1 PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Florence LACOSTE ITERG (Pessac, France)

f.lacoste@iterg.com

Contaminants in oils and fats: analysis and regulations

French Technical Industrial Center Authority : French Ministry of Industry

Staff : 80 2012 Budget : 6M€

ISO 9001:2008 & ISO 17025

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Polycyclic aromatic hydrocarbons

Mineral oil

Phthalates

3-MCPD esters & glycidol esters

Contents

2

Origins ?

Regulation ?

Recognized analytical methods ?

Risks ?

Risks Methods Regulation

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Polycyclic aromatic hydrocarbons

3

Environmental contamination

Production process

Regulation

(EC) n°1881/2006 – contaminants in foodstuffs

Risks Methods Regulation

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

PAHs origins in vegetable oils

PAHs

Extraction

v

Oilseeds Crude oil

+

HAP

Active carbon

Heavy PAHs

Light PAHs

Deodorisation

Drying

Refined oil

(+ e HAP)

4

* Grapeseed oil * Coconut oil * Pomace olive oil * Sunflower oil

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013 5

benzo(b)fluoranthene benzo(k)fluoranthene benzo(a)pyrene indeno(1,2,3-cd)pyrene

dibenz(ah)anthracene benzo(ghi)perylene

HEAVY PAHs

naphthalene

acenaphthene acenaphthylene fluorene phenanthrene anthracene

fluoranthene pyrene benz(a)anthracene chrysene

LIGHT PAHs

Structure of PAHs

GT

GT

GT GT GT

GT : genotoxic

GT GT

GT

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

PAHs regulation: (EC) n°1881/2006 contaminants in foodstuffs

PAHs Maximum

levels oils and fats (µg/kg)

Maximum levels coconut

oil (µg/kg)

Maximum levels cocoa

butter (µg/kg)

Sum of 4 PAHs: - benzo[a]pyrene - benz[a]anthracene - benzo[b]fluoranthene - chrysene

10,0 20,0

35,0 from 1.4.2013 until 31.3.2015

30,0 from 1.4.2015

benzo[a]pyrene 2,0 2,0 5,0

from 1.4.2013

6

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

PAHs: determination methods

7

Method ISO 15302 ISO 15753 ISO 22959

BaP LOQ (µg/kg) 0,1 0,2 0,1

Reproducibility (CVR%) 27 % (2,1µg/kg) 41% (3,21µg/kg) 10 % (2,6µg/kg)

Principle

PAH isolation: liquid chromatography (alumina, silica gel,C18-silica gel) or HPLC (donor-acceptor complex chromatography, size-exclusion chromatography)

Analysis: HPLC/fluorescence or GC/MS

ISO 15302 : benzo[a]pyrene in oils (LC + HPLC/FLD)

ISO 15753 : 12 PAHs in oils (2 SPE + HPLC/FLD)

ISO 22959 : 17 PAHs in oils (DACC on-line + HPLC/FLD)

EN xxxx (JRC) : 4 PAHs in foodstuffs (SEC + SPE + GC/MS)

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

0

10

20

30

40

50

60

70

80

90

100

<0,5 0,5 - 1 1 - 2 2 - 5 5 - 20 20 - 50 50 - 100 >100

% s

amp

les

Benzo[a]pyrene (µg/kg)

Virgin olive oil

Olive oil

Pomace olive oil

Grapeseed oil

Sunflower oil

EU limit

2 µg/kg

BaP : occurrence in vegetable oils

8

EU Scoop Task (Oct 2004)

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013 9

Edible oils: PAH4 Levels Screening 2012 : 21 vegetable oils

PAH4 en µg/kg

benzo(a)pyrene chrysene benz(a)anthracene benzo(b)fluoranthene

ITERG data (2012)

0

2

4

6

8

10

12EU limit PAH4 : 10 µg/kg

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Mineral oil

10

Environmental contamination (air, soil)

Crop protection

Transport & storage

Production process

Regulation

(EC) n°1151/2009 - import of sunflower oil from Ukraine

EFSA Scientific Opinion on Mineral Oil Hydrocarbons in food (2012)

Risks Methods Regulation

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

EFSA Scientific Opinion, 2012

Mineral oil: complex mixture of hydrocarbons

MOSH: straight or branched alkanes & alkylated cycloalkanes

MOAH: aromatic hydrocarbons including alkyl-substituted

Hydrocarbon compound number in mineral oil > 100 000 for those with less than 20 carbon atoms !

Different products & composition: diesel fuel, white oil, lubricant …

Technical grade mineral oil contain 15-35 % MOAH, which is minimised in food grade MOSH (white oils)

Mineral oil composition

11

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

2008 contamination of sunflower oil from Ukraine with a mineral oil from unknown origin

2009 contamination of walnut oil with a food grade lubricant oil during refining process

2010 identification of compounds eluted as mineral oil in grapeseed oils

2011 contamination of milk fat with a food grade lubricant oil during production

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Contaminated Sunflower oil (MOSH analysis)

Natural hydrocarbons

Mineral oil : unresolved complex mixture

12

Mineral oil detected in vegetable oils

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Fractionation of the sample by liquid chromatography on

silica gel impregnated with AgNO3

Quantification with an internal standard (C18 or C20)

GC/FID analysis on an short apolar column

MOSH determination: pr ISO17780

13

18

,5 g

Sili

ca g

el

Elution with hexane Solvent evaporation

Vide

40°C

GC analysis

On-column injection (2 µl) DB5 HT (15 m-0,25 mm-0,1 µm)

1 g oil

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

pr ISO17780 collaborative trial

14

2013 Trial 10 samples

virgin olive oil + mineral oil

refined pomace olive oil

crude palm oil + diesel oil

refined grapeseed oil

3 refined sunflower oil (+ mineral oil)

crude soja oil + mineral oil

margarine & mayonnaise + mineral oil

0

20

40

60

80

100

26 3 8 23 21 34 15 31 1 19 4 33 36 9 28 17 30 27 29 24 11 10 35 5 32 12 6 22 2 18 14 20

Lab

me

an v

alu

e (

mg/

kg)

Laboratories

Sample 2: 50 mg/kg spiked olive oil

50

70

90

110

130

150

170

26 10 34 3 23 15 31 8 27 9 30 33 19 35 29 11 21 4 1 20 32 5 14 22 28 6 36 12 2 24 18

Lab

me

an v

alu

e (

mg/

kg)

Laboratories

Sample 8: 100 mg/kg spiked refined sunflower oil

Mean value : 52 mg/kg

Mean value : 105 mg/kg

Mineral oil Sample 2 Sample 8

Number of laboratories retained (p) 31 30

Mean (m) (mg/kg) 51,8 105,0

REF Laboratory result (mg/kg) 54 87

Repeatability limit (r) 19,3 15,3

Reproducibility limit (R) 51,7 47,4

Horrat value 4,1 2,0 content < 100 mg/kg dispersed results

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Phthalates

15

Transport & storage

Production process

Regulation

EFSA Scientific Opinion on food additives, flavourings, processing aids and materials in contact with food (2005)

(EC) n°10/2011 - plastic materials and articles intended to come into contact with food

Risks Methods Regulation

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Chemical structures of phthalates

Di-methyl PHT DMP R1=R2=methyl

Di-ethyl PHT DEP R1=R2=ethyl

Di-isobutyl PHT DIBP R1=R2=isobutyl

Di-butyl PHT DBP R1=R2=butyl

Di-hexyl PHT DHexP R1=R2=hexyl

Benzyl butyl PHT BBP R1=benzyl R1=butyl

Di-n-heptyl PHT DHepP R1=R2=heptyl

Di-(2-ethyl hexyl) PHT DEHP R1=R2=ethyl-2 hexyl

Di-n-octyl PHT DNOP R1=R2=octyl

Di-isononyl PHT DINP R1=R2= isononyl

Di-isodecyl PHT DIDP R1=R2=isodecyl

16

C

C

O

O

O

OR1

R2

Oily visquous liquid

MW: 390,6 g/mol

BP: 385°C

Water solubility: 3 µg/l

High affinity for fat (log Kow : 7,5)

DEHP

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Flooring-Roofing- Wall covering Adhesives-Sealant-Rubber

Paints-Shower curtains Wires & cables-Fresheners

Cosmetics : Perfume Hairspray-Deodorant

Skin emollient-nail polish fingernail elongators

Car undercoating Dashboard-Door

panels-Safety glass

Shoes-Boots-Gloves Out-door & rainwear

Toys Child-care articles

Pharmaceuticals Medical devices :

Catheters-Blood bag

Phthalates are everywhere

17

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Specific migration limit in food

To be used as

BBP 30 mg/kg Plasticizer in single-use material containing non-fatty foods except infant formulae

DINP ∑ = 9 mg/kg

DIDP

DEHP 1,5 mg/kg Plasticizer in repeated use materials containing non-fatty foods DBP 0,3 mg/kg

(EC) n°10/2011 - Plastic materials & articles into contact with food

18

Material containing these phthalates cannot be used for oils & fats

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013 19

Phthalates: ITERG’s procedure

Advantages of the method :

No contamination, no solvent

Rapid analysis (45 min)

Sensitive LOQ < 0,1 mg/kg (excepted DINP & DIDP)

HS SPME

1 g oil

Analysis

DB5ms (30 m; 0,25 mm; 0,25 µm)

PDMS

m/z 111 (adipates)

m/z 149 (phthalates) GC/MS

DEHP DHexP

DNPP DBP

DEP

DMP

7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 minutes

0

1

2

3

4

5 50:500

DNOP

25 26 27 28 29 30 minute

s

0

50

100

150

200 kCounts

DINP

DIDP

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

0

1

2

3

4

543

DEHP in oils & fats

ITERG data, 2009

mg/kg

20

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

3-MCPD esters & glycidol esters

21

Production process (deodorisation)

Regulation for free 3-MCPD

(EC) n°1881/2006 – contaminants in foodstuffs (20 µg/kg in soya sauce)

Regulation for 3-MCPD esters

EFSA statement – related to 3-MCPD esters (2008)

EFSA Scientific Report on 3MCPD occurrence in food in Europe (2013)

Risks Methods Regulation

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

H2C

H2C

HC

O

AG

H2C

H2C

HC

O

OH

H2C

H2C

HC

OH

OH

Cl

H2C

H2C

CH

AG

AG

Cl

H2C

H2C

CH HO

Cl

AG

« free » 3-MCPD

Glycidol

3-MCPD monoesters « bound »

Glycidol esters (GEs)

3-MCPD diesters « bound »

49 7 14

3-MCPD esters & glycidol esters Structure

22

Hrncirik, OVID meeting, 2011

7 fatty acids

C12 - C14 C16 - C18

C18:1 - C18:2 C18:3

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Occurrence of 3-MCPD in food

EFSA Journal 2013; 11(9):3381

1 235 analytical results from 14 European countries 11 food groups

No distinction in the data analysis between unbound 3-MCPD and 3-MCPD esters

Vegetable fats and oils refined [MB (LB – UB)] = [530 (460 - 600) μg/kg]

Vegetable fats and oils unrefined [250 (200 - 310) μg/kg]

Walnut oils [4 750 (4 740 - 4 750) μg/kg]

Margarine and similar products [1 500 (1 480 – 1 530) μg/kg]

High single values reported Infant formulae powder (300 μg/kg, N = 1) & Follow-on formulae powder (800 and 1 200 μg/kg, N = 2)

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

3-MCPD esters & glycidol esters Analysis strategy

24

Direct methods Indirect methods

Pros No chemical reaction Individual quantification of species

Standards available Good sensitivity

Cons

Standards not available (60-70 different species) Expensive tools: HPLC/MS/MS Purification step needed

Reactivity of the species (glycidol & MCPD) Global quantification of glycidol esters and 3-MCPD esters

Standards AOCS-JOCS Cd 28-10 for GEs From Masukawa et al., JAOCS, 2010

AOCS Cd 29a-13 for 2- and 3-MCPD & GEs From Ermacora & Hrnirick, JAOCS, 2012

AOCS Cd 29b-13 for 2- and 3-MCPD & GEs From Kuhlmann, EJLST, 2011

AOCS Cd 29c-13 for 3-MCPD & GEs From DGF C-VI 18, 2010 & Weisshar, 2008

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013 25

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

3-MCPD esters & Glycidol esters Correlation between DAG & 3MCPD

26

0 2 4 6 8 10 12 14

Refined sunflower oil

Refined rapeseed oil

Refined peanut oil

Refined grapeseed oil

Walnut oil

Commercial mix of 4 refined oil

Refined palm oil

Crude Shea

Refined Shea

Hydrogenated Palm oil

Hydrogenated soyabean oil

DAG

3MCPD

Joffre et al. (2009)

Difficult to find a correlation between DAG & 3-MCP esters (Lack of information concerning chloride content may explain these results)

3-MCPD content

in mg/kg

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013

Conclusions

• Research of contaminants is part of multiple controls conducted by fat and oil industry to fulfill the EC regulation n°1881/2006.

• In the absence of regulation, the detection of contaminants must be addressed in partnership with authorities according to the toxicity of molecules.

• Risks are rather limited due to the efficient elimination during oil-refining steps

• However some contaminants can be formed during the production process of vegetable oils such as 3-MCPD & glycidyl esters.

27

PIPOC 2013 – Kuala Lumpur – 19-21 November 2013 28

A special thanks to….

ITERG Analysis Department team