The Development and Application of the Automated Method for the Quantification of 3-MCPD-,2-MCPD-, and Glycidylesters in Edible Oils and Fats Ralph Zwagerman, 21-06-2017, 3-MCPD Symposium, Berlin 1
Motivation for Development o Insufficient capacity o Problems with robustness o Necessity of processing optimization o Analysis time o Goals of automatization Decreasing analysis time Minimizing contact time Improving robustness Fast QC Service 2
Current Recommended Indirect Methods Reaction time Reaction conditions Components quantified Automation Problems Remarks AOCS Cd29a-13 AOCS Cd29b-13 AOCS Cd29c-13 16 hours 16 hours 5.5 min max Acidic, 40 C Alkaline, -25 C Alkaline, RT 3-MCPDe, 2-MCPDe and GE Precipitation of salts Solvent evaporation steps 3-MCPDe, 2-MCPDe and GE Very low temperatures Solvent evaporation steps 3-MCPDe and GE (Calculated) No direct GE detection No 2-MCPDe detection Solvent evaporation steps Basis for automation 3
Adaptions for automation Step AOCS Cd 29c-13 Automated method Sample Two Aliquots (A & B) One Aliquot Reaction alkaline, RT alkaline, 5 C Glycidol conversion 1) Sample clean-up Extraction Derivatives Analysis A: NaCl solution B: NaBr solution 3 x extraction + drying none iso-octane, after solvent evaporation PTV Splitless, GC-MS NaBr solution n-hexane PTV Splitless, GC-MS/MS 4
The Automated Method First Version Published in July 2016: Eur. J. Lipid. Sci. Technol. 2016, 118, 997-1006 Adapted version of AOCS Cd 29c-13 3-in-1 Analysis method Hardware: Thermo Scientific Triplus RSH / Trace 1300 / TSQ8000 Everything automated after weighing the sample 21 samples / 24 hours or 4 samples / 4.5 hours (1 series) Operated by 1 FTE 5
Chemistry of the automated method 3-MCPD Glycidol Conversion of esters to free 3-MCPD, Glycidol and 2-MCPD Release of FAMEs 2-MCPD 6
Chemistry of the automated method At -25 C No conversion (Kuhlmann, Eur. J. Lipid. Sci. Technol 2011, 113,335-344) Overestimation of Glycidylesters 7
Chemistry of the automated method 3-MBPD 2-MBPD 8
The 13 C-correction method m/z 240: m/z 243: m/z 245: Q1: Q2: Q3: m/z 147: Induced by: Glycidol 3-MCPD m/z 149: Induced by: 3-MCPD- 13 C 3 Implicit Hydrogens have been omitted m/z 150: Induced by: Glycidol-d 5 9
The 13 C-correction method Glycidol- 13 C 3 Result (ppm) = Area 3 MBPD 13 C 3 Area 3 MBPD d 5 glycidol-d 5 amount (µg) 1000 sample (mg) 3-MCPD induced glycidol (ppm) = 3 MCPD Result (ppm) C 3 MCPD 13 C 3 (ppm) glycidol- 13 C 3 Result (ppm) Glycidyl ester induced glycidol (ppm) = (Glycidol result 3-MCPD induced glycidol) Recovery factor 10
Optimization of Method Step First version Second version Sample 100-120 mg 100-120 mg Solvents Transest. 100 µl internal standard 100 µl toluene 200 µl of 0.5M NaOCH 3 /MeOH 100 µl internal standard 100 µl toluene 200 µl of 0.5M NaOCH 3 /MeOH Glycidol Conversion FAME extraction Derivatization 1000 µl 200g/L acidified NaBr sol. 2x 600 µl hexane 100 µl PBA solution 10 min at 80 C + shaking 1000 µl 650g/L acidified NaBr sol. 2x 600 µl hexane 100 µl PBA solution No heat, vortexing 10s Extraction 600 µl of hexane 600 µl of hexane 11
Days of operation Sample Capacity Increase Code fully re-written Customized Cooltray + Agitator, 6 Positions 20 positions 32 samples / 24 hours: 44% Faster than Original method 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Time vs. number of Samples 147 samples 264 samples 0 50 100 150 200 250 # of Samples prepared 12
Sample Capacity Increase First Version Second version Spike samples 2 2 QC samples 1 1 Unknown samples 3 17 Prep time 2 hrs, 35 min 6 hrs, 50 min GC analysis (inj to inj) 17 min 17 min Total time 4 hrs, 35 min 12 hrs, 54 min Per sample 69 min 38 min Single Sample n.a. 56 min Smart programming reduces reagent and time waste Samples are more clustered 13
Robot set-up Six automatically selectable syringes Three sample trays, one temperature controlled Agitator, vortexer and fast wash station 14
Robot set-up 15
Global Method Uniformity USA: 1 system in operation (Q3 2017)... Production location Sales / Regional office Bulk oil terminal........ The Netherlands: 2 systems in operation (2013-2016)..... Malaysia: 1 system in operation (2017). China: 1 system in operation (planned 2018).. Annual turnover > 1 billion 6 production locations / 9 sales offices 3 million MT of refining capacity / Over 1,000 employees 16 6
Global Control Sample 3-MCPDe (ppm) avg SD n remark The Netherlands 0.55 0.02 97 Malaysia 0.52 0.02 28 USA 0.54 0.03 5 Insufficient data SGS Hamburg 0.55-2 Glycidyl esters (ppm) avg SD n remark The Netherlands 0.06 0.02 97 Malaysia 0.07 0.02 28 USA 0.03 0.03 5 Insufficient data SGS Hamburg <0.10-2 17
Global Control Sample 2-MCPDe (ppm) avg SD n remark The Netherlands 0.30 0.02 97 Malaysia 0.32 0.02 28 USA n.a. n.a. - Not reported SGS Hamburg 0.28-2 Initial comparison looks very good Not enough data for US yet to compare Next: Extended Validation including all departments 18
20140103-098 20140113-183 20140124-138 20140215-120 20140307-034 20140409-044 20140512-128 20140601-015 20140622-052 20150602-041 20150704-068 20150801-120 20150823-118 20150921-137 20151010-113 20151024-126 20151118-162 20151220-120 20160112-164 20160125-176 20160208-231 20160225-129 20160304-025 20160327-155 20160422-106 20160521-013 20160619-055 20160714-173 20160809-115 20160903-105 20160923-013 20161009-066 20161024-090 20161107-189 20161128-003 20161208-128 20161225-014 20170108-006 20170205-020 20170311-086 20170407-143 (ppm) Practical Implications 6 Mitigated refined Palm Oil 5 4 2014 2015 2016 2014 2015 2016 2017 3 2 1 1 ppm 0 3-MCPDe Glycidylesters 19
20 (ppm) 1 ppm 0 1 2 3 4 5 6 20141202-006 20150228-003 20150513-002 20150726-081 20151007-007 20151219-002 20160109-019 20160116-018 20160123-051 20160129-201 20160203-249 20160210-209 20160216-103 20160222-216 20160226-018 20160304-140 20160311-009 20160319-001 20160327-130 20160412-026 20160424-096 20160807-061 20160824-033 20160905-216 20160921-162 20160928-142 20161006-050 20161011-164 20161016-120 20161021-197 20161029-008 20161107-026 20161117-248 20161202-047 20161215-216 20161224-049 20170104-179 20170112-179 20170125-046 20170206-066 20170219-096 20170227-049 20170304-107 20170312-076 20170317-078 20170325-043 20170404-144 20170415-080 20170421-083 20170429-076 Mitigated palm olein IV64 2016 2017 2015 Practical Implications
Further development Goal: Decreasing LOD/LOQ Improving accuracy at current LOQ levels Large Volume Injection (2µL 50-100µL?) Removal of large quantities of PBA from GC system experimental injector Impurities in internal standard might limit LOD decrease internal standard to counteract contribution cheaper Development still ongoing 21
Thank you for your attention 22