Michal Godula Thermo Fisher Scientific. The world leader in serving science

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1 Resolving the food authenticity challenges using advanced isotopic ratio and Thermo Scientific Orbitrap high resolution mass spectrometry tools in practice Michal Godula Thermo Fisher Scientific The world leader in serving science

2 Food Authentication Challenges Chemically identical foods or identical chemical entities Unique marker compounds rarely found - more often small analytical differences (isotopic patterns) Large natural variability based on climatic conditions, fertilizers used, variety, processing Techniques must be able to distinguish small differences Databases of authentic foods must be available to understand natural variability 2

3 Meet the Orbitrap TM : The Performance Leader Since 2006 What Orbitrap provides? Fundamental difference to other HRAM instruments Parameter measured is frequency, not time/voltage/et Resolution for more accurate m/z determination Less prone to ambient conditions changes Stability within <1-2 ppm during several days No need for lock mass in routine work Small footprint Easy setup Unmatched ultrahigh resolution, accurate mass performance Which applications? Accurate identification, structural analysis, and quantification of organic molecules, lipids, carbohydrates, peptides & proteins in complex mixtures 3

4 Selectivity Increases With Higher Mass Accuracy Relative Abundance Relative Abundance Relative Abundance RT: 8.45 RT: 8.45 RT: ppm Time (min) 20 ppm Time (min) ppm Time (min) m 4 4 { m 4 *Zachariasova M et al, Anal. Chim. Acta

5 Elemental Composition Confirmation Accurate mass and fragmentation are not the only tools available to us Accurate mass gives us access to elemental composition Very high resolutions bring even more power to our ability to determine correct elemental composition 100 Relative Abundance Relative Abundance 0 15 N C C 11 H 24 O 2 N 7 or C 15 H 28 O 4 N m/z VHRAM

6 High Resolution Essential for Fine Isotopic Pattern Determination L-Methionine C 5 H 11 NO 2 S (+ mode) Observed Simulated Relative Abundance R= ppm R= R= m/z C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:40) Chrg 1... Relative Abundance A R= ppm A1 R=500, R= m/z A R= Relative Abundance m/z C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:40) Chrg 1... Relative Abundance C 5 H 12 O 2 15 NS C 5 H 12 O 2 N 33 S C 4 13 CH 12 O 2 NS m/z R=30k R=60k R=120k R=240k R=500k C 5 H 12 O 17 ONS C 5 H 112 HO 2 NS 6

7 Comprehensive Workflow for Food Metabolomics Food Sample Samples Preparation Chromatography Mass Spectrometry Results Amino acids & amines, cationic metabolites MeOH extracts HILIC +/- mode MS Untargeted Profiling Discovery Translational Flux DAVIS, CA, USA Sugars, organic acids, nucleotides MeOH extracts Fatty acids, bile acids, lipid metabolites MeOH extracts Ion Chromatography - mode MS GC Q Exactive EI UHPLC C18 +/- mode MS Thermo Scientific Q Exactive HF Identification Structure ID De Novo ID Thermo Scientific Orbitrap Fusion Lumos Targeted Screening Profile LipidSearch Identify Glycerolipids Phospholipids Sphingolipids MTBE extract RPLC C18 or C30 +/- mode MS Pesticides Known EFS contaminants vdia Thermo Scientific Q Exactive Focus Quantify 7

8 Compound Discoverer 2.0: Unknown Workflow Powerful and flexible node based workflow Batch searching against mzcloud, ChemSpider databases Mass list search Unknown elemental composition Differential analysis 8

9 Compound Discoverer: Comprehensive Data Review USA Davis CA Isotope match and Mass error <0.5ppm Predicted formula, mass error, score 9

10 Automated ID Using mzcloud: Tartaric Acid (grapes) USA CA 0.3ppm 0.8ppm Query spectrum 0.4ppm 1.5ppm 0.1ppm Davis mzcloud Entry 10

11 Parallel Identification Through Multiple Reference Sources mzcloud Library Spectrum ChemSpider Search Results Local Library Results 11

12 Peak Detection and Candidate Matching with GC Orbitrap EI Deconvolve TIC Create clean spectrum Candidate Compounds Acquired Library Library search 12

13 High Resolution Filtering Candidate Compounds Subset formulae C 8 H 8 Cl 2 O 2 (m/z *Intensity) explained HRF Score = x 100% (m/z *Intensity) observed 13

14 Food Authenticity with Thermo Scientific GC Q Exactive Principle component analysis Clear differences apparent Bourbon J Single malt A PCA form SIEVE Software Bourbon J well separated from single malts Single malt S Except Single malt A more like bourbon than other single malts Pool 10 year 15 year 15 year 14

15 Ambient Ionisation Sources Coupled to Thermo Scientific Orbitrap MS

16 Sudan I Powder In Paprika At 50 ppm Relative Abundance [M+H] + External Cal -1.4 ppm Paprika Blank Paprika spiked with Sudan I NL: 3.13E7 NL: 2.28E m/z 16 Data provided by E.Crawford

17 DART- Orbitrap: Melamine In Milk At 50 mg/kg (A) DART TOFMS mass resolving power 3500 FWHM ( (C) FIA Orbitrap MS resolving power 50,000 FWHM 17 Vaclavik et al, Journal of Chromatography A, 1217 (2010)

18 Stable Isotope Ratio MS BSIA Bulk Stable Isotope Analysis Reactor Elemental Analyzer Conversion GC Separation Ref. gases Open Split DELTA V 2 H and 18 O or 13 C, 15 N and 34 S CSIA Compound Specific Isotope Analysis GC Separation of complex mixtures Capillary Reactor On-line Conversion Ref. gases Open Split DELTA V 2 H or 18 O or 13 C or 15 N of all Compounds 18

19 EA IsoLink IRMS: C analysis Of Ethanol δ 13 C can be used to determine if wines have been adulterated with sugar See Applications note AN

20 EA-IRMS: δ15n In Tomatoes (Organic vs. Non-Organic) Mineral fertilizer show low N values while organic fertilization by compost reults in higher N values. Organic Fertilization Mineral Fertilization N [ ]

21 EA IsoLink IRMS System: NEW in 2016 The new Thermo Scientific EA IsoLink IRMS System is an all-in-one fully automated, modular EA IRMS solution for all CNSOH applications. A modular EA-IRMS System fully software supported for CNSOH analyses The EA IsoLink IRMS System includes: Thermo Scientific Flash IRMS Elemental Analyzer Thermo Scientific ConFlo IV Universal Interface Thermo Scientific Isotope Ratio Mass Spectrometer 21

22 EA IsoLink IRMS System: Temperature Ramped GC NCS Analysis using Flash HT Plus System N 2 CO 2 SO Intensity (mv) Analysis time: 18 mins SO 2 peak width: 200 secs Sample: Sulfanilamide (400 μg) Time (secs) NCS Analysis using EA IsoLink IRMS System N 2 CO 2 SO 2 Analysis time: 10 mins SO 2 peak width: 60 secs Sample: Sulfanilamide (200 μg) 22

23 Analyze High C/S Ratio Samples In One Run Analyzing small amounts (< 20 μg) has become much simpler and more reliable 23

24 EA IsoLink IRMS System: Lowest Helium Usage 24

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