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Analysis of 3-MCPD

and Glycidyl Esters


in Palm Oil
Jimmy Wong
Marketing Program Manager
Gas Phase
2nd June 2015

Introduction
Free 3-MCPD (3-monochloropropane-1,2-diol or 3chloropropane-1,2-diol) is first identified on lipid hydrolyzed
by HCl

In 2006, 3-MCPD esters were reported in edible oils,


especially in refined oils but not in crude oils.

Introduction
The critical step of refining may be the deodorization. In this
step mono-and di-glycerides, chloride ion and temperature
led to the formation of 3-MCPD monoesters and 3-MCPD
diesters
It is one of the food safety concerns for oils and fats industry
Hot topic in Europe especially among the refiners of oils and
fats

Issue of concern
Dec 2007 German Risk Assessment Body (BfR) issued evaluation
dietary on 3-MCPD esters

Exposure up to tolerable daily intake (TDI) of 3-MCPD esters was


anticipated in infant formula

Refined oils - highest amount, hence food products using them will
contain elevated amount 3-MCPD esters

Toxicity level is not yet available

Possible formation oil refining process


Crude oil
Degumming
Bleaching
clay
is added

Bleaching
Filter Press

Deodorisation

Refined oil

Phosphoric acid
is added
Phosphoric acid
and gums

Gums/Clays and
phosphoric acid are
removed

(Siew W.L., 2012)

3-MCPD esters occurs during the refining of edible crude oils


mostly at the deodorization step

Formation mechanism hypothesis

O
OH

H+

-H2O
OR1
Acylglycerol

Cl

OR1
Cyclic acyl
oxonium ion

OR1

Cl

3-MCPD esters

R = alkyl
R1 = H or COR
3-MCPD

* International Life Sciences Institute (ILSI) Report, 2009

Summary of method:
AOCS Official Method Cd 29a:13
100 mg oil/fat sample

Add internal standards + THF

Conversion of GE to 3-MBPD monoesters in bromide salt

Conversion of 2- and 3-MCPD esters and 3-MBPD esters into free form in acid methanolic solution

Extraction of fatty acid methyl esters

Derivatization of 2- and 3-MCPD and 3-MBPD with Phenylboronic acid (PBA)

GC-MS Analysis

Agilent GC Configuration

7890B GC
Multimode inlet

5977B GCMSD
Extractor ion source
Triple Axis detector
Monolithic Quartz Quadrapole
Mass Hunter software

Chemicals and Reagents as per AOCS Cd 29a-13

Standards
1.
2.
3.
4.
5.

1,2- Dipalmitoyl-3-chloropropanediol (PP-3-MCPD)


1,3- Dipalmitoyl-3-Chloropropanediol( PP-2-MCPD
Pentadeuterated -1,2-Dipalmitoyl-3-chloropropanediol (PP-3-MCPD D-5)
Glycidyl Palmitate (Gly-P)
Pentadeuterated Glycidyl Palmitate (Gly-P-d5)

Reagents Preparation
Followed AOCS method- Below Screen shot

Standards Preparation
Followed AOCS method- Below Screen shot

Calibration Stock solution:


1) 550ul of 3-MCPD stock and 550ul of 2-MCPD stock diluted to 10 ml with toluene55ug/ml ( Calib 1)
2) 1ml of Calib 1 to 10 ml with toluene 5.5ug/ml ( Calib 2)
3) 400ul of 3-MCPD-d5 to 10 ml with toluene 40ug/ml ( Calib ISTD)

Standards Preparation- Continued

Standards Preparation- Continued


Calibration
level

Calib 1
(ul)

Calib 2
(ul)

Final
concentration
as 3-MCPD
(ug)

Calib
ISTD
(ul)

Final concentration as 3MCPD-d5(ug)

Level 0

50

0.39

Level 1

25

0.03

50

0.39

Level 2

50

0.05

50

0.39

Level 3

100

0.10

50

0.39

Level 4

20

0.21

50

0.39

Level 5

30

0.31

50

0.39

Level 6

70

0.72

50

0.39

Level 7

90

0.93

50

0.39

Method
Column Used- DB-1 , 30m x 0.25, 0.25mm FT capillary column
GC Conditions:
1. Injection Volume 1.0 ul
2. Inlet temperature- 250 deg C
3. Injection Mode- Pulsed splitless
4. Inlet Flow- 0.8 ml/min constant Flow
5. Oven Programming- 80C (1 min), from 80C to 170C at 10C/min, from 170C to 200C at
3C/min, from 200C to 300C at 15C/min, 15 min at 300C
MS Conditions
1. Transfer line temperature: 300C
2. Ion source temperature: 230C
3. Quadrupole temperature: 150C
4. Ionization mode: EI, SIM mode

Method Continued
Parameters for SIM (Selected Ion Monitoring) mode:
1. Phenylboronic derivative of 3-MCPD (m/z) 147 (quantifier ion); 196, 198 (qualifier ions);
2. Phenylboronic derivative of 2-MCPD (m/z) 196 (quantifier ion); 198 (qualifier ion);
3. Phenylboronic derivative of 3-MCPD-d5 (m/z) 150 (quantifier ion for 3-MCPD); 201
(quantifier ion for 2-MCPD); 203 (qualifier ion);
4. Phenylboronic derivative of 3-MBPD (m/z) 147 (quantifier ion); 240 (qualifier ion);
5. Phenylboronic derivative of 3-MBPD-d5 (m/z) 150 (quantifier ion); 245 (qualifier ion).

Chromatogram of 3-MCPD & 3-MBPD


Overlay of Cal 0, Cal 1, Cal 2, Cal 3, Cal 4, Cal 5, Cal 6, Cal 7 & Cal 8 of AOCS Method Cd 29a-13
x10

+ SIM(147.1) SIM 9Dec2015-C-Cal8.D

4.4

phenylboronic derivative of 3-MBPD

4.3
4.2
4.1
4
3.9
3.8
3.7
3.6
3.5
3.4

m/z 147

3.3
3.2
3.1
3
2.9
2.8
2.7
2.6
2.5
2.4
2.3
2.2
2.1
2
1.9
1.8
1.7
1.6
1.5

phenylboronic derivative of 3-MCPD

1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
10.3

10.4

10.5

10.6

10.7

10.8

10.9

11

11.1

11.2

Counts vs. Acquisition Time (min)

11.3

11.4

11.5

11.6

11.7

11.8

11.9

12

Chromatogram of 2-MCPD
Overlay of Cal 0, Cal 1, Cal 2, Cal 3, Cal 4, Cal 5, Cal 6, Cal 7 & Cal 8 of AOCS Method Cd 29a-13

x10 5
4.5
4.4
4.3
4.2
4.1
4
3.9
3.8
3.7
3.6
3.5
3.4
3.3
3.2
3.1
3
2.9
2.8
2.7
2.6
2.5
2.4
2.3
2.2
2.1
2
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

+ SIM(196.1) SIM 9Dec2015-C-Cal8.D

phenylboronic derivative of 2-MCPD

m/z 196
m/z 196
phenylboronic derivative of 3-MCPD

Overlay of Cal 0, Cal 1, Cal 2, Cal 3, Cal 4, Cal 5, Cal 6,


Cal 7 & Cal 8
of AOCS Method Cd 29a-13

10.3

10.4

10.5

10.6

10.7

10.8

10.9

11

11.1
11.2
Counts vs. Acquisition Time (min)

11.3

11.4

11.5

11.6

11.7

11.8

11.9

12

Chromatogram
x10

+ SIM(147.1) SIM 9Dec2015-C-Cal8.D

4.5
4

phenylboronic derivative of 3-MBPD

3.5
3
2.5

phenylboronic derivative of 3-MCPD

2
1.5
1
0.5
0
x10

+ SIM(150.1) SIM 9Dec2015-C-Cal8.D


9

phenylboronic derivative of 3-MBPD-d5

8
7

phenylboronic derivative of 3-MCPD-d5

6
5
4
3
2
1
0
x10

+ SIM(196.1) SIM 9Dec2015-C-Cal8.D

4.5

phenylboronic derivative of 2-MCPD

4
3.5
3
2.5
2
1.5
1
0.5
0
10.2

10.3

10.4

10.5

10.6

10.7

10.8

10.9

11

11.1

Counts vs. Acquisition Time (min)

11.2

11.3

11.4

11.5

11.6

11.7

11.8

11.9

3-MCPD- Linearity plot

2-MCPD- Linearity plot

Relative Response

Glycidyl Esters
Gly - 9 Levels, 9 Levels Used, 9 Points, 9 Points Used, 0 QCs
5

y = 1.390515 * x + 0.010148
R^2 = 0.99884432
Type:Linear, Origin:Ignore, Weight:None

4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

-0.2

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

2.2

2.4

2.6

2.8

3.2

3.4

3.6

Relative Concentra

Agilent Food Contamination Resource


http://www.agilent.com/about/features/food-contamination.html

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