Indirect Determination of Bound Glycidol & MCPD in...
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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
2•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
� 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 ?
3•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
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
4•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
� 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
5•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
� + 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”
6•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
� + 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
7•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
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+
8•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
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)
9•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
•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
10•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
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*
11•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
� 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
12•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
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”
Verification of the new method
13•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
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
Verification of the new method
� 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
14•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
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
Verification of the new method
� 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
15•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
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
Verification of the new method
� 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
?
16•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
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
17•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
� 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
18•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
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