Bidirectional conversion between 3-monochloro-1,2-propanediol and glycidol in course of the procedure of DGF standard methods

Article abstract of DOI:10.1007/s11746-011-1802-3

NMR observation revealed that bidirectional conversion occurred between 3-monochloropropane-1,2-diol (3-MCPD) and glycidol in the course of the analytical procedure of DFG standard method C-III 18 (09), option A; 3-MCPD was partly converted to glycidol at the transesterification step, and glycidol was converted partly to 3-MCPD at the derivatization step conducted at 80 °C under acidic condition in the presence of NaCl. Based on the proton numbers observed by ¹H NMR, the degrees of the conversion were estimated to be 37 and >70%, respectively. In addition, epoxide ring-opening of glycidol and its esters was found to be ca. 90% by the acid treatment described in the method, option B. Thus, it was concluded that the standard method, option A, did not correctly give the combined amount of 3-MCPD esters and glycidyl esters in oils containing glycidyl esters, and the difference of the values obtained by options A and B did not correspond to the amount of glycidyl esters, either. In addition, derivatives of 3-MCPD with phenylboronic acid were not observed by NMR at the derivatization step, although they were detected by GC-MS in the organic phase at the following extraction step.

Full text of DOI:10.1007/s11746-011-1802-3

J Am Oil Chem Soc (2011) 88:1143–1151
DOI 10.1007/s11746-011-1802-3
ORIGINAL PAPER
Bidirectional Conversion Between 3-Monochloro-1,2-propanediol
and Glycidol in Course of the Procedure of DGF Standard
Methods
Naoki Kaze
Yomi Watanabe
•
Hirofumi Sato
•
Hiroshi Yamamoto
•
Received: 27 December 2010 / Revised: 2 February 2011 / Accepted: 23 February 2011 / Published online: 25 March 2011
Ó AOCS 2011
Abstract NMR observation revealed that bidirectional
conversion occurred between 3-monochloropropane-1,2-
diol (3-MCPD) and glycidol in the course of the analytical
procedure of DFG standard method C-III 18 (09), option A;
Introduction
3-Monochloropropane-1,2-diol (3-MCPD) has recently
been a big issue due to the concerns to human health [1, 2].
The recommended guideline for its intake is 2 lg/kg body
weight per day. In order to estimate the daily intake from
the diet, a quantification method is essential. The amount of
3-MCPD and its fatty acid esters (referred to as esters
hereafter) in fat and oil products is currently measured by
the standard method established by the German Society for
Fat Science (DFG standard methods C-III 18 [3]). In the
method, it is stated that the method is not specific to
3-MCPD (esters) and that glycidol and its esters are known
to be detected as 3-MCPD. It was thus revised in 2009 to
remove them by the acid treatment (option B) prior to the
conventional procedure (option A). The values obtained by
option B are defined as the true amount of 3-MCPD,
whereas the difference between the values obtained by
option A and B is defined as the amount of glycidyl esters,
since glycidol is considered to be negligible in fats and oils.
Perplexingly, the amounts of glycidyl esters determined
in the revised standard method were not consistent with the
amounts of those determined directly by the LC-MS
method [4] when sample oils spiked with known amount of
glycidyl esters were analyzed in our laboratory. The
revised standard method is based on the assumption that
glycidyl esters were completely detected as 3-MCPD in
option A and that the removal of glycidyl esters were
complete in option B. However, there was a possibility that
the assumption might not be true. Moreover, the mecha-
nism of incorrect detection of glycidyl esters as 3-MCPD
by the standard method is not clearly understood. This
paper reveals that bidirectional conversion between
3
-MCPD was partly converted to glycidol at the transe-
sterification step, and glycidol was converted partly to
3
-MCPD at the derivatization step conducted at 80 °C
under acidic condition in the presence of NaCl. Based on
1
the proton numbers observed by H NMR, the degrees of
the conversion were estimated to be 37 and [70%,
respectively. In addition, epoxide ring-opening of glycidol
and its esters was found to be ca. 90% by the acid treatment
described in the method, option B. Thus, it was concluded
that the standard method, option A, did not correctly give
the combined amount of 3-MCPD esters and glycidyl esters
in oils containing glycidyl esters, and the difference of the
values obtained by options A and B did not correspond to
the amount of glycidyl esters, either. In addition, deriva-
tives of 3-MCPD with phenylboronic acid were not
observed by NMR at the derivatization step, although they
were detected by GC-MS in the organic phase at the fol-
lowing extraction step.
Keywords 3-Monochloropropane-1,2-diol (3-MCPD) Á
DFG standard method Á Glycidol Á Glycidyl ester Á NaCl
N. Kaze Á H. Yamamoto
Ueda Oils and Fats MFG Co. Ltd, 17 Uozakihamamachi,
Higashinada-ku, Kobe, Hyogo 658-0024, Japan
H. Sato Á Y. Watanabe (&)
Osaka Municipal Technical Research Institute,
1-6-50 Morinomiya, Joto-ku, 536-8553 Osaka, Japan
e-mail: yomi@omtri.or.jp
3-MCPD and glycidol was observed in the course of the
analytical procedure of DFG standard methods C-III 18
1
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1
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J Am Oil Chem Soc (2011) 88:1143–1151
(
09), and that the method was not suitable for fats and oils
J = 5.2 Hz, 1H), 3.62 (dd, J = 5.82, 11.3 Hz, 1H), 2.65 (bs,
1H), 2.35 (t, J = 7.5 Hz, 2H), 1.63 (quint, J = 7.4 Hz, 2H),
which include glycidyl esters.
1
.26 (m, 24H), 0.88 (t, J = 6.8 Hz, 3H); C NMR
3
1
(
75 MHz, CDCl ) d 173.1, 68.8, 68.4, 47.8, 33.9, 31.9,
3
Experimental Procedures
29.6–29.1 (10 9 CH ), 25.1, 22.8, 14.1; MS (ESI)
2
?
m/z 371.4 [M ? Na] ; Anal. Calcd for C H O Cl: C,
65.40; H, 10.69. Found: C, 65.44; H, 10.52.
1
9 37 3
Materials
3
-MCPD, 3-MCPD-d , glycidol, phenylboronic acid,
5
Procedure B
sodium methoxide/methanol solution, starting materials for
syntheses, and solvents were purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). NaCl and acetic
acid were purchased from Nakalai Tesque Co. Ltd. (Kyoto,
Japan). Soybean oil was the product of Ueda Oils and Fats
Palmitic acid (1.0 g, 3.90 mmol) was dissolved in dry
methylene chloride (20 mL) at 0 °C under argon
atmosphere. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCÁHCl) (0.75 g, 3.90 mmol), N,N-diiso-
propylethylamine (DIEA) (0.50 g, 3.90 mmol), and N,N-
dimethylaminopyridine (DMAP) (0.048 g, 0.39 mmol)
were added to the solution. The reaction mixture was stirred
for 5 min, and then cooled down. Glycidol (0.26 mL,
MFG Co. Ltd. (Kobe, Japan). D O (99.96 atom% D) and
2
CDCl3 (99.8 atom% D) were purchased from Aldrich
Chemical Company, Inc. (Milwaukee, WI, USA). Other
chemicals were of the analytical grade.
3
1
.90 mmol) was added to the mixture, and stirred for
0 min, then allowed to warm up to room temperature.
Synthesis of Glycidyl Esters
Glycidyl Palmitate
After the reaction was complete, the methylene chloride
solution was washed with water (20 mL), 0.1 M hydrogen
chloride solution (20 mL), sat. sodium hydrogen carbonate
solution (20 mL), and finally brine (20 mL). The solvent
was evaporated to dryness, and the residue was purified by
silica gel column chromatography (ethyl acetate/hexane =
1/1, vol/vol) to give glycidyl palmitate (0.96 g, 79%) as a
white solid.
Procedure A
Glycidol (0.27 g, 3.64 mmol) was dissolved in dry pyridine
(
20 mL) at 0 °C in a three-necked flask equipped with a
drying tube. Palmitoyl chloride (1.0 g, 3.64 mmol) was
added to the solution in four portions at intervals of
3-Chloro-2-Hydroxypropyl Palmitate
(3-MCPD Palmitate)
1
0 min. The reaction mixture was stirred overnight at room
temperature, and the solvent was removed by evaporation.
The residue was extracted with chloroform (20 mL), and
washed with water (20 mL), 0.1 M hydrogen chloride
solution (20 mL), sat. sodium hydrogen carbonate solution
3-Chloro-2-hydroxy propanol (0.40 g, 3.64 mmol) was
dissolved in dry pyridine (20 mL) at 0 °C in a three-necked
flask equipped with a drying tube. Palmitoyl chloride
(1.0 g, 3.64 mmol) was added to the solution in four por-
tions at intervals of 10 min. The reaction mixture was
stirred for 2 h at room temperature. The solvent was then
removed by evaporation. The residue was extracted with
chloroform (20 mL), washed with water (20 mL), 0.1 M
hydrogen chloride solution (20 mL), sat. sodium hydrogen
carbonate solution (20 mL), and finally brine (20 mL). The
solvent was evaporated to dryness. The residue was puri-
fied by flash chromatography on silica gel (ethyl acetate/
hexane = 1/2, vol/vol) to give 3-chloro-2-hydroxypropyl
palmitate (0.82 g, 64%) as a white solid, 3-chloropropyl
1,2-dipalmitate (0.040 g, 2%) as a white solid, and
3-chloro-1-hydroxypropyl palmitate (0.062 g, 5%) as a
white solid. 3-Chloropropyl 1,2-dipalmitate: IR (neat)
(
20 mL), and finally brine (20 mL). The solvent was
evaporated to dryness. The residue was purified by flash
chromatography on silica gel (ethyl acetate/hexane = 1/1,
vol/vol) to give glycidyl palmitate (0.80 g, 70%) as a white
solid and 3-chloro-2-hydroxypropyl palmitate (0.12 g, 9%)
as a white solid. Glycidyl palmitate: IR (neat) 2,912, 2,844,
1
1
4
1
2
1
,737, 1,471, 1,456, 846; H NMR (300 MHz, CDCl ) d
3
.40 (dd, J = 3.1, 12.2 Hz, 1H), 3.91 (dd, J = 6.3, 12.2 Hz,
H), 3.26 (m, 1H), 2.84 (t, J = 4.6 Hz, 1H), 2.65 (dd, J =
.6, 4.8 Hz, 1H), 2.35 (t, J = 7.6 Hz, 2H), 1.63 (m, 2H),
1
3
.26 (m, 24H), 0.88 (t, J = 6.5 Hz, 3H); C NMR
(
75 MHz, CDCl3) d 173.5, 64.7, 49.3, 44.6, 30.0, 31.9,
9.6–29.1 (10 9 CH ), 24.8, 22.6, 14.0; MS (ESI)
2
2
?
m/z 335.4 [M ? Na] ; Anal. Calcd for C H O : C, 73.03;
H, 11.61. Found: C, 73.06; H, 11.51. 3-Chloro-2-
1
9 36 3
1
2,913, 2,844, 1,728, 1,471, 1,253; H NMR (300 MHz,
hydroxypropyl palmitate (3-MCPD palmitate): IR (neat)
1
,436, 2,931, 1,737, 1,465, 1,180, 719; H NMR
CDCl ) d 5.22 (m, 1H), 4.28 (dd, J = 4.4, 11.8 Hz, 2H),
3
3
3.66 (dd, J = 5.4, 11.8 Hz, 2H), 2.33 (m, 4H), 1.63 (m, 4H),
1
1.26 (m, 48H), 0.88 (t, J = 6.8 Hz, 6H); C NMR
3
(
300 MHz, CDCl ) d 4.22 (d, J = 5.2 Hz, 2H), 3.91 (quint,
3
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J Am Oil Chem Soc (2011) 88:1143–1151
1145
(
75 MHz, CDCl ) d 173.2, 172.8, 70.25, 62.25, 42.2, 34.2,
at 80 °C for 20 min (step 4). Then, the extraction was
conducted using n-hexane (3 mL, step 5). The organic
phase was collected, evaporated to dryness, and was dis-
solved again to 2,2,4-trimethylpentane (2 mL). The sample
was filtered by paper before it was brought to GC-MS
analysis (step 6).
3
3
4.0, 31.9, 29.7–29.1 (20 9 CH ), 24.9, 22.7, 14.1; MS
2
?
(
ESI) m/z 609.2 [M ? Na] ; Anal. Calcd for C H O Cl:
35 67 4
C, 71.57; H, 11.50. Found: C, 71.66; H, 11.45. 3-Chloro-1-
hydroxypropyl palmitate: IR (neat) 3,434, 2,922, 1,737,
1
1
3
2
0
4
,460, 1,177; H NMR (300 MHz, CDCl ) d 5.06 (m, 1H),
3
.83 (d, J = 4.8 Hz, 2H), 3.70 (dd, J = 5.5, 11.5 Hz, 2H),
.37 (m, 2H), 1.64 (quint, J = 7.5 Hz, 2H), 1.26 (m, 24H),
Option B: soybean oils (0.1 g) spiked with glycidyl
esters were treated with 0.5% sulfuric acid/propanol solu-
tion (0.5 mL) at 45 °C for 15 min in the ultrasonic bath
(step 1). The samples were put through the above-men-
tioned procedure, steps 2–6.
1
3
.88 (t, J = 6.6 Hz, 3H); C NMR d 173.1, 81.3, 64.2,
4.3, 34.2, 31.9, 29.6–29.1 (10 9 CH ), 25.1, 22.8, 14.1;
2
?
MS (ESI) m/z 371.4 [M ? Na] ; Anal. Calcd for
C H O Cl: C, 65.40; H, 10.69. Found: C, 65.47; H,
1
9 37 3
1
3
0.47.
GC-MS
-Propoxypropane-1,2-diol (1-glyceryl 1-propyl ether)
GC-MS was carried out using a GCMS QP 2010 instru-
ment (Shimadzu, Kyoto, Japan) connected to a DB-5
capillary column (30 m, 0.25 lm, Agilent Technologies,
Tokyo, Japan). The column temperature was controlled as
follows; it was kept at 60 °C for 1 min, raised at 6 °C /min
to 190 °C, further raised at 20 °C/min to 280 °C, and
kept at 280 °C for 6 min. The temperature of the pro-
grammed-temperature vaporizer (PTV) injector was con-
trolled as follows; it was kept at 60 °C for 1 min, raised
at 10 °C/min to 180 °C and kept at 180 °C for 20 min.
The temperatures of the interface and the ion source were
set at 250 and 200 °C. Other conditions for GC-MS were
the same with those described in DGF standard methods
C-III 18.
Glycerol (9.7 g, 105 mmol) was dissolved in dry dimeth-
ylformamide (100 mL) at 0 °C in a three-necked flask with
a condenser. Sodium hydride (60% in oil, washed by
n-hexane, 0.49 g, 12.2 mmol) was slowly added to the
solution. After hydrogen gas was ceased, propyl bromide
(
1.0 g, 8.13 mmol) was added to the reaction mixture and
was stirred for 18 h at 80 °C. After the reaction mixture
cooled to the ambient temperature, ethyl acetate (100 mL)
was added and was washed with water (100 mL). The
aqueous phase was extracted twice with ethyl acetate
(
50 mL). The organic phase was combined, and was
evaporated to concentrate. The resulting residue was
purified by open column chromatography (hexane/ethyl
acetate/methanol = 4.5:4.5:1, vol/vol/vol) to give 1-glyc-
LC-MS
1
eryl 1-propyl ether (0.090 g, 8.3%). H NMR (300 MHz,
D O) d 3.84 (m, 1H), 3.69–3.40 (m, 7H), 1.55 (sext, J =
7
Glycidyl esters were treated with 0.5% sulfuric acid/pro-
panol solution (0.5 mL) at 45 °C for 15 min in an ultra-
sonic bath. To the sample, n-hexane (3 mL) and water
(3 mL) were added and mixed by vortex. The organic
phase was collected and dried over sodium sulfate. It was
evaporated to dryness and dissolved in acetonitrile
(1.5 mL). The resulting sample was then analyzed by API
2000 LC/MS/MS system (Life Technologies Japan, Tokyo,
Japan) connected to a YMC-Triart C18 column (2.0 9
50 mm, S-3 lm, 12 nm, YMC Co. Ltd., Kyoto, Japan).
The column temperature was set at 40 °C. Elution was
conducted at a flow rate of 0.2 mL/min, using mobile phase
A consisting of acetonitrile/methanol/water (=17:17:6, vol/
vol/vol) and mobile phase B consisting of 2-propanol. The
binary gradient program was as follows; mobile phase A,
98% and B, 2% at 0.0 min; a linear gradient elution to A,
85% and B, 15% from 0.0 to 15.0 min; an isocratic elution
with A, 5% and B, 95% from 15.1 to 25.0 min. Mass
chromatograms were recorded by a triple stage quadrupole
mass spectrometer equipped with an atmospheric pressure
chemical ionization (APCI) ionizer. The ion spray voltage
was ?4,500 V.
2
1
.2 Hz, 2H), 0.85 (t, J = 7.2 Hz, 3H); C NMR (75 MHz,
3
CDCl ) d 73.8, 71.8, 71.0, 63.4, 23.1, 10.3; MS (ESI) m/
3
?
z 135.4 [M ? H] ; HRMS (ESI) 135.1023 (C H O
requires 135.1021).
6
15
3
Determination of 3-MCPD Forming Substances
by DGF Standard Methods C-III 18 (09)
The contents of 3-MCPD forming substances were deter-
mined as described in DGF standard methods C-III 18(09)
with a slight modification. Option A: soybean oil (0.1 g)
mixed with 3-MCPD was dissolved in 0.5 mL solvent
consisted of tert-butyl methyl ether and ethyl acetate (=4:1,
vol/vol). To the sample, 3-MCPD-d (2 lg) and 0.5 M
5
sodium methoxide/methanol solution (1 mL) was added
and left for 10 min at room temperature (step 2). The
mixture was extracted using n-hexane (3 mL) and water
containing 16.7% NaCl and 3.3% acetic acid (3 mL). The
aqueous phase was rinsed with n-hexane (3 mL, step 3).
The aqueous phase was mixed with derivatization reagent
(
0.125 g/mL phenylboronic acid solution, 0.5 mL) and left
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J Am Oil Chem Soc (2011) 88:1143–1151
NMR
3-MCPD in the aqueous phase with phenylboronic acid, (5)
extraction of resulting derivatives with n-hexane, and (6)
GC-MS analysis. In order to verify the effectiveness of the
acid treatment (step 1), glycidyl esters were prepared first.
1
13
H- and C-NMR spectra were recorded on a JEOL AL-
3
00 spectrometer (Tokyo, Japan) at 300 and 75 MHz,
respectively and were referenced to internal tetramethyl-
silane (CDCl ) or to the residual protonated solvent (for
3
Synthesis of Glycidyl Fatty Acid Esters
D O and methanol-d ).
2
4
The Schotten-Baumann reaction, which is the one between
acid chlorides and alcohols under basic conditions, is
generally applicable for fatty acid ester synthesis. Palmi-
toyl chloride was reacted with glycidol to give not only the
corresponding glycidyl ester (70%) but also the unexpected
3-MCPD ester (9%). The epoxide ring of the ester was
nucleophilically substituted by the chloride ion which was
generated near the epoxide in the reaction. On the other
hand, esters are also obtained by the reaction between free
fatty acids and alcohols using condensation agents such as
carbodiimides under the basic conditions. The corre-
sponding glycidyl ester was afforded as the unique product
(79%) using 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide hydrochloride (EDCÁHCl) as the condensation agent
in spite of the chloride ion existing in the reaction system.
Results
DFG standard methods C-III 18 (09) describes that glycidol
and its fatty acid esters are detected as 3-MCPD in the
conventional procedure (option A) and that they should be
removed by acid treatment prior to the conventional pro-
cedure (option B). As diagrammatically described in
Fig. 1, it consists of the following steps; (1) treatment of oil
samples with 0.5% sulfuric acid/propanol to open epoxide
ring, (2) transesterification of glycerides and other esters
with sodium methoxide/methanol, (3) fractionation of fatty
acid esters from 3-MCPD using n-hexane/water containing
1
6.7% NaCl and 3.3% acetic acid, (4) derivatization of
Fig. 1 Flow diagram of DGF
standard methods C-III 18.
Major steps, reaction
conditions, and conversions of
target compounds were
described
3-MCPD esters, glycidyl esters, acylglycerols (0.1 g)
0.5% H SO /propanol (0.5 mL)
Step 1, epoxide ring opening
(Option B)
2
4
45
C, 15 min in ultrasonic bath
3-MCPD esters, 1-acyloxy-3-propyloxypropane-2-ol, acylglycerols
Step 2, transesterification
3-MCPD-d5 (2 µg)
(Option A)
0.5 M NaOMe/methanol (1 mL)
r.t. 10 min
3-MCPD, 3-propyloxypropane-1,2-diol, FAME, glycerol
Step 3, fractionation
n-hexane (3 mL)
16.7% NaCl and 3.3% acetic acid soln. (3 mL)
FAME/n-hexane
-MCPD etc./aqueous phase
n-hexane (3 mL)
3
3
FAME/n-hexane
-MCPD etc./aqueous phase
Step 4, derivatization
Step 5, extraction
0.125 g/mL phenylboronic acid soln. (0.5 mL)
C, 20 min
n-hexane (3 mL)
80
3
-MCPD phenylborate etc./organic phase
Dry-out
2,2,4-trimethylpentane (2 mL)
Step 6, quantification
GC-MS
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J Am Oil Chem Soc (2011) 88:1143–1151
1147
Therefore, the 3-MCPD ester formation by Schotten-Bau-
mann reaction progresses concurrently with the glycidyl
ester formation.
might be reduced to undetectable amount, but should
remain still in the samples after the acid treatment. The
observation was consistent with the observation of Shimizu
et al. [5], though they did not directly quantify the residual
glycidyl esters.
Palmitoyl chloride was reacted with 3-MCPD to give the
monoester of primary alcohol (3-MCPD ester, 64%), one of
the secondary alcohols (3-MCPD ester isomer, 5%), and
the diester (3-MCPD diester, 2%). This result suggested
that the first esterification took place at the less hindered
In order to confirm the incomplete ring-opening of
epoxides, the residual glycidyl esters after the acid treat-
ment were directly measured by LC-MS. As shown in
Table 2, 0.11, 0.42, and 1.72 ppm of glycidyl palmitate
remained in the oils spiked with 1, 5, and 20 ppm, even
after the acid treatment. Likewise, removal of glycidyl
oleate was not completed by the acid treatment. The
residual ratio was 10% approximately. Therefore, the
epoxide ring-opening of glycidyl esters was confirmed to
be ca. 90% by the acid treatment.
1
-position hydroxyl group. Interestingly, no glycidyl ester
production was observed in the EDCÁHCl system. The
glycidyl ester did not form due to too low basicity of
pyridine to deprotonate from the 2-position hydroxide.
Thus, the deprotonation may cause the conversion of
3
-MCPD ester to glycidyl ester, and the nucleophilicity of
the chloride ion may cause the conversion of the glycidyl
ester to the 3-MCPD ester.
Possibility of 1-Glyceryl 1-Propyl Ether Causing
the Incorrect Detection of 3-MCPD
Evaluation of Acid Treatment
The resulting glycidyl esters were used to verify the
effectiveness of the acid treatment (step 1). Soybean oil
spiked with glycidol or glycidyl esters were treated
according to the standard method, option B, which inclu-
ded the treatment with 0.5% sulfuric acid/propanol solution
The treatment of glycidyl esters by sulfuric acid/propanol
gives the corresponding 1-acyloxy-3-propyloxypropane-2-
ol. Here, 1-acyloxy-propane-2,3-diol (monoacylglycerol) is
not expected due that sulfuric acid/propanol contains little
water. Based on the result above, 90% of glycidyl esters
should be converted to 1-acyloxy-3-propyloxypropane-2-
ol. Its deacylation by the following treatment with sodium
methoxide (step 2) gives 3-propyloxypropane-1,2-diol (1-
glyceryl 1-propyl ether). In general, 1-glyceryl 1-propyl
ether is stable under basic conditions. However, glycidol,
which is responsible for the incorrect detection as
(
Table 1). 3-MCPD was detected by GC-MS in the oil
samples spiked with C10 ppm of glycidols but was not in
that with 5 ppm. Similar results were obtained with oils
spiked with glycidyl palmitate and oleate. These results
indicated that the removal of glycidol in addition to its
esters were incomplete. The glycidyl esters of B5 ppm
3-MCPD, could be generated from the ether by the attack
of the neighboring 2-position alkoxide on the epoxide-
carbon if the alkoxide is generated under basic conditions.
In order to investigate the possibility of 1-glyceryl 1-propyl
ether causing the incorrect detection of 3-MCPD, the
compound was synthesized, and its behavior under treat-
Table 1 Detection of 3-MCPD in oils spiked with glycidol or glyc-
idyl esters by DGF standard methods C-III 18(09), option B
Spiked compound
Spiked amount
Detected amount
a
as 3-MCPD
(ppm)
(
ppm)
5
1
3
b
ment with sodium methoxide was monitored by NMR. C-
NMR spectra were identical before and after the treatment
Glycidol
n.d.
1
2
0
0
5
0
0
5
0
0
1.17 ± 0.44
1.68 ± 0.65
b
Glycidyl palmitate
Glycidyl oleate
n.d.
Table 2 Residual amount of glycidyl esters after treatment with
0
.5% sulfuric acid/propanol
1
2
0.64 ± 0.23
1.04 ± 0.11
a
Compound
Amount
(ppm)
Detected amount
(ppm)
Residual
ratio (%)
b
n.d.
1
2
0.63 ± 0.17
1.31 ± 0.30
Glycidyl palmitate
1.0
0.11
0.42
1.72
0.11
0.52
11.5
8.5
5
.0
Soybean oil was spiked with glycidol or its esters. The oil samples
were treated as described in DGF standard method C-III 18(09),
option B
20.0
1.0
8.6
Glycidyl oleate
10.6
10.3
5
.0
a
The amount in soybean oil without any spike was 0.32 ppm. The
amount was subtracted from those detected in the spiked oil samples.
All analyses were conducted 3–4 times. The mean values and the
standard deviations were presented
Glycidyl esters were treated with 0.5% sulfuric acid/propanol solution
at 45 °C for 15 min in the ultrasonic bath as described in DGF
standard method C-III 18(09), option B
b
a
Not detected. The minimum limit of detection was 0.2 ppm
Glycidyl esters were analyzed by LC-MS
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1
148
J Am Oil Chem Soc (2011) 88:1143–1151
(
Fig. 2), since the peak at 49 ppm belonged to methanol. It
45.9 ppm, Fig. 3a) were the only peaks observed before the
treatment. After the treatment, the peaks derived from
was thus clarified that glycidol was not generated by the
sodium methoxide treatment of 1-glyceryl 1-propyl ether.
Consequently, the acid treatment of 1-acyloxy-3-propy-
loxypropane-2-ol (step 1) did not cause the incorrect
detection of 3-MCPD by the standard procedure, option B.
1
3
glycidol (61.5, 53.0, 45.0 ppm) appeared on the C-NMR
chart (Fig. 3b). The conversion ratio was 37% calculated
1
on their proton numbers obtained by H NMR.
The behavior of glycidol in the standard method option
A was also monitored by NMR. Glycidol was treated in the
similar way to the standard method steps 2–4 (transesteri-
fication to derivatization steps), except n-hexane and
Bidirectional Conversion of 3-MCPD and Glycidol
When soybean oil (0.1 g) spiked with 3-MCPD (1 lg) was
treated according to the DGF standard method, option A
phenylboronic acid was not added and D O was used
2
instead of water (details of reaction conditions were given
in the legend Fig. 3d). pH of the reaction mixture was 4.2.
In addition to the peaks derived from glycidol, those from
3-MCPD were newly observed by NMR after the treatment
(Fig. 3c, d). The conversion ratio from glycidol to
3-MCPD was 70%, when it was calculated on their proton
numbers. Interestingly, the conversion was not detected
when the derivatization step (step 4) was conducted at
room temperature instead of 80 °C. On the other hand, the
conversion increased to nearly 100% when glycidol was
(
without acid treatment, steps 2–6), the peak area of
phenylboronic acid derivatives of 3-MCPD detected by
GC-MS was 600. On the other hand, it was 2,600 when
the sample was treated in the same procedure without the
transesterification step. The peak areas of 3-MCPD-d , the
5
internal standard, were 1,100 and 4,800 with or without
transesterification. It was therefore indicated that 77% of
3
-MCPD was lost somehow due to the step. Weisshaar
estimated that sodium methoxide might decompose
-MCPD [6]. However, the details of the decomposition
3
directly dissolved in D O containing 16.7% NaCl and 3.3%
2
have not yet been understood.
acetic acid (pH 1.9), and stood at 80 °C for 20 min. It was
thus indicated that the heating under the acidic conditions
at the derivatization step accelerated the conversion of
glycidol to 3-MCPD greatly.
Thus, the behavior of 3-MCPD under the transesterifi-
13
1
cation step was directly monitored by H and C NMR.
-MCPD was dissolved in sodium methoxide/methanol
3
solution, kept at room temperature for 10 min. After the
addition of D O, the sample was analyzed by NMR mea-
2
Production of 3-MCPD Derivatives
at the Derivatization Step
surement. The peaks derived from 3-MCPD (71.1, 62.5,
As shown Fig. 3d, treatment of glycidol with steps 2–4
without phenylboronic acid generated 3-MCPD with 70%
of conversion. The treatment was then conducted with
phenylboronic acid. The reaction conditions were the same
with the standard method steps 2–4 except n-hexane was
not added and D O was used instead of water. The con-
2
1
3
version of glycidol to 3-MCPD was again observed by
C
NMR (Fig. 4b). The degree of conversion was 74% cal-
1
culated on the proton numbers observed by H NMR.
1
Suppose that the errors of integration value measured in H
NMR were ± 5%, the conversion ratio were nearly the
same with or without phenylboronic acid at the derivati-
zation step. On the other hand, the 3-MCPD phenylborate
was not detectable (Fig. 4b). Association constant of
phenylboronic acid and diols was reported to drastically
change at pH *7.5, and the phenyl borate was hardly
formed below pH 6.5 [7]. It was therefore speculated that
the 3-MCPD phenyl borate were produced only in a small
amount in the experimental conditions of pH 4.2, and thus
were undetectable by NMR.
1
3
Fig. 2 C-NMR spectra of 3-propoxypropane-1,2-diol. a 3-prop-
oxypropane-1,2-diol (standard) dissolved in D O; b 3-propoxypro-
2
The reaction mixture was further extracted by n-hexane
as the procedure step 5. After the removal of the hexane
phase, the aqueous phase was analyzed by NMR again.
pane-1,2-diol (0.01 g) was dissolved in 0.5 M sodium methoxide/
methanol solution (0.2 mL) and kept at room temperature for 10 min.
2
Then, D O (0.6 mL) was added to the solution. All reactions were
1
3
conducted in NMR tubes
Both of glycidol and 3-MCPD were observed by C NMR
1
23

J Am Oil Chem Soc (2011) 88:1143–1151
1149
(
A)
(C)
(D)
(
B)
7
5
70
65
60
55
50
45
ppm
75
70
65
60
55
50
45
ppm
1
3
Fig. 3 C-NMR spectra of 3-monochloropropane-1,2-diol (3-
MCPD, inverted triangles), glycidol (closed circles), and their
d glycidol (0.01 g) was dissolved in 0.5 M sodium methoxide/
methanol solution (0.2 mL) and kept at room temperature for 10 min.
products. a 3-MCPD (standard) dissolved in D
(
(
2
O; b 3-MCPD
0.13 g) dissolved in 0.5 M sodium methoxide/methanol solution
1 mL) was kept at room temperature for 10 min. Then, D O (1 mL)
O;
2
Then, D O containing 16.7% NaCl and 3.3% acetic acid (0.6 mL),
was added to the mixture, and allowed to stand at 80 °C for 20 min.;
All reactions were conducted in NMR tubes
2
was added to the solution. c glycidol (standard) dissolved in D
2
(A)
(
B)
MeOH→
(
C)
Acetone→
←AcOH
←
PhB(OH)2
←
AcOH
Fig. 4 The conversion of glycidol to 3-MCPD under the condition of
option A. The circles and triangles represent the peaks of glycidol and
3-MCPD, respectively. a Glycidol (standard) dissolved in D O;
2
b glycidol (0.01 g) was dissolved 0.5 M sodium methoxide/methanol
6
acetone-d containing 12.5% (w/v) phenylboronic acid (0.133 mL)
were added to the mixture, and allowed to stand at 80 °C for 20 min.;
c After treatments described in b, the reaction mixture was washed by
hexane (1 mL). The resulting aqueous phase was analyzed; all
reactions were conducted in NMR tubes
solution (0.2 mL) and kept at room temperature for 10 min. Then,
2
D O containing 16.7% NaCl and 3.3% acetic acid (0.6 mL) and of
(
Fig. 4c), with the ratio of 13:87, calculated from the
3-MCPD phenyl borate, 3-MCPD, and glycidol in the
aqueous phase. As a result, the ratio of 3-MCPD against
glycidol increased from 74 to 87%. It should be noted that
glycidol and 3-MCPD remained in the aqueous phase even
after the hexane extraction at step 5 of the standard method.
1
proton numbers obtained by H NMR. These results indi-
cated that the extraction of 3-MCPD phenyl borate, which
was more hydrophobic than 3-MCPD, to the organic phase
shifted the equilibrium of the three compounds, namely
1
23

1
150
J Am Oil Chem Soc (2011) 88:1143–1151
The low efficiency of the 3-MCPD phenyl borate formation
and extraction might explain the relatively high standard
deviations given in Table 1.
were abandoned chloride ions with high nucleophilicity,
and few hydroxyl ions in the solution. What was important
was that the degree of conversion from glycidol to
3
2
-MCPD depended on the conditions of the procedure steps
–5 (transesterification, derivatization, and extraction), and
Discussion
was not 100%. This observation contradicted that of
Kuhlmann cited in Ref. [8] namely that the conversion was
nearly complete. Based on our observation, the standard
method, option A, did not give the combined amount of
3-MCPD esters and glycidyl esters correctly. The removal
of epoxides by the acid treatment described in option B was
not complete either, as shown in Table 2. Therefore, the
difference of the values obtained by options A and B did
not correspond to the amount of glycidyl esters.
It has been described that the epoxide ring-opening in
glycidol and its esters was incomplete by the acid treatment
described in the DGF standard methods C-III 18 (09),
option B, and that the bidirectional conversion between
3
-MCPD and glycidol was observed by NMR in the course
of the method. The behaviors of 3-MCPD esters and
glycidyl esters, which were supposed to be in fats and oils,
in the course of the standard method are schematically
shown in Fig. 5. 3-MCPD produced by the transesterifi-
cation using sodium methoxide was partly converted to
glycidol in the step (37%). There also is a possibility that
The conversion of 3-MCPD to glycidol at the transe-
sterification step was estimated to be 37% by NMR,
whereas that of glycidol to 3-MCPD at the following steps
was 74%. The loss of 3-MCPD in total was thus ca. 10%,
which was not consistent with the observation described in
the section ‘bidirectional conversion of 3-MCPD and
glycidol’, where the loss was estimated to be 77% under
the influence of the transesterification step. In another
report of ours, it was clarified that the loss was also caused
by the low extraction capability of n-hexane used in the
extraction step (step 5) [9]. The substitution of n-hexane
with a more polar solvent such as chloroform increased the
recovery of 3-MCPD derivatives. In the mean time, the
derivatives of 3-MCPD with phenylboronic acid were not
observed in NMR analysis in the aqueous phase at the
derivatization step (step 4), although they were detected by
GC-MS in the hexane phase obtained in step 5. It was thus
estimated that the derivatives were rather formed in the
course of hexane extraction (Fig. 5), than at the derivati-
zation step, and the polarity of the solvent might affect the
production as well as the recovery of the derivatives.
2
-MCPD were converted to glycidol, though it has to be
proven. The resulting glycidol, in addition to glycidol
derived from glycidyl esters, were partly converted to
3
-MCPD in the following steps, which were conducted in
the presence of saturated NaCl under acidic conditions at
0 °C (74%). Glycerol was not generated from glycidol
under the conditions, which could be explained that there
8
3
-MCPD ester
a
Glycidyl ester
a
H+
a
b
O
HO
Conclusion
Cl–
This paper reports that bidirectional conversion was con-
firmed between 3-MCPD and glycidol in the course of the
analytical procedure of DFG standard methods C-III 18
c
c & d
(09), option A; 3-MCPD was partly converted to glycidol
at the transesterification step, and glycidol was converted
partly to 3-MCPD at the derivatization step conducted at 80
°C under acidic condition in the presence of NaCl. In
addition, epoxide ring-opening of glycidol and its esters
was shown to be incomplete by the acid treatment descri-
bed in the method, option B. Thus, the standard method,
option A, did not give the combined amount of 3-MCPD
esters and glycidyl esters correctly, and the difference
between the values obtained by options A and B did not
correspond to the amount of glycidyl esters, either. The
Ph
B
O
O
Cl
Fig. 5 Behaviors of 3-MCPD esters and glycidyl esters under the
procedure of DGF standard method C-III 18(09). R represents fatty
acyl group. Arrows with solid lines represent the conventionally
known/believed routes, whereas those with dotted lines represent
routes newly confirmed in this study. Reagents and conditions:
a Sodium methoxide/methanol, rt, 10 min. b Acetic acid, NaCl,
water. c Phenylboronic acid, 80 °C, 20 min. d Hexane extraction
1
23

J Am Oil Chem Soc (2011) 88:1143–1151
1151
restricted application of the standard method, option A, to
glycidyl ester-free samples is recommended. In addition,
the conversion of 3-MCPD to phenylboronic acid was not
observed by NMR at the derivatization step. The deriva-
tization was estimated to occur rather in the following
hexane extraction step. The observations presented in this
paper are important for our understanding of the standard
method, and for the interpretation of the values so far given
by the standard method.
vegsource.com/articles/carrageenan/JECFA_2001_Carrageenan_
Report.pdf. Accessed Oct 2010
. (2009) Ester-bound 3-chloropropane-1,2-diol (3-MCPD esters) and
glycidol (glycidol esters). Deutsche Gesellschaft fuer Fettwissens-
chaft: DGF Standard Methods (14. Supplement) C-III 18
4. Masukawa Y, Shiro H, Nakamura S, Kondo N, Jin N, Suzuki N,
Ooi N, Kudo N (2010) A new analytical method for the
quantification of glycidol fatty acid esters in edible oils. J Oleo
Sci 59:81–88
5. Shimizu M, Kudo N, Shiro H, Yasunaga K, Masukawa Y,
Katsuragi Y, Yasumasu T (2010) Comparison of indirect and
direct quantification of glycidol fatty acid ester in edible oils.
J Oleo Sci 539:535–539
3
6
. Weisshaar R (2008) Determination of total 3-chloropropane-1,
-diol (3-MCPD) in edible oils by cleavage of MCPD esters with
2
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