Analysis of MCPD- and glycidyl-esters in edible oils

1 Workshop on 3-MCPD IN EDIBLE VEGETABLE OILS OCCURRENCE AND ANALYTICAL METHODS,Udine, June 8th 2016 Jan Kuhlmann / SGS Germany GmbH

Analysis of MCPD- and glycidyl-esters in edible oils:

Method improvements and restrictions

SGS Germany GmbH J. Kuhlmann


Ø  Introduction 2- & 3-MCPD, Glycidol

Ø  General analytical approaches

Ø  Methods for analysis of oils and fats

Ø  Methods for analysis of oils/fats extracted from complex foods

Ø  Method comparison

Ø  Conclusions

Contents

Structure of the presentation


3-MCPD 3-mono-chloropropane-1,2-diol 3-Chloropropane-1,2-diol

2-MCPD 2-mono-chloropropane-1,3-diol 2-Chloropropane-1,3-diol

Free analytes

Glycidol (2,3-Epoxi-1-propanol) Glycidylpalmitate

3-MCPD-1,2-bis-palmitoylester

just examples, all fatty acids of an oil/fat might be present (Fatty acid) Bound analytes

2-MCPD-1-oleoyl-3-stearoylester

3-MCPD-1-palmitoylester

Free & bound 2-MCPD, 3-MCPD & glycidol - structures

Introduction

+/- H+Cl-

Δ


Potential hazards of 2-MCPD, 3-MCPD & glycidol

Introduction

Fatty acid esters of 2- & 3-MCPD and glycidol (“bound 2-/3-MCPD; bound glycidol”) represent a complex group of process induced contaminants.

Containing chloride or an epoxy group they might have toxic impacts similar to free MCPD/glycidol when the core compounds are released during digestion.

glycidol: genotoxic carcinogen

3-MCPD: non-genotoxic carcinogen

2-MCPD: No official classification available

In regard to risk assessment (and product quality) glycidol is the more problematic compound in comparison to 3-MCPD!

Consumers intake should be “As-Low-As-Reasonably-Achievable” (ALARA-priciple)

3-MCPD 2-MCPD Glycidol

OR, OR`= OH; various fatty acids

Fatty acid esters of 2- & 3-MCPD and glycidol (“bound 2-/3-MCPD; bound glycidol”) represent a complex group of process induced contaminants.

Containing chloride or an epoxy group they might have toxic impacts similar to free MCPD/glycidol when the core compounds are released during digestion.


Regulations and recommendations

Introduction

2-MCPD and glycidol are not included in the TDI.

e.g. BfR-estimation on max. glycidol contents in infant formulae:

≤  67 µg/kg fat ≅ 17 µg/kg final product (fat content ca. 25 %)

BfR Opinion 007/2009 from10th March 2009.

EU-Regulations are set only for free 3-MCPD:

free 3-MCPD ≤ 20 µg/kg in soy sauce (or HVP); EU 466/2001

free 3-MCPD ≤ 100 µg/kg in glycerol used as food additive; EU 232/2012

Bound 3-MCPD: Tolerable Daily Intake (TDI) = 2 µg/kg x bw x d until 05/2016

! EFSA opinion on 2- & 3-MCPD and glycidol; j.efsa.2016.4426 ! Ø From toxicological perspective the free and bound analytes are considered to be equivalent on molar base.

Ø  The CONTAM panel established a Tolerable Daily Intake (TDI) lowered from 2 µg to 0.8 µg 3-MCPD/kg x bw x d

Example: 70 kg bw ⇒ TDI = 56 µg 3-MCPD

≈ 2.8 kg soy sauce @ 20 ppb 3-MCPD

≈ 255 g nut-nougat spread @ 220 ppb 3-MCPD ≈ 22.4 g of frying fat @ 2.5 mg/kg 3-MCPD


Introduction

EFSA reaction on the story


Introduction

Sources of glycidol & MCPD

1.  Source: Bound MCPD & glycidol are generated mainly during the refining of edible oils & fats (deodorisation is the most critical step)

In general every refined edible oil/fat contains bound MCPD & glycidol The potential of contaminant formation is dependant upon the oil type & quality

The degree of formation or removal is dependant upon refining conditions

crude oil refining refined oil


Introduction

Further sources of glycidol & MCPD

2. Source: The preparation of food may cause the formation of free/bound MCPD & glycidol ! (e.g. frying, smoking, roasting, toasting, barbecueing ….)

3. Source: free MCPD might migrate from contact materials into foods. (Paper and plastic wrappings, surface coatings, tea- and coffee filters, paper kitchen towels)


Analytical approaches

Direct analysis; determination of the single original esters

Hypothetic oil Contains only 3 relevant fatty acids

Matrix removal in the

majority of applications

Chromatogram displays up to 27 analytes!

This yields up to 27 analytes

3 Glycidyl ester 9-MCPD mono-ester

15 MCPD di-ester

glycidol

3-MCPD

2-MCPD

Fatty acid(s)

LC-MS / LC-MS² / LC-MS-TOF / GC-MS


Hypothetic oil Contains only 3 relevant fatty acids

Analytical approaches

matrix clean-up (e.g. l/l-extraction)

glycidol

3-MCPD

2-MCPD

fatty acid(s)

Indirect analysis; determination of the released analytes

ester cleavage (alkaline, acidic, enzymatic) chemical modification

glycidol conversion (into MXPD)

Derivatisation* (e.g. HFBA/acetone/PBA)

GC-MS

2-MCPD

3-MCPD

Glycidol* Glycidol as

3-MBPD


Analytics

Indirect determination (ester cleavage releases the 3 core analytes, GC-MS)

Many analytical approaches available for the bound analytes in oils and fats.

Direct determination (determination of a selected number of contaminant esters)

Early DGF C-III 18 (09) Σ  3-MCPD + glycidol DGF C-VI 17 (10); fast

alkaline

Late DGF C-III 18 (09) A,B A: Σ 3-M + g, B: 3-MCPD Withdrawn by DGF

DGF C-VI 18 (10) A, B A: Σ 3-M + g, B: 3-MCPD AOCS Cd 29c-13; fast

Küsters et al. 2010 3-MCPD, Glycidol fast

SGS “3-in-1” Kuhlmann 2011 3-MCPD, 2-MCPD, Glycidol AOCS Cd 29b-13; slow

acidic

Divinova et al. 2004 Zelinkova et al. 2006 3-MCPD; slow

enzymic

Koyama et al. 2015 3-MCPD, Glycidol fast

Myasaki et al. 2012 3-MCPD, 2-MCPD, Glycidol fast

“Unilever” Ermacora et al. 2013 3-MCPD, 2-MCPD, Glycidol AOCS Cd 29a-13; slow

Blumhorst et al. 2011 GE LC-MS²

Dilute & shoot

Haines et al. 2011 3-MCPD-E, GE LC-MS²

SPE or SPE²

Masukawa et al. 2010/11 GE SPE²; LC-MS: AOCS Cd 28-10

Dubois et al. 2011 3-MCPD-E, 2-MCPD-E, GE SPE²; LC-MS²

Steenbergen et al. 2013 GE l/l; LC; GC/MS

Granvogl et al. 2011 GE SPE; LC-MS²

MacMahon et al. 2013 3-MCPD-E, 2-MCPD-E, GE 2 x SPE²; LC-MS²

BfR method 09 3-MCPD fast

BfR method 08 3-MCPD slow EFSA recommends to use the

AOCS Official Methods Cd 29a,b,c-13 not only for analysis of bound 2- & 3-MCPD and glycidol in oils and fats

but also in oil- & fat containing foods. LOQ = 0.1 mg/kg in the oil/fat fraction

No officially validated methods available for the analysis of free 2- MCPD or

bound 2- & 3-MCPD and glycidol in oil- & fat containing foods (except AOCS Cd 30-15 for spreads & margerine)


General approaches for the analysis of complex composed foods

Two principal ways might be used for routine analysis of complex matrices:

No fat extraction: taking whole samples into an

AOCS method.

Fat extraction prior to analysis with an

AOCS method.

New methodological applications

Some points have to be checked!

No fat extraction: taking whole samples into an

AOCS method.

Impact on ruggedness/trueness? Free MCPD included?

Fat extraction prior to analysis with an

AOCS method. Extraction efficiency?

Impact on ruggedness/trueness? Free MCPD included?


New applications for the analysis of spreads & margarine

Cd 30-15: Method validation trial (to be published): “Analysis of 2‐ and 3‐MCPD Fatty Acid Esters and Glycidyl Fatty Acid Esters in Oil‐Based Emulsions“

Principle:

l/l extraction + AOCS Cd 29a-13


How do the other validated methods perform for analysis of spreads & margarine?

Is the fat extraction step necessary?


SGS: Method comparison in regard to the analysis of spreads & margarine part I

The ring test samples were analysed with all three methods for oils and fats (“Unilever”- slightly modified, “DGF”, “3-in-1”):

A): with l/l - fat extraction (acc. AOCS Cd 30-15) prior to analysis

B): direct analysis without fat extraction

Method comparison

0,00

0,25

0,50

0,75

1,00

Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11

mg/

kg

2-MCPD: results with & without fat extraction extracted fat + Unilever

extracted fat + "3-in-1"

extracted fat + DGF

Whole sample + Unilever

Whole sample + "3-in-1"

Whole sample + DGF

0,00

0,25

0,50

0,75

1,00


mg/

kg

2-MCPD: results with & without fat extraction extracted fat + Unilever


extracted fat + DGF



Whole sample + DGF


0,00

0,50

1,00

1,50

2,00


mg/

kg

3-MCPD with & without fat extraction

fat extraction + Unilever

fat extraction + "3-in-1"

fat extraction + DGF



Whole sample + DGF

0,00

0,50

1,00

1,50

2,00


mg/

kg

3-MCPD with & without fat extraction

extracted fat + Unilever


extracted fat + DGF



Whole sample + DGF

SGS: Method comparison in regard to the analysis of spreads & margarine part II

Method comparison

Overestimations of 3-MCPD


0,00

1,25

2,50

3,75

5,00


mg/

kg

Glycidol with & without fat extraction



extracted fat + DGF



Whole sample + DGF

Method comparison

All three methods gave similar results when the fat was extracted prior to analysis but the “DGF”- & “Unilever”-methods showed restrictions when the whole samples were proceeded without fat extraction.

SGS: Method comparison in regard to the analysis of spreads & margarine part III

0,00

1,25

2,50

3,75

5,00


mg/

kg

Glycidol with & without fat extraction



extracted fat + DGF



Whole sample + DGF

Overestimations of glycidol


0,0

0,2

0,4

0,6

0,8

1,0

1,2


aged oilblend: RBP palm oil / rape seed oil

Unilever-method (n = 5)

"3-in-1"-method (n = 5)

Method comparison

SGS method comparison with oil blends of different age

SGS: Method comparison in regard to the analysis of aged oils and fats

At SGS laboratory the glycidol results differ when using the “Unilever” vs. the “3-in-1”-method for analysis of aged oils & fats.

0,0

0,2

0,4

0,6

0,8

1,0

1,2


Ana

lyte

[mg/

kg]

fresh oilblend: RBP palm oil / rape seed oil

Unilever-method (n =5)

"3-in-1"-method (n = 5)

0,0

0,2

0,4

0,6

0,8

1,0

1,2


medium aged oilblend: RBP palm oil / rape seed oil

Unilever-method (n = 7)

"3-in-1"-method (n = 7)


Method comparison

Preliminary results from an interlaboratory comparison focusing on fats extracted from infant formula: „Unilever“ vs. „3-in-1“ & direct LC-MS² method.

Interlaboratory comparison in regard to the analysis of extracted oils and fats by different methods

The “Unilever-method” gave inconsistent glycidol values in fat extracted from aged infant formula.

0,00

0,10

0,20

0,30

0,40

0,50

0,60

3-MCPD 2-MCPD glycidol

mg/

kg

Infant formula sample B: data comparison 2014-2015 2 Laboratories: various extractions & 3 methods applied

"Unilever"

"3-in1"

direct LC-MS²

0,00

0,10

0,20

0,30

0,40

0,50

0,60

3-MCPD 2-MCPD glycidol

mg/

kg

Infant formula sample B: data comparison 2014-2015 2 Laboratories: various extractions & 3 methods applied

"Unilever"

"3-in1"

direct LC-MS²


New methods

Further trials in purpose to reflect the EFSA specifications

EU reference laboratory JRC/IRMM: “In-house-validated method for the separate analysis of free 2-/3-MCPD & bound 2-/3-MCPD and glycidol in foods1)

1): EFSA supporting publication 2015: EN-779: T. Wenzel et al.

Extraction not suitable vor infant formulae or

microencapsulated products. Complete separation free vs.

bound MCPD remains unclear.

BfR approach for infant formula: ASE + AOCS Cd 29a,b-13

FDA approach for infant formula: l/l-Extraction + LC-MS² analysis

AOCS Cd 29a-13

Bound analytes

Free analytes



SGS new “5-in-1” approach for complex composed foods

Heat-Ultrasonic-Pressure supported Solvent Extraction (HUPsSE) A new SGS approach for separate analysis of free & bound MCPD/glycidol in a

broad range of composited foods including infant formula and microencapsulated products.

HUPsSE: Triple extraction in ultrasonic bath at 65°C

Solvent removal by flow of N2

l/l-Separation non-polar polar

Derivatisation & GC-MS analysis

Analogue to “3-in-1”

Free 2- & 3-MCPD LOQ 5 µg/kg each

„3-in-1“ = AOCS Cd29b-13 modified

bound 2- & 3-MCPD & glycidol

LOQ 10 µg/kg each

1 g / 2 g Sample + free & bound internal standards


Ø  2-MCPD, 3-MCPD and glycidol are relevant food contaminants and attracted increasing attention by EFSA

Take home messages

Ø  Recent findings indicate, that AOCS methods (Cd 29a,b,c-13) might show different performance in other matrices and should be used only after careful verification of

their applicability!

Conclusions

Ø  Validated methods (AOCS Cd 29a,b,c-13) do show satisfying comparability and performance when applied for the analysis of fresh oils and fats.

Ø  When edible oils/fats are extracted from foods, it has to be evaluated if the extraction technique is sufficient and if free MCPD is included or not.


SGS Germany GmbH Dr. Jan Kuhlmann

Weidenbaumsweg 137 D-21035 Hamburg

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

www.de.sgs.com [email protected]

Thank you for your kind attention!

Documents

Analysis of MCPD- and glycidyl-esters in edible oils