NON TARGETED SEARCHING FOR FOOD

Transcription

1 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

2 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

3 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

4 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

5 Example: Analysis of Milk Powder Milk sample 2g Add 20 ml Acetonitrile/Water 70:30 v/v Extraction 20 min Centrifugation 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

6 Real sample chromatogram: where is the information? RT: SM: 7B TIC NL: 1.58E8 TIC F: FTMS {1,1} + p ESI Full ms [ ] MS Non_Target_Milk_PosC1 8ESI_250809Sample Relative Abundance BPC NL: 3.70E7 Base Peak F: FTMS {1,1} + p ESI Full ms [ ] 00 00] MS Non_Target_Milk_PosC1 8ESI_250809Sample Time (min) 6

7 Persistent background subtraction Background subtracted file Clusters extracted (RT and m/z) 7

8 Blank milk vs. spiked milk RT: SM: 7B Relative Abundance Time (min) 8

9 Manual data extraction Non_Target_Milk_PosC18ESI_250809Sample 8/26/2010 1:52:43 AM S5 spike 10ppm B RT: SM: 7B Relative Abundance RT: NL: 4.47E7 TIC F: FTMS {1,1} + p ESI Full ms [ ] MS Non_Target_Milk_PosC1 8ESI_250809Sample Time (min) Non_Target_Milk_PosC18ESI_250809Sample #3077 RT: AV: 1 SB: , NL: 3.52E6 T: FTMS {1,1} + p ESI Full ms [ ] Relati ve Abundance m/z hits < 5 ppm CHNOS P Cl Br 9

10 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 11 Sieve Framing Process

12 Sieve Database Search Option ChemSpider search Web based Number of databases available Targeted search MZ Lookup Custom build library Adducts searched 12

13 Using Sieve to screen spiked milk samples 10 mg/kg 13

14 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 15 PCA analysis of data

16 Sieve frame generation and data comparison Difference in data found ChemSpider search Hits reported Confirmation? 16

17 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

18 Q-Exactive DDMS2 screening workflow Sample Ion Accumulation Sample preparation Orbitrap Full Scan Analysis 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 resolution 18

19 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 ms @hcd35.00 [ ] Data dependent MS/MS confirmation ative Abundance Rela m/z 19

20 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