Innovazioni tecnologiche per l¢â‚¬â„¢analisi GC-MS in bassa ed ... Innovazioni tecnologiche per l¢â‚¬â„¢analisi

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  • The world leader in serving science

    Innovazioni tecnologiche per l’analisi GC-MS in bassa ed alta risoluzione con il nuovo GC-Orbitrap Dott.ssa Cristina Neri, Ph.D Sales Specialist GC-GC&MS 1°Seminario di Spettrometria di Massa. Università degli studi di Milano 23/06/2017

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    • Bassa Risoluzione vs Alta Risoluzione

    • Il GC-Orbitrap

    • Untargeted Metabolomics using Orbitrap GC-MS • Conclusioni

    • Q&A

    Agenda

  • 3 The world leader in serving science

    Bassa Risoluzione vs Alta Risoluzione

  • 4

    Resolution- what is it?

    • Ability of a mass spectrometer to distinguish between ions of nearly equal m/z ratios (isobars).

    • m - measured mass • Δm - peak width measured at 50% peak intensity (Full Width Half Maximum)

    - or the mass difference between two adjacent peaks of equal intensity, in this case pw @ 10% valley definition is used.

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    Resolution- what is it?

    • Ability of a mass spectrometer to distinguish between ions of nearly equal m/z ratios (isobars).

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    Resolution- what is it? • At minimum the resolution of the mass analyzer should be sufficient to separate two

    ions differing by one mass unit anywhere in the mass range scanned (unit mass resolution).

    • typical values of resolution for low resolution mass analyzers (e.g. quadrupoles and ion traps) are below 5000.

    • High resolution instruments have a resolution exceeding 15000.

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    • Selectivity:

    • High mass resolution can greatly reduce matrix interference.

    • It can also reduce interference between ions of the same compound (e.g. fine isotopic structure).

    Why resolution is important?

    ±0.00157 amu ±0.5 amu

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    Resolving Power: Selectivity

    False negative

    False negative

    Positive Detection

    High Selectivity ∴ high sensitivity and confidence in identification

    Pyrimethanil in leek at 10 μg/Kg < 5 ppm ID criteria

    R el

    at iv

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    Ab un

    da nc

    e R

    el at

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    Ab

    un da

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    R el

    at iv

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    Ab un

    da nc

    e

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    Mass Accuracy: what is it?

    • Mass accuracy is the accuracy of which the mass is measured by the mass spectrometer. • Typical way of reporting mass error in ppm (relative mass error): • Absolute mass error can be used (mDa). • Main advantage: the possibility to determine the elemental composition of individual molecular or fragment ions, a powerful tool for the structural elucidation or confirmation.

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    1 1,5 2 5 10 50

    N o

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    ul ae

    Mass Accuracy (ppm)

    Chemical element No

    C 50 H 100 O 10 N 10 Cl 10

    147

    2 5 7

    16

    30

    Compound ID confirmation

  • 11 The world leader in serving science

    Il GC-Orbitrap

  • 12

    Introduzione del GC-Orbitrap

    “An Illustrated History of Gas Chromatography: Thermo Scientific Q Exactive GC”

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    Redefining Routine GC-MS

    RP 60,000 (FWHM @ m/z 200)

    EI/CI; Full-scan; Timed-SIM

    Thermo Scientific™ Exactive™ GC system

    New Addition to the Orbitrap GC-MS Family

    Thermo Scientific Q Exactive GC system

    Unprecedented Depth in Analysis

    RP 120,000 (FWHM @ m/z 200)

    EI/CI; Full-scan, Timed-SIM

    MS/MS capability

  • 14

    Exactive GC system: The Technology Inside

    Thermo Scientific™ ExtractaBrite™ Ion Source technology

    Routine grade robustness

    Patented RF lens

    Removable without breaking vacuum

    Orbitrap mass analyzer

    Incredible HRAM performance

    Highly regarded Q Exactive GC system platform

    Thermo Scientific™ TRACE™ 1310 GC System

    Unique modular injector and detector design

    Rapid heat cycling

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    Bringing GC and Orbitrap Technology Together

    C-TRAP

    HCD Cell

    AQT Quadrupole

    Orbitrap Mass Analyzer

    Bent Flatapole

    ExtractaBrite™ Ion Source

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    What the Orbitrap Looks like

    Inner-electrode Split outer-electrode

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    • Ions are stored and cooled in the RF-only C-trap

    • Ions are ejected along lines converging to the pole of curvature (which coincides with the Orbitrap entrance).

    • As ions enter the Orbitrap, they are picked up and squeezed by its electric field

    Pulsing Ions into the Orbitrap: Curved Linear Trap (C-trap)

    Push Trap Pull Lenses Orbitrap

    Gate Deflector

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    zm k /

    Retaining the Ions in the Orbitrap

    • Frequency of axial oscillations are independent of initial conditions of ions entering trap • Therefore these oscillations used for mass determination • Image current measured by outer split electrodes (no electron multiplier to replace!) • Ion frequencies determine by complex superposition of measured ring oscillations

    through Fourier transform

  • 19

    GC-MS Until Now

    Challenge

    Compromised Options

    Multi-instrument approach • Can be inefficient, convoluted

    and complicated

    • GC single and triple quadrupole MS for target quantitation

    • GC- Time-of-flight (Tof) MS with limited performance

    Limited GC-MS analysis • No comprehensive

    quantitative & qualitative analysis

    • For quantitation: targeted approaches required

    • HR/AM Qualitative approaches compromised

  • 20

    Breakthrough in GC-MS Performance

    Sensitivity

    ppt

    Resolution

    Up to 120,000 at m/z 200

    • Highest available

    • Maximum selectivity

    • Fast enough for GC

    Mass accurancy

    < 1 ppm

    • Every scan

    • All concentrations

    • In complex matrix

    • Across the mass range

    • Everyday!!

    • In full scan

    • High selectivity

    • High spectra fidelity

    Dinamic Range

    >6 Orders

    • Excellent coverage in sample profiling

    • “Triple quad grade” quantitation in full scan

    &

  • 21

    Exactive GC System Main Workflows

    Targeted Quantitation

    expected

    1x Full-scan

    Scope

    Non-targeted Screening

    unexpected

    Targeted Screening

    suspected

  • 22

    Non-targeted Screening Overview

    • Sensitive and selective peak detection

    • High resolution spectral deconvolution

    • Clean spectrum

    detect and refine

    1 generate candidates

    2 filter and identify

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    • Search spectra against spectral libraries

    • HRAM or unit mass

    • Candidates list generated

    • High resolution filtering of candidates

    • Putative identifications made

    Process semi/fully automated as preferred

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    Identify the compound – searching NIST 14

    26 Hits from NIST are sorted based on: 1. Spectral matching.

    2. High Resolution Filtering (HRF) score. % of the ions in the

    spectrum that can be explained by the element in the proposed compound.

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    Identify the compound – searching NIST 14

    Combined SI and HRF values give an overall score (%) to quickly and confidently identify the compound. Eliminates other hits that would be valid if only SI used.

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    Fragments can be explained with < 1ppm mass accuracy

  • 26

    Q Exactive GC: Compound Discovery and Identification

    Search

    Library

    Identify

    AM filtering

    Known Unknown

    Unknown Unknown …

  • 27

    Q Exactive GC: Compound Discovery and Identification

    Remove entire ion source or change to CI source in under 2 minutes without venting …

  • 28

    EI & PCI spectra for peak at 15.17 mins.

  • 29

    08OCT15_01 #2053-2062 RT: 15.65-15.66 AV: 10 NL: 1.19E7 T: FTMS + p CI Full ms2 325.14@hcd10.00 [50.00-350.00]

    60 80 100 120 140 160 180 200 220 240 260 280 300 m/z

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    R el

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    un da

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    223.07545 C 15 H11 O 2 = 223.07536

    0.42975 ppm

    251.10672 C 17 H15 O 2 = 251.10666

    0.26844 ppm195.08049 C 14 H11 O = 195.08044

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