Finding a Needle in a Haystack: Using High Resolution Mass Spectrometry in Targeted and Non Targeted Searching for Food

1. Finding a Needle in a Haystack: Using High Resolution Mass Spectrometry in Targeted and Non Targeted Searching for Food Contaminants Erik Verschuuren Strategic Account Manager, Food Safety and Environmental Thermo Fisher Scientific

2. Melamine in milk powder Illegal dyes in chilli powder Acrylamide in crisp bread & French fries BADGE in canned fish 2-Isopropylthioxanthone (ITX) in packaged foods Illegal addition of proteins to milk Industrial chemicals & pharmaceuticals in environment Where Target Analysis has Failed?

3. Different Modes of Analysis Pure Quantitative Pure Qualitative Detect & Quantify Determine structure Targeted Analysis Non-Target Analysis • Structural ID • Compound Confirmation • Reaction Monitoring • Process Monitoring • Metabolism • Proteomics • Metabolomics Screening Profiling Fingerprinting, Statistical Database searching Quantification Confirmation Find your needles Identification of unknown

4. TSQ Quantum Access Max Triple QuadrupolesUsed for Targeted Analysis TSQ Quantum Access TSQ Quantum Ultra TSQ Vantage Ultimate Value Ultimate Performance

5. However…. Technology is evolving… ….direction glaring in the mist

6. Shift Towards HRAM : Crossover Point Official Food Control Authority of the Canton of Zurich

7. Shift Towards HRAM : Crossover Point - cont

8. High Resolution Analyzers Are Claiming Their Share Exactive QExactive LTQ Orbitrap Bench-top HRMS Ultimate Performance

9. 340 350 360 370 380 390 400 410 m/z How Much Mass Accuracy is Needed? [M+H]+ 381.07828 381.07828 C H O N 27 11 2 100 90 80 Relative Abundance 70 60 50 40 • Compounds containing • C[5-15]N[0-10]O[0-15]H[0-40] 30 20 10 0

10. Selectivity Increases With Higher Mass Accuracy 207.1489± 0.1µ 207.1489± 0.05µ 207.1489± 0.005µ

11. 289.1424 289.1390 100 100 100 289.1314 289.1547 289.1329 289.1022 289.0514 90 90 90 80 80 80 289.1215 289.1424 70 70 70 289.1329 289.1021 289.0514 60 60 60 289.1215 Relative Abundance Relative Abundance Relative Abundance 50 50 50 289.1424 289.1329 289.1022 40 40 40 289.0513 289.1215 30 30 30 20 20 20 10 10 10 0 0 0 289.00 289.00 289.00 289.05 289.05 289.05 289.10 289.10 289.10 289.15 289.15 289.15 289.20 289.20 289.20 m/z m/z m/z Detection of Isobaric Compounds in Mixtures Element Exact Mass H 1.007825 C 12.000000 N 14.003074 O 15.994915 Is a simultaneous measurement possible? R = 100,000 R = 20,000 R = 10,000 0.1033µ

12. 239.15181 C H O N 11 19 2 4 6.50 ppm 100 80 60 Relative Abundance 40 20 0 239.00 239.05 239.10 239.15 239.20 239.25 m/z Detection of Analytes in Heavy Matrix Pesticide Mixture in Horse Feed Matrix 239.15033 C H O N 11 19 2 4 0.32 ppm 100 80 R = 15,000 Error = 6.50 ppm 60 Relative Abundance 40 20 0 239.00 239.05 239.10 239.15 239.20 239.25 m/z R = 80,000 Error = 0.32 ppm

13. Target Screen Approach Raw Data Targeted Screen Report Library of “Knowns”

14. Non Targeted Analysis – What’s the challenge? • High resolution MS data acquisition- Unknown is unknown…- Matrix presence- Variability of samples- Small concentrations of potential threats- Extraction procedures: polarity, pH • Data processing?

15. MS Detectors for Unknowns Unknowns High Resolution Orbitrap MS Ion Trap MS • Full scan mode acquisition • Confirmation by MSn • Exact mass and high resolution data • Confirmation by isotopic pattern and MS/MS • Confirmation and Quantitation in 1 run

16. Key Requirements: Mass Accuracy + Resolution m/z 127.07263 C, N, O, H m/z 707.21625 C, N, O, H

17. Example: Analysis of Milk Powder Milk sample 2g Add 20 ml Acetonitrile/Water 70:30 v/v Extraction 20 min Centrifugation 14000 rpm 10 minutes Filtration through 0.2 µm PTFE into LC vial UHPLC/Exactive MS analysis SIEVE data processing Poster: NEW STRATEGIES FOR FOOD PROFILING AND MONITORING USING HIGH RESOLUTION MASS SPECTROMETRY APPROACH, M. GODULA1, A. CHARLTON2, D. ROBERTS2 AND K. MITTENDORF1

18. TIC and BPC of Milk Extract - No Difference… TIC (Total Ion Current) BPC (Base Peak Chromatogram)

19. Automated Component Detection - ExactFinder Background subtracted file Components extracted

20. Comparing Contaminated Sample (10ppm) With Blank The only visually identified difference Fenthion Sudan dyes MelamineCyanuric acid Urea

21. Blank Milk vs. Spiked Milk – Visual Comparison Only 20 hits < 10 ppm CHNOSP

22. SieveTM –Data Comparison Tool Blank sample Alignment ChemSpider Search Framing Real sample Table of positive hits Table of identified frames Compensation of RT changes

23. Sieve™ – Framing Process

24. Sieve™ – Database Search Option • ChemSpider search • Web based • Number of databases available • Targeted search MZ Lookup • Custom build library • Adducts searched

25. Advanced Algorithms – Elemental Composition Calculation MH+ ExactFinder considers isotopic pattern as a confirmation criteria ASMS 2010 Poster-T501: High-Resolution LC-MS Analysis of Compounds for Early Drug Discovery: Quantitative and Qualitative Screening in One Pass, Caroline Ding1, Jim Shofstahl1, Karen Salomon1, Hans Pfaff2, and Thomas McClure1

26. Isotopic Pattern Fitting is Influenced by Resolution Table of candidates • Isotopic patterns compared • Resolution is needed to separate overlapping peaks

27. Conclusions – Future View • Minimize Sample preparation • Simplified sample preparation strategies • Direct analysis where possible • Advanced data processing algorithms must be used • Data extraction and background subtraction • Comparative approaches • Libraries of accurate masses and fragments required • Isotopic pattern fitting • Validation • Validation for diverse matrices required