg kg1 . Since
the commercial fatty acid esters of 2-MCPD, 1,3-DCP and 2,3-DCP

are not available, 2-MCPD, 1,3-DCP and 2,3-DCP were spiked into
blank oil samples. Average recoveries of the eight 3-MCPD esters
and the four chloropropanols ranged from 70.7% to 113.3%. The
reproducibility of this method represented by the relative standard
deviation (RSD) percentage is less than 15.0% at each spiked level
(Table 4).
The recoveries of 3-MCPD esters are comparable with that
of 3-MCPD, meaning the complete cleavage of ester bond in
chloropropanols esters. Consequently, the recoveries of the chloropropanols can represent that of the corresponding chloropropanol

214

Q. Liu et al. / J. Chromatogr. A 1314 (2013) 208215

Table 4
Average recoveriesa of the chloropropropanol esters, chloropropanols and glycidyl esters at three spiked levels.
Compound

Fortication levels (mg kg1 )

0.1

Oleoyl-3-MCPD
Linoleoyl-3-MCPD
Linolenoyl-3-MCPD
1,2-Dilinolenoyl-3-MCPD
1,2-Dilinoleoyl-3-MCPD
Palmitoyl-3-MCPD
1,2-Dioleoyl-3-MCPD
1,2-Distearoyl-3-MCPD
3-MCPD
2-MCPD
1,3-DCP
2,3-DCP
Glycidyl-oleate
Glycidyl-linoleate
Glycidyl-linolenate
Glycidyl-stearate
Glycidyl-palmitate
a

0.5

Recovery

RSD

Recovery

113.3
99.8
87.5
89.5
98.7
95.6
98.8
105.7
94.9
96.6
90.0
93.9
ND
ND
ND
ND
ND

3.8
3.3
10.2
0.8
2.2
8.8
4.0
3.8
12.4
7.3
3.9
7.7

109.2
89.9
89.4
77.0
70.7
76.9
72.5
97.2
96.9
86.5
97.6
104.8
ND
ND
ND
ND
ND

2
RSD
5.6
3.2
5.4
1.0
7.0
2.1
3.3
1.5
9.8
2.6
3.9
10.1

Recovery

RSD

112.8
84.8
77.9
93.1
76.6
84.0
82.2
108.2
95.5
85.8
106.2
98.3
8.8
2.1
7.3
7.0
8.0

3.9
3.1
5.9
6.6
4.7
4.8
8.7
12.1
8.5
10.9
10.1
3.5
2.4
5.0
8.5
2.2
12.4

Results are the mean of ve replicates (%), and RSD % are given in parentheses. ND: not detected.

Fig. 4. FAPAS prociency test result (Lab No. 21 is the present method).

esters. Additionally, the recoveries of ve glycidyl esters were very

low (ND-8.8%), which can draw the same conclusion that the determination of fatty acid esters of chloropropanols was not interfered
by glycidyl esters in the proposed method.
Additionally, this method was applied to the rened palm oil
sample of prociency test 2631 of Food Analysis Performance
Assessment Scheme (FAPAS) [40] for the analysis of 3-MCPD esters.
The value submitted (4.53 mg kg1 ) by this novel method falls well
within the scope of specied satisfactory range (assigned value,
4.99 mg kg1 ), with Z-score obtained at 0.5 (Fig. 4). In addition,
the level of 2-MCPD esters in the palm oil sample determined with
the novel method (2.32 mg kg1 ) was also submitted, which was
not a compulsive analyte in this performance test.

oil, sesame oil, virgin olive oil, peanut oil, linseed oil, tea-seed oil,
tea-seed olive blend oil, sunower-seed oil and blend oil were analyzed.
The purication process was demonstrated to be efcient, and
no baseline shift appeared after several repetitious analyses. The
3-MCPD esters and 2-MCPD esters were detected in all samples
except virgin olive oil. Total concentrations of 3-MCPD esters in all
the 10 oil samples ranged from ND to 1.54 mg kg1 , and that for 2MCPD esters were generally in the range of ND-1.06 mg kg1 . The
esters of 2,3-DCP was found in a sunower oil and a tea-seed oil,
with the concentration of 0.300 mg kg1 and 0.340 mg kg1 , respectively, while no esters of 1,3-DCP was detected in any of the tested
oil samples.

3.4. Method application

4. Conclusions

To further demonstrate the utility and performance of the proposed methodology, different edible oils including soybean oil, corn

This paper proposed a novel analytical procedure for the routine analysis of total fatty acid esters of chloropropanols in edible

Q. Liu et al. / J. Chromatogr. A 1314 (2013) 208215

oils based on GCMS. The proposed method has robust resistance

to the matrix of edible oil, and is suitable for the determination of
total fatty acid esters of chloropropanols by GCMS without the
PTV injectors. The method validation data including calibration,
LOD/LOQ, accuracy and repeatability and prociency test results
(Z-score: 0.5) of the ofcial FAPAS indicated that the present
quantitative method could be successfully applied to the determination of total chloropropanols esters in various edible oils.
According to results of sample determination, fatty acid esters of 3MCPD and 2-MCPD are found at signicant levels in various edible
oils, while esters of 1,3-DCP and 2,3-DCP might also present in oils,
but at relatively low concentrations. The results obtained will be
useful for future studies focusing on the formation and occurrence
of fatty acid esters of chloropropanols.
Acknowledgements
We would like to thank all the donors who provided edible oil
samples. This study was supported by the National Basic Research
Program (973, No. 2012CB720804) and the National High-Tech Program (863, No. 2010AA023003). No funding bodies had any role in
study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
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