Gesundheitsdirektion Moving from targeted towards non-targeted approaches Anton Kaufmann Official Food Control Authority of the Canton of Zurich () Switzerland
2 Overview I From single residue to multi residue II Data dependent acquisition (DDA) III Data independent acquisition (DIA) IV Deconvolution based approaches V Data mining of compound lists
3 Overview I From single residue to multi residue II Data dependent acquisition (DDA) III Data independent acquisition (DIA) IV Deconvolution based approaches V Data mining of compound lists
4 Food matrices can be extremely complex Even the combination of UHPLC and HRMS will not resolve all trace analytes from the matrix peaks Product ion information is essential for unequivocal identification of trace analytes
5 Single Target 1 SRM Product ion
6 Multiple Targets many SRM s
We got a hit! Kanton Zürich 7
Positive findings frequently generate new questions Kanton Zürich 8 Now; since drug A is present, how about related drugs, how about metabolites?
9 Tandem quadrupole is not the technique to navigate in an ocean of data
10 Overview I From single residue to multi residue II Data dependent acquisition (DDA) III Data independent acquisition (DIA) IV Deconvolution based approaches V Data mining of compound lists
Data dependent acquisition (DDA) Kanton Zürich 11 - Checking the chromatogram for the accurate mass of suspected analytes (inclusion list) - MS/MS triggering of any sufficiently abundant chromatographic peak
12 Data dependent acquisition (DDA)
Signal intensity Point of triggering Kanton Zürich 13 time
Correct triggering Kanton Zürich 14 - Complex set-up - Inclusion & exclusion list (targeted) - Thresholds (un-targeted) - No guarantee that every compound of interest will be triggered (how to validate limits of detection?)
Signal intensity Library based Ion ratio Kanton Zürich 15 Two ions for the calculation of an ion ratio DDA based Ion ratio Product ion 1 Peak area based Ion ratio Product ion 2 time
16 Overview I From single residue to multi residue II Data dependent acquisition (DDA) III Data independent acquisition (DIA) IV Deconvolution based approaches V Data mining of compound lists
Data independent acquisition (DIA) Kanton Zürich 17 - Monitoring different MS experiments Sequentially isolating mass ranges which afterwards undergo fragmentation (SWATH) Low and high collision energy traces (all ion fragmentation) e.g. assisted by ion mobility separation
SWATH Kanton Zürich 18 m/z
19 SWATH Scan 1 Scan 2 Scan 3 Scan 4 Scan 5 Scan 6 Scan 7 Scan 8 m/z : 100 200 300 400 500 600 700
20 Overview I From single residue to multi residue II Data dependent acquisition (DDA) III Data independent acquisition (DIA) IV Deconvolution based approaches V Data mining of compound lists
Deconvolution Kanton Zürich 21 5 4.5 Chart Title 4 3.5 TIC 3 2.5 2 1.5 1 Analyte A Analyte B 0.5 0 0 1 2 3 4 5 6 Retention time
Principle of Deconvolution Kanton Zürich 22 Ions originating from the same compound will be correlated across the time range of a chromatographic peak - Based on calculation of coefficient of correlation - Based on principle component analysis (All techniques require a prior peak alignment)
rel. Response rel. Response Kanton Zürich 23 5 µg/kg methyltestosterone in bovine liver unit mass resolution based product ion (PRM) spectrum MRM (peak @ Peak apex) Apex 10000 9000 8000 7000 All ion fragmentation (AIF) spectrum (peak AIF @ Peak apex) Apex 10000 9000 8000 7000 6000 6000 5000 5000 4000 4000 3000 3000 2000 2000 1000 1000 0 50 150 250 350 450 m/z 0 50 150 250 350 450 m/z
rel. Response rel. Response 4000 Correlation across a chromatographic peak versus spectra at the peak apex MRM (PRM) e 10000 Kanton Zürich 24 AIF (AIF) e 3500 9000 8000 3000 7000 2500 6000 2000 5000 1500 4000 1000 500 3000 2000 1000 0 50 150 250 350 450 m/z 0 50 100 150 200 250 300 350 400 450 m/z
rel. Response rel. Response Kanton Zürich 25 SWATH produces DIA data with MRM spectra quality 4000 (PRM) MRM e 10000 (SWATH) e 3500 3000 9000 8000 7000 2500 6000 2000 5000 1500 4000 3000 1000 2000 500 1000 0 50 150 250 350 450 m/z 0 50 150 250 350 450 m/z
The Potential Kanton Zürich 26 - Deconvolution algorithms isolate all chromatographic peaks from the three dimensional landscape (e.g. UNIFI) - Spectra clean up by ion mobility assisted AIF - Scan based or nested window SWATH
The Limitations Kanton Zürich 27 - Fast acquisition speed is essential in order to: Do as many experiments as possible (narrow SWATH windows and possibly multiple collision energies))
28 Extracting all components (compounds) and not just spectra precursor + isotopes
29 Cutting the mountain tops above a certain threshold (deconvolution)
30 Grouping the mountains into ridges (componentization)
Peak deconvolution and componentization Synchronizing drift time with m/z Kanton Zürich 31 Column bleed m/z noise Retention time
32 Componentization does more than only finding isotopes - Product ions - In source fragments of labile precursor ions - Adducts (e.g Na + ) or dimers
33 Component list instead of mass traces Reten tion time Precursor ion m/z Product ions m/z 2.67 257.156 0.2 156.102 0.5 68.1256 0.3 56.024 0.4 2.56 356.058 0.7 256.098 0.2 198.025 1.2 98.624 0.8 2.57 286.105 0.2 198.064 0.9 125.025 2.2 97.0125 1.6 2.58 346.112 0.25 324.106 2.0 187.101 1.5 87.0215 2.5 m/z min m/z Abundance Abundance Abundance Abundance Possibly 10 000 s of components (compounds)
Data Mining Kanton Zürich 34
35 Data mining tools - Filters - MS/MS Library search
36 Filters - Halogen & Sulfur - Characteristic product ion - Neutral loss filter - Removal of multiple charged ions (e.g. peptides) - Mass defect filter
MS/MS library based search Kanton Zürich 37 - MS/MS Library search ( red, exogenous compounds) - In silico fragmentation for suspect screening - TIC clean-up (not simple baseline subtraction but removal of green endogenous compounds)
38 The problem: Finding the needle in the haystack
39 Solution step 1: Getting rid of endogenous signals
Orthogonal filter Kanton Zürich 40
41 Solution step 2: Utilizing the specific properties of the exogenous compound
42 True non-target in complex matrices still needs some additional innovations In the future we shall travel in the air