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Food Additives & Contaminants: Part A

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A new, direct analytical method using LC-MS/MS for

fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD
esters) in edible oils
a a a b b a
K. Yamazaki , M. Ogiso , S. Isagawa , T. Urushiyama , T. Ukena & N. Kibune
a
Japan Food Research Laboratories, 7-4-41, Saito-Asagi, Ibaraki-shi, Osaka 567-0085, Japan
b
Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries,
Kasumigaseki, Chiyoda-ku, Tokyo, Japan
Accepted author version posted online: 24 Jul 2012.Version of record first published: 21 Aug
2012.

To cite this article: K. Yamazaki , M. Ogiso , S. Isagawa , T. Urushiyama , T. Ukena & N. Kibune (2013): A new, direct
analytical method using LC-MS/MS for fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD esters) in edible oils, Food
Additives & Contaminants: Part A, 30:1, 52-68

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 Food Additives & Contaminants: Part A, 2013
Vol. 30, No. 1, 52–68, http://dx.doi.org/10.1080/19440049.2012.713031

A new, direct analytical method using LC-MS/MS for fatty acid esters of
3-chloro-1,2-propanediol (3-MCPD esters) in edible oils
K. Yamazakia, M. Ogisoa, S. Isagawaa, T. Urushiyamab, T. Ukenab and N. Kibunea*
a
Japan Food Research Laboratories, 7-4-41, Saito-Asagi, Ibaraki-shi, Osaka 567-0085, Japan; bFood Safety and Consumer
Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, Kasumigaseki, Chiyoda-ku, Tokyo, Japan
(Received 1 June 2012; final version received 12 July 2012)

A new, direct analytical method for the determination of 3-chloro-1,2-propanediol fatty acid esters (3-MCPD
esters) was developed. The targeted 3-MCPD esters included five types of monoester and 20 types of diester.
Samples (oils and fats) were dissolved in a mixture of tert-butyl methyl ether and ethyl acetate (4:1), purified
Downloaded by [University of Chicago Library] at 22:03 23 April 2013

using two solid-phase extraction (SPE) cartridges (C18 and silica), then analysed by liquid chromatography-
tandem mass spectrometry (LC-MS/MS). Five monoesters and five diesters with the same fatty acid group could
be separated and quantified. Pairs of 3-MCPD diesters carrying the same two different fatty acid groups, but at
reversed positions (sn-1 and sn-2), could not be separated and so were expressed as a sum of both compounds.
The limits of quantification (LOQs) were estimated to be between 0.02 to 0.08 mg kg1, depending on the types
of 3-MCPD ester. Repeatability expressed as relative standard deviation (RSDr%) varied from 5.5% to 25.5%.
The new method was shown to be applicable to various commercial edible oils and showed levels of 3-MCPD
esters varying from 0.58 to 25.35 mg kg1. The levels of mono- and diesters ranged from 0.10 to 0.69 mg kg1 and
from 0.06 to 16 mg kg1, respectively.
Keywords: 3-MCPD esters; LC-MS/MS; direct determination; edible oils

Introduction information on the occurrence of 3-MCPD esters.

3-Chloropropane-1,2-diol (3-MCPD) is a food-proces-
sing contaminant most commonly present in acid-
hydrolysed vegetable protein (HVP) and its derived
products. It is considered to be carcinogenic in vitro,
but in vivo it is a non-genotoxic compound (Hamlet
et al. 2002). A provisional maximum tolerable daily
intake (PMTDI) of 2 mg kg1 body weight day1 was
established by the Joint FAO/WHO Expert Committee
on Food Additives (JECFA) (2007). The presence of
3-MCPD fatty acid (3-MCPD) esters in different
refined oils and fats and in infant formula was recently
reported (Zelinková et al. 2006, 2009; Weißhaar 2009).
The potential human health risks of these compounds
require investigation because 3-MCPD esters can be
metabolised to release free 3-MCPD into the human
gastrointestinal system. It is thus very important
to know the accurate content and composition of
3-MCPD esters in foods.
Currently, most analytical methods for determining
the accurate concentration of 3-MCPD esters in food
are indirect methods based on the measurement of free
3-MCPD after transesterification. These methods
include those used by Zelinková et al. (2006, 2009)
and Weißhaar (2009), and lead to the above
The transesterification included methanolysis catalysed
by acidic (Divinová et al. 2004; Zelinková et al. 2006,
2009) or alkaline (Weißhaar 2009) conditions, and
hydrolysis by lipase (Hamlet and Sadd 2004). Thus, the
data leading to concerns regarding the safety of
3-MCPD esters were mainly obtained using indirect
methods.
The indirect methods developed by Weißhaar
(2008), called DGF C-III 18(09) (DGF 2009), used
alkaline methanolysis for transesterification to release
free 3-MCPD prior to derivatisation and quantifica-
tion using gas chromatography-mass spectrometry
(GC-MS). These methods have been widely used to
study the presence or reduction of 3-MCPD esters
and glycidyl esters (Franke et al. 2009; Weißhaar et al.
2010; Hrncirik et al. 2011; Hrncirik and van Duijn
2011; Matthäus et al. 2011; Strijowski et al. 2011).
These alkaline transesterification methods were based
on the assumption that ‘‘3-MCPD esters and 3-MCPD
forming substances’’ were generated exclusively from
3-MCPD esters and glycidyl esters. Various sugges-
tions were directed against the DGF methods
(ILSI Europe 2009; Karasek et al. 2010; Shimizu
et al. 2010; Fiebig et al. 2011; Haines et al. 2011;

*Corresponding author. Email: kibunen@jfrl.or.jp

ß 2013 Taylor & Francis


Hrncirik and van Duijn 2011; Kaze et al. 2011;
Kuhlmann 2011; Küsters et al. 2011), leading to the
current C-VI 18(10) version (DGF 2011).
On the other hand, analytical methods for
3-MCPD esters based on acidic transesterification,
including alcoholysis using 1.8% sulphuric acid/meth-
anol, were reported prior to alkaline methanolysis
methods (Divinová et al. 2004; Svejkovská et al. 2004;
Zelinková et al. 2006). Hamlet and Asuncion (2011)
validated this method in various foods, including
edible oils, and confirmed the method’s robustness
and that 3-MCPD is not generated during transester-
ification. This method was improved to be more
accurate (AOCS 2011; Ermacora and Hrncirik 2012).
Indirect methods require the derivatisation of
free 3-MCPD prior to quantification by GC-MS.
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Phenylboronic acid (PBA) is generally used as the
derivatising reagent in both alkaline and acidic condi-
tions, although Hamlet et al. (2002), Hamlet and
Asuncion (2011), and Dubois et al. (2012) employed
heptafluorobutyrylimidazole (HFBI). Indirect methods
were improved at several points, therefore a recent
collaborative study showed that both methods, alka-
line or acidic transesterification, provided relatively
consistent results (Fiebig et al. 2011).
Indirect analytical methods for quantifying
3-MCPD esters are relatively sensitive as all 3-MCPD
esters are converted to 3-MCPD and thus provide
information on the total amount of 3-MCPD. However,
indirect methods do not provide significant information
on what kinds of fatty acids are present in 3-MCPD
esters, and whether monoesters or diesters are present.
The in vivo metabolism of monoesters and diesters of 3-
MCPD may be different, with monoesters perhaps
metabolised more rapidly than diesters (Schilter et al.
2011). Furthermore, depending on the substrate speci-
ficity of lipases, sn-2-monoesters were absorbed and
used in the re-synthesis of triglycerides in vivo (Seefelder
et al. 2008, 2011; Schilter et al. 2011). More recently, it
was also reported that 3-MCPD-1-monoester was
hydrolysed in differentiated Caco-2 cell, while diester
was absorbed and metabolised by the cells (Buhrke et al.
2011). Since the chemical structure of 3-MCPD ester
might influence its toxicity (Hamlet et al. 2011), it is very
important to characterise individual 3-MCPD esters
present in foods.
To quantify simultaneously and directly both
3-MCPD esters and glycidy esters (GEs), Haines
et al. (2011) used liquid chromatography coupled
with time-of-flight mass spectrometry (LC-TOF/MS).
This method required the use of a specific LC-TOF/
MS and frequent maintenance of the ion source due to
the accumulation of sodium salt used for forming the
adduct ion. Hori et al. (2012) also reported a method
for simultaneously quantifying 3-MCPD esters and
Food Additives & Contaminants: Part A 53
H2C Cl H2C Cl
O
HC OH HC O C
R2
H2C O C R1 H2C O C
R1
O O
3-MCPD-1- mono-ester 3-MCPD-1,2- diester
Figure 1. Structure of 3-MCPD esters: R1 or R2 means
either the palmitoyl, stearoyl, oleoyl, linoleoyl or linolenoyl
group.
GEs as sodium adduct ions using LC-TOF/MS.
Dubois et al. (2012) conducted the comparative study
of the direct and indirect method for 3-MCPD esters.
They also used LC-TOF/MS for direct determination
of 3-MCPD esters but detected as ammonium
adduct ion.
However, liquid chromatography-tandem mass
spectrometry (LC-MS/MS), which is more commonly
available than LC-TOF/MS, has seldom been used
to quantify 3-MCPD esters directly (Pinkston and
Stoffolano 2011). Additionally, in the method of
Pinkston and Stoffolano there were disadvantages
such as the use of the standard addition method
being unsuitable for routine analysis or the higher
LOQs for some diesters. A new method that is more
convenient and applicable to the direct quantification
of many species of 3-MCPD fatty acid esters is
urgently required.
The development of a new analytical method to
determine individual 3-MCPD esters using LC-MS/
MS has begun. First of all, we selected the C16–C18
fatty acid esters which were known to be usually
abundant in vegetable oils, putting off other esters with
short-chain fatty acid (e.g. C12 or C14). We focused on
identifying the chemical species of 3-MCPD esters.
Thirty standards were obtained, including five mono-
esters, five diesters with a pair of the same fatty acid
(defined as ‘‘homo-diesters’’) and 20 diesters with a
pair of different fatty acids (defined as ‘‘hetero-
diesters’’). The skeletal structures of the target com-
pounds are shown in Figure 1. The list of standards
and ISs of 3-MCPD ester examined in this study is
shown in Table 1. This paper reports both a method
for the quantification of 3-MCPD esters in edible oils
using LC-MS/MS and the concentration of 3-MCPD
esters in several edible oils.
Materials and methods
Chemicals
3-MCPD ester standards
The 3-MCPD ester standards were supplied by
Wako Chemical Industries (Osaka, Japan) and
 54 K. Yamazaki et al.

Table 1. 3-MCPD standards.

Compound Abbreviation Formula MWa

Monoesters
1-Palmitoyl-3-chloro-1,2-propanediol 3-MCPD-P C19H37ClO3 348.24
1-Stearoyl-3-chloro-1,2-propanediol 3-MCPD-S C21H41ClO3 376.27
1-Oleoyl-3-chloro-1,2-propanediol 3-MCPD-O C21H39ClO3 374.26
1-Linoleoyl-3-chloro-1,2-propanediol 3-MCPD-L C21H37ClO3 372.24
1-Linolenoyl-3-chloro-1,2-propanediol 3-MCPD-Ln C21H35ClO3 370.23
Homo-diestersb
1,2-Dipalmitoyl-3-chloropropanediol 3-MCPD-PP C35H67ClO4 586.47
1,2-Distearoyl-3-chloropropanediol 3-MCPD-SS C39H75ClO4 642.53
1,2-Dioleoyl-3-chloropropanediol 3-MCPD-OO C39H71ClO4 638.50
1,2-Dilinoleoyl-3-chloropropanediol 3-MCPD-LL C39H67ClO4 634.47
1,2-Dilinolenoyl-3-chloropropanediol 3-MCPD-LnLn C39H63ClO4 630.44
Hetero-diestersc
1-Palmitoyl-2-stearoyl-3-chlorppropanediol 3-MCPD-PS C37H71ClO4 614.50
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1-Palmitoyl-2-oleoyl-3-chloropropanediol 3-MCPD-PO C37H69ClO4 612.49

1-Palmitoyl-2-linoleyl-3-chloropropanediol 3-MCPD-PL C37H67ClO4 610.47
1-palmitoyl-2-linolenoyl-3-chloropropanediol 3-MCPD-PLn C37H65ClO4 608.46
1-Stearoyl-2-palmitoyl-3-chloropropanediol 3-MCPD-SP C37H71ClO4 614.50
1-Stearoyl-2-oleoyl-3-chloropropaenediol 3-MCPD-SO C39H73ClO4 640.52
1-Stearoyl-2-linoleoyl-3-chloropropaenediol 3-MCPD-SL C39H71ClO4 638.50
1-Stearoyl-2-linolenoyl-3-chloropropaenediol 3-MCPD-SLn C39H69ClO4 636.49
1-Oleoyl-2-palmitoyl-3-chloropropanediol 3-MCPD-OP C37H69ClO4 612.49
1-Oleoyl-2-stearoyl-3-chloropropanediol 3-MCPD-OS C39H73ClO4 640.52
1-Oleoyl-2-linoleoyl-3-chloropropanediol 3-MCPD-OL C39H69ClO4 636.49
1-Oleoyl-2-linolenoyl-3-chloropropanediol 3-MCPD-OLn C39H67ClO4 634.47
1-Linoleoyl-2-palmitoyl-3-chloropropanediol 3-MCPD-LP C37H67ClO4 610.47
1-Linoleoyl-2-stearoyl-3-chloropropanediol 3-MCPD-LS C39H71ClO4 638.50
1-Linoleoyl-2-oleoyl-3-chloropropanediol 3-MCPD-LO C39H69ClO4 636.49
1-Linoleoyl-2-linolenoyl-3-chloropropanediol 3-MCPD-LLn C39H65ClO4 632.46
1-Linolenoyl-2-palmitoyl-3-chloropropanediol 3-MCPD-LnP C37H65ClO4 608.46
1-Linolenoyl-2-stearoyl-3-chloropropanediol 3-MCPD-LnS C39H69ClO4 636.49
1-Linolenoyl-2-oleoyl-3-chloropropanediol 3-MCPD-LnO C39H67ClO4 634.47
1-Linolenoyl-2-linoleoyl-3-chloropropanediol 3-MCPD-LnL C39H65ClO4 632.46
Internal standard
d5-1-Palmitoyl-3-chloro-1,2-propanediol d5-3-MCPD-P C19H32D5ClO3 353.27
d5-1,2-Dipalmitpyl-3-chloro-1,2-propanediol d5-3-MCPD-PP C35H62D5ClO4 591.50
d5-1,2-Distearoyl-3-chloro-1,2-propanediol d5-3-MCPD-SS C37H70D5ClO4 647.57
d5-1,2-Dilinoleoyl-3-chloro-1,2-propanediol d5-3-MCPD-LL C39H62D5ClO4 639.50
3-Chloro-1,2-propanediol 3-MCPD C3H7ClO2 110.01

Notes: aMolecular weight, calculated using 35 Da as the mass of chlorine.

b
The term ‘‘hetero-diesters’’ is used for compounds that have a pair of different fatty acid groups.
c
The term ‘‘homo-diesters’’ is used for compounds that have a pair of identical fatty acid groups.

consisted of five monoesters, five homo-diesters and
20 hetero-diesters, as shown in Table 1. All standards
were racemic mixtures due to asymmetry of the carbon
on the glycerol backbone (Myher et al. 1986; Velı́šek
et al. 2002). Four deuterium-labelled 3-MCPD esters
for use as internal standards (IS) were supplied by
Wako Chemical Industries: d5-1-palmitoyl monoester
(d5-3-MCPD-P), d5-1,2-dipalmitoylyl diester (d5-3-
MCPD-PP), d5-1,2-distearoyl diester (d5-3-MCPD-
SS), and 1,2-dilinolenoyl diester (d5-3-MCPD-LnLn).
Both these 3-MCPD ester standards and ISs were the
custom-made products with 50–100 mg of content.
Currently, these are commercially available from the
above supplier. The purity was 498% for monoesters,
homo-diesters and ISs (498%D), whereas hetero-
diesters were the products without purity indication
confirmed by the shipment standard of the supplier
(tested by IR and 1H-NMR).
Reagents
Ethyl acetate, tert-butyl methyl ether (MTBE), meth-
anol, ethanol, hexane acetonitrile and 2-propanol were
all of analytical grade. Sep-pakTM C18 Env. cartridges
(900 mg) and Sep-pakTM Vac silica cartridges (500
and 1000 mg) were obtained from Nihon Waters K.K.
(Tokyo, Japan).
 Food Additives & Contaminants: Part A 55

Apparatus
LC-MS/MS was conducted using a Waters 2695
LC systems connected to a Quattro Premier XE
(Waters Corporation, Milford, MA, USA).
Samples
Refined lard and 10 edible oils were used to test the
methodology. The oils were soybean, rapeseed, rice,
safflower, sesame, olive, grape seed, perilla and palm.
All were purchased from local supermarkets in Osaka.
Extra virgin olive oil for method validation was also
purchased from a supermarket.
Standard solutions
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Diesters
Oil samples (1 g) were weighed and dissolved in a
mixture of MTBE and ethyl acetate (4:1 v/v, 10 ml) and
spiked with 100 ml (1 mg) of each of the three ISs
(10 mg ml1) using a microsyringe. A portion (0.1 ml)
of the 10 ml sample was removed and charged into a
Sep-pakTM vac silica cartridge (1000 mg) pre-condi-
tioned with hexane (5 ml). The target compounds were
eluted with a mixture of ethyl acetate and hexane (5 ml,
5:95 v/v). The eluate was evaporated to dryness and
the residue was dissolved in a mixture of MTBE and
ethyl acetate (4:1 v/v, 0.1 ml) and then in a mixture
of methanol and 2-propanol (1 ml, 1:1 v/v) prior to
charging in a Sep-pakTM C18 Env. cartridge pre-
conditioned with methanol (5 ml). After rinsing the
vessel with a mixture of methanol and 2-propanol

Each standard 3-MCPD ester was dissolved in hexane (1 ml, 1:1 v/v) and charging in a C18 cartridge, the
to prepare 0.5 mg ml1 stock solutions. The working analytes in the cartridge were eluted with a mixture of
standard solutions for calibration were prepared ethanol, acetonitrile and methanol (50 ml, 5:30:65 v/v).
by diluting the stock solution using 2-propanol. The eluate was evaporated to dryness and the residue
Mixed working solutions for calibration were pre- was re-dissolved in 1 ml of 2-propanol; the 3-MCPD
pared with five monoesters, five homo-diesters and diesters were quantified by LC-MS/MS.
10 hetero-diesters. Ten of the 20 hetero-diesters had
essentially identical characteristics, e.g. 3-MCPD-PL
and 3-MCPD-LP cannot be distinguished by this Analysis by LC-MS/MS
assay, so only 3-MCPD-LP was used in the mixed Operating conditions
standard solutions. The working standard solutions for HPLC separation was performed using a reversed-
calibration were prepared at concentrations of 0.0005, phase Luna 3 u C18(2) column (2.0 mm I.D., length
0.001, 0.0025, 0.005 and 0.01 mg ml1. LC vials con- 50 mm, particle size 3.2 mm). The mobile phase was
taining the working standard solutions were spiked composed of water (A), 0.01 mol l1 ammonium ace-
immediately before LC-MS/MS with 0.1 mg ml1 tate in methanol (B), methanol (C) and 2-propanol
(d5-3-MCPD-P) or 0.01 mg ml1 (d5-3-MCPD-PP, (D). The gradient system consisted of four pumps.
d5-3-MCPD-SS and d5-3-MCPD-LnLn) as ISs. A typical gradient elution programme is shown in
Table 2. The flow rate was 0.2 ml min1; the column
oven was set at 40 C’ the injection volume was 10 ml.
Sample preparation
Mass data were obtained using electrospray ionisa-
Monoesters tion (ESI) operated in positive mode. The ion source
Oil samples (1 g) were weighed and dissolved in a temperature was 120 C; cone gas flow was 50 l h1; the
mixture of MTBE and ethyl acetate (4:1 v/v, 10 ml) argon collision gas flow was 0.2 ml min1. Nitrogen
and spiked with 100 ml (10 mg) of IS (d5-3-MCPD-P;
100 mg ml1) using a microsyringe. A portion (0.1 ml)
of the 10 ml sample was removed and charged into Table 2. Composition and gradient programme for the
a Sep-pakTM Env. C18 cartridge pre-conditioned with mobile phase for HPLC.
methanol (5 ml). The target compounds were eluted
Mobile phase
with methanol (10 ml), evaporated to dryness, and the (linear gradient) (%)
residue was dissolved in a mixture of ethyl acetate and
hexane (2 ml, 5:95 v/v), then charged into a Sep-pakTM Time (min) A B C D Remark
vac silica cartridge (500 mg) pre-conditioned with a
mixture of ethyl acetate and hexane (2 ml, 5:95 v/v). 0–3 30 10 50 10 –
3–10 2 10 88 0 Monoester fraction
After rinsing the vessel with a mixture of ethyl acetate 10.1–11.0 2 10 48 40 –
and hexane (20:80 v/v, 2 ml) and charging into silica 11.0–22.0 1 10 89 0 Diester fraction
cartridge, the analytes in the cartridge were eluted with 22.1–30.0 0 10 85 5 Diester fraction
a mixture of ethyl acetate and hexane (8 ml, 20:80 v/v). 30.1–35.0 0 0 0 100 Wash column
The eluate was evaporated to dryness and re-dissolved 35.1–40.0 30 10 50 10 Return to initial
in 2-propanol (1 ml) for LC-MS/MS quantification of Note: A, water; B, 0.01 mol l1 ammonium acetate solution;
3-MCPD monoesters. C, methanol; and D, 2-propanol.
 56 K. Yamazaki et al.

was used as the desolvation gas at 300 C at a flow rate
of 800 l h1. 3-MCPD esters were quantified by
selected reaction monitoring (SRM). Cone voltage,
collision energy and monitoring ion (m/z) are provided
in Table 3.
monoesters and d5-3-MCPD-PP was used for diesters,
except for 3-MCPD-LnLn and 3-MCPD-SS, which
used d5-3-MCPD-LnLn and d5-3-MCPD-SS as ISs,
respectively.

Quantification of 3-MCPD esters Results and discussion

The final solution described under ‘‘sample prepara- Separation of each ester by HPLC
tion’’ was analysed by LC-MS/MS. Quantification Earlier experiments attempted to separate all mono-
was performed using external calibration normalised and diesters during the same HPLC run, but resulted
against the IS. In general, d5-3-MCPD-P was used for in poor fractionation. In this study, mono- and diester

Table 3. Monitoring ions (m/z) and acquiring conditions for 3-MCPD esters.

Cone Collision Precursor ion Product Retention

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Compounda voltage (V) energy (eV) [M þ NH4] (m/z)b ion (m/z)c time (min)

Monoesters
3-MCPD-Ln 20 10 388 261 7.76
3-MCPD-L 20 10 390 263 8.18
3-MCPD-P 10 10 366 239 8.48
3-MCPD-O 20 10 392 265 8.66
3-MCPD-S 20 10 394 267 9.24
Hetero-diesterse
3-MCPD-LnL 30 20 650 353 d 15.64
3-MCPD-LLn 30 20 650 353 –
3-MCPD-LnO 30 20 652 357 16.73
3-MCPD-OLn 30 20 652 353 –
3-MCPD-LP 30 20 628 331 17.87
3-MCPD-PL 30 20 628 355 –
3-MCPD-LO 30 20 654 357 18.42
3-MCPD-OL 30 30 654 355 –
3-MCPD-LnP 30 20 626 331 19.00
3-MCPD-PLn 30 20 626 261 –
3-MCPD-PO 30 20 630 357 20.04
3-MCPD-OP 30 20 630 331 –
3-MCPD-LnS 30 20 654 261 20.84
3-MCPD-SLn 30 20 654 359 –
3-MCPD-SL 30 20 656 355 21.06
3-MCPD-LS 30 20 656 359 –
3-MCPD-SP 30 20 632 331 23.14
3-MCPD-PS 30 20 632 359 –
3-MCPD-SO 30 20 658 357 23.86
3-MCPD-OS 30 20 658 359 –
Homo-diestersf
3-MCPD-LnLn 30 20 648 353 14.90
3-MCPD-LL 30 20 652 355 16.67
3-MCPD-PP 30 20 604 331 19.45
3-MCPD-OO 30 10 656 357 20.66
3-MCPD-SS 30 20 660 359 27.76
IS
d5-3-MCPD-P 10 10 371 244 8.46
d5-3-MCPD-PP 30 20 609 336 19.36
d5-3-MCPD-LnLn 30 20 653 358 14.87
d5-3-MCPD-SS 30 20 665 364 27.61

Notes: aCompounds shown in italics were not measured because the corresponding isomer was measured.
b
The precursor ion was the ammonium adduct.
c
Product ions were the [R1 þ H]þ ion for monoesters, and the [(CH2  Cl-CH-O-R2-CH3 þ H]þ ion for diesters.
d
[(CH2  Cl-CH2-CH-O-R1]þ ion (m/z 353) was used instead of the [(CH2  Cl-CH-O-R2-CH3 þ H]þ ion
(m/z 355) in order to avoid interference.
e
The term ‘‘hetero-diesters’’ is used for compounds that have a pair of different fatty acid groups.
f
The term ‘‘homo-diesters’’ is used for compounds that have a pair of identical fatty acid groups.

mixtures were injected separately but separated using
the same HPLC conditions. It proved impossible to
separate hetero-diesters that contained the same pair of
fatty acids but at different positions (e.g., 3-MCPD-PO
and 3-MCPD-OP). Furthermore, these hetero-diesters
have the same calculated exact mass and cannot
distinguish even with mass selectivity such as TOF/MS.
Several analytical columns were tested: ZORBAX
Eclipse XDBTM C-8 (Agilent Technologies Inc., CA,
USA), Luna 3uTM C18(2) (Phenomenex, Inc., CA,
USA), InertsilTM ODS-P (GL Sciences Inc., Tokyo,
Japan), and DevelosilTM C30-UG-3 (Nomura
Chemical Inc., Seto, Japan). The Luna 3 u C18(2)
column and the operating conditions shown in Table 2
provided good separation of the mono- and diesters
of 3-MCPD, except for hetero-diesters, as described
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above. The analysis run was 40 min and the last analyte
peak to elute, 3-MCPD-SS, had a retention time of
approximately 28 min.
A slow increase in concentration of the mobile
phase was required to avoid high pressure and subse-
quent shutdown of the LC pumps caused by the high
viscosity of 2-propanol. 2-Propanol was required to
wash the analytical column and remove non-polar
components such as triacylglycerides in the sample
matrixes. These components appeared as interference
(both during chromatography and ionisation) during
LC-MS/MS measurements. The mobile phase con-
tained 30% water at the beginning of the run to
improve the peak shape of the mono-esters. Also,
2-propanol was added in the initial mobile phase,
considering the solubility of analytes, and the propor-
tion of 2-propanol at the intermediate of the gradient
cycle was increased in order to accelerate the elution
of diesters from the column. Considering carefully,
the presented condition may be able to simplify
by, for example, the combination of mobile phases A
and B.
Detection by LC-MS/MS
It is very difficult to detect the protonated precursor ion
of 3-MCPD esters. Haines et al. (2011) and Hori et al.
(2012) detected 3-MCPD esters by forming a sodium
adduct, whereas Pinkston and Stoffolano (2011) and
Dubois et al. (2012) formed ammonium adduct ions of
3-MCPD esters for detection. We chose the latter
adduct ion to avoid troublesome maintenance issues
caused by the use of sodium salt (Haines et al. 2011).
The ammonium adduct ion was formed at the
primary ionisation step and used as a precursor ion to
form the product ions in the collision cell. It was
presumed that the most abundant monoester product
ions were detected as the [M – (CH2Cl-CH(OH)-CH2-
O)]þ ion (i.e., the ion corresponding to the fatty acid
group). For the diesters, the main product ion was
Food Additives & Contaminants: Part A 57
detected as a [M – (R1-C(O)O þ 2H]þ ion (i.e., the fatty
acid at sn-1 was defragmented and chlorine atom
remained). The presumed transition is shown in
Figure 2. Monitoring ions were selected as the most
abundant ions. However, only the 1-linolenoyl-2-
linoleoyl ester of 3-MCPD (3-MCPD-LnL) was mon-
itored using the [M – (R2-C(O)O) þ 2H]þ ion (i.e., fatty
acid at sn-2, linoleoyl group defragmented; m/z 353)
as the product ion due to interference at m/z 355
(i.e., fatty acid at sn-1, linolenoyl group defragmented)
(Figure 2). In addition, the hetero-diesters produced
product ion profiles in which both the sn-1 and sn-2
fatty acids were present and had relative intensities
of between 1:2.5 and 1:4 (ratio of sn-1 versus sn-2
fatty acid).
It might be possible to use a different monitor
ion in MS to distinguish compounds that cannot be
separated on HPLC. However, although the product
ions formed from a specific hetero-diester were specific
to some extent (because the product ion contained
both the sn-2 fatty acid group and chlorine), another
ion from the same hetero-diester but containing the
sn-1 fatty acid group had the same m/z but lower
intensity than the major product ion. Thus, satisfac-
tory separation remained difficult. Representative
mass spectra of 3-MCPD esters are shown in Figure 3.
As described above, two hetero-diesters with the
same pair of fatty acids but at reversed positions were
impossible to separate, so the pair was used together
for quantification or calibration. Thus, 3-MCPD-PO,
-LnS, -LnP, -LP, -SP, -LnL, -LnO, -LO, -SL and -SO
were used, and 3-MCPD-OP, -SLn, -PLn, -PL, -PS,
-LLn, -OLn, -OL, -LS and -OS were not used
(see Tables 1 or 3 for abbreviations). It may be
possible to distinguish these pairs of hetero-diesters
from their individual product ion spectra, but this was
not conducted in this study and so this possibility
remains unclear.
Purification procedure
Direct dissolution and injection was attempted first, but
was unsuccessful due to possible suppression by co-
extracts from the oil samples during the ionisation step
on LC-MS/MS. The extracts were therefore purified on
a Sep-pakTM C18 plus Sep-pakTM silica column using a
method based on the determination of glycidyl esters
reported by Masukawa et al. (2010) and Shiro et al.
(2011). The method involved removing triacylglycerides
using the C18 cartridge, whereas removal of diacylgly-
cerides was achieved with a silica cartridge.
Several general findings are described below.
The eluting solvent for the Sep-pakTM C18 column
was a mixture of ethanol and methanol (1:4), since
although 90% of monoesters could be recovered using
methanol alone, only approximately 70% of diesters
 58 K. Yamazaki et al.

NH4+
H2C Cl
+
C C15H31
HC OH
O
H2C O C C15H31
3-MCPD-P product ion (m/z 239)
O
3-MCPD-P precursor ion (m/z 366)
(ammonium adduct ion)

NH4+
H2C Cl H2C Cl
O O
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HC O C C15H31 HC O C C15H31

+
H2C O C C15H31 H3C H

O 3-MCPD-PP product ion (m/z 331)

3-MCPD-PPprecursor ion (m/z 604)
(ammonium adduct ion)

NH4+
H2C Cl H2C Cl
O O

HC O C C17H31 HC O C C17H31

+
H2C O C C 17H29 H3C H

O or 3-MCPD-LnL product ion (m/z 355)

3-MCPD-LnLprecursor ion (m/z650)

(ammonium adduct ion) H2C Cl

+
H3C H O

H3C O C C 17H29

3-MCPD-LnL product ion (m/z 353)

Figure 2. Proposed precursor ion and product ion for 3-MCPD-P, 3-MCPD-PP and 3-MCPD-LnL.

were recovered with pure methanol. However,
increased ethanol reduced the apparent recovery due
to ion suppression during detection caused by co-
eluted triacylglycerides. Therefore, for diesters we used
a larger C18 cartridge (900 mg) and took into account
the excessive loading beyond the cartridge’s capacity,
as well as the use of a mixture of ethanol, acetonitrile
and methanol (5:30:65) as the eluting solvent to
improve recovery. For the monoester, methanol was
used as the eluting solvent.
Both 3-MCPD mono- and diesters loaded on a
Sep-pakTM silica cartridge were eluted with a mixture
of ethyl acetate and hexane (20:80 v/v), whereas ethyl
acetate and hexane (5:95 v/v) resulted in the elution
of only diesters. However, interfering components
were more eluted simultaneously when the 20:80 (v/v)
solvent was used. In order to facilitate quantification
by LC-MS/MS, we decided first to elute diesters
with the 5:95 mixture, and then the monoesters
with the 20:80 mixture. LC-MS/MS quantification
of the monoesters still in a difficult condition, so the
order of the silica and C18 cartridges was exchanged
to reduce the amount loaded into silica cartridge.
This methodology allowed the monoesters to be
 Food Additives & Contaminants: Part A 59
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Figure 3a. Mass spectrum of precursor ion (A) and product ion (B) from 3-MCPD-LnL.

Figure 3b. Mass spectrum of precursor ion (A) and product ion (B) from 3-MCPD-LLn.

analysed stably and with relatively little ion was obtained by spiking the IS into the working
suppression. standard solution just before injection onto LC-MS/
MS. It remains unclear whether isotope exchange
actually occurred.
Quantification
The calibration curve was initially linear but became
unstable with time, probably due to changes in the Method validation
mixed ISs, such as isotope exchange (between deute- The method was validated in the laboratory in terms
rium and hydrogen). A more stable calibration curve of precision (as repeatability), trueness (as recovery),
 60 K. Yamazaki et al.
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Figure 3c. Mass spectrum of precursor ion (A) and product ion (B) from 3-MCPD-LL.

Figure 3d. Mass spectrum of precursor ion (A) and product ion (B) from 3-MCPD-Ln.

limit of detection (LOD) and limit of quantification
(LOQ) as performance characteristics.
Trueness (recovery test)
The recoveries are shown in Table 4 for analysed
soybean oil spiked with 20 analytes (five monoesters,
five homo-diesters and 10 hetero-diesters) at 5 mg kg1.
The relative recoveries adjusted with the IS were
overall satisfactory (78–163%), but 3-MCPD-SS and
3-MCPD-LL provided somewhat unusual results.
It is possible that the distribution of 3-MCPD-SS
on the silica cartridge varied depending on the sample
(judging from its late elution), and that for 3-MCPD-
LL, the interaction of native with IS compound
occurred such as the isotope exchange (between
 Food Additives & Contaminants: Part A 61
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Figure 3e. Mass spectrum of precursor ion (A) and product ion (B) from d5-3-MCPD-LnLn.

Table 4. Recoveries of 3-MCPD esters from soy bean oil.

Recovery

Absolute recovery (%)a Relative recovery (%)b

Compounds Duplicates Mean Duplicates Mean

Mono-esters
3-MCPD-Ln 87, 108 98 101, 100 101
3-MCPD-L 97, 125 111 112, 115 114
3-MCPD-P 94, 121 108 109, 111 110
3-MCPD-O 77, 111 94 90, 102 96
3-MCPD-S 87, 106 97 101, 97 99
Hetero-diestersc
3-MCPD-LnL 64, 81 73 86, 74 80
3-MCPD-LnO 74, 85 80 118, 107 113
3-MCPD-LP 73, 81 77 116, 102 109
3-MCPD-LO 61, 74 69 102, 93 98
3-MCPD-LnP 73, 73 73 98, 84 91
3-MCPD-PO 81, 85 83 129, 107 118
3-MCPD-LnS 78, 97 88 103, 104 104
3-MCPD-SL 98, 115 107 131, 124 128
3-MCPD-SP 114, 87 101 144, 91 118
3-MCPD-SO 85, 100 93 133, 126 130
Homo-diestersd
3-MCPD-LnLn 73, 90 82 127, 130 129
3-MCPD-LL 95, 99 97 168, 157 163
3-MCPD-PP 59, 82 71 90, 115 103
3-MCPD-OO 71, 64 68 115, 94 105
3-MCPD-SS 44, 97 71 53, 103 78

Notes: aAbsolute recovery results were derived from measurements of individual native compounds
calculated using an external calibration method.
b
Relative recovery results were derived using an internal adjusted calibration method (the method
described in this paper).
c
The term ‘‘hetero-diesters’’ is used for compounds that have a pair of different fatty acid groups.
d
The term ‘‘homo-diesters’’ is used for compounds that have a pair of identical fatty acid groups.
 62 K. Yamazaki et al.

Table 5. Repeatability (precision), LOD and LOQ of 3-MCPD esters in olive oil.

Spiked level Recovery RSDr LOD LOQ

Compound (mg kg1) (%)a (%) (mg kg1)b (mg kg1)b

Mono-esters
3-MCPD-Lnc 0.05 104 10.6 0.021 0.055
3-MCPD-Lc 0.05 109 25.5 0.053 0.139
3-MCPD-P 0.1 109 16.4 0.068 0.181
3-MCPD-O 0.1 117 10.9 0.048 0.128
3-MCPD-S 0.1 93 14.2 0.050 0.132
Hetero-diestersd
3-MCPD-LnL 0.1 91 14.3 0.049 0.131
3-MCPD-LnO 0.05 131 8.6 0.021 0.056
3-MCPD-LP 0.05 131 14.0 0.035 0.091
3-MCPD-LO 0.05 118 12.6 0.028 0.075
3-MCPD-LnP 0.05 105 12.7 0.025 0.066
3-MCPD-PO 0.05 136 9.8 0.025 0.066
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3-MCPD-LnS 0.05 105 21.9 0.044 0.115

3-MCPD-SL 0.1 66 18.6 0.047 0.124
3-MCPD-SP 0.05 120 17.5 0.040 0.105
3-MCPD-SO 0.1 66 8.1 0.020 0.053
Homo-diesterse
3-MCPD-LnLn 0.05 119 15.7 0.035 0.093
3-MCPD-LL 0.05 118 6.8 0.015 0.040
3-MCPD-PP 0.05 135 5.5 0.014 0.037
3-MCPD-OO 0.1 79 23.5 0.071 0.186
3-MCPD-SS 0.05 130 12.6 0.031 0.082
Total monoestersa 0.24 0.64
Total diestersb 0.50 1.32

Notes: aThe recoveries are shown as the mean from eight trials.
b
The LOD and LOQ were estimated from the standard deviation(s) of eight measurements using the
following equation: LOD ¼ 2t(n–1,0.05)s, LOQ ¼ 10 s.
c
Instead of olive oil, MCPD esters were spiked to soy bean oil.
d
The term ‘‘hetero-diesters’’ is used for compounds that have a pair of different fatty acid groups.
e
The term ‘‘homo-diesters’’ is used for compounds that have a pair of identical fatty acid groups.

deuterium and hydrogen), although the latter remains
unclear.
Precision (repeatability)
The precision at levels near the expected LOQ was
examined from the deviation of eight replicate mea-
surements. Eight samples of extra virgin olive oil were
spiked at 0.05 or 0.1 mg kg1 (partially soy bean oil
instead of olive oil was used). The precision in terms
of relative standard deviation (RSDr%) is shown in
Table 5 and varied from 5.5% (3-MCPD-PP) to 25.5%
(3-MCPD-L).
LOD and LOQ
LOD and LOQ were estimated from the deviation of
replicate measurements at levels near the LOQ (Currie
1995; US Environmental Protection Agency (USEPA)
2003). The calculated equation is:
LOD ¼ 2tðn1, 0:05Þ s or LOQ ¼ 10s
where s is the standard deviation of measurements;
and t(n1,0.05) is the Student’s t-value at n  1 degrees
of freedom (in this case, 1.895 for eight trials). The
estimated values for LOD and LOQ, together with
repeatability, are provided in Table 5. The LODs
and LOQs for monoesters were 0.021–0.068 and
0.055–0.181 mg kg1, and for diesters 0.014–0.071 and
0.037–0.186 mg kg1, respectively. The minimum
detectable concentrations at the actual conditions
used were approximately 0.005–0.002 mg ml1.
3-MCPD-P, -O, -S, -SL and -SO were detected with
low sensitivity, and 3-MCPD-OO was detected with
poor sensitivity. Given the ratio of dilution in the
above procedure, these LOD and LOQ values
were considered adequate. Ultimately, either 0.05 or
0.10 mg kg1 was taken as the LOD, depending on the
estimated value for individual 3-MCPD esters
(reflected in Table 6).
Applicability of the method for determining 3-MCPD
esters in retailed oils
The results of quantifying 3-MCPD esters in 10
different commercial edible oils are shown in
Table 6. Examples of SRM chromatograms are
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Table 6. Content of 3-MCPD esters in commercial edible oils.

Concentration (mg kg1)a

IS applied Soybean Rapeseed Rice Safflower Sesame Olive Grape Perilla Palm Refined
Compounds for calculation oil oil oil oil oil oil seed oil oil oil lard

3-MCPD-Ln d5-3-MCPD-P 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05
3-MCPD-L d5-3-MCPD-P 50.05 50.05 50.05 50.05 50.05 50.05 0.54 50.05 0.23 50.05
3-MCPD-P d5-3-MCPD-P 50.10 c 50.10 50.10 50.10 50.10 50.10 50.10 50.10 0.44 50.10
3-MCPD-O d5-3-MCPD-P 50.10 50.10 0.12 50.10 50.10 0.29 0.14 50.10 0.69 50.10
3-MCPD-S d5-3-MCPD-P 50.10 50.10 50.10 50.10 50.10 50.10 50.10 50.10 0.10 50.10
Total monoesters n.d.c n.d. 0.12 n.d. n.d. 0.29 0.68 n.d. 1.46 n.d.
3-MCPD-LnLn d5-3-MCPD-LnLn 50.05 0.11 50.05 50.05 50.05 50.05 50.05 0.89 50.05 50.05
3-MCPD-LL d5-3-MCPD-PP 50.05 50.05 0.35 50.05 0.07 50.05 16 0.08 0.20 50.05
3-MCPD-PP d5-3-MCPD-PP 50.05 50.05 50.05 50.05 50.05 0.07 50.05 50.05 2.4 0.06
3-MCPD-OO d5-3-MCPD-PP 50.10 0.17 0.61 0.50 0.11 2.2 0.60 50.10 2.3 0.17
3-MCPD-SS d5-3-MCPD-SS 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05
Total homo-diesterse n.d. 0.18 0.96 0.50 0.18 2.27 16.6 0.97 4.90 0.23
3-MCPD-LnL (LLn)b d5-3-MCPD-PP 0.58 0.39 0.11 0.16 0.30 0.21 0.77 0.38 50.10 0.15
3-MCPD-LnO (OLn)b d5-3-MCPD-PP 50.05 50.05 50.05 50.05 50.05 50.05 1.2 0.08 50.05 50.05
3-MCPD-LO (OL)b d5-3-MCPD-PP 50.05 50.05 0.28 0.11 0.10 0.50 3.2 50.05 0.53 50.05
3-MCPD-LP (PL)b d5-3-MCPD-PP 50.05 50.05 0.11 50.05 50.05 0.09 0.92 50.05 0.74 0.09
3-MCPD-LnP (PLn)b d5-3-MCPD-PP 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05
3-MCPD-PO (OP)b d5-3-MCPD-PP 50.05 50.05 0.20 0.06 50.05 0.78 0.53 50.05 5.6 0.15
3-MCPD-LnS (SLn)b d5-3-MCPD-PP 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05
3-MCPD-SP (PS)b d5-3-MCPD-PP 50.05 50.05 50.05 50.05 50.05 50.05 50.05 50.05 0.28 0.14
3-MCPD-SL (LS)b d5-3-MCPD-PP 50.10 50.10 50.10 50.10 50.10 50.10 1.2 50.10 0.19 50.10
3-MCPD-SO (OS)b d5-3-MCPD-PP 50.10 50.10 50.10 50.10 50.10 0.20 0.25 50.10 0.70 50.10
Total hetero-diestersf 0.58 0.39 0.70 0.33 0.40 1.78 8.07 0.46 8.04 0.53
Sum of 3-MCPD esters (mono- plus di-esters) 0.58 0.50 1.78 0.83 0.58 4.34 25.35 1.43 14.40 0.76
Conversion to free 3-MCPDd 0.102 0.118 0.328 0.146 0.102 0.800 4.545 0.252 2.714 0.134

Notes: aMean of duplicate samples.

b
For hetero-diesters, the results are expressed as the corresponding compound in Table 6 (not in parentheses). Thus, these values refer to the sum of two compounds, though only
one compound may be present.
c
The term ‘‘50.05’’ means below the LOD (0.05 mg kg–1). The total ‘‘n.d.’’ was calculated, assuming that the value of 5LOD was zero.
d
The conversion factor to free 3-MCPD was 0.299 for monoesters and 0.176 for diesters calculated from the mean value derived from the molecular ratio of individual esters.
e
The term ‘‘hetero-diesters’’ is used for compounds that have a pair of different fatty acid groups.
f
The term ‘‘homo-diesters’’ is used for compounds that have a pair of identical fatty acid groups.
Food Additives & Contaminants: Part A
63
 64 K. Yamazaki et al.
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Figure 4a. SRM chromatogram from (A) standard solution (0.005 mg ml1) and (B) olive oil for 3-MCPD monoesters (3-MCPD-
P, -Ln, -L, -S, -O, d5-3-MCPD-P).
shown in Figure 4. Haines et al. (2011) reported that
3-MCPD monoesters were not detected in several
vegetable oils and fats (LOD ¼ 1 mg kg1) and total
diesters were present at concentrations of between
3.7 and 6.2 mg kg1 in palm-based oils (LOD ¼
0.5 mg kg1).
In this study, 3-MCPD monoesters were found in
four samples tested (40%) and ranged from trace levels
(0.10 mg kg1) to 0.69 mg kg1, with the highest con-
centration of 3-MCPD-O being found in palm oil.
For diesters, at least one compound was detected in
all the samples tested (100%). The highest concentra-
tion of diesters found was 16 mg kg1 (3-MCPD-LL
in grape seed oil). Of the monoesters, 3-MCPD-P
(0.44 mg kg1 only in palm oil), 3-MCPD-O (0.12–
0.69 mg kg1 in four oil samples) and 3-MCPD-L
(0.23 and 0.54 mg kg1 in two oil samples) were found,
while 3-MCPD-Ln and 3-MCPD-S were not detected.
Homo-diesters of 3-MCPD (with the same pair of
fatty acids) were found in nine of the 10 edible
oils (absent from soy bean oil), at concentrations
of 0.11–16 mg kg1. The predominant homo-diester

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Figure 4b. SRM chromatogram from (A) standard solution (0.005 mg ml1) and (B) olive oil for 3-MCPD-hetero-diesters
(3-MCPD-LP, -PO, -LnO, -LO, -SO, d5-3-MCPD-PP).
found was 3-MCPD-OO (0.11–2.3 mg kg1 in eight
samples), followed by 3-MCPD-LL (0.08–16 mg kg1
in four samples). 3-MCPD-PP (0.06–2.4 mg kg1)
and 3-MCPD-LnLn (0.11 and 0.89 mg kg1) were
found in three and two samples, respectively, but
no sample contained 3-MCPD-SS. In hetero-diesters
of 3-MCPD, a higher occurrence (nine samples) of
3-MCPD-LnL (or -LLn) was noticed at concentrations
between 0.11 and 0.77 mg kg1, followed by 3-MCPD-
PO (or -OP) and 3-MCPD-LO (or -OL) in six sam-
ples at 0.06–5.6 and 0.10–3.2 mg kg1, respectively.
Five samples contained 3-MCPD-LP (or -PL) at
Food Additives & Contaminants: Part A 65
0.09–0.92 mg kg1, whereas 3-MCPD-SP (or -PS),
3-MCPD-LnO (or -OLn) 3-MCPD-SL (or -LS) and
3-MCPD-SO (or OS) were found only in two sam-
ples at 0.08–1.2 mg kg1. Neither 3-MCPD-LnS (or
-SLn) nor 3-MCPD-LnP (or -PLn) were detected
(50.05 mg kg1). Discrepancies between Haines et al.
(2011) and this study are likely due to differences in the
LOD for monoesters and differences in the types of oils
tested for diesters. However, the concentrations of 3-
MCPD ester in palm oils were similar to those obtained
using LC-TOF/MS by Hori et al. (2012) and Dubois
et al. (2012). Also, the types of 3-MCPD ester were in
 66 K. Yamazaki et al.
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Figure 4c. SRM chromatogram from (A) standard solution (0.005 mg ml1) and (B) olive oil for 3-MCPD homo-diesters
(3-MCPD-PP, -LnLn, -LL, -OO, -SS, d5-3-MCPD-PP, d5-3-MCPD-LnLn, d5-3-MCPD-SS).
accordance with the finding reported by Svejkovská
et al. (2004) and Zelinková et al. (2009), although the
3-MCPD-SS described in bakery products by Zelinková
et al. (2006) was not found in the current study.
As reported by Svejkovská et al. (2004) and Ilko
et al. (2011), potato products fried in edible oils might
contain a significant amount of 3-MCPD esters (up to
about 2 mg kg1). Also, Küsters et al. (2011) found
3-MCPD esters to be contained in some foods (e.g.
cookies and gravy powder). Thus, a high consumption
of potato-derived products or cereal-based foods might
significantly contribute to the intake of 3-MCPD esters.
Further research is required to determine the levels of 3-
MCPD esters in edible oils and other products.
In addition, according to Dubois et al. (2012),
2-MCPD were found in most of the palm oil samples
tested as determined by an indirect method using acid
transesterification and the mean rate to the total
amounts of MCPD was 35%. Therefore, 2-MCPD
fatty acid esters should also be characterised by the
direct method. Also, concerning other oils with short-
chain fatty acid, such as palm kernel oil or coconut oil,
the direct method for MCPD esters should be consid-
ered to extend the range of application. For this
purpose, it is essential to compare the results obtained
from both of direct and indirect methods.
Conclusion
A new, direct analytical method for quantifying
3-MCPD esters in edible oils and fats using LC-MS/
MS and without the need for ester cleavage was

developed and validated in our laboratory. Its appli-
cability was tested using a variety of edible oils.
The results showed that this method can be used to
determine significant amounts of 3-MCPD esters in
several commercial edible oils and fats, including lard.
Furthermore, it may be possible to determine the true
concentration and composition of 3-MCPD esters
in various edible oils. Further studies are required to
compare the results of this direct analytical method
with previous indirect analytical methods. In addition,
further investigation is required not only to determine
the concentration of 3-MCPD esters in foods other
than edible oils, including potato products and cereal
products, but also to characterise 2-MCPD esters or
other esters with short-chain fatty acids.
Downloaded by [University of Chicago Library] at 22:03 23 April 2013
Acknowledgements
This work was performed as part of the Regulatory Research
Projects for Food Safety, Animal Health, and Plant
Protection of the Ministry of Agriculture, Forestry and
Fisheries, Japan.
References
AOCS. 2011. AOCS 7th Meeting: AOCS Expert Panel
on Process Contaminants; Rotterdam, the Netherlands;
19 September; [cited 2012 Apr 21]. Available from:
http://www.aocs.org/files/ResourcesPDF/notes%207th%
20expert%20panel.pdf/
Buhrke T, Weibhaar R, Lampen A. 2011. Absorption
and metabolism of the food contaminant 3-chloro-1,2-
propanediol (3-MCPD) and its fatty acid esters by human
intestinal Caco-2 cells. Archiv Toxicol. 85:1201–1208.
Currie LA. 1995. Nomenclature in evaluation of analytical
methods including detection and quantification capabili-
ties (IUPAC Recommendations 1995). Pure Appl Chem.
67:1699–1723.
Deutsche Gesellschaft für Fettwissenschaft (DGF): DGF
Standard Method: C-III 18(09). 2009. Ester-bound 3-
chloropropane-1,2-diol (3-MCPD-esters) and glycidol
(glycidyl esters) – determination in fats and oils by GC-
MS; [cited 2009 Nov 18]. Stuttgart (Germany): WVG.
Deutsche Gesellschaft für Fettwissenschaft (DGF): DGF
Standard Method: C-VI 18(10). 2011. Fatty-acid-bound
3-chloropropane-1,2-diol (3-MCPD) and 2,3-epoxipro-
pane-1-ol (glycidol) – determination in oils and fats by
GC/MS (differential measurement); [cited 2011 Jun 1].
Stuttgart (Germany): WVG.
Divinová V, Svejkovská B, Doležal M, Velı́šek J. 2004.
Determination of free and bound 3-chloropropane-1,2-diol
by gas chromatography with mass spectrometric detection
using deuterated 3-chloropropane-1,2-diol as internal
standard. Czech J Food Sci. 22:182–189.
Dubois M, Tarres A, Goldmann T, Empl AM,
Donaubauer A, Seefelder W. 2012. Comparison of indirect
and direct quantification of esters of monochloropropa-
nediol in vegetable oil. J Chromatogr A 1236:189–201.
Food Additives & Contaminants: Part A 67
Ermacora A, Hrncirik K. 2012. Evaluation of an improved
indirect method for the analysis of 3-MCPD esters based
on acid transesterification. J Am Oil Chem Soc.
89:211–217.
Fiebig HJ. 2011. Determination of ester-bound 3-chloro-1,2-
propanediol and glycidol in fat and oils – a collaborative
study. Eur J Lipid Sci Tech. 113:393–399.
Franke K, Strijowski U, Fleck G, Pudel F. 2009. Influence
of chemical refining process and oil type on bound
3-chloro-1,2-propanediol contents in palm oil and rape-
seed oil. LWT – Food Sci Tech. 42:1751–1754.
Haines TD, Adlaf KJ, Robert M, Pierceall RM, Lee I,
Venkitasubramanian P, Collison MW. 2011. Direct deter-
mination of MCPD fatty acid esters and glycidyl fatty
acid esters in vegetable oils by LC-TOFMS. J Am Oil
Chem Soc. 88:1–14.
Hamlet CG, Asuncion L. 2011. Single-laboratory validation
of a method to quantify bound 2-chloropronpane-1,3-diol
and 3-chloropropane-1,2-diol in foodstuffs using acid
catalyzed transesterification, HFBI derivatisation and
GC-MS detection. Eur J Lipid Sci Tech. 113:344–355.
Hamlet CG, Asuncion L, Velı́šek J, Doležal M, Zelinková Z,
Crews C. 2011. Formation and occurrence of esters of
3-chloropropane-1,2-diol (3-MCPD) in foods: what we
know and what we assume. Eur J Lipid Sci Tech.
113:279–303.
Hamlet CG, Sadd PA. 2004. Chloropropanols and their
esters in cereal products. Czech J Food Sci. 22S:259–262.
Hamlet CG, Sadd PA, Crews C, Velišek J, Baxter DE. 2002.
Occurrence of 3-chloropropane-1,2-diol (3-MCPD) and
related compounds in foods: a review. Food Addit
Contam. 19:619–631.
Hori K, Koriyama N, Omori H, Kuriyama M, Arishima T,
Tsumura K. 2012. Simultaneous determination of
3-MCPD fatty acid esters and glycidol fatty acid esters
in edible oils using liquid chromatography time-
of-flight mass spectrometry. LWT – Food Sci Tech.
48:204–208.
Hrncirik K, van Duijn G. 2011. An initial study on
the formation of 3-MCPD esters during oil refining.
Eur J Lipid Sci Tech. 113:374–379.
Hrncirik K, Zelinkova K, Ermacora A. 2011. Critical factors
of indirect determination of 3-chloropropane-1,2-diol
esters. Eur J Lipid Sci Tech. 113:361–367.
Ilko V, Zelinková Z, Doležal M, Velı́šek J. 2011.
3-Chloropropane-1,2-diol fatty acid esters in potato prod-
ucts. Czech J Food Sci. 29(4):411–419.
ILSI Europe. 2009. 3-MCPD esters in food products.
Summary Report of a Workshop held in February 2009
in Brussels, Belgium. ILSI Europe Report Series.
Joint FAO/WHO Expert Committee on Food Additives
(JECFA). 2007. 3-Chloro-1,2-propane-diol. In: Safety
Evaluation of Certain Food Additives and Contaminants,
Prepared by the Sixty-seventh Meeting of the Joint
FAO/WHO Expert Committee on Food Additives, WHO
Food Additives Series 58. p. 239–266.
Karasek L, Wenzl T, Ulberth F. 2010. Proficiency test on
the determination of 3-MCPD esters in edible oils.
Luxembourg: Publications Office of the European Union.
Kaze N, Sato H, Yamamoto H, Watanabe Y. 2011.
Bidirectional conversion between 3-monochloro-1,2-
propanediol and glycidol in course of the procedure of
 68 K. Yamazaki et al.
DGF standard methods. J Am Oil Chem Soc.
88:1143–1151.
Kuhlmann J. 2011. Determination of bound 2,3-epoxy-
1-propanol (glycidol) and bound monochloropropanediol
(MCPD) in refined oils. Eur J Lipid Sci Tech. 113:
335–344.
Küsters M, Bimber U, Reeser S, Rainer Gallitzendörfer R,
Gerhartz M. 2011. Simultaneous determination and differ-
entiation of glycidyl esters and 3-monochloropropane-1,2-
diol (MCPD) esters in different foodstuffs by GC-MS.
J Agricult Food Chem. 29:6263–6270.
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. 2:81–88.
Matthäus B, Pudel F, Fehling P, Vosmann K,
Freudenstein A. 2011. Strategies for the reduction of
Downloaded by [University of Chicago Library] at 22:03 23 April 2013
3-MCPD esters and related compounds in vegetable oils.
Eur J Lipid Sci Tech. 113:380–386.
Myher JJ, Kuksis A, Marai L, Cerbulis J. 1986.
Stereospecific analysis of fatty acid esters of chloropropa-
nediol isolated from fresh goat milk. Lipids. 21:309–314.
Pinkston JD, Stoffolano PJ. 2011. Update on the develop-
ment of a sensitive, accurate, and user-friendly method for
the direct determination of 3-MCPD Esters. In: 6th
Meeting of Expert Panel at the AOCS 102nd Annual
Meeting & Expo in Cincinnati, OH, USA; May 2011.
Presentations; [cited 2012 Apr 21]. Available from:
http://www.aocs.org/files/ResourcesPDF/5_AOCS_2011_
Pinkston_110405.pdf/
Schilter B, Scholz G, Seefelder W. 2011. Fatty acid
esters of chloropropanols and related compounds in
food: toxicological aspects. Eur J Lipid Sci Tech.
113:309–313.
Seefelder W, Scholz G, Schilter B. 2011. Structural
diversity of dietary fatty acid esters of chloropropanols
and related substances. Eur J Lipid Sci Tech. 113:
319–322.
Seefelder W, Varga N, Studer A, Williamson G, Scanlan FP,
Stadler RH. 2008. Esters of 3-chloro-1,2-propanediol
(3-MCPD) in vegetable oils: significance in the formation
of 3-MCPD. Food Addit Contam. 25:391–400.
Shimizu M, Kudo N, Shiro H, Yasunaga K, Masukawa Y,
Katsuragi Y, Yasumatsu T. 2010. Comparison of indirect
and direct quantification of glycidol fatty acid ester in
edible oils. J Oleo Sci. 59:535–539.
Shiro H, Kondo N, Nobuyuki K, Masukawa Y. 2011. Direct
method for quantification of glycidol fatty acid esters in
edible oils. Eur J Lipid Sci Tech. 113:356–360.
Strijowski U, Heinz V, Franke K. 2011. Removal of
3-MCPD esters and related substances after refining by
adsorbent material. Eur J Lipid Sci Tech. 113:387–392.
Svejkovská B, Novotoný O, Divinová V, Réblová Z,
Doležal M, Velišek J. 2004. Esters of 3-chloropropane-
1,2-diol in foodstuffs. Czech J Food Sci. 22:190–196.
US Environmental Protection Agency (USEPA). 2003.
40 CFR Part 136; Guidelines establishing test procedures
for the analysis of pollutants; procedures for detection and
quantification. Federal Reg. 68:11770–11790.
Velı́šek J, Doležal M, Crews C, Dvorak T. 2002. Optical
isomers of chloropropanediols: mechanisms of their
formation and decomposition in protein hydrolysates.
Czech J Food Sci. 22:190–196.
Weißhaar R. 2008. Determination of total 3-chloropropane-
1,2-diol (3-MCPD) in edible oils by cleavage of MCPD
esters with sodium methoxide. Eur J Lipid Sci Tech.
110:183–186.
Weißhaar R. 2009. Fatty acid esters of 3-MCPD: overview
of occurrence in different types of foods. 3-MCPD esters
in food products. Brussels (Belgium): ILSI Europe;
[cited 2011 Aug]. Available at: http://www.ilsi.org/Europe/
Documents/E2009MCPD-7.pdf/
Weißhaar R, Perz R. 2010. Fatty acid esters of glycidol
in refined edible oils. Eur J Lipid Sci Tech. 112:158–165.
Zelinková Z, Doležal M, Velı́šek J. 2009. Occurrence of
3-chloropropane-1,2-diol fatty acid esters in infant and
baby foods. Eur Food Res Toxicol. 228:571–578.
Zelinková Z, Svejkovská B, Velı́šek J, Doležal M. 2006.
Fatty acid esters of 3-chloropropane-1,2-diol in edible oils.
Food Addit Contam. 23:1290–1298.