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1 PIPOC 2013 – Kuala Lumpur – 19-21 November 2013
Florence LACOSTE ITERG (Pessac, France)
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
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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0
1
2
3
4
543
DEHP in oils & fats
ITERG data, 2009
mg/kg
20
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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
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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
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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)
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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
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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
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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
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A special thanks to….
ITERG Analysis Department team