indirect determination of bound glycidol & mcpd in...

Indirect Determination of Bound Glycidol & MCPD in Indirect Determination of Bound Glycidol & MCPD in

1•102nd AOCS Annual Meeting & Expo May 1-4,2011 Cincinnati, Ohio / Emerging Analytical Issues in Process Contaminants / Jan Kuhlmann: Indirect Determination of Bound Glycidol and MCPD in Refined Oils

Indirect Determination of Bound Glycidol & MCPD in Indirect Determination of Bound Glycidol & MCPD in Refined OilsRefined Oils

SGS Germany GmbHJan Kuhlmann

� Introduction

� Analytical methods (main focus: indirect analysis)

Contents


� The new SGS method “3 in 1”

� Comparison of results achieved with different methods

� Conclusions

Introduction

Glycidyl- & MCPD esters are process contaminants(Anaytes origin mainly from the oil refining process (critical step: deodorisation)

� 2- & 3-MCPD ester, bound MCPD: 2- & 3-Monochloro-1,2-propanediol derivatives

3-MCPD derivatives

*Cl OR

OR

Cl

OROil refining / Deo-dorisation / T > 200°C

+ Cl- + X ?


ORO

Glycidyl

derivatives

*

� Glycidyl ester, bound glycidol: 2,3-Epoxy-1-propanol derivatives

MCPD

derivatives

2-MCPD derivatives

RO OR

Cl

Glycerol

derivatives

RO OR

R = H, Acyl, …?

dorisation / T > 200°C

Cl-

� Free glycidol, toxic effect in vivo & in vitro: EU classifications:

mutagenic 3 / carcinogenic 2 / reproductive toxicity 2

� Free 3-MCPD: The EU Commission Regulation N°466/2001 sets a

Toxicological data indicates the need for monitoring

Introduction


� Free 3-MCPD: The EU Commission Regulation N°466/2001 sets a

MRL (Maximum Residue Limit) of 0,02 mg/kg in HVP & HVP products

� Bound 3-MCPD: BfR (Federal Institute for Risk Assesment) Assumption that 3-MCPD is

released completely from 3-MCPD esters during human digestion:

TDI (Tolerably Daily Intake) level of 0,002 mg/kg body weight day

� Free & bound 2-MCPD: no toxicological data is available

Analytical methods

Direct methods - general approach: “single ester analysis”

� A) Determination of single esters

� - calibration, validation and quantification is work-intensive

� - several reference compounds are required

� - unknown or unconsidered derivatives may be not detected !

� + exact information about the composition of the contaminants


� + exact information about the composition of the contaminants

� + no chemical reaction during sample preparation is required !

Chromatogramexhibiting up to 27 single analytes

Hypothetic oil sample containing just three different fatty acids

2-/3-MCPD Monoester (3/6)

2-/3-MCPD Diester (6/9)

Glycidyl Monoester (3)

Sample

preparation

Analysis

Analytical methods

� B) Determination of the released agents

� + easy information about the overall content of bound analytes

� + few reference compounds are required (available in isotopic labelled form)

� + calibration, validation and quantification is simple

� + unknown or unconsidered cleavable derivatives are included !

Indirect methods - general approach: “released agent analysis”


� + unknown or unconsidered cleavable derivatives are included !

� - chemical reactions during sample preparation may effect trueness !

Sample

preparation

Analysis

Chromatogramexhibiting up to 3 released analytes

Hypothetic oil sample containing just three different fatty acids

2-/3-MCPD Monoester (3/6)

2-/3-MCPD Diester (6/9)

Glycidyl Monoester (3)

Analytical methods

Indirect analysis: Acidic catalysed ester cleavage

16 h H+

2-/3-MCPDEster

Glycidyl Ester

Sample

e.g.

Ester cleavage

(ec)

Ester cleavage &

glycidol conversion


derivatisation

GC-MS Analysis [BfR 008]

Free2-/3-MCPD

e.g. Glycerol,2,3-MXPD

Free2-/3-MCPD

Clean up; Matrix removal

[ring tested method]

Analytical methods

Indirect analysis: Alkaline catalysed ester cleavage

3 – 10 min2 % NaOR

RT

2-/3-MCPD Ester

Glycidyl Ester

Free 2-/3-MCPD

Free Glycidol

Aliquot

A

Alkaline treatment

2-/3-MCPD Ester

Glycidyl Ester

Free 2-/3-MCPD

Free Glycidol

Alkaline treatment

Aliquot

B

DGF C-III 18 (09) B / H+


2-/3-MCPDGlycidol 2-/3-MCPDGlycidol

Matrix removal; derivatisation

GC-MS Analysis A [DGF C-III 18 (09) A, SGS A]

Free 2-/3-MCPDFree 2-/3-MCPDinduced !

H+ / Cl-: Reaction stop AGlycidol transformation A

Calculative approach for the glycidol determination: Glycidol ≈≈≈≈ (A – B)

Matrix removal; derivatisation

GC-MS Analysis B [BfR 009, SGS B]

Free 2-/3-MCPD

H+ / X- ≠ Cl-: Reaction stop BGlycidol transformation B

e.g. Glycerol or2-/3-MXPD

Analytical methods

Indirect analysis: DGF methods

•DGF C-III 18 (09) A: new name: C-VI 17 (10)


•DGF C-III 18 (09) B: rejected

•SGS A & B: will become DGF C-VI 18 (10)

Analytical methods

Transformation of 3-MCPD into glycidol by alkaline treatment

75

100

an

aly

te [

%]

Transformation of 3-MCPD during

alkaline treatment (2 % NaOH) atroom temperature

3-MCPD

Glycidol98 %

75

100

an

aly

te [

%]

Transformation of 3-MCPD duringalkaline treatment (0,25 % NaOH) at

- 25 °C

3-MCPD


0

25

50

0 5 10 15

an

aly

te [

%]

reaction time [min]

⇒⇒⇒⇒ Sufficiant ester cleavage

� The undesired conversion of 3-MCPD into glycidol can be minimised by using diluted alkaline solutions and performing the reaction in the cold.

0

25

50

0 10 20 30 40 50an

aly

te [

%]

reaction time [h]

3-MCPD

Glycidol

⇒⇒⇒⇒ Sufficiant ester cleavage

New SGS method

Conception of the new SGS method “3 in 1”

Bound2- & 3-MCPD

Free 2- & 3-MCPD

Free 2- & 3-MCPD

Free

Mild alkaline

ec

Acidicreactionstop withH+ / Br-

Matrix removal,

derivatisation with PBA*

* : Phenyl boronic

acid

3-MCPD*

2-MCPD*


� A mild alkaline cleavage minimises the transformation of MCPD into glycidol

� Free glycidol: instable & commonly poor chromatographic performance monobromopropanediol (MBPD): excellent stability and GC performance

� Isotopic labelled reference compounds are available

� The determination of 2-MCPD is covered by this method

Boundglycidol

Free glycidol

Free 3-MBPD

2-MBPD

acid

Glycidol as

3-MBPD*

GC-MS Analysis

All analytes < LOD in crude/pristine oil or fat from:

Avocado Butter Cacao

Castor plant Caraway Fish (caplin, salmon)

Hazelnut Lard Linseed

Bound glycidol & MCPD in crude/pristine edible oils (Data achieved by the new SGS method “3 in 1”)

Verification of the new method


Hazelnut Lard Linseed

Macadamia nut Manila Olive

Palm Paprika Para nut

Pomegranate kernel Pumpkin-seed Rapeseed

Sesame Sun-flower Walnut

LODs: bound glycidol < 0.025 mg/kg

bound 3-MCPD < 0.05 mg/kg

bound 2-MCPD < 0.05 mg/kg

Bound glycidol & 3-MCPD in refined edible oils (Comparison of the conventional and the new SGS method “3 in 1”)

Refined oilBound 3-MCPD

[mg/kg]Bound glycidol

[mg/kg]Analytical method

[N° of samples] Maximum Mean > 0.15 Maximum Mean > 0.15 SGS

Soybean

[10]

0.33 0.21 0.38 0.34 A & BGlycidol calculated

0.33 0.24 0.32 0.32 New “3 in 1”



0.33 0.24 0.32 0.32

Sunflower

[13]

2.0 0.71 0.37 0.24 A & BGlycidol calculated

2.1 0.72 0.31 0.26 New “3 in 1”

Palm

[57]

10 2.4 18 2.4 A & BGlycidol calculated

10 2.4 18 2.4 New “3 in 1”

Interlaboratory comparison of indirect methods


� 2009 BfR ring test : 3-MCPD esters in 5 different refined oils/fat

�SGS Method “A & B”: z-scores - 0.65 ↔↔↔↔ + 0.16�SGS Method “3 in 1”: z-scores - 0.62 ↔↔↔↔ + 0.35

� 2011: 10 refined palm oils - analysed in parallel by Nestle´ and SGS

bound 3-MCPD & bound 2-MCPD


ec = ester cleavage

0

2

4

6

0 1 2 3 4 5 6 7 8 9 10 11

[mg

/kg

]

Sample-N°

Dots: SGS „3 in 1“ alkaline ec

Triangles: Nestle´ acidic ec

3-MCPD

2-MCPD

Interlaboratory comparison indirect vs. direct methods


� 2011: 10 refined palm oils- analysed in parallel by Nestle´ and SGS

6

bound glycidol

SGS indirect "3 in 1" / alkaline ec

Nestle` direct LC-MS


0

2

4

1 7 4 2 6 9 3 8 5 10

[mg

/kg

]

Sample-N°

Nestle` direct LC-MS

Samples 1, 7, 4

< 0.1 mg/kg

Interlaboratory comparison indirect vs. direct methods


� 05/2011: 7 refined oil samples - analysed in parallel by M. Granvogl/DFA (German Research Centre for Food Chemistry) and SGS

8

10

[mg

/kg

]

bound glycidol

SGS indirect "3 in 1" / alkaline ec

DFA direct LC-MS

Value x 10 =

70.9 / 70.3 mg/kg

?


0

2

4

6

[mg

/kg

] DFA direct LC-MS ?

Conclusions

� Different indirect (validated) methods are in use for the determination

of true & induced MCPD. Some of these methods include a calculative

determination of bound glycidol


� A new SGS method “3 in1” for the simultaneous determination of

bound glycidol, 2- & 3-MCPD in various oils & fats has shown good

matching results with other direct and indirect methods in first

comparative studies

� Further improvement & comparison of direct & indirect methods is

always recommended


Thank you for your

attention!

Dr. Jan Kuhlmann

Laboratory Services Hamburg

SGS Germany GmbH

Weidenbaumsweg 137

D-21035 Hamburg / Germany

+ 49(0)40 88309-423 + 49(0)172 413 8446

[email protected]

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