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

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 So where the targeted analysis failed: few examples, the use of illegal proteins (kolagene from leather waste) being the recent one. The reason: cost cutting in most cases (melamine, dyes, protein…) The others are unexpected contamination issues like the residues of inks and various modifiers in the cardboards, acrylamide in crisps and many others…

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

Triple Quadrupoles Used for Targeted Analysis TSQ Quantum Access Max TSQ Quantum Access TSQ Quantum Ultra TSQ Vantage The following is what Thermo Scientific has to offer in the TSQ line. For the ultimate value we have the TSQ Quantum Access Max. For the utmost detection needed TSQ Vantage which was introduce in 2008. Ultimate Value Ultimate Performance

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

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

Shift Towards HRAM : Crossover Point - cont

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

How Much Mass Accuracy is Needed? 340 350 360 370 380 390 400 410 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 381.07828 C 27 H 11 O 2 N [M+H]+ 381.07828 Mass accuracy Number of hits 100 ppm 34 50 ppm 17 30 ppm 11 10 ppm 4 5 ppm 2 3 ppm Compounds containing C[5-15]N[0-10]O[0-15]H[0-40]

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

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? 289.00 289.05 289.10 289.15 289.20 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 289.1314 289.0513 289.1022 289.1215 289.1329 289.1424 R = 10,000 289.00 289.05 289.10 289.15 289.20 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 289.1390 289.1021 289.0514 289.1215 289.1329 289.1424 R = 20,000 289.00 289.05 289.10 289.15 289.20 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 289.1424 289.1547 289.1329 289.1022 289.0514 289.1215 R = 100,000 288.0441 C9H21O2P1S3 Terbufos 288.0949 C13H21O3P1S1 Iprobenfos 288.1142 C15H17N4Cl1 Myclobutanil 288.1256 C11H20N4O3S1 Epronaz 288.1351 C11H21N4O3P1 Pirimethaphos 288.1474 C16H20N2O3 Imazamethabenz How would this be used for residue analysis in food? These six pesticides have the same nominal mass, but different exact mass. The six compounds are isobaric compounds. Is a simultaneous measurement possible for the six compounds? At 20,000 resolution, which TOF instrument can reach, impossible. But at 100,000 resolution, total separation, which TOF unable to reach. 0.1033µ 11

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

Targeted Screen Report Target Screen Approach Raw Data Targeted Screen Report This slide shows the main targeted screen approach: GC or LC HRMS data acquisition (non targeted) Analysis of raw data by searching against database of known compounds (ExactFinder) Library of “Knowns”

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?

MS Detectors for Unknowns Ion Trap MS High Resolution Orbitrap MS For non-target screening, ion trap MS can be applied. Ion Trap MS ID peaks of unknowns by full scan mode. Confirmation by MSn in a second run. More often, HRMS are used because of the accurate mass and high resolution offered by these instruments. The same, when use HRMS for non-target screening, analytes defined after data acquisition ↑Selectivity by exact mass Confirmation by accurate mass & ion ratio deviation Quantitation is available for the instrument with good dynamic range, such as Orbitrap Exactive. Click 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

Key Requirements: Mass Accuracy + Resolution m/z 127.07263 C, N, O, H ppm error Number of hits 150 11 50 4 10 1 5 2 m/z 707.21625 C, N, O, H ppm error Number of hits 150 38 50 12 10 3 5 2 Mass accuracy and resolution are important factors but strongly depend on the mass... See example of two ions, and number of potential hits based on various mass errors: 10 ppm with mass 127 means 1 hit, while it means 3 hits with mass 700. And gets worse when other elements are considered. Conclusion: the mass accuracy and resolution is important but needs additional information.

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 This is an example of the attempt to use a simplistic sample preparation approach in conjunction with HRMS to search for “unknowns” in milk powder. Various contaminants (pesticide, dyes, adulterants) were spiked into milk and processed as shown above. The results obtained are described below. Poster: NEW STRATEGIES FOR FOOD PROFILING AND MONITORING USING HIGH RESOLUTION MASS SPECTROMETRY APPROACH, M. GODULA1, A. CHARLTON2, D. ROBERTS2 AND K. MITTENDORF1

TIC and BPC of Milk Extract - No Difference… TIC (Total Ion Current) BPC (Base Peak Chromatogram) The most critical issue in unknown analysis is to find a presence of a “foreign” peak. However by looking on the TIC or base peak chromatogram (BPC) we can see some peaks but certainly not to say if they are natural of contaminants. The key question is: where is the information?

Automated Component Detection - ExactFinder Background subtracted file Components extracted Some of the new data processing algorithms like to automated component detection in ExactFinder can reduce the data complexity, remove background and show the presence of a chromatographic peak: picture below shows clearer chromatogram when data reduction is performed but info is still limited - too many unknowns. Now it is important to decide what is the peak we look for.

Comparing Contaminated Sample (10ppm) With Blank The only visually identified difference Fenthion Sudan dyes Melamine Cyanuric acid Urea We can compare the contaminated (or real) sample with a typical blank sample. When looking on the chromatograms shown above, we can find only one visible difference while more compounds are spiked into sample at reasonably high concentration of 10 ppm. By only manual data processing it is impossible to reveal any difference between samples. We have identified only one visible difference – blue rectangle. Let’s look at this one in more detail: click

Blank Milk vs. Spiked Milk – Visual Comparison Only 20 hits < 10 ppm CHNOSP We can look at the peak, subtract backgroung, take spectra and what we see is a mix of peaks. Let’s assume the highest one is the pseudomolecular ion. Large list of positive hits is generated – no confidence in the results unless more parameters are involved. We can now apply spectral searching, isotopic pattern fitting an so on and hope we have the right one! But what about the others? We cannot even see them on the chromatogram. click

SieveTM –Data Comparison Tool Blank sample Alignment Framing ChemSpider Search Real sample Compensation of RT changes Table of identified frames Table of positive hits Sieve is the software tool originally developed for identification of metabolites in metabolomic studies. It is designed to make alignment of retention times of blank (reference) sample and real (contaminated) sample. After alignment the Sieve identifies frames within the 3D space of retention time, m/z and abundance. Each frame is unique and defined by retention time, mass and abundance. After generating list of frames, those are compared between 2 sets of data and differences are found. Those are then subjected to search of accurate masses against the web based databases. Click

Sieve™ – Framing Process Explain the Sieve framing process (frame extraction) – describes how Sieve creates frames in the 3D space of the chromatographic data and classifies them according to abundance.

Sieve™ – Database Search Option ChemSpider search Web based Number of databases available These are new features built in the Sieve: possibility to search ChemSpider in multiple databases and for multiple molecular modifications (adducts) or use Targete MZ Lookup feature where the search through custom built database makes the search more targeted and faster: but library is necessary. Targeted search MZ Lookup Custom build library Adducts searched

Advanced Algorithms – Elemental Composition Calculation ExactFinder considers isotopic pattern as a confirmation criteria MH+ Empowering confidence by elemental composition confirmation is essential. By applying isotopic pattern recognition algorithm available in ExactFinder the predicted elemental composition can be confirmed. 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

Isotopic Pattern Fitting is Influenced by Resolution The picture shown here documents one more important factor. If we want to fit the elemental composition to the isotopic pattern, resolution of the acquired data is essential. On the picture shown here the pattern looks OK but if we look at the least abundant isotope (282.0811) we can see by zooming in Click That the correct isotope is much less abundant then adjacent matrix peak. Without ultra high resolution applied the isotopic pattern would NOT fit.: Argument for Exactive or Orbitrap. Table of candidates Isotopic patterns compared Resolution is needed to separate overlapping peaks

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 Self explanatory.