MCPDs Esters & Glycidyl Esters: Validation of the Analytical Method and Occurrence in Vegetable Oils

Transcription

1 MCPDs Esters & Glycidyl Esters: Validation of the Analytical Method and Occurrence in Vegetable Oils Florence LACOSTE, ITERG Recherche. Innovation. Qualité SISSG Workshop on 3-MCPD in Vegetable Oils UDINE, 8th June

2 Agenda Chemical Structures EFSA Scientific Opinion on MCPDs Esters & Glycidyl Esters Methods of analysis & Standardization In-house Validation of the Analytical Procedure Occurrence in Vegetable oils FEDIOL Position

3 Chemical structures EFSA Journal 2016;14(5): monochloro-propanol-1,2-diol (3-MCPD) Glycidol Esters of 3-MCPD (MCPDE) and esters of glycidol (GE)

4 EFSA Scientific Opinion EFSA Journal 2016;14(5):4426 CONTAM Panel conclusions: GE, 3-MCPDE & 2-MCPDE are formed during the deodorisation step of edible oil refining, from the reaction of chloride present naturally in the oil. 3-MCPDE, 2-MCPDE & GE were found at the highest levels in palm oil, but most vegetable oil contain substantial quantities. 3-MCPDE, 2-MCPDE & GE are hydrolysed to their respective free forms in the gastrointestinal tract. The experts confirmed that the toxicity of 3-MCPD esters should be considered equivalent to that of the parent compound. 3-MCPD & 2-MCPD and glycidol bound as esters are determined by the methods used for the free compounds after chemical cleavage of the ester bonds. Average exposure to 3-MCPDE & GE for young age groups including adolescents is a potential concern for health.

5 EFSA Scientific Opinion EFSA Journal 2016;14(5):4426 CONTAM Panel recommendations: The inclusion of samples from all food groups potentially contaminated by MCPDs and glycidol is recommended. Both forms, free and ester bound, should be measured. Further studies are recommended on the release of the free compounds from mono- & diesters to clarify the metabolic fate of the compounds. Analysis of the enantiomeric composition of 3-MCPDE is recommended for foods containing high levels of these compounds. The mechanism of action and the long-term toxicity of 2-MCPD need to be investigated to provide a basis for quantitative risk assessment.

6 Methods & Standardization Pros Direct methods No chemical reaction Individual quantification of species Standards available Good sensitivity Indirect methods Cons Standards not available Expensive tools: HPLC/MS/MS Purification step needed Reactivity of the species Global quantification of species Standards AOCS-JOCS Cd 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

7 Standardization work : AOCS Methods Samples: 2013 AOCS Collaborative Study Sample 1: Refined rapeseed oil spiked with 3-MCPDE and GE Sample 2: Refined rapeseed oil spiked with GE Sample 3: Refined rapeseed oil spiked with 3-MCPDE Sample 4: Refined rapeseed oil spiked with 3-MCPDE and GE Sample 5: Refined rapeseed oil Sample 6: Refined palm oil Sample 7: Refined palm oil spiked with 3-MCPDE and GE Participation: AOCS Cd 29a laboratories AOCS Cd 29b laboratories AOCS Cd 29c-13 9 laboratories

8 Standardization work : AOCS Methods 2013 AOCS Collaborative Study 3-MCPD - AOCS METHODS - MEAN VALUES (MG/KG) Cd 29a-13 Cd 29b-13 Cd 29c ,0 1,5 1,0 0,5 5,82 5,93 5,86 3,41 3,46 3,39 1,95 1,93 2,09 0,49 0,51 0,47 0,50 0,50 0,51 Sample 2 Sample 5 Sample 6 Sample 7 Sample 3 2-MCPD- AOCS METHODS - MEAN VALUES (MG/KG) Cd 29a-13 Cd 29b-13 1,61 1,73 0,93 0,95 0,32 0,32 0,21 0,24 0,22 0,24 0,26 0,27 0,22 0,24 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 G LYCIDOL - AOCS METHODS - MEAN VALUES (MG/KG) Cd 29a-13 Cd 29b-13 Cd 29c-13 5,34 4,69 5,27 4,92 4,75 4,99 1,46 1,34 1,42 0,28 0,18 0,44 0,32 0,17 0,14 Sample 3 Sample 5 Sample 2 Sample 4 Sample 7 Comparable results for the 3 indirect methods

9 Standardization work : AOCS Methods 2013 AOCS Collaborative Study 3MCPD - AOCS Methods Reproducibilty (Horrat) Glycidol - AOCS Methods Reproducibilty (Horrat) 4,0 4,0 3,0 3,0 2,0 2,0 1,0 1,0 Sample 2 Sample 5 Sample 6 Sample 7 Sample 3 Sample 4 Sample 1 Sample 2 Sample 5 Sample 6 Sample 7 Sample 3 Sample 4 Sample 1 Cd 29a-13 Cd 29b-13 Cd 29c-13 Cd 29a-13 Cd 29b-13 Cd 29c-13 2MCPD - AOCS Methods Reproducibilty (Horrat) 4,0 3,0 During 2013 collaborative study 2,0 1,0 Methods did not give reliable results for Glycidol concentrations < 1 mg/kg Sample 2 Sample 3 Sample 5 Sample 4 Sample 1 Sample 6 Sample 7 Cd 29a-13 Cd 29b

10 AOCS Cd 29b-13 - Assay B - 3-MCPD/2-MCPD Determination 3-MCPD-d5-diC16 3-MCPD esters GE NaOH/MeOH -18 C/12h glycidol-d5 3-MCPD-d5 3-MCPD Glycidol NaBr acid 3-MBPD-d5 3-MCPD-d5 3-MCPD 3-MBPD To quantify % of 3-MCPD conversion into Glycidol 3-MCPD content Kuhlmann, EJLST, 113: (2011) 10

11 AOCS Cd 29b-13 - Assay A - Glycidol determination 3-MCPD esters GE GE-d5 NaOH/MeOH -18 C/12h Glycidol 3-MCPD Glycidol Glycidol-d5 NaBr acid 3-MBPD 3-MCPD 3-MBPD 3-MBPD-d5 3-MCPD conversion Glycidol quantified in assay B GE content Kuhlmann, EJLST, 113: (2011) 11

12 Mean Area (n=2) Mean Area (n=2) Mean Area (n=2) In-house Validation Impact of the Acidity of the Oil Impact of Acidity on 3-MCPD Area Impact of Acidity on Glycidol Area MCPD d5 3-MCPD Glycidol d5 Glycidol Free Fatty acids (%) Free fatty acids (%) Impact of Acidity on 2-MCPD Area MCPD d5 2-MCPD Free Fatty acids (%) For samples with acidity > 5 % conc (NaOH) needs to be increased (3 x) 12 -

13 In-house Validation - Sensitivity Content in mg/kg 3-MCPD 2-MCPD Glycidol LOD (ratio S/N =3) LOQ (ration S/n = 10) Content in mg/kg 3-MCPD 2-MCPD Glycidol Concentration (mg/kg) 90 0,103 0,152 Recovery % 103 % 99 % 105 % Reproducibility RSD % 4,9% 2,7% 2,9% Horrat Reprod % 0,22 0,12 0,14 Limit of quantifications fixed at 0,10 mg/kg for 3MCPD - 2MCPD - Glycidol 13 -

14 Recovery (%) Recovery (%) Recovery (%) In-house Validation Recovery MCPD- Recoveries MCPD - Recoveries Glycidol - Recoveries Concentration (mg/kg) Refined Palm Oil Crude Rapeseed Oil Refined Sunflower Oil Recoveries between 70 % and 120 %

15 In-house Validation Precision Crude Rapeseed Oil 3MCPD - Precision of the Method 2MCPD - Precision of the Method 6% 6% 5% 5% 4% 4% 3% 3% 2% 2% 1% 1% 0% 9 0,21 1,13 5,58 16,77 0% 0,10 0,25 1,20 5,89 17,71 Repeatability RSD % Reproducibility RSD % Repeatability RSD % Reproducibility RSD % 6% 5% 4% 3% 2% 1% 0% Glycidol - Precision of the Method 0,15 0,37 1,46 7,18 21,60 In-house precision data are very good even at the level of 0,1 mg/kg Incertainty based on the AOCS Standard data : 30 % of the concentration with a minimum of 0,4 mg/kg Repeatability RSD % Reproducibility RSD % 15 -

16 Occurrence in Vegetable Oils EFSA Journal 2016;14(5):4426 Evolution across the years of the average level (µg/kg) of 3-MPD & 2-MCPD from esters and glycidol from esters in different types of oils and fats 16 -

17 brute raffinée Occurrence of 3-MCPDE & GE in vegetable oils brute raffinée Screening ITERG ,0 8,0 3MCPD (mg/kg) 1,2 1,0 GLYCIDOL (mg/kg) 7,0 6,0 5,0 grapeseed 0,8 0,6 grapeseed 4,0 3,0 2,0 1,0 sunflower rapeseed 0,4 0,2 sunflower rapeseed Industrial specidifcation 3MCPD = 2 mg/kg Industrial specification glycidol = as low as possible 17 -

18 mg/kg Occurrence of 3-MCPD esters in vegetable oils mg/kg SOJA BRUTE SOJA RAFFINÉE PALME BRUTE PALME RAFFINÉE MAÏS RAFFINÉE Screening ITERG MCPD Sunflower & Rapeseed 3-MCPD - Peanut & Grapeseed & Other 2,5 6,0 2,0 1,5 1,0 Sunflower Oil 0,88 mg/kg Rapeseed Oil 0,21 mg/kg 5,0 4,0 3,0 2,0 Peanut Oil 0,20 mg/kg Grapeseed Oil 2,20 mg/kg 1,0 0,5 Very low content for rapeseed oils & peanut oils & soya oils 3MCPE > 1 mg/kg for 37 % of the sunflower oils & 100 % of the grapeseed oils 18 -

19 mg/kg Occurrence of 2-MCPD esters in vegetable oils mg/kg SOJA BRUTE SOJA RAFFINÉE PALME BRUTE PALME RAFFINÉE MAÏS RAFFINÉE Screening ITERG MCPD - Sunflower & Rapeseed 2-MCPD - Peanut & Grapeseed & Other 1,4 3,0 1,2 2,5 1,0 0,8 0,6 0,4 Sunflower Oil 0,48 mg/kg Rapeseed Oil 0,11 mg/kg 2,0 1,5 1,0 0,5 Peanut Oil 0,12 mg/kg Grapeseed Oil 1,18 mg/kg 0,2 Very low content for rapeseed oils & peanut oils & soya oils Pattern of concentrations similar to 3-MCPD concentrations 19 -

20 mg/kg Occurrence of Glycidol esters in vegetable oils mg/kg SOJA BRUTE SOJA RAFFINÉE PALME BRUTE PALME RAFFINÉE MAÏS RAFFINÉE Screening ITERG 2015 Glycidol - Sunflower & Rapeseed Glycidol - Peanut & Grapeseed & Other 0,6 0,5 0,4 Sunflower Oil 0,26 mg/kg 3,0 2,5 2,0 Grapeseed Oil 1,08 mg/kg 0,3 0,2 Rapeseed Oil 0,10 mg/kg 1,5 1,0 Peanut Oil 0,25 mg/kg 0,5 0,1 Pattern of concentrations different from MCPD concentrations Glycidol > 0,5 mg/kg for 6 % of the sunflower oils & 100 % of the grapeseed oils 20 -

21 2MCPD ESTERS (MG/KG) GLYCIDOL ESTERS (MG/KG) Correlation between MCPD esters content in vegetable oils 1,4 1,2 1 0,8 Sunflower Oils y = 0,5837x R² = 0, ,8 0,6 Sunflower Oils 0,6 0,4 0, ,5 1 1,5 2 2,5 3MCPD ESTERS (MG/KG) 0,4 0, ,5 1 1,5 2 2,5 3MCPD ESTERS (MG/KG) Very good correlation between 3-MCPD & 2-MCPD (sunflower : ratio 37/63) No correlation between 3-MCPD and Glycidol 21 -

22 FEDIOL POSITION FEDIOL Q&A on 2-MCPDE &3-MCPDE & GE (24 February 2016) Vegetable oil and fat refiners: have been actively working on effective mitigation technologies; have collected a substantial amount of data according to validated test methods; have, as a priority, committed to reduce the levels of GE of all oils to 1 mg/kg. Mitigation on 3MCPDE has proven to require more time to implement, but FEDIOL members will continue researching and implementing ways to prevent and reduce these substances as prompted by the recent EFSA Opinion. 22 -

23 Pour nous contacter : ITERG 11 rue Monge Parc Industriel Bersol PESSAC Tél : iterg@iterg.com Recherche. Innovation. Qualité 23