Simultaneous quantitative determination and screening of pesticides using Orbitrap MS Technology. Hans Mol, Paul Zomer, Marc Tienstra
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1 Simultaneous quantitative determination and screening of pesticides using Orbitrap MS Technology Hans Mol, Paul Zomer, Marc Tienstra 1st International Symposium on Recent Developments in Pesticide Analysis, Prague April 215
2 Outline Introduction pesticide residue analysis Instrument and method (LC) Quantitative analysis Identification Qualitative screening What about GC? Conclusions
3 Pesticide residue analysis in food The analytical challenge: theory Pesticide Manual: 163 entries World: ~7 in use, others obsolete EU: 462 approved But: residues imported illegal pesticides but not gone 1s of different food matrices of varying complexity MRLs:.1-1 mg/kg
4 Pesticide residue analysis in food The analytical challenge: practice Detection rate of pesticides amenable to LC-MS based multi-residue analysis number of pesticides (targeted measurement: 231) never found, 15 only once, in 1, samples Quantitative analysis with extensive AQC = waste of time Qualitative analysis with automated detection more appropriate 127 pesticides 2x found in 1, samples Quantitative analysis with extensive AQC justified: Manual check XICs LOQ Linearity Recovery Repeatability Measurement uncertainty detection frequency in ~1, fruit/veg samples Data compiled from NVWA,
5 The challenge and the solution Analysis request: a) are any pesticides present; b) if so: at what level? Current New solution: solutions: Solution 1: (majority of routine labs) Solution 2: Solution 3: LC-MS/MS (triple quad) LC-MS/MS (triple quad) LC-MS/MS (triple quad) + LC-Q-HRMS + LC-MS/MS (triple quad) LC-fs-HRMS (TOF, Orbitrap technology) Quantitative analysis for usual suspects Qualitative analysis for others quantitative analysis ~25 pesticides quantitative analysis ~5 pesticides quantitative analysis ~25 pesticides qualitative analysis >5 pesticides
6 Outline work flow LC-Q-HRMS analysis Sample homogenisation homogenised sample extraction/cleanup Extract quantification + identification < LOQ or xx mg/kg Raw data detection (identification) positive or negative Re-run with calibrants positive?
7 Outline Introduction pesticide residue analysis Instrument & method (LC) Quantitative analysis Identification Qualitative screening What about GC? Conclusions
8 Instrument used Thermo Scientific Exactive Plus MS Thermo Scientific Q Exactive MS Thermo Scientific Q Exactive Focus MS Thermo Scientific Q Exactive Plus MS Thermo Scientific Exactive Plus EMR MS Thermo Scientific Q Exactive HF MS Resolution m/z 2 (scan speed) 17,5 (12 Hz); 35, (6 Hz); 7, (3 Hz); 14, (1.5 Hz) [Focus: up to 7,] m/z 5-6 (2) Mass accuracy: internal < 1 ppm RMS; external < 3 ppm RMS Polarity switching: one full cycle pos&neg in <1 sec (R=35,) Variable precursor ion isolation width selection from.4 Da to full mass range
9 Various acquisition options Non-target acquisition without fragmentation (Full Scan) with fragmentation in HCD cell AIF = all-ion-fragmentation vdia = variable Data Independent Acquistion Targeted acquisition without fragmentation (SIM = Selected Ion Monitoring) with fragmentation ddms/ms = data-dependent MS/MS with inclusion list t-ms/ms = targeted MS/MS PRM = Parallel Reaction Monitoring Combinations of the above
10 Full scan acquisition FS: m/z 1-1 FS: m/z 1-1 FS: m/z 1-1 FS: m/z 1-1 Upper & lower m/z cut-off
11 Set up of acquisition method: full scan General source parameters, AGC settings: TFS default recommendations Full scan measurement: m/z range: mass resolution: 5, for reliable mass accuracy in complex samples to ensure for selectivity and quantification* Here: 7, m/z 2 % of 151 pesticides Mass accuracy at different resolving power settings* ' 25' 5' 1' Resolving power 25 ppb in highly complex matrix < 2 ppm 2-5 ppm 5-1 ppm 1-25 ppm > 25 ppm/nd * Kellmann et al, JASMS, 29, 2,
12 Extracting pesticides from the raw data Extract signal of exact mass ± x Da (ppm), e.g. Dimethoate [M+H] ± 5 ppm (±.12 Da) Relative Abundance Exception N m FT 5 Exception [ Q Time (min) 3 T p [1 1 Q 2 m/z C 5 H 12 NO 3 PS 2 dimethoate m/z C 9 H 6 F 3 N 3 O flonicamid m/z C 19 H 25 ClN 2 OS pyridaben m/z C 7 H 12 ClN 5 simazine m/z C 12 H 14 N 4 O 4 S 2 Thiophanate-methyl Leek 1 ppb, Full scan m/z 135-1; Res = 7,
13 Set up of acquisition method: fragmentation Generation of fragments: 1) needed for identification, 2) improve screening selectivity For optimum detection and identification: full scan acquisition without and with fragmentation in 1 run Non-targeted fragmentation: two options: AIF and vdia
14 Combined Full scan + AIF acquisition FS: m/z 1-1 AIF Ϟ m/z 1-1 FS: m/z 1-1 AIF Ϟ m/z 1-1 Upper & lower m/z cut-off
15 Combined Full scan + AIF acquisition FS: m/z 1-1 AIF Ϟ m/z 1-1 FS: m/z 1-1 AIF Ϟ m/z 1-1 Upper & lower m/z cut-off
16 Combined Full scan + vdia acquisition FS: m/z 1-1 Ϟ: 1-2 Ϟ: 2-3 Ϟ: 3-4 Ϟ: 3-4 Ϟ: 5-1 FS: m/z 1-1 Upper & lower m/z cut-off m/z isolation window
17 Combined Full scan + vdia acquisition FS: m/z 1-1 Ϟ: 1-2 Ϟ: 2-3 Ϟ: 3-4 Ϟ: 3-4 Ϟ: 5-1 FS: m/z 1-1 Upper & lower m/z cut-off m/z isolation window
18 Combined Full scan + vdia acquisition FS: m/z 1-1 Ϟ: 1-2 Ϟ: 2-3 Ϟ: 3-4 Ϟ: 3-4 Ϟ: 5-1 FS: m/z 1-1 Upper & lower m/z cut-off m/z isolation window
19 Combined Full scan + vdia acquisition FS: m/z 1-1 Ϟ: 1-2 Ϟ: 2-3 Ϟ: 3-4 Ϟ: 3-4 Ϟ: 5-1 FS: m/z 1-1 Upper & lower m/z cut-off m/z isolation window
20 AIF vs. vdia Dimethoate 1 ppb in wheat Carbaryl 1 ppb in wheat FS m/z RP = 7, AIF m/z 67-1 RP = 7, vdia m/z RP = 35, [M+H] Fragment m/z Fragment m/z Time (min) Relative Abundance [M+H] Fragment m/z Fragment m/z Time (min) vdia preferred: improved selectivity & sensitivity + beneficial for identification
21 Dealing with exception 1: interfering analytes Simazine C 7 H 12 ClN 5 and Carbaryl C 12 H 11 NO 2 : difference [M+H] + =.9 mda (4 ppm) 1 5 XIC ± 5 ppm: Simazine [M+H] Relative Abundance XIC ± 5 ppm: Simazine Fragment Carbaryl [M+H] Carbaryl Fragment Time (min) Spiked Leek FS 7K, vdia 35K
22 Dealing with exception 2: interfering matrix Thiophanate-methyl in leek ppb from full scan from fragmentation (vdia m/z ) Relative Abundance Time (min) [M+H] + = [(M+1)+H] + = ( 13 C, 15 N) [(M+2)+H] + = ( 34 S) Fragment 1: (C 7 H 7 N 2 S + ) Fragment 2: (C 6 H 7 N + ) Spiked Leek FS 7K, vdia 35K C 12 H 14 N 4 O 4 S 2
23 Method used Sample preparation: QuEChERS (AOAC version) 1 g homogenised sample + 1 ml Acetonitrile/1% HAc Shake 3 min 4 g MgSO g NaAc, centrifuge (no dspe cleanup) Dilute acetonitrile phase 1:1 with water LC: Thermo Scientific Dionex UltiMate 3 system: Injection: 5 µl Column: 1 3 mm ID, 3 μm Atlantis T3; T=35 C Gradient: water/methanol, 2 mm NH 4 HCOO Flow:.3 ml/min HRMS: Q Exactive MS with H-ESI-II source Heated capillary: 32 C FS+vDIA Cycle time 978 ms full scan: no fragmentation m/z 135-1@7K HCD: 3 and 8 NCE, ACG: 1 6 Fragments of 95-25@35K Fragments of @35K Data handling: Thermo Scientific TraceFinder 3.2 software Fragments of @35K Fragments of @35K Fragments of @35K
24 Outline Introduction pesticide residue analysis Instrument and method (LC) Quantitative analysis Identification Qualitative screening What about GC? Conclusions
25 Quantitative validation Frequently found + others to widen range of phys/chem properties Abamectin Carbofuran DNOC Fluroxypyr Mesotrione Pirimicarb Spiroxamine Acephate Carfentrazone-ethyl Dodemorph Flutolanil Metalaxyl Pirimiphos-methyl Sulcotrione Acequinocyl Chlorantraniliprole Dodine Foramsulfuron Metamitron Prochloraz Tebuconazole Acetamiprid Chlorbromuron Emamectin B1a Fosthiazate Metazachlor Profenofos Tebufenpyrad Aclonifen Chloridazon Epoxiconazole Haloxyfop Metconazole Propamocarb Tepraloxydim Aldicarb Clodinafop-propargyl Ethirimol Haloxyfop-etotyl Methabenzthiazuron Propiconazole Terbuthylazine Ametoctradin Clofentezine Ethoprophos Hexythiazox Methamidophos Propyzamide Terbutryn Aminopyralid Clomazone Etoxazole Imazalil Methiocarb Prosulfocarb Tetraconazole Amisulbrom Clopyralid Famoxadone Imidacloprid Methomyl Pymetrozine Thiabendazole Asulam Clothianidin Fenamidone Indoxacarb Methoxyfenozide Pyraclostrobin Thiacloprid Azadirachtin Cyazofamid Fenamiphos Iodosulfuron-methyl Metolachlor Pyridaben Thiamethoxam Azamethiphos Cybutryne Fenhexamid Ioxynil Metoxuron Pyridalyl Thiophanate-methyl Azoxystrobin Cymoxanil Fenoxaprop-p-ethyl Iprovalicarb Metrafenone Pyridate Tolylfluanid Bendiocarb Cyproconazole Fenoxycarb Isoproturon Metribuzin Pyrimethanil Triallate Bentazone Cyprodinil Fenpropidin Isopyrazam Metsulfuron-methyl Pyriproxyfen Tribenuron-methyl Bifenazate Cyromazine Fenpropimorph Isoxaben Mevinphos Pyroxsulam Triclopyr Bifenthrin Cythioate Fipronil Isoxaflutole Myclobutanil Quinmerac Trifloxystrobin Bixafen D 2 4- Flonicamid Kresoxim-methyl Nicosulfuron Quinoclamine Triflumizole Boscalid Dichlofluanid Florasulam Lenacil Omethoate Quinoxyfen Triflumuron Brodifacoum Difenoconazole Fluazinam Linuron Oxamyl Quizalofop-ethyl Triflusulfuron-methyl Bromadiolone Diflubenzuron Flubendiamide Lufenuron Oxydemeton-methyl Rimsulfuron Triforine Bromoxynil Diflufenican Flucycloxuron Malathion Paclobutrazol Silthiofam Trinexapac-ethyl Bupirimate Dimethenamid Fludioxonil Mandipropamid Penconazole Simazine Buprofezin Dimethoate Flufenacet MCPA Pencycuron Spinosyn-A Carbaryl Dimethomorph Flufenoxuron MCPP Phenmedipham Spinosyn-D Carbendazim Dinoterb Fluopicolide Mepanipyrim Picoxystrobin Spirodiclofen Carbetamide Diuron Fluoxastrobin Mesosulfuron-methyl Pinoxaden Spiromesifen in red = ESI -
26 Quantitative data review Review by pesticide (compound view): XIC mass extraction window: ±5 ppm For each quan pesticide: click through the samples and check assignment/integration of quantifier (main adduct) and qualifier (fragment), adjust when needed Quantifier OK Qualifier OK
27 Quantitative data review delete
28 Verification of linearity ng/ml Solv. Lett Oran 5-18% -17% -9% 1-7% -2% -9% 5 6% 4% 5% 1-1% -1% -1% +9% -7% ng/ml Solv. Lett Oran 5 1% % % 1-1% -1% % 5-2% % % 1 % % % ng/ml Solv. Lett Oran 5 2% 19% 5% 1 3% % -7% 5-1% -4% 2% 1 % 1% % -16% ng/ml Solv. Lett Oran 5 8% 16% 2% 1 % 4% -5% 5-1% -4% 1% 1 % 1% % -16% -5%
29 Recoveries and RSDs
30 Outline Introduction pesticide residue analysis Instrument and method (LC) Quantitative analysis Identification Qualitative screening What about GC? Conclusions
31 Identification Guidance document: EU SANCO/12571/213 Chromatography: t r > 2t ; retention time deviation < ±.2 min Mass spectrometry ±3% (relative)
32 Identification EU SANCO/12571/213 under revision... Table below: under construction/discussion...
33 Ion ratio Full scan acquisition with/without fragmentation: Various options for ratio determination: area F2 area F1 area F2 area [M+H] + area F1 area [M+H] + area F1 area [M+Na] + area F1 area [(M+2)+H] + Relative Abundance difenoconazole C 19 H 17 Cl 2 N 3 O 3 in Lettuce (1 ppb) [M+H] + = [M+Na] + = [(M+2)+H] + = 48.6 ( 37 Cl) Fragment 1: [C 13 H 9 OCl 2 ] + Fragment 2: [C 12 H 9 Cl ] Time (min) FS FS FS vdia vdia
34 Identity confirmation Examples: isopyrazam and clofentezine Solvent standards isopyrazam ng/ml ion ratio (%) Reference ion ratio 5.11 tolerance -3% 3.58 tolerance +3% 6.65 Lettuce Orange µg/kg ion ratio (%) Solvent standards clofentezine ng/ml ion ratio (%) Reference ion ratio 51.9 tolerance -3% tolerance +3% Lettuce Orange µg/kg ion ratio (%) [M+H] + F % FS vdia
35 Outcome Quantitative Method Validation Selectivity: no significant response in blank lettuce and orange Adequate linearity in most cases Recovery and RSD r meet requirements for majority of pesticides exceptions included: acequinocyl, aminopyralid, clopyralid, quinmerac, fluroxypyr, triclopyr Quantitative performance and identification capabilities similar to triple quadrupole MS/MS / fit-for-purpose => Q Exactive suitable to replace triple quad
36 Outline Introduction pesticide residue analysis Instrument and method (LC) Quantitative analysis Identification Qualitative screening What about GC? Conclusions
37 Qualitative screening: method set up Same raw data, different data review High number of target pesticides, low probability of detection Manual verification of all XICs too time consuming Automated pesticide detection by the software Various options: TraceFinder SW (screening module), Thermo Scientific ToxFinder ID software,... Here: quan module (but without any quan) Default settings for pesticide detection: Mass extraction window: exact m/z ±5 ppm Time window: database RT.5 min Requirement: signal found for pre-set adduct AND fragment ion Output: report of samples showing only pesticides found
38 Screening: data review Review by sample (sample view): For each sample, click through the pesticides found: Check: 2 peaks present? Matching peak profile/rt? Optional: isotope pattern, additional fragments Prosulfocarb? Reject X
39 Screening: data review Terbutylazine? (upon quantification: << 1 ppb)
40 Screening method: validation Guidance document: EU SANCO/12571/213* Initial validation: Required for each individual pesticide, for each commodity group Establish SDL: screening detection limit = lowest concentration for which it has been demonstrated that a pesticide can be detected in 95% of the samples 2 samples (m matrices in n-fold, with n 2) reflecting scope of laboratory Spike each sample at anticipated SDL Include a blank for each matrix Supplemented by on-going validation (QC sample added to routine analysis): Cover additional matrices Demonstrate performance over time/routine conditions Criteria: False negative rate 5% False positive rate: no requirement (any detect triggers identification/quantification/confirmatory analysis) * /docs/qualcontrol_en.pdf
41 Screening method: validation Validation set: 11 matrices: Non-fortified, 3 fortifications:.1,.5 and.2 mg/kg Test set for fortification: 183 pesticides Analysed on 2 different days (4 weeks in between) => 22 samples Validation parameters: Count # pesticides found in each sample detection rate / false negatives blank samples: false positives
42 Screening method: validation Overall detections in spiked samples (426 pesticide/matrix combinations per level):.1 mg/kg: 91.9%.5 mg/kg: 97.2%.2 mg/kg: 98.3% Detection rates in % of spiked pesticides / sample: Orange Grapes Apple Necatarine Leek Orange Grapes Apple Necatarine Leek 2 2 ppb 85 Carrot Tomato 5 ppb 1 ppb Carrot 8 Tomato Beans Broccoli Beans Broccoli Celery Lettuce Celery Lettuce
43 Screening method: validation Screening detection limits: number of pesticides (out of 183) ?? Fortification level (µg/kg)
44 Outline Introduction pesticide residue analysis Instrument and method (LC) Quantitative analysis Identification Qualitative screening What about GC? Conclusions
45 GC-full scan MS Required for further coverage + highly useful complementary technique > 199s: GC-EI-single quad / GC-ion trap / TOF > mid 2s GCxGC-EI-hs-TOF-MS > mid 2s GC-EI-hr-TOF-MS (RP 5-1K) > 21 GC-EI-hr-TOF-MS and GC-EI-Q-TOF-MS (RP 15-25K) GC-APCI-Q-TOF-MS (RP > 2K) APCI: con: can t use EI-MS libraries pro: molecular ion or adduct ion generation of fragment ions, same approach as in LC-ESI-HRMS EI: pro: simple, one acquisition event to get multiple accurate mass ions use of existing EI-MS libraries 1thousands of compounds con: molecular ion not always present > 215 GC-EI-Orbitrap MS (RP m/z 2)
46 Mass accuracy in complex matrix RP 15, F:\GC-Orbitrap\...\2April15_15K_2 4/3/15 1:12:57 Spices 1 RT: SM: 7B 1 Relative Abundance GC-EI-Orbitrap MS RP = 15, m/z 2 XIC m/z ± 25 ppm Diazinon fragment C 1 H 15 N 2 O + # individual scans <1 ppm 2 out of 24 <2 ppm 4 out ot 24 <5 ppm 5 out of 24 <1 ppm 12 out of 24 <25 ppm 24 out of 24 <5 ppm 24 out of 24 NL: 3.84E5 m/z= F: FTMS + p EI Full ms [5.-5.] MS 2April15_15K_ Time (min) 2April15_15K_2 #6616 RT: AV: 1 NL: 9.E5 T: FTMS + p EI Full lock ms [5.-5.] Relative Abundance MS spectrum profile m/z ppm m/z
47 Mass accuracy in complex matrix RP 6, F:\GC-Orbitrap\...\3March15_6K_2 3/31/15 4:49:47 Spices 1 RT: SM: 7B 1 Relative Abundance GC-EI-Orbitrap MS RP = 6, m/z 2 XIC m/z ± 5 ppm Diazinon fragment C 1 H 15 N 2 O + # individual scans <1 ppm 14 out of 14 <2 ppm 14 out ot 14 <5 ppm 14 out of 14 <1 ppm 14 out of 14 <25 ppm 14 out of 14 <5 ppm 14 out of 14 NL: 3.25E5 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3March15_6K_ Time (min) 3March15_6K_2 #3128 RT: AV: 1 NL: 5.7E5 T: FTMS + p EI Full lock ms [5.-5.] Relative Abundance MS spectrum profile m/z ppm m/z
48 GC-Orbitrap MS: example kresoxim-methyl 1 p [ ] Relative Abundance m/z Exact mass most abundant fragment ions: C 11 H 12 NO C 9 H 9 N C 8 H 6 N
49 Simulated unit resolution MS: MEW: ± 5 mda F:\GC-Orbitrap\...\3April15_6K_1 4/3/15 19::16 L1 (1g/mL); 1-3-'15 RT: Relative Abundance SM: 5B TIC ± 5 mda ± 5 mda ± 5 mda Time (min) NL: 8.E9 TIC F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 7.7E6 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 1.4E8 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 2.27E7 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 1 µl inj. GC-Orbitrap MS Leek 1 ppb, Full scan m/z 5-5; Res = 6,
50 Narrowing down the MEW: ± 1 ppm F:\GC-Orbitrap\...\3April15_6K_1 4/3/15 19::16 L1 (1g/mL); 1-3-'15 RT: Relative Abundance SM: 5B TIC ± 1 ppm (21 mda) ± 1 ppm (13 mda) ± 1 ppm (12 mda) Time (min) NL: 8.E9 TIC F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 6.15E5 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 1.1E8 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 1.98E6 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 1 µl inj. GC-Orbitrap MS Leek 1 ppb, Full scan m/z 5-5; Res = 6,
51 Narrowing down the MEW: ± 25 ppm F:\GC-Orbitrap\...\3April15_6K_1 4/3/15 19::16 L1 (1g/mL); 1-3-'15 RT: Relative Abundance SM: 5B ± 25 ppm TIC ± 25 ppm ± 25 ppm Time (min) NL: 8.E9 TIC F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 6.12E5 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 9.57E5 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 1.81E6 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 1 µl inj. GC-Orbitrap MS Leek 1 ppb, Full scan m/z 5-5; Res = 6,
52 Narrowing down the MEW: ± 5 ppm F:\GC-Orbitrap\...\3April15_6K_1 4/3/15 19::16 L1 (1g/mL); 1-3-'15 RT: Relative Abundance SM: 5B TIC ± 5 ppm ± 5 ppm ± 5 ppm NL: 8.E9 TIC F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 6.12E5 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 9.57E5 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_1 NL: 1.81E6 m/z= F: FTMS + p EI Full ms [5.-5.] MS 3April15_6K_ Time (min) 1 µl inj. GC-Orbitrap MS Leek 1 ppb, Full scan m/z 5-5; Res = 6,
53 Comparison with GC-MS/MS (triple quad) F:\GC-QQQ feb215 runcorn\2feb_qqq_38 2/21/15 9:56:25 Leek 1 RT: SM: 5B Relative Abundance RT: m/z Relative Abundance RT: m/z Time (min) Kresoxim-methyl in 1 ppb 1 µl inj. GC-MS/MS (TSQ 8 Evo)
54 Outline Introduction pesticide residue analysis Instrument and method (LC) Quantitative analysis Identification Qualitative screening What about GC? Conclusions
55 Conclusions Acquisition: Full scan combined with vdia: optimum way of non-targeted measurement; provides best sensitivity, selectivity, fragments without sacrificing scope Quantification [top 1-15 frequently found, with calibrants]: Performance comparable with triple quadrupole instruments, sensitivity fit-for-purpose for pesticide residue analysis Identification: Meets EU requirements (SANCO/12571/213) Screening [for the other 1s, without calibrants]: Fully automated output, low # false positives, easy manual accept/reject of hits Overall detection rate 1 ppb SDLs 1 ppb for majority of pesticides tested GC-Orbitrap MS highly promising to complement LC-based quan/qual analysis
56 Acknowledgement RIKILT: Paul Zomer, Marc Tienstra, Ruud van Dam Thermo Fisher Scientific: Olaf Scheibner, Markus Kellmann, Dominic Roberts, Cristian Cojocariu, Paul Silcock Thank you for your attention!
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