for new contaminants at ultra trace level by using high resolution mass spectrometry

Transcription

1 Non-targeted screening for new contaminants at ultra trace level by using high resolution mass spectrometry Dr Igor Fochi LSMS Product Specialist Thermo Fisher Scientific

2 What is Environmental Analysis Challenge? Analysis of thousands of pollutants High diversity of chemical characteristics Different matrices Screening Workflows with Quan/Qual capabilities needed New issues: Hydraulic fracturing Regulatory EU Water Framework Directive Emerging Contaminants in The Environment USGS study F HO O Pharmaceuticals, Pesticides, Endocrine Disruptors, I I O N HN HN I OH O O Personal Care Products Cl N HO O O O O O O O O Cl O OH O O OH O N N NH N HO HO O OH O N N O OH OH O Contamination of ground and drinking water is a risk for human health 2

3 Status quo: Classic targeted workflow Sample EQuan SPE Time consuming and very compound class specific Issues: LC/MS/MS Manual double check to avoid false positive results (unit resolution) Limited to 4 to transitions Pre-selection of compounds needed Manual Data Evaluation Peaks Found Confirmation 3 min total for LC-MS/MS Integration, Quantitation 2 nd transition Reporting 3

4 Analytical Challenges for Traditional Mass Spectrometry Detect and identify almost isobaric compounds Isolate compounds from matrix components Searching for unknowns Here targeted data acquisition (SIM, SRM ) fails Large number of compounds High Resolution High mass accuracy (HRAM) MS can do the job 4

5 Mass accuracy Mass accuracy m m m / z = meas true 1 m true 6 Quadrupole MS.1. m / z = 1 6 = 2 ppm Orbitrap MS TOF MS m / z = 1 6 = 2ppm

6 Drinking water sample (:1) - Atrazine.8 ppb C:\Xcalibur\...\Test\blanksample_liv 1/9/ blanksample bianco campione arpal RT: SM: 7G Relative Abundance RT: 8.8 MA: Time (min) NL: 4.E7 TIC MS blanksample_liv NL: 4.63E3 m/z= F: FTMS {1,1} + p ESI Full ms [.-6.] MS blanksample_liv Relative Abundance Relative Abundance Monoisotopic mass Measured Theoretical m/z [ 37 Cl] Measured Theoretical m/z NL: 2.69E3 blanksample_liv# RT: AV: 7 T: FTMS {1,1} + p ESI Full lock ms [.-6.] NL: 1.6E4 C 8 H 14 ClN 5 +H: C 8 H 15 Cl 1 N 5 p (gss, s /p:4) Chrg 1 R: NL: 6.73E2 blanksample_liv# RT: AV: 9 T: FTMS {1,1} + p ESI Full lock ms [.-6.] NL: 5.11E3 C 8 H 14 ClN 5 +H: C 8 H 15 Cl 1 N 5 p (gss, s /p:4) Chrg 1 R: 6

7 Selectivity increases with higher mass accuracy lative undance 4 Rel Abu Relative Abundance RT: 8.45 NL: 1.2E5 m/z= F: FTMS {1,} + p APCI Full ms [3.-8.] MS pivo_5_uh RT: ppm Time (min) 2 ppm Time (min) NL: 1.2E5 m/z= F: FTMS {1,} + p APCI Full ms [3.-8.] MS pivo_5_uh Relative Abundance RT: ppm Time (min) NL: 1.25E5 m/z= F: FTMS {1,} + p APCI Full ms [3.-8.] MS pivo_5_uh 7 *Zachariasova M et al, Anal. Chim. Acta

8 Resolution (resolving power) Resolution R m = m (FWHM) m m 1% Quadrupole MS m R = m = = m R = m =.5 Orbitrap MS = 8

9 Detection of analytes in heavy matrix Pirimicarb in Matrix C 11 H 19 O 2 N 4 6. ppm Relative Abundance R = 17, Error = 6. ppm m/z C 11 H 19 O 2 N 4.32 ppm R = 7, Error =.32 ppm 9

10 Resolution influence (17. vs. 7.) Resolution_17_5K #1823 RT: 2.3 AV: 1 NL: 1.6E6 T: FTMS + p ESI Full ms [12.-.] Resolution_7K #123 RT:.54 AV: 1 NL: 1.8E6 T: FTMS + p ESI Full ms [12.-.] Name Molecular Formula M+H Relative Abundance Carbofuran C12H15NO Formetanate C11H15N3O Relative Abundance m/z m/z 1

11 Where s the crossover point? 11 Kaufmann et al: Anal. Chim. Acta 673, (21) 6-72

12 Skip the matrix: Dummy transitions concentrations 12 Kaufmann et al: Anal. Chim. Acta 673, (21) 6-72

13 Skip the matrix: Dummy transitions concentrations the selectivity of LC-HRMS exceeds LC-MS/MS, if high resolution mass spectrometry (HRMS) data is recorded with a resolution of, full width at half maximum (FWHM) 13 Kaufmann et al: Anal. Chim. Acta 673, (21) 6-72

14 Q Exactive TM Hardware Innovations Quadrupole mass filter Advanced signal processing for Orbitrap data processing Predictive automatic gain control (pagc) and parallel filling & detection C-trap directly interfaced to HCD Possibility of multiple fills for spectrum multiplexing S-lens for higher transmission with rugged optics C-trap HCD cell Orbitrap Mass Analyzer Quadrupole Mass Filter S-lens 14

15 Resolution vs. Scan Speed (Full MS) Exactive Q-Exactive Resolution Scan Speed [Hz] Improved resolution & increased speed 15

16 Deviation [ppm] Long-term Mass Accuracy with External Calibration Realistic conditions of an average lab Temperature variations up to 3 C m/z 391 m/z Time [h] 16

17 17 Linearity

18 Spectrum Multiplexing Principle Demonstration of 1-fold spectrum multiplexing 2113 JPH TargetSIM MSX1_ #4 RT:.34 AV: 1 NL: 1.35E8 T: FTMS + ESI SIM msx ms [ , , , , , , , R= Relative Abundance R= R= R= R= R= R= m/z 18 See poster MP13 J.-P.Hauschild et al Multiple C-Trap Fills as a Tool for Massive Parallelization of Orbitrap Mass Spectrometry- a new Concept for Targeted Mass Analysis

19 What do we gain by using the quadrupole: mx-sim N= NL: 1.94E8 [1.-2.] Full MS S/N = 745 Lowest detected signal/scan 233 Relative Abundance N= NL: 1.12E8 [ ] mx-sim (1amu) S/N = 54 Lowest detected signal/scan Gain in sensitivity (7x) S/N (spectrum) Caffeine 195,82 195,84 195,86 195,88 195,9 195,92 195,94 S/N (FMS) S/N (SIM1) Sensitivity gain 5 1 x with mx-sim mode 19

20 Data dependent MS/MS capabilities 256ms 64ms 1 full 7K res K Apex Triggered MSMS Relative Abundance Time (min)

21 Data dependent MS/MS confirmation Relative Abundance Imazalil Desmedipham Diuron Benoxacor Fluridone Azoxystrobin Fenamidone Time (min) NL: 1.98E7 m/z= F: FTMS + p ESI Full ms [.-7.] MS 4xloq_2 NL: 4.67E6 m/z= F: FTMS + p ESI Full ms [.-7.] MS 4xloq_2 NL: 8.97E7 m/z= F: FTMS + p ESI Full ms [.-7.] MS 4xloq_2 NL: 1.15E7 m/z= F: FTMS + p ESI Full ms [.-7.] MS 4xloq_2 NL: 2.21E7 m/z= F: FTMS + p ESI Full ms [.-7.] MS 4xloq_2 NL: 1.42E7 m/z= F: FTMS + p ESI Full ms [.-7.] MS 4xloq_2 NL: 4.23E7 m/z= F: FTMS + p ESI Full ms [.-7.] MS 4xloq_2 Relative Abundance m/z 21

22 Comparison of AIF vs MS/MS: Trimethoprim and IS c:\documents and settings\...\1pg_3 8/24/211 4:2:26 PM 1pg_3 #113 RT: 5.78 AV: 1 NL: 2.33E6 F: FTMS + p ESI Full ms2 275.@hcd35. [8.-4.] All Ion Fragmentation spectra of Thrimethoprim and its IS ( 13 C3) Relative Abundance m/z Relative Abundance Data dependent MSMS Trimethoprim- 13 C NL: 1.7E5 1pg_5#131 RT: 5.84 AV: 1 F: FTMS + p ESI d Full ms @hcd35. [.-32.] m/z Trimethoprim NL: 1.71E4 1pg_5#1283 RT: 5.72 AV: 1 F: FTMS + p ESI d Full ms @hcd35. [.-315.] 22

23 Result: Q Exactive Screening Procedure Inclusion list up to compounds per run with specific time windows, and Collision Energies Sample Benefits: Quadrupole selection MS/MS High Resolution and several confirmation parameters avoid false positives Retrospective data analysis possible LC/Q Exactive Identification / Quantitation Confirmation Reporting LC/MS/MS Precursor Ion + RT Isotopic pattern match, ddmsms or AIF spectra 23

24 Target & Non-Target trace Analysis side by side SAMPLE Target Analysis Non-Target Analysis Spectral Libraries Screening Elemental composition Quantitation Quanfirmation Identification of unknown molecules 24

25 Benefits of EQuan-Orbitrap combination Screening and targeted workflow for trace detection Ease of use; no compound optimization needed for quantitation or confirmation Quantitation based on full scan acquisition Confirmation based on targeted data dependent MS2, using ExactFinder software Automated online sample preparation High volume injection up to 2 ml 25

26 Data mining issues: What s the issue with data mining? Large data files All information contained Most of the data is ballast We need to extract relevant information! 26

27 ExactFinder - Screening and Quantitation Software What it does? Qualitative and quantitative HR/AM data processing and review Database & Library Search capabilities for screening experiments Quanfirmation Targeted & Unknown Screening Quanfirmation Reporting 27

28 ExactFinder finds real peaks in your data Original raw file CDL raw file 28

29 29 ExactFinder finds real peaks in your data

30 Screening: Method Setup screen 3

31 Screening: Method Setup screen 31

32 Screening: Method Setup screen 32

33 ExactFinder Library screen More than substances Include: Names Accurate masses Formulas Fragments Spectral Information more than 6 spectra (CID, HCD, variable CE, Positive/Negative) Contains information for: Pesticides, hormones, mycotoxins, veterinary drugs, biotoxins, pharmaceuticals Delivered free of charge 33

34 Targeted Screen: Environmental Application screen 34

35 Targeted Screen: Environmental Application screen theoretical measured 35

36 Unknown Screen: Environmental Application screen 36

37 Unknown Screen: Environmental Application screen 37

38 Unknown Analysis: Sieve TM Data comparison tool Blank sample Alignment Framing ChemSpider Search Real sample Compensation of RT changes Table of identified frames Table of positive hits 38

39 Quantitative Data Review quan 39

40 Quantitative Data Review quan 4

41 Conclusion Equan MAX-Q Exactive provides excellent sensitivity and low LODs Minimum of 7,K resolution is recommended to obtain best selectivity High confidence confirmation by Isotopic Pattern Match or ddms2 fragmentation Screening and quantitation is feasible on one instrument Full-MS screening data files could be re-processed for additional compounds (retrospective data analysis) Used in many routine labs around the world 41

42 Customers made in Applied Markets References : 1. Dr Heinz Singer, EAWAG, Zurich, Switzerland 2. Prof Sebastien Sauve, Montreal, Canada 3. Heritage Laboratories, USA (Exactive/TSQ/EQuan) 4. Dr Jacques Poinsot, LDA, France 5. Dr Luc Zwank, CRTE, Luxembourg 6. Korea Water, Korea (Exactive/EQuanMAX) 7. KIWA, Belgium 8. GE, Singapore 9. Daegu Water Quality Research Lab Korea (Exactive/EQuanMax) 42