GC-MS Evolutionary Technology & the Art of POPs Analysis Chris Cheah Senior Laboratory Manager - SEA The world leader in serving science
Persistent Organic Pollutants Proprietary & Confidential 2
Dioxin Incidents” – Huge Economical Impact Proprietary & Confidential 3
“Dioxin Incidents” – Huge Economical Impact • Dioxin and PCB incidents Repeated “Dioxin” cases from only recent years: • … are expensive. • 1998 Milk from citrus pulp pellet feed • Egg production enterprise with 5 million hens • 1999 Belgian PCB/dioxin in eggs, poultry would cost in excess of $30 million. • 1999 Clay and zeolithes for feed • Broiler enterprise producing 3 million broilers • 2000 Choline chloride per week would exceed $85 million. • 2002 “Carbosan Copper” • Times Beach, Missouri, road dust covered by oil spread. The cleanup cost with a total of • 2004 Potato pulp $110 million. • 2005 Hydrochloric acid • Sources to the Environment • 2007 Indian Guar Gum thickener • 2008 Irish Pork Meat • Combustion processes • 2008 Italian Mozarella Cheese … • Landfill sites • ... still on today around Naples. • Improper waste managment • Sewedge sludge • Accumulation in Food Chain • Regular control required • In environment, feed, and food Proprietary & Confidential 4
Global Regulation & Response Proprietary & Confidential 5
Global Response - Stockholm Convention Proprietary & Confidential 6
EU Commission Regulation – Past/Present Proprietary & Confidential 7
EU Commission Regulation – GC-MS/MS Requirements Proprietary & Confidential 8
POPs Analysis Workflow Proprietary & Confidential 9
POPs Analytical Tools Proprietary & Confidential 10
DFS GC-HRMS: Gold Standard for Dioxins Thermo Scientific DFS Magnetic Sector GC-HRMS • Worldwide method compliance • EPA 1613 ,1668,1614 & others • Use PFK/PFTBA , lock mass trace, etc. • Analytical Performance • best Dioxin spec available • large ion source : ultimate sensitivity combined with highest robustness • Unique Dual GC configuration • References (10 years DFS!) • Worldwide use in Dioxin expert labs Proprietary & Confidential 11
Confirmation Analysis with High Resolution Mass peaks at Different Resolution Settings: Red: TCDD mass trace at R 1.000 (GC-MS/MS) Low Resolution TCDD Green: TCDD mass trace at R 10.000 (DFS) Blue: another chlorinated compound mass trace High Resolution Accurate mass SIM is used for Yet another chlorinated Confirmation Analysis compound Proprietary & Confidential 12
DFS Ion Source – Robustness by Size DFS ion source - designed for combining ultimate sensitivity with robustness, because sensitivity alone is not enough Proprietary & Confidential 13
DFS Common Instrument Control Software Makes the DFS as easy to operate as a Benchtop MS • Common User Interface known from DSQ, Polaris Q or LCQ/LTQ. • AutoTune • Resolution setting Proprietary & Confidential 14
2fg -100 fg TCDD on 60 m column RT: 21.36 - 26.69 x5 RT: 25.80 NL: 2.13E3 - zoomed - AA: 8787 m/z= 100 321.8876-321.9004 Relative Abundance MS 80 Dioxin_2-5-10-25- Inj. 1 RT: 25.22 50- 100fg_TCDD_1ul_0 60 AA: 4505 1 RT: 22.98 RT: 24.69 40 RT: 22.68 MA: 356 AA: 2042 AA: 181 RT: 24.30 20 AA: 651 0 NL: 1.93E3 RT: 25.81 m/z= AA: 8332 100 321.8876-321.9004 Relative Abundance MS ICIS Inj. 2 80 Dioxin_2-5-10-25- RT: 25.25 50- AA: 4356 60 100fg_TCDD_1ul_0 2 RT: 23.01 RT: 24.72 40 RT: 22.68 AA: 472 AA: 2250 AA: 165 RT: 24.32 20 AA: 843 0 NL: 2.04E3 RT: 25.81 m/z= AA: 8621 100 321.8876-321.9004 MS Relative Abundance 80 Dioxin_2-5-10-25- Inj. 3 50- RT: 25.22 60 100fg_TCDD_1ul_0 AA: 4231 3 RT: 23.00 RT: 24.69 40 AA: 440 RT: 22.66 AA: 2097 RT: 24.30 20 MA: 130 AA: 918 0 NL: 2.29E3 RT: 25.79 AA: 8982 m/z= 100 321.8876-321.9004 Relative Abundance MS Inj. 4 80 Dioxin_2-5-10-25- 50- RT: 25.23 100fg_TCDD_1ul_0 60 AA: 4343 4 RT: 22.99 RT: 24.70 40 RT: 22.68 AA: 419 AA: 2172 RT: 24.29 MA: 157 20 AA: 923 0 RT: 25.80 NL: 2.24E3 AA: 8955 m/z= 100 321.8876-321.9004 Relative Abundance MS ICIS Inj. 5 80 Dioxin_2-5-10-25- 50- RT: 25.24 100fg_TCDD_1ul_0 60 AA: 4324 5 RT: 22.98 40 RT: 24.71 AA: 392 RT: 22.71 RT: 24.30 AA: 1797 20 AA: 165 AA: 928 0 NL: 2.12E3 RT: 25.79 m/z= AA: 8448 100 321.8876-321.9004 Relative Abundance MS ICIS 80 Inj. 6 Dioxin_2-5-10-25- 50- RT: 25.23 60 100fg_TCDD_1ul_0 AA: 4063 RT: 22.97 6 RT: 24.70 40 AA: 515 RT: 22.66 AA: 2075 RT: 24.29 AA: 152 20 AA: 968 0 21.4 21.6 21.8 22.0 22.2 22.4 22.6 22.8 23.0 23.2 23.4 23.6 23.8 24.0 24.2 24.4 24.6 24.8 25.0 25.2 25.4 25.6 25.8 26.0 26.2 26.4 26.6 Time (min) Proprietary & Confidential 15
Dioxin & Furan Analysis D:\Demo\...\calib8290\100514PM\HRCC3 5/14/2010 7:25:30 PM RT: 21.75 - 59.62 RT: 56.25 NL: AA: 1199571 2.47E5 100 TIC MS Octa- ICIS DD/DF HRCC3 90 80 Hepta- DD/DF 70 RT: 52.55 Relative Abundance Hexa- AA: 591456 60 DD/DF Tetra- Penta- RT: 40.42 50 DD/DF AA: 895215 DD/DF RT: 25.84 RT: 32.04 40 AA: 517411 AA: 538684 RT: 44.30 30 AA: 643508 RT: 34.35 AA: 342445 20 RT: 45.37 10 AA: 119680 0 25 30 35 40 45 50 55 Time (min) Proprietary & Confidential 16
PBDE Brominated Flame Retardants (BFRs) PBDE Brominated Flame Retardants (BFRs) 8.42 100 8.17 8.17: BDE-17 8.42: BDE-28 3-BDE 50 m/z 405.803 0 9.83 10.50 100 9.60: BDE-49 9.64 4-BDE 9.64: BDE-71 10.09 9.83: BDE-47 50 m/z 485.711 10.09: BDE-66 10.50: BDE-77 0 11.13 Relative Abundance 12.76 100 11.64 11.13: BDE-100 5-BDE 11.34: BDE-119 12.51 11.64: BDE-99 50 m/z 563.622 12.51: BDE-85 12.76: BDE-126 0 13.00 13.85 13.00: BDE-154 100 Instrumental Parameters: 13.85: BDE-153 6-BDE 14.93 DFS: 14.93: BDE-138 50 m/z 483.696 EI ionization, 40 eV, source 280 o C 0 R = 10,000 (10 % valley) 16.28 100 7-BDE BDE-183 Trace GC Ultra: 50 m/z 561.606 TR-5MS (15/0.25/0.1) 0 constant flow 1 ml/min 23.92 100 splitless injection, 280 o C 10-BDE BDE-209 50 120 o C/2 min – 230 o C/15 – 270 o C/5 m/z 799.333 – 330 o C/10 0 transfer line temperature 280 o C 23 24 8 9 10 11 12 13 14 15 16 Time (min) Proprietary & Confidential 17
TSQ 8000 EVO – State of Art GC-MS/MS Proprietary & Confidential 18
EU Regulation 589/2014 Requirements Proprietary & Confidential 19
Methods for DioxinFurans & PCBs Measurement SCREENING: • GC-MS • GC-MS/MS • Magnetic Sector • Bioassay (e.g. CALUX) CONFIRMATION: • Magnetic Sector – MLs and background • GC-MS/MS – MLs as outlined in EU regulations 709/2014 and 589/2014 Proprietary & Confidential 20
Experimental conditions (GC & MS) TRACE 1310 GC Parameters TSQ 8000 Evo Mass Spectrometer Parameters Injection Volume ( µ L): 2.0 Transfer line ( ° C): 280 Liner SSL Single tapered P/N: 453A2342 Ionization type: EI Inlet ( ° C): 280 Ion source( ° C): 300 Inlet Module and Mode: SSL splitless Electron energy (eV): 40 Carrier Gas, (mL/min): He, 1.2 Acquisition Mode: SRM (Timed Acquisition) Oven Temperature Program: Collision gas: Argon (60 psig) Temperature 1 ( ° C): 120 Resolution Q1: 0.7 u (FWHM) Hold Time (min): 2 Resolution Q3: 0.7 u (FWHM) Temperature 2 ( ° C): 250 Rate ( ° C/min) 25 Hold Time (min): 0 Temperature 3 ( ° C): 285 Rate ( ° C/min) 2.5 Hold Time (min): 0 Temperature 4 ( ° C): 320 Rate ( ° C/min) 10 Hold Time (min): 15 Column Type Length (m) I.D.(mm) Film (µm) P/N TG-5SilMS 60 0.25 0.25 26066-1540 All 17 PCDD/F and 12 dl-PCBs native and labeled congeners can be acquired in one method Proprietary & Confidential 21
Sample Preparation • PCDD/Fs and dl-PCB standards containing the native and the 13 C-labelled compounds were obtained from Wellington Laboratories Inc. The following food and feed extracted samples were provided by the Institute of Environmental Assessment and Water Research, CSIC Barcelona, Spain: − Adipose tissue − Fish liver dl-PCBs − Fish oil − Dry fishspiked feed sample − milk powder sample (certified reference material). − 3x dry fish samples (previously used in inter-laboratory studies) PCDD/Fs − one feed sample (internal reference material). − one milk powder sample (certified reference material). • Extraction and clean-up of the matrix samples was performed either by PowerPrep™ SPE system (feed sample) or using a manual clean-up with multilayer silica, followed by basic alumina and a final carbon column (milk, tissue and fish samples). Proprietary & Confidential 22
Chromatography - PCDD/Fs Excellent resolution of the isomeric pair 1,2,3,4,7,8 and 1,2,3,6,7,8 HxCDF RT: 22.11 - 25.01 RT: 22.70 NL: 2.58E6 100 RT: 23.42 TIC F: + c EI SRM ms2 95 RT: 22.83 373.820@cid28.00 RT: 24.46 [310.855-310.865] 90 MS ICIS project48 85 80 75 70 Limit as defined in 65 EPA 1613 60 55 Relative Abundance 50 45 40 <5% valley 35 resolution 30 25 20 15 10 5 0 22.2 22.4 22.6 22.8 23.0 23.2 23.4 23.6 23.8 24.0 24.2 24.4 24.6 24.8 25.0 Time (min) 2µL EPA 1613 CS3 standard Proprietary & Confidential 23
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