analysis & occurrence of bound glycidol and mcpd in oil

1•103rd Annual AOCS Meeting & Expo April 29 – May 2 21012, Long Beach, California, USA Jan Kuhlmann / SGS Germany GmbH

Analysis & occurrence of bound glycidol and MCPD in oil containing foods

SGS Germany GmbHJ. Kuhlmann

Introduction

2-MCPD, 3-MCPD & Glycidol: Glycerolderivates

3-MCPD(3-Chloropropane-1,2-diol)

2-MCPD(2-Chloropropane-1,3-diol)

+ HCl

- H2O


Glycerol

(3-Chloropropane-1,2-diol) (2-Chloropropane-1,3-diol)

Glycidol- H2O

+ HCl- HCl

Introduction

(fatty acid) bound Glycidol & MCPD in oils & fats


Bound MCPD & Glycidol are generated mainly during

deodorisation.

crude oil refining refined oil

The formation of these process contaminants depends upon the oil composition and process control.

Introduction

Generation of bound Glycidol & MCPD from natural oil components

Mono- & Diacylglycerides (Glycerol mono- & di-fatty acid esters = „bound glycerol“)

Deo-Temp. > 200 °C

natural (!) HCl-sources: e.g. FeCl , chlorinated phytosphingosides1)

starting at Temp. < 200 °C


1) K. Nagy et al.: Mass-defect filtering of isotope signatures to reveal the source of chlorinated palm oil contaminants; Food Addit. Contam. 2011, 28, 1492–1500

Glycidyl ester

� fatty acid mono-esters

representative to fatty acid composition

� „low“ boiling points

� reactive epoxy-funktion; limited stability

Glycidylpalmitate

2- & 3-MCPD-ester� fatty acid 1-/2-Mono- & 1,2-/1,3-Di-esters

� „high“ boiling points ; persistant compounds

FeClx, chlorinated phytosphingosides1)

3-MCPD-1,2-bis-pamitoyl-ester

� free 3-MCPD

Some in-vivo studies indicated toxic effects especially to kidneys and testes after oral uptake.

MRL (Maximum Residue Limit): 20 µg/kg in HVP & HVP-products

EU Commission Regulation N°466/2001

Toxicological impact of MCPD

Introduction

� bound 3-MCPD


� bound 3-MCPD

TDI (Tolerably Daily Intake): 2 µg/kg bw d

Recent in-vivo studies have shown that the majority of 3-MCPD is released from its bound

form during digestion. In-vivo released 3-MCPD causes similar toxic effects like the uptake

of free 3-MCPD 2)

2) EFSA (2011). Scientific report submitted to EFSA ‘Comparison between 3-MCPD and its palmitic esters in a 90-day toxicological study’ prepared by E. Barocelli, A. Corradi, A. Mutti and P.G. Petronini, University of Parma, Parma, Italy. CFP/EFSA/CONTAM/2009/01.

� free & bound 2-MCPD

Same story as last year: still very little analytical and almost no toxicological data available

� free glycidolEpoxides are highly reactive an combine with many chemical groups

⇒ Several serious toxic effects have been proven in in-vitro & in-vivo studies

Toxicological impact of glycidol

Introduction

Classification according to Regulation (EC) No 1272/2008 [EU-GHS/CLP]Acute toxicity, Oral (Category 4)

Acute toxicity, Inhalation (Category 3)

Acute toxicity, Dermal (Category 4)


� Bound glycidolSo far no data available (actual studies by the German Federal Institute of Risk Assessment (BfR) in progress)

The release of free glycidol from its bound form

Acute toxicity, Dermal (Category 4)

Skin irritation (Category 2)

Eye irritation (Category 2)

Germ cell mutagenicity (Category 2)Carcinogenicity (Category 1B)Reproductive toxicity (Category 1B)Specific target organ toxicity - single exposure (Category 3)

Classification according to EU Directives 67/548/EEC or 1999/45/ECToxic by inhalation. May cause cancer. May impair fertility. Harmful in contact with skin and if swallowed. Possible risk of irreversible effects. Irritating to eyes, respiratory system and skin.

excerpt material safety data sheed Sigma-Aldrich Co. G5809 04-2012

Analytics

Direct analysis; determination of the original esters

Hypothetic oil Contains only 3 relevant fatty acids

This yields up to

3 Glycidyl ester9-MCPD-mono-ester

15 MCPD-di ester

glycidol

3-MCPD

2-MCPD

Fatty acid(s)


Chromatogram displays up to 27 analytes!

maybe Matrix-clean-up

LC-MS

Analytics

Hypothetic oil Contains only 3 relevant fatty acids

This yields up to

3 Glycidyl ester9-MCPD-mono-ester15 MCPD-di -ester

Indirect analysis; determination of the released analytes


Chromatogram displays up to 3 analytes!

Derivatisation

GC-MS

Matrix removal

Ester cleavage

glycidol

3-MCPD

2-MCPD

glycidol

3-MCPD

2-MCPD

� acidic ester cleavage; 2- & 3-MCPD are stable; glycidol is instable

EFSA (2011). Scientific report submitted to EFSA ‘Comparison between 3-MCPD and its palmitic esters in a 90-day toxicological study’ prepared

Analytics

Indirect analysis; different methods

2- & 3-MCPD-ester

Glycidyl-ester

H+ 2-MCPD3-MCPD

Glycidol

� alkaline ester cleavage; glycidol is stable but 3-MCPD reacts to glycidol

bound freeca. 16 h

shorter (Unilever)


H+ / chloride freeB 2-MCPD

3-MCPD

Glycidol

B) acidic reaction stop in absence of chloride

H+ / Cl- 2-MCPD

3-MCPD

A

A: Sum 3-MCPD & Glycidol

B: True 3-MCPD- value

� alkaline ester cleavage; glycidol is stable but 3-MCPD reacts to glycidol

A) acidic reaction stop in presence of chloride; original and induced glycidol reacts to 3-MCPD.

(A-B)xTf = Glycidol

DGF C-VI 18 (10) A,B

3-10 min

OH-2- & 3-MCPD-ester

Glycidyl-ester

2-MCPD

3-MCPDGlycidol

Tfbound

free

Bound

3- & 2-

MCPD

Bound

Glycidol-trans-

formationwith

H+ / Br-

Free

Free

3- & 2-

MCPD

Indirect analysis - SGS method “3-in-1”

Analytics

Glycidol as

3-MBPD*

3-MCPD*

2-MCPD*

GC/MS-Analysis

Derivatisation with PBA** Phenyl Boronic Acid

Free

3- & 2-

MCPD

Mild alkaline

estercleavage

ca. 16 h

Matrix removal


� Mild reaction conditions minimize the undesired transformation

of 3-MCPD into glycidol (artefact minimisation, high MCPD sensitivity)

� The transformation of the instable glycidol into 3-MBPD generates a highly stable derivative

with excellent gas chromatographic performance (robust & sensitive glycidol determination)

� Isotopic labelled & esterified analytes for internal standard purposes are commercially

available (best quantification approach)

� Micro size sample preparation results in low organic solvent consumption of approximately

8 mL/sample (sustainable laboratory practise)

BoundGlycidol

Free

3- & 2-

MBPD

2-MCPD*Free

Glycidol

Vegetable baking & frying fats

Occurrencem

g/k

g

Determination of bound glycidol & MCPD in vegetable baking & frying

fats from the local German market

15,00

18,00

Fat N°11

3-MCPD = 3.7 mg/kg


mg

/kg

0,00

3,00

6,00

9,00

12,00

N°1 N°2 N°3 N°4N°5

N°6N°7

N°8N°9

N°10N°11

N°12N°13

0,1

0,2 0,40,7 1,1

1,5 2,6

0,30,3 0,9 1,5 2,5 3,7

5,50,9

0,6 1,5 0,9

2,3

11,1

0,3

bound glycidol

bound 3-MCPD

bound 2-MCPD

3-MCPD = 3.7 mg/kg

70 kg ≈ TDI 140 µg/d = 38 g fat/d

Σ analytes = 16.3 mg/kg

if assumed as 3-MCPD impact = 8.6 g fat/d

70 kg

Occurrence of bound Glycidol & MCPD in other foodstuff

Occurrence

Almost all foods containing refined oils and fats may contain Chocolate & nut-

nougat spreadsFrench fries, fried

potatoes, chips,

Infant formulaCoffee

creamer


bound glycidol and MCPD

Positive findings are frequently in the middle µg/kg up to the low mg/kg range (German market products)

ω-3

Cookies, cakes, cruller

nougat spreadspotatoes, chips,

mayonnaise

Spreads, dressings,

margarine

Dietary

supplement

oilsInstant

soups

Tofu meals

vegetarian

sausage/lard/etc. Chocolates

Occurrence

Influence of customary frying

5

6

7bound 2-MCPD

bound 3-MCPD

bound glycidol

Olive oil in parts refined

fryin

go

il

Olive oil extra virgin ButterLiquid

paraffine

x 3 !


0

1

2

3

4

an

aly

te m

g/k

g f

ryin

g

afo: after frying of m.T.: moderate temperature h.T.: high temperature

start

start

Summary

� Bound MCPD & glycidol are toxicologically relevant process contaminants;

bound glycidol can be assumed to be more hazardous.

� The analysis of these compounds is sophisticated but feasible.

� SGS has developed two methods for the determination of bound MCPD &


� SGS has developed two methods for the determination of bound MCPD &

glycidol (DGF C-VI 18 (10) and “3-in-1”).

� Bound MCPD & glycidol may occur widespread in foods containing refined oils

and fats.

�Bound 2-MCPD and glycidol together with 3-MCPD should be taken into

account to evaluate toxicological relevant over-all exposure.

� Very first preliminary results of frying experiments indicate that bound MCPD

may be generated during the frying of meat and fish with edible oils or fats.

SGS Germany GmbHDr. Jan Kuhlmann

Weidenbaumsweg 137D-21035 Hamburg

Tel.: +49 (0)40 88 309 423mobile: +49 (0)172 413 8446

www.de.sgs.com

Many thanks to the lab team:Many thanks to the lab team:Many thanks to the lab team:Many thanks to the lab team:

Svetlana BarantsevaSvetlana BarantsevaSvetlana BarantsevaSvetlana Barantseva

Laetitia MarchalLaetitia MarchalLaetitia MarchalLaetitia Marchal

Juliane DircksJuliane DircksJuliane DircksJuliane Dircks

Lan Ly & Alexandra ReisigLan Ly & Alexandra ReisigLan Ly & Alexandra ReisigLan Ly & Alexandra Reisig


[email protected]

Thank you for your kind attention!Thank you for your kind attention!Thank you for your kind attention!Thank you for your kind attention!

analysis & occurrence of bound glycidol and mcpd in oil

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