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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

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Bassa Risoluzione vs Alta Risoluzione

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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

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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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Mass Accuracy (ppm)

Chemical element No

C 50 H 100 O 10 N 10 Cl 10

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2 5 7

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Compound ID confirmation

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Il GC-Orbitrap

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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

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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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zmk/

=ω

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

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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

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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

&

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Exactive GC System Main Workflows

Targeted Quantitation

expected

1x Full-scan

Scope

Non-targeted Screening

unexpected

Targeted Screening

suspected

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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

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Q Exactive GC: Compound Discovery and Identification

Search

Library

Identify

AM filtering

Known Unknown

Unknown Unknown …

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Q Exactive GC: Compound Discovery and Identification

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

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EI & PCI spectra for peak at 15.17 mins.

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08OCT15_01 #2053-2062 RT: 15.65-15.66 AV: 10 NL: 1.19E7T: FTMS + p CI Full ms2 325.14@hcd10.00 [50.00-350.00]

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223.07545C 15 H11 O 2 = 223.07536

0.42975 ppm

251.10672C 17 H15 O 2 = 251.10666

0.26844 ppm195.08049C 14 H11 O = 195.08044

0.24907 ppm

105.03358C 7 H5 O = 105.03349

0.83676 ppm

145.02854C 9 H5 O 2 = 145.02841

0.92080 ppm

279.10166C 18 H15 O 3 = 279.10157

0.30906 ppm117.0701277.03894 161.90226

MS/MS m/z 325.14 to support proposed formula

>13 fragments can be explained based on C20H20O4

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No. of proposed formulae for m/z 324.13541

1 ppm 3 ppm 5 ppm 20 ppm 10 ppm

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25 Top hit = C20H20O4

Elements used: C (1-30), H (1-60, N (1-5), O (1-5), P (1-2), S (1-2)

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Untargeted Metabolomics using Orbitrap GC-MS

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• Metabolomics aims to characterize and quantify the complete small molecule complement (the metabolome) of a biological system.

• The metabolome is very diverse mixture of small molecules (amino acids, sugars and phosphosugars, biogenic amines and lipids).

• Untargeted metabolomics - challenging due to the requirement to identify and quantify hundreds of different compounds with limited a priori knowledge.

• Ideally - use MS analytical platforms able of sensitive detection of molecules, in addition to providing accurate mass information for structural analysis of unknowns.

Introduction

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Introduction

• The goal of metabolomics is a comprehensive and systematic understanding of all metabolites in biological samples.

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• Targeted • Define specific metabolites • Detect them with appropriate parameters • Identity is unambiguous • Quantitation often absolute • Interpretation relatively easy • Validation

• Untargeted

• More challenging, less quantitative • Identity ambiguous due to breadth of separation • Greater coverage • Detect unexpected or new compounds • Interpretation and statistical analysis challenging • Discovery

Challenges in metabolomics

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Metabolic Profiling of bacterial ‘quorum sensing’

Developing a metabolomic toolbox for pathogen-pathogen interactions

1. Candia albicans: cells & media 2. Staphylococcus aureus: cells & media 3. C. albicans & S. aureus co-culture: cells & media

Q Exactive GC MS • Full MS • Relative quantitation and

identification • Compound ID using

NIST and pure standards

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Candida albicans and Staphylococcus aureus dual-species biofilms

Staphylococcus aureus

Candida albicans

• Leading pathogens in bloodstream and systemic infections

• Major cause of morbidity and

mortality

• Form biofilms

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Staphylococcus aureus

Candida albicans

Staphylococcus aureus + Candida albicans

Carlson, E., (1983), Infection and Immunity, 42 (1) 285-92.

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C. albicans S. aureus

S. aureus added to C. albicans biofilm

SEM images: 4000x magnification

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Aims

• Characterise the dual-species biofilm • Develop metabolomics methods for the analysis of

polymicrobial biofilms • Apply methods to determine the interaction between

Candida albicans and Staphylococcus aureus

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RT: 8.82 - 9.87 SM: 3B

8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8Time (min)

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8.978.92 9.659.12 9.55 9.859.609.48 9.808.87 9.769.18 9.33 9.71

9.04 9.24

9.419.06

9.55 9.659.48 9.809.138.92 8.98 9.619.349.18 9.709.24

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8.98 9.659.55 9.799.489.138.928.84 9.629.359.19 9.719.24

9.069.41

9.12 9.659.559.47 9.858.92 9.798.978.86 9.619.18 9.35 9.71

NL:2.84E7TIC MS MO3

NL:3.57E7TIC MS CAM3

NL:3.48E7TIC MS SAM2

NL:3.57E7TIC MS SCM3

Media blank

Candida media

S. aureus media

Co-culture

Media samples Chromatogram from GC-Orbitrap

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Principal Component Analysis

Media samples

Cells samples

SCC SAC

CAC MO SCM

SAM

CAM

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Amino Acid Uptake: media samples Fresh Medium CAM SAM SCM

L-Methionine, 2TMS 0 -6.8 -6.0 -6.1 L-Proline, 2TMS 0 -6.3 -1.0 -3.9 L-Histidine, 3TMS 0 -2.1 -0.2 -1.1 L-Threonine, 3TMS 0 -2.0 -3.5 -4.5 L-Glutamic acid, 3TMS 0 -1.9 -0.9 -3.6 L-Serine, 3TMS 0 -1.8 -2.8 -3.3 L-Leucine, 2TMS 0 -1.7 -2.8 -3.3 L-Isoleucine, 2TMS 0 -1.5 -3.6 -2.9 L-Phenylalanine, 2TMS 0 -1.0 -2.1 -2.2 L-Alanine, 2TMS 0 -1.0 -0.4 -0.7 L-Aspartic Acid, 3TMS 0 -1.0 -0.8 -2.6 L-Ornithine (and L-Argininine), 3TMS 0 -0.8 -0.7 -0.8 L-Valine, 2TMS 0 -0.7 -2.0 -1.8 L-Tryptophan, 3TMS 0 -0.3 -2.0 -1.3 L-Homoserine, 3TMS 0 -0.2 3.0 2.7 L-Tyrosine, 3TMS 0 0.0 -5.1 -1.7 Glycine_3TMS 0 0.0 0.1 0.0 L-Hydroxyproline, 3TMS 0 0.1 0.2 0.3 L-Cysteine, 3TMS 0 0.3 -1.6 0.1

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Central carbon metabolism: media samples

Fresh Medium CAM SAM SCM

D-Glucose, 6TMS + Oxime 0 -6.3 -8.2 -7.8

Succinic acid, 2TMS 0 0.2 0.4 0.3

Lactic Acid, 2TMS 0 0.1 -0.06 -0.76

Fucose + 4TMS + Oxime 0 0.04 -0.03 -0.06

Myo-Inositol + 6TMS 0 0.04 -0.1 -0.06

D-Lactose + 8TMS + Oxime 0 -0.6 -1.7 1.3

D-Arabinose + 4TMS + Oxime 0 -0.19 -0.3 -0.04

D-Ribose + 4TMS + Oxime 0 0.1 -0.1 -0.18

Sucrose + 8TMS 0 2.0 -0.2 0.01

Maltose + 8TMS + Oxime 0 0.5 -1.6 -1.7

D_Threose + 4TMS + Oxime 0 1.1 1.7 ND

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Intracellular sugar phosphates: cells samples

Normalised CAC SAC SCC

Myo-Inositol-1-phosphate + 7TMS 0 -4.5 0.1

D-Glucose 6-phosphate + 7TMS + Oxime 0 -3.0 0.1

Sedoheptulose 7-phosphate + 7TMS + Oxime 0 -0.1 1.0

D-ribose 5-phosphate + 5TMS + Oxime 0 1.3 -0.1

D-Erythrose 4-phosphate +5TMS 0 ND ND

D-Fructose 1,6-bisphosphate + 6TMS + Oxime 0 ND ND

D-Fructose 1-phosphate + 7TMS + Oxime 0 ND ND

D-Fructose 6-phosphate + 6TMS + Oxime 0 ND 0.7

Ribulose-5-phosphate + 5TMS + Oxime 0 ND ND

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Compound Discoverer 2.0 metabolomics workflow

Experiment definition (sample grouping)

Data processing (peak alignment & extraction)

Data export further statistical analysis using 3rd party software

Data review (based on %CV, p-values)

import .raw data (full scan EI or CI)

Statistical analysis (trend plot, PCA)

Compound ID (NIST or user database)

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Compound Discoverer results browser

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Untargeted Metabolomics

Dr. Karl Burgess

Glasgow Polyomics

University of Glasgow, UK

Application: Automated, untargeted metabolomics of decaying muscle tissue from various species Aim: Discovery of bio markers for time of death Results: PCA for time of death in Rats allowed group separation suggesting a prediction model for time of death can be obtained. Automatic identification of significant metabolites and potential biomarkers was possible with Q Exactive GC and intelligent deconvolution and identification software

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GC-MS chromatograms of a rat muscle tissue sample RT: 14.68 - 22.86

15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)

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15.32 16.63

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16.59 16.8918.53 20.5115.66 17.6615.00

21.0418.0915.81 17.0416.17 22.5420.4119.88 20.97 22.0119.60 21.9221.2618.64 19.17

17.4415.66

16.6317.0416.58 18.53

15.8217.77

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21.0416.1714.99 20.5119.62 19.88 20.41 22.4021.9917.12 18.64 21.9621.5318.90 19.4218.19

17.4216.36 18.5217.03

17.7620.49

18.0815.81

19.58 20.4015.17 19.8716.16 22.4021.0320.9218.63 21.9920.12 21.8921.2517.22 18.95 19.4118.3916.63 17.04

18.5317.7816.58

15.8216.38

18.0916.1814.82 19.6215.00 20.5119.88 21.04 22.4120.4015.88 18.64 18.9117.12 21.26 22.0021.5419.52 20.6718.19

NL:1.00E9TIC MS t0r1r

NL:1.00E9TIC MS t1r3r

NL:1.00E9TIC MS t2r5r

NL:1.00E9TIC MS t3r7r

T0

T1

T2

T3

Immediately post-mortem

Day 1 post-mortem

Day 2 post-mortem

Day 3 post-mortem

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• Automated peak picking using TraceFinder resulted in 1193 distinct peak clusters detected, 156 peaks as TMS derivatives.

• Example of deconvoluted peak cluster putatively identified as glutamate:

Peak Deconvolution

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Quantitation matrix of detected peak clusters

Base Peak Mass.

RT max

intensity T0 T1 T2 T3

ttest: T0

ttest: T1

ttest: T2

ttest: T3

244.09515 12.70 14200000 1.0 8.0 4.2 3.0 1.000 0.017 0.012 0.007

174.10451 20.93 3514752 1.0 4.1 2.4 2.9 1.000 0.048 0.131 0.019

115.08180 16.82 641130 1.0 2.3 2.8 2.8 1.000 0.032 0.083 0.043

246.14626 15.31 870807 1.0 2.4 2.0 2.4 1.000 0.004 0.014 0.010

267.07825 12.93 1375261 1.0 3.3 2.6 2.4 1.000 0.032 0.156 0.132

157.05594 18.64 1901910 1.0 3.9 3.0 2.4 1.000 0.023 0.044 0.066

204.10023 21.64 1306996 1.0 4.7 2.1 2.3 1.000 0.049 0.396 0.163

304.24032 23.89 1282461 1.0 4.2 2.6 2.2 1.000 0.049 0.248 0.218

409.16741 23.76 63600000 1.0 2.2 2.7 1.6 1.000 0.037 0.266 0.445

297.13302 23.76 1791507 1.0 2.3 2.5 1.5 1.000 0.031 0.252 0.515

220.07711 6.12 30600000 1.0 0.5 0.6 0.8 1.000 0.044 0.089 0.403

238.08772 13.80 662692 1.0 0.6 0.6 0.6 1.000 0.003 0.005 0.004

189.07643 8.96 1444272 1.0 0.4 0.4 0.6 1.000 0.046 0.067 0.141

179.04016 9.16 1710471 1.0 0.4 0.4 0.6 1.000 0.028 0.027 0.060

188.07996 8.76 1890000000 1.0 0.4 0.4 0.4 1.000 0.024 0.036 0.039

133.01916 8.76 1820872 1.0 0.3 0.4 0.4 1.000 0.024 0.035 0.035

230.07952 16.63 7380595 1.0 0.3 1.2 0.3 1.000 0.037 0.502 0.045

240.11144 14.63 1063520 1.0 0.2 0.3 0.3 1.000 0.022 0.033 0.026

214.04808 15.85 883184 1.0 0.3 0.3 0.2 1.000 0.047 0.063 0.041

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• Multivariate analysis that collapses high-dimensional data to principal components responsible for the majority of variance in the dataset.

Partial Least Squares-Discriminant Analysis

PC1

PC

2

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• Example of changing intensities of detected chemicals across biological groups (T0 T3).

Variables trend

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Putative compound ID RT (min)

NIST Forward Match

Fold increase

compared to T0

Base Peak Fragment elemental

composition

Ppm accuracy

(base peak)

ppm accuracy (Mol. ion)

L-Threonine, 3TMS 10.71 795 2.8 C9H24ONSi2 0.27 0.13

L-Aspartate, 3TMS 11.78 707 7.0 C9H22NO2Si2 0.18 0.34

L-Methionine, 2TMS 12.40 749 15.0 C7H18NSSi 0.24 0.04

L-Glutamine-3TMS 15.32 815 2.0 C7H14NOSi 0.53 0.21

Putrescine, 4TMS 16.18 870 2.0 C7H20NSi2 0.05 N/A

Untargeted metabolomics

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Conclusioni

• GC/MS market gap exists for simultaneous targeted and untargeted analysis with sensitivity and selectivity comparable to that of a triple quadrupole

• An Orbitrap based GC system provides the technology to fill this gap based on its sensitivity and ability to operate routinely at very high resolutions

• The very high resolution and accurate mass data the Orbitrap based GC system produces can increase confidence in unknown identification.

Q Exactive GC is an easy-to-use, dedicated GC-MS that provides the highest confidence in compound discovery, identification and quantitation for a comprehensive understanding of your samples. This is achieved through the superior resolving power, mass accuracy and sensitivity that only Orbitrap technology can deliver.

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Grazie per l’attenzione!!!