second collaborative study - results...

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Methods for the Determination of 3-MCPD-fatty acid esters

Second Collaborative Study

Dr. Angelika Preiß-WeigertDr. Angelika Preiß-Weigert

Angelika Preiß-Weigert 18 May 2010 3rd Meeting AOCS Expert Panel on Process Contaminants page 2

December 2007 Federal Ministry of Food, Agricultureand Consumer Protection

established two Working Groups on 3-MCPD-Fatty Acid Esters in Refined Oils

1 „Working Group on Research and Measures“2 „Working Group for Analytical Aspects“

to be chaired by the BfR (Federal Institute for RiskAssessment)

Mandate to test the analytical method developed in CVUA Stuttgart in a collaborative study

Weißhaar et al, EJLST, 2008, 110, 183-186


First BfR Collaborative Study (2008)Results of Method Validation Study and Proficiency-Test

0

2

4

6

8

10

12

Geh

alt 3

-MC

PD

[mg/

kg]

Variante A (20) Variante A*/B (10) Variante C (4)R ih 1 Oli Oli

Rapsöl Olivenöl Distelöl Traubenkernöl FettRape Seed Oil Oilve Oil Safflower Oil Grape Seed Oil Soild Fat

Original methodCVUA Stuttgart (20)

Modified methods(6+4)

In-house methods(4)

3-M

CPD

(mg/

kg)


First Collaborative Study (2008) Conclusions

Working Group for Analytical AspectsMethods providing chloride ions lead to excessive 3-MCPD fattyacid values. It was concluded that chloride ions in combination with an unknown substance x can react to 3-MCPD during the analysis.• this substance x could be esterified glycidyl.

MRI and DGF (March 2009, DGF C-III 18 (09))published this method for the determination of 33--MCPD FA Esters and MCPD MCPD FA Esters and MCPD formingforming substancessubstances……

BfR positionDue to the different toxicological effects of 3-MCPD and glycidolthe tested method is not an appropriate tool for risk assessmentpurposes.


Strategy for the Development of BfR methods

General General ObjectivesObjectivesto determine exclusively 3-MCPD FAEto offer a sufficient robustness for adoption in routine labs

DirectDirect MethodMethod versusversus IndirectIndirect MethodMethod??Direct methods like LC-MS/MS of the esters

- guarant high specificity- require the availability of the complete „set of standard materials“- require very low LOD/LOQ for each single 3-MCPD FAE- offer a limited robustness

Indirect methods like GC-MS of the free 3-MCPD - offer an increased robustness- involve hydrolysis and derivatization- might induce chemical modifications of the analyte

Final Final DecisionDecision forfor thethe developmentdevelopment of of indirectindirect methodsmethodsHowever, with the additional obligation that severalseveral methodsmethods should betested applyingapplying different different typestypes of of hydrolysishydrolysis.


Requirements fulfilled by the BfR methods

1. Without addition of sodium chloride

2. Hydrolysis of the (di)esters• alkaline• acidic

3. Derivatisation of 3-MCPD with• Phenylboronic acid• Heptafluorbutyric Anhydride

4. Increase in robustness • additional cleanup / change of solvent

5. Increase in practicability• reduction of volumes


Characteristics of the methods

H2SO4

Fat/Oil sample (100-200 mg), Addition of d5-3-MCPD

2 h + 16 h

GC-MS

phenylboronic acid

NaHCO3

Hydrolysisreagent

Derivatisationreagent

BfR Method 9 BfR Method 10

NaOCH3

9-10 min

(NH4)2SO4 / H2SO4

HFBA

NaOCH3

9-10 min

(NH4)2SO4 / H2SO4

BfR Method 8

phenylboronic acid

Stopreagent


Germany 29 labsSwitzerland 2 labsAustria 1 labItaly 2 labsBelgium 1 labThe Netherlands 1 lab

Participants of the Second Collaborative Study

40 laboratories received samples36 laboratories reported results

2 laboratories applied 4 different methods1 laboratory applied 3 different methods2 laboratories applied 2 different methods

Official control laboratories13 labs (all from Germany)

Free market labs and industrial:23 labs


3-MCPD-Content

Quality control sample 3.0 ± 0.5 mg/kg(solid fat (first coll.study))

Vegetable oil ~ 0.2 mg/kg Vegetable fat (hydrogenated) ~ 1.0 mg/kgFrying oil (non-hydrogenated) ~ 1.6 mg/kgVegetable fat (hydrogenated) ~ 3.2 mg/kgGrape seed oil ~ 3.9 mg/kg

Sample materials Second Collaborative Study


48 datasets- BfR-Method 8 6 Labs- BfR-Method 9 27 Labs- BfR-Method 10 6 Labs- in-house methods 9 Labs

⇒ Validation of „BfR Method 9“ 27 DatasetsProficiency Test 48 Datasets

Evaluation data


3-MCPD-fatty acid esters [mg/kg] in a solid fat sample

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8,00

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13,00

Nummer Serie

3-M

CPD

(mg/

kg)

original method CVUA Stuttgart modified method CVUA StuttgartIn-house methods BfR-Method 10BfR-Method 9 BfR-Method 8respecive mean values 2.Collaborative Study - Part I, all methods

First Collaborativestudy

BfR-Methods inhouse validation Second Collaborativestudy


Results Second Collaborative Study - method validation -“BfR-Method 9” Statistical data for all sample materials (ISO 5725-2 / -3)

L_Oel B_Fett F_Oel P_Fett T_Oel Cont

Mean value of 3-MCPD [mg/kg] 0.30 0.91 1.72 3.46 4.04 2.96*)

Rel. SD according to Horwitz [%] 19.17 16.23 14.74 13.27 12.96 13.59

Rel. reproducibility SDsR (rel.sR )[%] 55.22 22.87 16.38 15.77 15.26 12.82

Number of datasets(after elimination of outliers) 19 25 25 25 25 23

Total of datasets 27**) 27 27 27 27 27

Number of outliers ***) 2 1 1 1 1 3

Ratio sr/sR 0.29 0.50 0.49 0.47 0.45 0.48

HorRat 2.9 1.4 1.1 1.2 1.2 0.9*) the concentration of the control sample was previously specified to be 3.0 ± 0.5 mg/kg **) five laboratories stated values as <LOD or <LOQ***) one data set was completely excluded from evaluation due to the laboratory‘s systematic deviation from the method


all methods 48 datasets

BfR Method 9 27 datasetsBfR Method 8 6 datasets

Second Collaborative Study (2009)Method Validation Study and Proficiency-Test

mean values and reproducability standard deviations of the different sampel materials obtained for "BfR Method 8", "BfR Method 9"

and the proficiency test (all methods)

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0,50

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sample materials

3-M

CPD

(mg/

kg)

all methods (n=47) BfR-Method 9 (n=26) BfR-Methode 8 (n=6) R ih 4 R ih 5 R ih 6

Plant Oil Vegetable Fat Frying Oil Solid plant fat Grape seed oil Control sample


Results Second Collaborative StudyProficiency Test: Participant‘s z-Scores for the Frying Oil sample

mean value 1,69 mg/kg

-3,50

-3,00

-2,50

-2,00

-1,50

-1,00

-0,50

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

LC00

32

LC00

18

LC00

29

LC00

01

LC00

21

LC00

08

LC00

04

LC01

11

LC02

25

LC00

13

LC03

11

LC00

30

LC00

11

LC00

39

LC02

11

LC02

13

LC03

25

LC03

02

LC00

16

LC01

02

LC00

31

LC02

03

LC00

03

LC00

28

LC02

02

LC00

12

LC01

01

LC00

09

LC00

19

LC00

27

LC00

22

LC00

24

LC00

02

LC00

40

LC00

25

LC00

38

LC00

35

LC00

17

LC00

06

LC00

05

LC00

20

LC00

34

LC00

07

LC00

33

LC04

32

LC00

10

LC00

15

LC00

26

Labcode

z-Sc

ore

BfR Method 8 BfR Method 10BfR Method 9 In-house methods


Results Second Collaborative StudyProficiency Test: Participant‘s z-Scores for the Grape Seed Oil sample

BfR Method 8 BfR Method 10BfR Method 9 In-house methods

mean value 3,39 mg/kg

-3,50

-3,00

-2,50

-2,00

-1,50

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1,50

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3,50

LC00

32LC

0018

LC00

08LC

0021

LC00

24LC

0004

LC00

11LC

0311

LC01

11LC

0029

LC00

28LC

0302

LC00

19LC

0213

LC00

01LC

0020

LC00

13LC

0225

LC00

17LC

0211

LC00

16LC

0102

LC00

39LC

0027

LC00

05LC

0009

LC00

02LC

0012

LC00

22LC

0035

LC02

02LC

0040

LC02

03LC

0432

LC03

25LC

0025

LC00

31LC

0003

LC00

30LC

0033

LC00

07LC

0006

LC00

34LC

0010

LC01

01LC

0038

LC00

15LC

0026

Labcode

z-Sc

ore


Conclusions of Second Collaborative Study

ResultsResults of of thethe MethodMethod Validation and Validation and thethe ProficiencyProficiency Test Test 1. „BfR Method 9“ delivered reproducible results, which are

comparable with the results of all applied „in-house“ methodsacidic hydrolysis „BfR Method 8“HFBA derivatization „BfR Method 10“

2. more than 90% of the laboratories reached a z-score < |2| for all five samples


Third Collaborative Study in fat containing foodsSchedule

Types of Food Infant formulaMargarineHazelnut or Chocolate Spread

The work for preparing the study - like method development and homogeneity studies - is still ongoing

Shipment of samples October 2010Report of results December 2010Finalization of evaluation March 2011

You are kindly invited to participate in this study.You are kindly invited to participate in this study.

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Thank you for your attention

Angelika Preiß-WeigertFederal Institut for Risk AssessmentThielallee 88-92 D-14195 BerlinTel. 0 30 - 8412 - 0 Fax 0 30 - 8412 [email protected] [email protected]


Second Collaborative Study - Proficiency Test -Overview of z-Scores for all samples and for each laboratory

BfR Method 8 BfR Method 10BfR Method 9In-house methods

z-Scores of all sample materials

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LC00

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LC00

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LC02

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LC04

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LC00

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LC00

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LC00

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LC00

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LC01

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LC01

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LC00

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LC00

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LC00

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LC00

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LC00

26

LC00

27

LC00

30

LC00

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LC00

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LC00

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LC00

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LC00

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LC00

40

LC01

01

LC02

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LC02

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LC02

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LC02

25

LC00

08

LC00

15

LC00

31

LC03

02

LC03

11

LC03

25

Labcode

z-Sc

ore

B_Fett L_Oel F_Oel P_Fett T_Oel


Methode „BfR 8“saure Hydrolyse. Phenylboronsäure-Derivatisierung

1.8 ml H2SO4

100 mg Probe in 0.5 ml tert. Butylmethylether 20 µl d5 MCPD

2 h + 16 h

GC-MS

PBA Derivatisierung in Aceton

2 x 1 ml Hexan

0.5 ml NaHCO3

3 x 1 ml Cyclohexan

2 – 3 min

N2. Lösen in i-Oktan


Methode „BfR 9“Umesterung im Alkalischen. Phenylboronsäure-Derivatisierung


9 – 10 min

GC-MSPBA – Derivatisierung

in Diethylether

2 x 1 ml Hexan

0.5 ml (NH4)2SO4 / H2SO4

2 x 0.6 ml EtOAc

2 – 3 min N2. 40 CLösen in Aceton

0.2 ml NaOCH3 - Lösung


Methode „BfR 10“Umesterung im Alkalischen. Heptafluorbuttersäure-Derivatisierung

0.2 ml NaOCH3 - Lösung


HFBA – Derivatisierungin i-Oktan GC-MS

2 x 1 ml Hexan

0.5 ml (NH4)2SO4 / H2SO4

2 x 0.6 ml EtOAc

20 min

250 µl H2O

10 min

second collaborative study - results...

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