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
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
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
Selectivity Increases With Higher Mass Accuracy 100 90 80 70 60 50 40 30 20 10 0 Relative Abundance 100 90 80 70 60 Relative Abundance 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 Relative Abundance RT: 8.45 RT: 8.45 RT: 8.45 2 ppm 7.26 7.41 7.49 7.76 7.91 7.99 8.55 7.67 9.46 10.16 10.31 10.50 10.61 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 Time (min) 20 ppm 7.62 8.21 7.59 7.85 7.37 7.40 7.81 7.92 7.99 8.25 7.26 7.30 8.61 8.67 8.88 9.11 9.46 10.09 10.16 10.31 10.50 10.61 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 Time (min) 8.72 9.29 8.67 8.99 8.78 8.21 9.11 7.62 7.85 8.13 8.83 7.59 7.38 7.81 8.10 8.35 7.33 100 ppm 7.27 9.46 9.50 9.64 10.09 10.16 10.34 10.50 10.61 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 Time (min) m 4 4 { m 4 *Zachariasova M et al, Anal. Chim. Acta
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 286.1999 100 Relative Abundance 0 15 N 287.2033 13 C 287.1970 287.2063 287.20 287.22 287.2033 287.2028 C 11 H 24 O 2 N 7 or C 15 H 28 O 4 N 0 287 288 m/z VHRAM 288.2067 288.2051 5
High Resolution Essential for Fine Isotopic Pattern Determination L-Methionine C 5 H 11 NO 2 S (+ mode) Observed Simulated Relative Abundance 100 90 80 70 60 50 40 30 20 10 150.0584 R=627006 0.7ppm 151.0618 R=621502 152.0542 R=601802 0 150.0 150.5 151.0 151.5 152.0 m/z C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:40) Chrg 1... Relative Abundance 100 90 80 70 60 50 40 30 20 10 0 A0 150.0583 R=499553 0.0ppm A1 R=500,000 151.0617 R=499368 150.0 150.5 151.0 151.5 152.0 m/z A2 152.0541 R=499443 Relative Abundance 100 80 60 40 20 0 151.05 151.06 m/z C5H11NO2S +H: C5 H12 N1 O2 S1 p(gss, s/p:40) Chrg 1... Relative Abundance 100 80 60 40 20 0 151.0554 C 5 H 12 O 2 15 NS 151.0577 C 5 H 12 O 2 N 33 S 151.0617 C 4 13 CH 12 O 2 NS 151.050 151.055 151.060 151.065 m/z R=30k R=60k R=120k R=240k R=500k 151.0625 C 5 H 12 O 17 ONS 151.0646 C 5 H 112 HO 2 NS 6
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
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
Compound Discoverer: Comprehensive Data Review USA Davis CA Isotope match and Mass error <0.5ppm Predicted formula, mass error, score 9
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
Parallel Identification Through Multiple Reference Sources mzcloud Library Spectrum ChemSpider Search Results Local Library Results 11
Peak Detection and Candidate Matching with GC Orbitrap EI Deconvolve TIC Create clean spectrum Candidate Compounds Acquired Library Library search 12
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
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
Ambient Ionisation Sources Coupled to Thermo Scientific Orbitrap MS https://phytronix.com/ldtd/ http://www.prosolia.com/products/desi http://www.ionsense.com/ 15
Sudan I Powder In Paprika At 50 ppm Relative Abundance 100 80 60 40 20 0 100 80 60 40 20 0 116.07076 127.03903 146.08092 146.08090 156.10171 180.08637 198.09690 [M+H] + External Cal -1.4 ppm 160.09660 199.10754 298.27359 216.12267 360.98689 225.14805 279.23139 312.28901 230.10196 249.10189 268.10352 Paprika Blank Paprika spiked with Sudan I 291.15450 NL: 3.13E7 NL: 2.28E7 100 150 200 250 300 350 400 m/z 16 Data provided by E.Crawford
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) 4204 4211
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
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 AN30147 19
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 12.0 10.0 8.0 15 N [ ] 6.0 4.0 2.0 0.0 20
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
EA IsoLink IRMS System: Temperature Ramped GC NCS Analysis using Flash HT Plus System N 2 CO 2 SO 2 7000 Intensity (mv) 6000 5000 4000 3000 2000 1000 Analysis time: 18 mins SO 2 peak width: 200 secs Sample: Sulfanilamide (400 μg) 0 200 400 600 800 1000 1200 1400 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
Analyze High C/S Ratio Samples In One Run Analyzing small amounts (< 20 μg) has become much simpler and more reliable 23
EA IsoLink IRMS System: Lowest Helium Usage 24
Thank you! michal.godula@thermofisher.com The world leader in serving science