NON TARGETED SEARCHING FOR FOOD CONTAMINANTS USING ORBITRAP HIGH RESOLUTION MASS SPECTROMETRY Michal Godula 1, Adrian Charlton 2 and Klaus Mittendorf 1 1 Thermo Fisher Scientific, Dreieich, Germany 2 Food and Environment Research Agency, York UK
Strategies for Screening of Organic Contaminants Organic Contaminants Known Knowns Known Unknowns Unknown Unknowns QQQ QQQ? High Resolution >50,000 Target screening Non-target screening Rapid and sensitive screening methods able to assign hits positively to particular organic compounds 2
Where Target Analysis has Failed? 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 3
Non targeted analysis using mass spectrometry Data acquisition: Chromatography-MS High Resolution + Accurate Mass-HRAM Other options: NMR, FTIR, FTNIR, Raman Data processing - relevant data extraction (peak, m/z) - libraries of accurate masses/fragments/adducts - sample-to-sample comparison approach - statistical models and database of normal samples needed Sample preparation? 4
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. GODULA 1, A. CHARLTON 2, D. ROBERTS 2 AND K. MITTENDORF 1 5
Real sample chromatogram: where is the information? RT: 0.39-60.03 30 25 SM: 7B TIC 44.46 38.18 43.82 46.56 NL: 1.58E8 TIC F: FTMS {1,1} + p ESI Full ms [50.00-1000.00] MS Non_Target_Milk_PosC1 8ESI_250809Sample Relative Abundance 20 15 10 5 14.81 41.80 48.62 10.26 41.27 48.98 9.82 13.82 19.81 33.20 49.72 8.56 5.47 16.73 37.76 21.12 50.40 22.71 27.95 31.41 34.90 51.07 27.60 51.69 53.57 55.96 0 30 25 BPC 38.20 44.46 NL: 3.70E7 Base Peak F: FTMS {1,1} + p ESI Full ms [50.00-1000.00] 00 00] MS Non_Target_Milk_PosC1 8ESI_250809Sample 20 10.25 46.56 14.81 41.80 15 33.14 10 5 46.81 48.85 2.03 41.46 50.18 9.30 19.84 37.76 50.83 2.59 8.32 12.78 37.52 51.22 15.18 21.10 22.62 30.21 52.36 54.95 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min) 6
Persistent background subtraction Background subtracted file Clusters extracted (RT and m/z) 7
Blank milk vs. spiked milk RT: 8.71-60.03 SM: 7B 105 100 95 38.18 43.82 44.46 90 46.56 85 80 75 14.81 14.81 38.23 41.80 41.80 46.58 48.62 70 Relative Abundance 65 60 55 50 45 40 35 30 25 20 15 10.26 10.29 13.82 12.79 41.27 48.98 40.53 49.28 33.20 49.72 19.81 33.16 37.53 50.18 16.73 37.76 50.40 21.12 22.71 27.95 50.79 22.95 34.90 31.41 49.48 51.07 25.01 35.96 19.87 27.60 51.22 26.12 26.32 52.98 16.71 27.89 51.69 17.75 22.58 28.14 53.57 54.09 54.95 55.96 58.20 10 10 15 20 25 30 35 40 45 50 55 6 Time (min) 8
Manual data extraction Non_Target_Milk_PosC18ESI_250809Sample 8/26/2010 1:52:43 AM S5 spike 10ppm B RT: 42.64-46.16 SM: 7B Relative Abundance 110 105 100 95 90 85 80 75 70 65 60 55 RT: 43.82 44.46 45.36 45.69 42.91 42.94 44.22 44.69 45.03 43.24 43.27 43.59 45.76 46.09 45.91 45.22 NL: 4.47E7 TIC F: FTMS {1,1} + p ESI Full ms [50.00-1000.00] MS Non_Target_Milk_PosC1 8ESI_250809Sample 42.8 43.0 43.2 43.4 43.6 43.8 44.0 44.2 44.4 44.6 44.8 45.0 45.2 45.4 45.6 45.8 46.0 Time (min) Non_Target_Milk_PosC18ESI_250809Sample #3077 RT: 43.84 AV: 1 SB: 31 43.35-43.61, 44.09-44.25 NL: 3.52E6 T: FTMS {1,1} + p ESI Full ms [50.00-1000.00] 93.05714 100 90 Relati ve Abundance 80 70 60 50 40 30 20 10 0 128.04893 156.04360 9 249.10082 116.98526 188.06968 232.09821 149.01094 133.03367 160.07487 84.95951 184.07246 70.98044 101.00794 177.00567 195.08568 225.36233 214.91702 243.59590 253.32847 279.64330 297.00241 60 80 100 120 140 160 180 200 220 240 260 280 300 m/z hits < 5 ppm CHNOS P Cl Br 9
Sieve TM Data Comparison Tool Blank sample Real sample Alignment Framing ChemSpider Search Compensation of RT changes Table of identified frames Table of positive hits 10
11 Sieve Framing Process
Sieve Database Search Option ChemSpider search Web based Number of databases available Targeted search MZ Lookup Custom build library Adducts searched 12
Using Sieve to screen spiked milk samples 10 mg/kg 13
Untargeted analysis detecting differences Compound 1 Compound 2 Compound 4 Compound 3 Response ratio in different sample vs. P-value shows statistically significant differences 14
15 PCA analysis of data
Sieve frame generation and data comparison Difference in data found ChemSpider search Hits reported Confirmation? 16
Total Unknown Screen and Confirmation High Resolution Full Scan Best Match for HR AM / MS 2 SIEVE Blank/Sample Sample Blank Library Search (Int or Ext) Generate Inclusion List High Res MS ddms 2 17
Q-Exactive DDMS2 screening workflow Sample Ion Accumulation Sample preparation Orbitrap Full Scan Analysis 70.000000 resolution UHPLC/Q-Exactive Compound Inclusion List, up to 5000 cpds Precursor Selection (List) Peaks Found MS/MS precursor Confirmation Reporting Integration, Quantitation, Confirmation (2 ions + MS/MS spectra) Automated (15 min/run*) Fragmentation in HCD Orbitrap Full Scan MS 35.000 resolution 18
Metconazole in pepper MS/MS Confirmation 74 ppb Metconazole in Pepper Sample: (74 ppb) Pepper_025 #4119 RT: 9.74 AV: 1 NL: 6.86E4 F: FTMS + p ESI d Full ms2 320.15@hcd35.00 [70.00-345.00] 100 320.2950 Data dependent MS/MS confirmation ative Abundance Rela 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 91.0550 107.0499 111.1174 212.2374 215.9721 228.2325 287.9568 125.0156 233.9826 95.0863 177.0467 81.0708 133.10151015 197.19051905 259.9621 171.0740 243.9674 274.2249 139.1119 191.0626 303.2325 80 100 120 140 160 180 200 220 240 260 280 300 320 340 m/z 19
Summary Simplified sample preparation strategies or direct analysis Highly accurate mass and high resolution data acquisition is a necessary requirement Advanced data processing algorithms needed: Data extraction and background subtraction Comparative approach (Sieve) Libraries of accurate masses and fragments Isotopic pattern fitting Final confirmation my MS/MS is a must But. 20