Redefining GC-MS in the Laboratory with GC Orbitrap Michal Godula, Ph.D. Thermo Fisher Scientific michal.godula@thermofisher.com The world leader in serving science
A New Chapter in GC-MS Thermo Scientific TM Q Exactive TM GC Hybrid Quadrupole-Orbitrap TM GC-MS/MS System Offering Unprecedented Depth in GC-MS Analysis • Hugely powerful HR/AM performance for GC-MS • Uncompromised quantitative and qualitative capability • Reliable, robust and easy to operate 2
Makarov A. Anal. Chem. 2000, 72 , 1156-1162. Orbitrap Principles 3
Bringing GC and Orbitrap Technology Together AQT Bent Quadrupole Flatapole C-TRAP HCD Cell ExtractaBrite™ Ion Source Orbitrap Mass Analyzer 4
Breakthrough in GC-MS Performance Unrivalled Resolving Power • Up to 120k @ m/z 200 • Allows excellent selectivity in complex matrix, maintaining confidence in detection and identification • Drives highly resolved data sets and high capacity peak detection • Fast scanning Orbitrap for well defined, reproducible GC peaks 5
Breakthrough in GC-MS Performance Chlorprophram • 10 ng/g QuEChERS extract of leek • Full scan with resolving power of >30k (FWHM @ m/z 200) provides interference free detection • Excellent mass accuracy for confident identification 6
Pesticides screening Secure screening False negatives at 15,000 RP • 5 ppm identification threshold • Higher resolving power allows confident detection and identification • Mid- resolution of 15k causes false negatives • Q Exactive GC routinely operates at 60k (FWHM @ m/z 200) 7
Breakthrough in GC-MS Performance XIC of dieldrin in baby food ( m/z 262.85642) 100ppb XIC of dieldrin in baby food ( m/z 262.85642) 100ppb Fast acquisitions 9 • Important for 8 10 accurate profiling of 60,000 RP (m/z 200) 7 narrow GC peaks 2.4 s peak 11 • Full scan with resolving power of 6 17 scans 12 60 (FWHM @ m/z 200) generates 17 scans 5 13 • Fast enough for 4 14 GC! 3 15 2 16 17 1 8
Breakthrough in GC-MS Performance XIC of dieldrin in baby food ( m/z 262.85642) 100ppb XIC of dieldrin in baby food ( m/z 262.85642) 100ppb < 1ppm (internal) Scan to-scan Scan to-scan 0.3 < 3ppm (external) 0.5 0.3 • Scan-to-scan 60,000 RP (m/z 200) 0.6 2.4 s peak 0.3 • Low level in matrix 0.5 ppm RMS 0.3 • Over full mass 0.5 range 0.5 • Over full 0.6 concentration range 0.6 0.6 • No need for 0.7 0.7 continuously 0.7 0.5 calibrating in sequence 9
Breakthrough in GC-MS Performance Mass accuracy over >6 Over full concentration range Over full concentration range orders • Hexachlorobenzene • 0.2 – 250,000 ng/mL • Mass accuracy within 1 ppm • Orbitrap spectral fidelity independent of concentration Data courtesy of Hans Mol and Marc Tienstra, RIKILT 10
Orbitrap GC-MS Family Q Exactive GC system Unprecedented Depth in Analysis RP 120,000 (FWHM @ m/z 200) EI/CI; Full-scan, Timed-SIM MS/MS capability 11
Orbitrap GC-MS Family Thermo Scientific TM Q Exactive TM GC Orbitrap TM GC-MS/MS System Unprecedented Depth in Analysis RP 120,000 (FWHM @ m/z 200) EI/CI; Full-scan, Timed-SIM MS/MS capability Redefining Routine GC-MS RP 60,000 (FWHM @ m/z 200) EI/CI; Full-scan; Timed-SIM Thermo Scientific TM Exactive TM GC Orbitrap TM GC-MS System 12
Insight into the Orbitrap GC-MS Systems 13
Insight into the Orbitrap GC-MS Systems Thermo Fisher TM TRACE TM 1310 GC system Unique modular injector and detector design Short cycle time 14
Insight into the Orbitrap GC-MS Systems TRACE 1310 GC system Unique modular injector and detector design Short cycle time Thermo Scientific™ ExtractaBrite™ ion source technology Routine grade robustness Patented RF lens Removable without breaking vacuum through VPI Vacuum-free column replacement through VPI 15
Insight into the Orbitrap GC-MS Systems TRACE 1310 GC Orbitrap mass system analyzer Unique modular injector and Incredible HRAM performance detector design Extended Short cycle time Dynamic Range Thermo Scientific™ ExtractaBrite™ ion source technology Routine grade robustness Patented RF lens Removable without breaking vacuum through VPI Vacuum-free column replacement through VPI 16
Insight into the Orbitrap GC-MS Systems Sensitivity Dynamic Range Mass Accuracy Resolving Power Up to 120,000 at < 1ppm ppt >6 orders m/z 200 • Maximum • Every scan • In full-scan • Excellent selectivity coverage in • All concentrations • High selectivity sample profiling • Fast enough for • In complex matrix • High spectral GC • “Triple quad fidelity grade” • Across the mass quantitation in range full-scan • Everyday! 17
Analytical Workflows Overview Non- targeted full scan acquisition Known unknown ID Targeted Targeted Non-targeted screening screening quantitation “the things you “the things you “the things you may suspect” expect” don’t expect” Unknown unknown ID i.e. Commonly used i.e. Unusual, banned or i.e. Other contaminants or pesticides for a alternate pesticides for compounds, could be particular commodity particular commodity known or unknown 18
Pharmaceutical O-Rings Extractables Study • 4 O-ring samples • Solvents • Water • A Red • 5M NaCl • B Brown • 50% Ethanol • C White • 100% Ethanol • D Black • 1% PS-80 • Blank (control) • 0.5N NaOH • 0.1M Phosphoric Acid • 40 ° C for 30 days 19
GC-MS Instrument Conditions TRACE 1310 GC Parameters Injection Volume (mL): 1 Liner Single gooseneck Inlet ( ° C): 280 Carrier Gas, (mL/min): He, 1.2 Oven Temperature Program: Temperature 1 ( o C): 40 Hold Time (min): 1 Temperature 2 ( o C): 320 Rate ( o C/min) 15 Hold Time (min): 10 Q Exactive GC MS Parameters Thermo Scientific™ Q Exactive™ GC Transfer line ( ° C): 280 Hybrid Quadrupole-Orbitrap GC-MS/MS Ionization type: EI Ion source( ° C): 230 Thermo Scientific™ Trace™ 1310 GC Electron energy (eV): 70 Thermo Scientific™ TraceFinder™ Acquisition mode: Full scan Mass range (Da): 50-650 Mass resolution (FWHM): 60k Lockmass (m/z): 207.03235 20
TraceFinder Non-targeted Screening Overview 1. Detect and refine 2. Generate candidates 3. Filter and identify • Sensitive and • Search spectra against • High resolution selective peak spectral libraries filtering of candidates detection • HRAM o low resolution • Putative • High resolution spectral libraries (NIST, identifications made spectral Wiley…) deconvolution • Candidates list • Clean spectrum generated Automatic compound identification 21
Example Results RT: 4.10 - 27.88 SM: 7B NL: 5.66 100 6.86E8 Blank control 90 TIC MS 03June15_0 80 08 70 Relative Abundance 4.78 60 21.02 21.86 22.45 23.25 20.64 24.83 26.44 50 20.03 19.59 40 7.65 19.16 30 6.66 18.56 8.84 10.33 17.88 20 11.65 16.62 12.80 13.82 10 0 NL: 17.48 100 2.54E9 90 TIC MS Brown O-Ring 03june15_01 80 1 70 60 50 40 30 5.67 24.62 20 21.86 24.39 20.80 20.37 25.02 26.04 5.10 15.42 5.70 11.10 19.19 10 0 6 8 10 12 14 16 18 20 22 24 26 Time (min) 22
Step 1: Deconvolution of the Data • TraceFinder first performs an accurate mass deconvolution of the data. • Extracts all of the peaks in the chromatogram. • Provides a cleaned spectrum for library matching. 23
Step 2: Quickly Isolate the Peaks of Interest • 2051 peaks were extracted from the brown O-ring sample Heat Map indicates the peaks elevated in the sample . 24
Top 10 Differential Peaks in Brown O-ring Peaks list can also be sorted by fold difference compared with control to isolate the differential peaks that could be low or high intensity. 25
Step 3: Identify the Compound – Searching NIST 14 26 Hits from NIST are sorted based on: • Spectral matching • High Resolution Filtering (HRF) score. 26
Step 3: 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. 27
Step 4: Fragments Can Be Explained With < 1ppm Mass Accuracy 28
NEW : VeV Electron Ionization Technology • Top: spectrum acquired with classic 70eV • Bottom: spectrum acuquired with low 12eV, showing enhancement of molecular ion The key benefits of VeV are: Fully automated for optimum performance: VeV set-up is very simple and easy with fast, fully automated tuning. Increased confidence in identification: VeV promotes molecular ion and diagnostic high mass signals, important information for compound identification and confirmation. 29
NEW : HRAM Library for Metabolomics Database •High resolution, high mass accuracy spectra acquired at 60,000 RP ( m/z 200) •Spectra refined and curated with elemental composition of each EI fragment verified •Kovats retention index for each entry •PubChem ID for unique metabolites •Can be used in combination with existing unit mass libraries 30
Q Exactive GC: Compound Discovery and Identification Remove entire ion source or change to CI source in under 2 minutes without venting … 31
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