New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Dr. Nuria Cortés-Francisco Servei de Química Laboratori Agència Salut Pública Barcelona Vancouver, August 22 nd, 217
Summary Laboratori Agència de Salut Pública de Barcelona Orbitrap technology Brominated Flame Retardants: PBDEs Polychloronaphtalenes Ndl-PCBs New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 2/26
Laboratori Agència de Salut Pública Food Safety Regulations Accreditation The official control labs should: - help to ensure food safety - fulfill regulations - work under accreditation ISO/IEC 1725 New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 3/26
Laboratori Agència de Salut Pública Regulations INSTRUMENTATION VALIDATION New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 4/26
Laboratori Agència de Salut Pública Food Safety Accreditation New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 5/26
Laboratori Agència de Salut Pública Method matrix matched standard calibration curve (MMSCC) SCC (Standard calibration curve): calibration using standards in solvent MSCC (Matrix matched standard calibration curve): addition of standards to sample extracts from blank matrix. MMSCC (Method matrix matched standard calibration curve): addition of standards to blank matrix prior to extraction. New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 6/26
Laboratori Agència de Salut Pública Method matrix matched standard calibration curve (MMSCC) Matrix effect evaluation: egg analysis Blue: SCC standards in solution Red: MSCC Matrix matched Green: MMSCC Matrix matched New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 7/26
Methods based on Orbitrap at LASPB Analytical methods already in use: ANALYSIS OF VETERINARY DRUGS: Antibiotics Hormones Chloramphenicol Beta agonists Nitrofurans Nonsteroidal anti-inflammatory drugs.. ANALYSIS OF MARINE BIOTOXINS ANALYSIS OF CONTAMINANTS FROM FOOD PACKAGING ANALYSIS OF PROCESS CONTAMINANTS IN FOODSTUFF New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 8/26
New instrumentation: Orbitrap GC-MS Q-Exactive Exactive * Installed 14/12/215 Brominated Flame Retardants: PBDEs Polychloronaphtalenes (PCNs) Ndl-Polychlorobiphenyls (ndl-pcbs) Thermo Scientific Q Exactive GC system Unprecedented Depth in Analysis RP 12, (FWHM @ m/z 2) EI/CI; Full-scan, Timed-SIM MS/MS capability New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 9/26
Bringing GC and Orbitrap Technology Together C-TRAP AQT Quadrupole Bent Flatapole HCD Cell ExtractaBrite Ion Source Orbitrap Mass Analyzer * Courtesy of Thermo Fisher Scientific New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 1/26
Polybrominated diphenyl ethers (PBDEs) (a) Polybrominated diphenyl ethers (PBDEs) BDE 28 BDE 47 BDE 49 BDE 99 BDE 1 BDE 138 BDE 153 BDE 154 BDE 183 BDE 29 Brx O x=1-5, y=-5 Bry eggs and egg products milk and dairy products meat and meat products animal and vegetable fats and oils fish and other seafood products for specific nutritional uses food for infants and small children Analytical methods with a limit of quantification of,1 ng/g wet weight or lower 1 ppt in the sample New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 11/26
Polybrominated diphenyl ethers (PBDEs) 1 5 RT: 6.76 AA: 72862 BDE-28 1 5 1 5 1 BDE-49 RT: 7.3 AA: 112726 RT: 7.21 AA: 6831 BDE-1 BDE-47 RT: 7.7 AA: 75285 RT: 7.8 AA: 48941 BDE-99 RT: 8.1 AA: 76896 RT: 8.44 AA: 68971 TUNA sample spiked with.1 ng/g for all the PBDEs 5 1 5 BDE-154 BDE-153 BDE-138 RT: 8.67 AA: 56967 BDE-183 1 5 RT: 1.29 AA: 2293 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 Time (min) BDE-29 New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 12/26
HRMS approach With the new HRMS instruments we are capable of acquiring the full isotopic pattern without losing sensitivity and working at R: 3, (FWHM, m/z 2) or higher. 799.3334 C 12 O Br 4 81 Br 4.5961 ppm Quantification m/z 2.3 After cleanup, the extract is concentrated to 2 µl and labeled injection internal standards are added. An aliquot of the extract is injected into the gas chromatograph (GC). The analytes are separated by the GC and detected by a high-resolution (>5) mass spectrometer. Two exact m/z s are monitored at each level of bromination (LOB) throughout a predetermined retention time window. 797.3351 C 12 O Br 5 81 Br 3.1834 ppm Confirmation m/z * 81.3311 C 12 O Br 3 81 Br 5.341 ppm 795.3378 83.3299 793.3385 85.3267 795 8 85 m/z New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 13/26
HRMS approach Quantification m/z 799.3334 C 12 O Br4 81 Br4.5961 ppm MOLECULAR ION 959.1678 C12 O Br5 81 Br5.2624 ppm 797.3351 C12 O Br5 81 Br3.229 ppm * 81.339 C12 O Br3 81 Br5.553 ppm Confirmation m/z TUNA sample spiked before extraction at,1 ng/g with BDE-29 957.168 961.1643 795.337 963.164 83.331 953.1734 811.3714 89.3762 793.3462 84.3342 785 79 795 8 85 81 815 m/z 95 955 96 965 97 m/z New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 14/26
Optimization of EI Source Parameters 5 25 ev 4 ev 5 ev 7 ev 45 4 35 Response 3 25 2 15 1 5 BDE-28 BDE-49 BDE-47 BDE-1 BDE-99 BDE-154 BDE-153 BDE-138 BDE-183 BDE-29 New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 15/26
Acquisition modes.5 µg/l PBDE standard 1 5 RT: 7.32 AA: 526447 RT: 7.4 AA: 898734 Full Scan m/z 4-1 1 5 RT: 1.42 AA: 159338 1 5 RT: 7.32 AA: 191545 RT: 7.41 AA: 334622 SIM 4 Da 1 5 RT: 1.43 AA: 445931 1 RT: 7.41 AA: 4268 1 RT: 1.43 AA: 513755 5 RT: 7.32 AA: 24495 SIM 1 Da 5 1 5 RT: 7.32 AA: 3474 RT: 7.41 AA: 52322 SIM 25 Da 1 5 RT: 1.46 AA: 3416 RT: 1.42 AA: 75762 1 5 RT: 7.32 AA: 337592 RT: 7.4 AA: 591524 SIM 5 Da 1 5 RT: 1.42 AA: 84771 7.15 7.2 7.25 7.3 7.35 7.4 7.45 7.5 7.55 Time (min) 9.8 1. 1.2 1.4 1.6 1.8 11. Time (min) New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 16/26
Acquisition modes TUNA sample spiked with,1 ng/g 1 RT: 6.86 AA: 28948 Full Scan m/z 4-1 1 RT: 6.87 AA: 3316 SIM 5 Da 5 5 1 5 RT: 7.31 AA: 1988 RT: 7.39 AA: 7133 1 5 RT: 7.31 AA: 29875 RT: 7.4 AA: 62815 1 RT: 7.79 AA: 5166 1 RT: 7.8 AA: 39342 5 5 1 5 RT: 8.2 AA: 2467 RT: 1.45 AA: 6371 1 5 RT: 8.2 AA: 2187 1 5 1 RT: 8.19 AA: 27111 RT: 8.54 AA: 26621 RT: 8.77 AA: 3263 RT: 1.44 AA: 1966 1 5 1 RT: 8.2 AA: 25795 RT: 8.54 AA: 16627 RT: 8.78 AA: 1253 5 5 1 RT: 1.45 AA: 55774 1 RT: 1.46 AA: 312582 5 5 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 Time (min) 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 Time (min) New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 17/26
Acquisition modes TUNA sample spiked with,1 ng/g 1 RT: 6.86 AA: 28948 Full Scan m/z 4-1 1 RT: 6.87 AA: 3316 SIM 5 Da 5 BDE-28 5 BDE-28 1 5 BDE-1 RT: 7.79 AA: 5166 BDE-99 1 5 BDE-1 RT: 7.8 AA: 39342 RT: 7.91 AA: 1975 BDE-99 1 RT: 8.77 AA: 3263 1 RT: 8.78 AA: 1253 5 BDE-183 5 BDE-183 1 5 matrix co-elution BDE-29 RT: 1.45 AA: 55774 1 5 BDE-29 RT: 1.46 AA: 312582 1 5 Total Ion Chromatogram 9.16 1 5 6.92 7.21 9.25 Total Ion Chromatogram 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 Time (min) 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 Time (min) New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 18/26
Acquisition modes TUNA sample spiked with,1 ng/g 1 AA: 5166 Full Scan m/z 4-1 1 AA: 39342 SIM 5 Da Relative Abundance 1 Relative Abundance 8 6 4 2 8 6 4 2 BDE-1 BDE-99 AA: 3263 BDE-183 Relative Abundance Relative Abundance 8 6 4 2 1 8 6 4 2 BDE-1 AA: 1975 BDE-99 BDE-183 AA: 1253 Ion Time: 11 ms Ion Time: 119 ms C- trap saturation due to matrix is overcome using the Q! New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 19/26
Polybrominated diphenyl ethers (PBDEs) Monkfish extracted matrix-matched calibration curve.1 ng/g to.3 ng/g PBDEs BDE-29 Quantification m/z Confirmation m/z New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 2/26
Polychloronaphtalenes (PCNs) Samples analyzed Fish and seafood Eggs and egg products Instrumental Analysis PTV injection HP-5MS, 3 m, i.d..25 mm,,25 µm GC column Optimized Tune parameters High Resolution ( 6, FWHM m/z 2) Wide SIM: 25 m/z window to monitor the whole isotopic pattern Mass accuracy < 1ppm External calibration Listed under Annex A and C with specific exemptions for use RT: 12.77-21.91 SM: 7G RT: 14.11 AA: 37333 1 Relative Abundance Relative Abundance Relative Abundance Relative Abundance Relative Abundance 8 6 4 2 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 PCN-48 (tetra) RT: 15.5 AA: 2583 PCN-52 (penta) RT: 17. AA: 5512 PCN-73 (hepta) PCN-66/67 (hexa) RT: 19.16 AA: 21479 PCN-75 (octa) RT: 2.89 AA: 39545 13 14 15 16 17 18 19 2 21 Time (min) New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 21/26
Polychloronaphtalenes (PCNs) If m=4 and n=4 PCN-75: Octachloronaphthalene LOQ=,1 ng/g wet weight Quantification m/z Confirmation m/z New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 22/26
Non-dl-Polychlorobiphenyls (n-dl-pcbs) Analytical requirements for the official control of the levels of non-dioxin-like PCBs: PCB-18,8 ng/g wet weight,1 ng/g fat,25 ng/g wet weight,31 ng/g fat GC-MS/MS (QqQ) + MRM (393,7 -> 324,) 174417.D 1742724.D Smooth + MRM (395,7 -> 326,) 174417.D 1742724.D Smooth x1 2 29,346 29,321 min. min. x1 21 Ratio = 56,5 46, (1, (74, %) %) 1 2,87 29,344 * 29,327 min. min. 3,5,9 2,6 6,5 2,4,8 3 2,2 6 5,5 2 2,5,7 1,8 5,6 1,6 2 1,4 4,5,5 1,5 1,24,4 3,5 1 1,8,3,63,5,2,4 2,5,2,1 2 28,9 28,9 29 29 29,1 29,129,2 29,29,3 29,3 29,4 29,4 29,5 29,5 29,6 29,6 29,7 29,7 29,8 28,9 28,9 29 29 29,1 29,129,2 29,29,3 29,3 29,4 29,4 29,5 29,5 29,6 29,6 29,7 29,7 29,8 Acquisition Time (min) Acquisition Time (min) New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 23/26
Ndl-Polychlorobiphenyls (Ndl-PCBs) Analytical requirements for the official control of the levels of non-dioxin-like PCBs: PCB-18 Full Scan SIM PRM PCB-28 Total Ion Chromatogram Meat sample spiked at.25 ng/g Total Ion Chromatogram Standard New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 24/26
Conclusions GC-Orbitrap is a useful tool for control labs: outstanding sensitivity high selectivity when analyzing complex matrices high robustness helps to fulfill new Regulations with extremely low limits (e.g. PBDEs) helps to confirm doubtful results But some expertise and optimization of instrumental parameters is needed. New Methodologies for POPs Analysis using Orbitrap GC-MS in Official Food Control Vancouver, August 22 nd, 217 25/26
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