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    Analysis of 3-MCPDand Glycidyl Estersin Palm Oil

    Jimmy Wong

    Marketing Program Manager

    Gas Phase2nd June 2015

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    Introduction

    Free 3-MCPD (3-monochloropropane-1,2-diol or 3-

    chloropropane-1,2-diol) is first identified on lipid hydrolyzedby HCl

    In 2006, 3-MCPD esters were reported in edible oils,especially in refined oils but not in crude oils.

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    Introduction

    The critical step of refining may be the deodorization. In this

    step mono-and di-glycerides, chloride ion and temperatureled 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

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    Issue of concern

    Dec 2007 German Risk Assessment Body (BfR) issued evaluationdietary 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

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    Possible formation oil refining process

    Crude oil

    Refined oil

    Degumming

    Bleaching

    Phosphoric acidis added

    Phosphoric acid

    and gums

    Gums/Clays and

    phosphoric acid are

    removed

    Bleaching

    clay

    is addedFilter Press

    Deodorisation

    (Siew W.L., 2012)

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

    mostly at the deodorization step

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    Formation mechanism hypothesis

    Acylglycerol Cyclic acyl

    oxonium ion

    3-MCPD esters

    R O

    OOH

    OR1

    H+

    -H2O

    R

    O

    O

    OR1

    Cl-

    O

    OR1

    O

    Cl

    R

    3-MCPD

    R = alkyl

    R1 = H or COR

    * International Life Sciences Institute (ILSI) Report, 2009

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    Summary of method:AOCS Official Method Cd 29a:13

    GC-MS Analysis

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

    Extraction of fatty acid methyl esters

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

    Conversion of GE to 3-MBPD monoesters in bromide salt

    Add internal standards + THF

    100 mg oil/fat sample

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    Agilent GC Configuration

    7890B GC Multimode inlet

    5977B GCMSD

    Extractor ion source Triple Axis detector

    Monolithic Quartz Quadrapole

    Mass Hunter software

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    Chemicals and Reagents as per AOCS Cd29a-13

    Standards

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

    2. 1,3- Dipalmitoyl-3-Chloropropanediol( PP-2-MCPD

    3. Pentadeuterated -1,2-Dipalmitoyl-3-chloropropanediol (PP-3-MCPD D-5)4. Glycidyl Palmitate (Gly-P)

    5. Pentadeuterated Glycidyl Palmitate (Gly-P-d5)

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    Reagents Preparation

    Followed AOCS method- Below Screen shot

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    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 toluene-

    55ug/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)

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    Standards Preparation- Continued

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    Standards Preparation- Continued

    Calibrationlevel

    Calib 1(ul)

    Calib 2(ul)

    Finalconcentration

    as 3-MCPD

    (ug)

    CalibISTD

    (ul)

    Final concentration as 3-MCPD-d5(ug)

    Level 0 0 0 0 50 0.39

    Level 1 0 25 0.03 50 0.39

    Level 2 0 50 0.05 50 0.39

    Level 3 0 100 0.10 50 0.39

    Level 4 20 0 0.21 50 0.39

    Level 5 30 0 0.31 50 0.39

    Level 6 70 0 0.72 50 0.39

    Level 7 90 0 0.93 50 0.39

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    MethodColumn 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: 230C3. Quadrupole temperature: 150C

    4. Ionization mode: EI, SIM mode

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    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).

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    6x10

    0

    0.1

    0.2

    0.3

    0.4

    0.5

    0.6

    0.7

    0.8

    0.9

    1

    1.1

    1.2

    1.3

    1.4

    1.5

    1.6

    1.7

    1.8

    1.9

    2

    2.1

    2.2

    2.3

    2.4

    2.5

    2.6

    2.7

    2.8

    2.9

    3

    3.1

    3.2

    3.3

    3.4

    3.5

    3.6

    3.7

    3.8

    3.9

    4

    4.1

    4.2

    4.3

    4.4

    +SIM(147.1)SIM9Dec2015-C-Cal8.D

    phenylboronic derivative of 3-MCPD

    phenylboronic derivative of 3-MBPD

    Countsvs.AcquisitionTime(min)

    10.3 10.4 10.5 10.6 10.7 10.8 10.9 11 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12

    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

    Chromatogram of 3-MCPD & 3-MBPD

    m/z 147

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    Chromatogram of 2-MCPD

    m/z 196

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

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

    5x10

    0

    0.1

    0.2

    0.3

    0.4

    0.5

    0.6

    0.7

    0.8

    0.9

    1

    1.1

    1.2

    1.3

    1.4

    1.5

    1.6

    1.7

    1.8

    1.9

    2

    2.1

    2.2

    2.3

    2.4

    2.5

    2.6

    2.7

    2.82.9

    3

    3.1

    3.2

    3.3

    3.4

    3.5

    3.6

    3.7

    3.8

    3.9

    4

    4.1

    4.2

    4.3

    4.4

    4.5

    +SIM(196.1)SIM9Dec2015-C-Cal8.D

    phenylboronic derivative of 2-MCPD

    Countsvs.AcquisitionTime(min)

    10.3 10.4 10.5 10.6 10.7 10.8 10.9 11 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12

    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

    m/z 196

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    Chromatogram

    6x10

    0

    0.5

    1

    1.5

    2

    2.5

    3

    3.5

    4

    4.5

    +SIM(147.1)SIM9Dec2015-C-Cal8.D

    phenylboronic derivative of 3-MCPD

    phenylboronic derivative of 3-MBPD

    5x10

    0

    1

    2

    3

    4

    5

    6

    7

    8

    9

    +SIM(150.1)SIM9Dec2015-C-Cal8.D

    phenylboronic derivative of 3-MCPD-d5

    phenylboronic derivative of 3-MBPD-d5

    5x10

    0

    0.5

    1

    1.5

    2

    2.5

    3

    3.5

    4

    4.5

    +SIM(196.1)SIM9Dec2015-C-Cal8.D

    phenylboronic derivative of 2-MCPD

    Countsvs.AcquisitionTime(min)

    10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 11 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9

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    3-MCPD- Linearity plot

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    2-MCPD- Linearity plot

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    Glycidyl Esters

    Gly-9Levels,9LevelsUsed,9Points,9PointsUsed,0QCs

    RelativeConcentra

    -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6

    RelativeR

    esponse

    0

    0.5

    1

    1.5

    2

    2.5

    3

    3.5

    4

    4.5

    5=1.390515*x+0.010148R^2=0.99884432Type:Linear,Origin:Ignore,Weight:None

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    Agilent Food Contamination Resource

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

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    THANK YOU!