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Die Nadel im Heuhaufen Workflow zur Identifizierung unerwarteter Komponenten in LC Q-Tof Daten Umwelt & Lebensmittel Seminar Tour Andreas Reimann Produktspezialist LC-MS Agilent Technologies

Instrumentation

Quantifizierung Sulfamethoxazole ESI-positive-Modus

Qualitative und Quantitative Analyse unknown screening was ist in der Probe? Nicht-Selektiv Target Screening sind bestimmte Substanzen in der Probe und wenn ja wieviel? Quantifizierung wie hoch ist die Konzentration bestimmter Komponenten in der Probe? Selektiv

Workflows: Screening vs. Profiling

Screening

processing Different algorithms to remove background, extract compounds Molecular feature extraction (MFE) by Formula (used for MS data) by Auto MSMS (used for data-dependent MSMS data) by Target MSMS ( used for data from targeted experiments) base search of accurate masses with or without retention time Used for MS data from MFE, by Formula Also used for MS data from Auto MS/MS and targeted MS/MS Library search for accurate mass MS/MS spectra Used for msms spectra from data-dependent Auto MS/MS Used for MS/MS spectra from targeted experiments Used for MS/MS spectra assigned to MFE compounds

by Formula Ion Information Ion chromatogram drawn from information Spectra extracted from integrated peak Compound information from formula or base Spectral performance calculated

by Auto MS/MS or Targeted MS/MS by Auto MS/MS or Targeted MS/MS automatically extract the MS and MS/MS information, aligns the spectra sorts and display the according spectra as a single compound. The data can be used to calculate empirical formulas for MS and MS/MS. In addition the information can be used to search the database with the MS data and library search the MS/MS data using the accurate mass MS/MS spectral library. Qual B.04 extracts MFE from Auto MSMS and aligns possible product ion spectra to found features. This allows for a database search of accurate MFE compounds and an MSMS library search of the attached product ion spectra.

Molecular Feature Extractor (MFE) MFE is extremely fast Does not use exhaustive EIC extraction Uses total ion count abundance information, not just base peak Can yield more false negatives than exhaustive approach chromatographic peaks Subtract background Locate the groups of co-variant ions in each chromatogram; Adducts, dimers, isotopes ([M+H] +, [M+H+1] +, Sum all ion signals into one value Raw data Background noise removed Individual m/z peaks grouped into isotope clusters Isotopes grouped into features

bases and Libraries base Consists of Name Empirical formula Accurate monoisotopic mass Optional Structure CAS_Number Chemspider Number Retention time Library Same as database plus structure In addition it contains Spectra from Different collision energies Different ion sources Different polarities

base searching From by Formula: Software extracts ion chromatogram based on information from database. Retention time is used to separate isobaric compounds and to speed up the process. From Auto MSMS data: MS data is compared with the database. No MSMS information used. From MFE data: After MFE a list of masses is compared with the database. Optional retention time can be used to separate isobaric compounds. Can be used with pos/neg switching. Positive and negative results are aligned and displayed together

base searching Results Compound list with name and formula. If additional calculations are used coloring helps to find same or different results in different alogrithms MFE spectrum with structure and formula Results with score and individual scores for mass, abundance and spacing

base searching Results pos/neg switching Compound list with name and formula. If additional calculations are used coloring helps to find same or different results in different alogrithms. Please see polarity column saying both if compound is identified in both ionization modes. MFE spectrum with structure and formula. Both spectra are shown but fidelity and structure only shown in positive spectrum and only for M+H+ if MFG calculated Results with score and individual scores for mass, abundance and spacing

Library searching Acquiring data for a MSMS library Collaboration with leading labs to acquire targeted MSMS data at 3 different collision energies MSMS spectra are imported automatically into the library. A tool calculates the theoretical MSMS masses and corrects the masses, not the intensities in the library Background in MS/MS spectra removed by algorithm This makes library indepentent from lab or instrument used Library alogrithm based on NIST/PBM search. Alternatively you can do it yourself! Open System!

Library build PCDL Tool Spectra from different modes can be stored and searched

Library searching Spectra example Caffeine 10 Volt 20 Volt 40 Volt

Profiling

compounds in MassHunter Qual by Molecular Feature (MFE) by Formula/ ion Naïve Targeted

Compound Exchange File format (.cef) A compound contains Mass RT/RI Acquisition metadata Polarity Fragmentor voltage Saturation warning SNR MS1 peak lists m/z, abund (and/or volume) Ion species (i.e. [M+H]) Identification results Compound name Formula Accession # s (CAS) XML Portability Human readable Compressible (usually > 50% via ZIP) Standards for binary encoding of data One neutral mass, one RT MFE spectra: ~10 (mz, abund) data points Forwards/Backwards compatible

Import of compound into MassProfiler Professional 1.Filter 2.Alignment -with or without standard 3.Grouping -several groups are possible

Import of compound into MassProfiler Professional 4.Import Summary 5.Normalization - With standard - With scalar 6. Baselining - Several options

Import of compound into MassProfiler Professional Green box: one hit wonders : Can be filtert out Improvments of the rest of the data by using recursive workflow

Mass Profiler Professional: Intuitive Analysis Workflow

Mass Profiler Professional: Intuitive Analysis Workflow PCA neg PCA pos Maize spike is statistically different to Maize Blank.

ID Browser Compound Structure Composite Spectra Spectra Details Detailed Compound Table

LCMS GCMS Agilent s Profiling Workflow Separate & Detect Feature ing Quantitate Alignment & Statistics Mass Profiler (MP) GC/MSD GC-QQQ MassHunter Qual AMDIS or by chromatographic deconvolution ID Browser LC-TOF/QTOF LC-QQQ MassHunter Qual MFE, by Formula, by Ion Mass Profiler Professional (MPP)

MassHunter PCDs, PCDLs and PCDL Manager Integrated system for ID via accurate mass MS + MS/MS Personal Compound base (PCD) Compound names Accurate masses (AM), molecular formulas and structures Optional retention times (AMRT) Located on the local PC or intranet ( Personal ) Personal Compound base and Library (PCDL) Same as PCD Plus accurate mass MS/MS spectra for some or all compounds Accurate mass MS/MS library search provides higher confidence MassHunter PCDL Manager software One software to view and edit all local PCDs and PCDLs Pioneered by and unique to Agilent! Q-TOF

Comprehensive offering of PCDs and PCDLs The 2 first accurate mass MS/MS libraries on market (!) G6854AA Pesticides PCD B.04.00 AM database of 1609 compounds G6855AA Forensics and Toxicology PCD B.04.00 AM database of 7360 compounds G3876AA Forensics and Toxicology PCDL B.04.00 In addition contains 8263 accurate mass MS/MS spectra from 2720 compounds at up to 3 collision energies mostly in positive mode. G6825AA METLIN Metabolite PCD B.04.00 AMRT database of 24768 compounds, 670 with RT on a RP HPLC column G3874AA METLIN Metabolite PCDL B.04.00 To analyze the food you eat! Crime Scene Investigation! Acute poisoning! biomarkers for early disease detection! In addition contains 8712 accurate mass MS/MS spectra from 2286 compounds at up to 3 collision energies in positive and/or negative mode.

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