Orbitrap Technology: New Frontiers in Pesticide Residue Analysis
Michal Godula
Special Solutions Center Europe
Thermo Scientific
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• No compromise MS, MS/MS and MSn
• Speed, sensitivity, ultrahigh resolution, precise mass accuracy, and dynamic range - at the same time
Orbitrap: The Performance Leader since 2006
What Orbitrap provides
Unmatched ultrahigh resolution, accurate mass performance
Which applications are optimized• Virtually all molecular analyses:
accurate identification, structural analysis, and quantification of organic molecules, lipids, carbohydrates, peptides & proteins in complex mixtures
• Proteomics, drug discovery, toxicology, forensics, environmental, food safety and many more
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Orbitrap FTMS
Makarov A. Anal. Chem. 2000, 72, 1156-1162.
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Orbitrap Mass Analyzer Features
• Fundamental difference to other HRAM instruments• Parameter measured is frequency, not time/voltage/current• Resolution allows more accurate m/z determination• Less prone to ambient conditions changes• Usually stable within <2 ppm during several days• No need for lock mass in “routine work”• Small footprint• Easy to setup
How accurate is your mass?
Mass accuracy
610/
true
truemeas
m
mmzm
• Quadrupole MS
• Orbitrap MSTOF MS
ppmzm 20010500
0.5001.500/ 6
ppmzm 21010314.500
10214.50010314.500/ 6
Selectivity increases with higher mass accuracy
7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6Time (min)
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RT: 8.45
9.298.728.678.99
8.789.118.217.85 8.838.137.627.59 7.81 8.10 8.357.38
7.337.27 9.46 10.6110.509.50 10.3410.169.64 10.09
100 ppm
20 ppm
2 ppm
7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6Time (min)
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RT: 8.45
8.217.62 7.857.598.257.407.37 7.81 7.92 7.99
8.617.30 10.617.26 10.508.88 10.318.67 9.11 10.1610.099.46
7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6Time (min)
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RT: 8.45
8.557.997.49 7.917.76 10.617.417.26 10.5010.3110.167.67 9.46
NL: 1.20E5m/z= 484.25313-484.25507 F: FTMS {1,0} + p APCI Full ms [300.00-850.00] MS pivo_5_UH
*Zachariasova M et al, Anal. Chim. Acta
Resolution (resolving power)
• Resolution
m
mR
m (FWHM)
m 10%
8336.0
500
m
mR• Quadrupole MS
• Orbitrap MS 100000005.0
500
m
mR
Does resolution matter?
RAFA 2013: Łukasz Rajski, María del Mar Gómez Ramos, Amadeo R. Fernández-Alba; EURL for Pesticide Residues in Fruits and Vegetables. Pesticide Residue Research Group. University of Almeria, Spain. e-mail: [email protected]
Q Exactive MS - a 3D view
Hyperbolic Quadrupole Mass filter
HCD Cell
Orbitrap Mass Analyzer
C-Trap
RF-Lens
Q Exactive FS-ddMS2 screening process
Inclusion List
Ion Accumulation
Full Scan Orbitrap Analysis
Precursor Selection
Precursor accumulation
Fragmentation in HCD
MS2 Orbitrap Analysis
Full Scan MS Data: Screening/Quantitation
Full Scan MS/MS Data:Confirmation
Multi-residue screening by Q Exactive LC-MS
• Complete solution for quantitiative analysis of LC amenable compounds• Implementation method for most relevant pesticides/mycotoxins and
pyrrolizidine alkaloids covering different chemistries• In 3 relevant matrices• Critical evaluation of established method performance criteria vs. current
regulation
strawberry leek wheat flour
Quechers extraction
10 g of homogenized sample is weight into Quechers extraction tube
+ 20 mL of water
+ 10 mL of ACN
Centrifugation 5 min @ 5000 rpm
Analysis
shaking 10 min
Ext
ract
ion
UHPLC instrumental method
Thermo ScientificTM UltiMate® 3000 RSLC : • column: Accucore aQ 100 mm x 2.1 mm x 2.6 µm • Column oven T: 25 °C • Injection Volume: 1 µL• Mobile phase A: Water/MeOH (98:2) 5mM Ammonium
formate, 0.1% FA• Mobile phase B: Water/MeOH (2:98) 5mM Ammonium
formate, 0.1% FA• Flow rate: 300 µL/min
Q Exactive Conditions – ddMS2 experiment
• Q-ExactiveTM MS conditions: Spray voltage 3800V, Capillary temperature 295°C, sheath gas 32 au, Aux gas 7 au, S-lens RF level 55
• Full MS/data dependent MS2
• full MS:resolution 70,000 FWHM
scan range 120-1200 m/z
• ddMS2: resolution 35,000 FWHM
Inclusion list on
isolation 1 m/z
exclusion 5 s
List of 556 target analytes
1234567891011121314151617181920212223242526 … … …556
Isotopes
Fragments
LibrarySpectrum
Chromatogram
Target analytes
TraceFinderTM Screening Data Review
Thermo EFS Screening Database: 1729 substances
• Targeted screening in “known RT” compounds• Untargeted in the rest of database (RT not known)
Screening Example – Acetamiprid in honey sample
Acetamiprid confirmation
Isotopic Pattern
+ Main fragment
Acetamiprid details
Standard 50 ug/kg
Sample – determined concentration 5.3 ug/kg
SampleMS2 spectra
Standard MS2 spectra
Isotopic pattern fitting is influenced by resolution
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Q Exactive Focus: Introducing vDIA
• Variable Data independent acquisition (vDIA) acquires MS2 spectra systematically and independent of previously acquired scans
• You obtain:• complete record of data
• very good reproducibility
• high throughput comprehensive quantification acquisition methods with quantitative information
• no requirement for detailed sample knowledge prior to data acquisition
• Your routinely acquired data is ready to go for any non-target screening approach
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Variable DIA – alternative to All Ions Fragmentation
Presented by P Zomer, RIKILT at EPRW 2014:
Traditional AIF
Variable Data Independent Analysis
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AIF vs. vDIA
AIF
vDIA
Oxacillin in Pig Kidney @ 2 ppb background
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Running 380 Compounds with the Q Exactive Focus
Data Dependent-MS2 vDIA
Quan/Qual Quantitation Targeted Screening
# of targeted compounds
Total: 380345 Pesticides, 35 Mycotoxins and Alkaloids
Selectivity Retention time, fragment ions, ion ratios, isotope ratio, library match
Linearity – working range [ppb]
1-500 (6 levels and 2 replicates)
Matrix Black Tea/Honey
Sample Prep Buffered QuEChERS method clean-up made by MgSO4, PSA and C18
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vDIA sensitivity in honey
80%
20%
Number of compounds with <1ppb sensitivity
Peaks in honey sample spiked to 1ppb
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345 target pesticides in tea matrix
Data still being processed
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A New Chapter in GC-MS
Thermo Scientific Q Exactive GC Hybrid Quadrupole-Orbitrap 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
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Bringing GC and Orbitrap Technology Together
C-TRAP
HCD Cell
AQT Quadrupole
Orbitrap Mass Analyzer
BentFlatapole
ExtractaBrite™ Ion Source
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Breakthrough in GC-MS Performance
Highest selectivity and confidence with high resolving power
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
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Breakthrough in GC-MS Performance
Highest selectivity and confidence with high resolving power
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
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Pesticides screening
High resolving power crucial to avoid false negatives
False negatives at 15,000 RP
Secure screening
• 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)
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Breakthrough in GC-MS Performance
Highest selectivity and confidence with high resolving power
XIC of dieldrin in baby food (m/z 262.85642) 100ppb
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3
4
5
6
7
89
10
11
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1516 17
60,000 RP (m/z 200) 2.4 s peak
Fast acquisitions
• Important for accurate profiling of narrow GC peaks
• Full scan with resolving power of 60 (FWHM @ m/z 200) generates 17 scans
• Fast enough for GC!
17 scans
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Scan to-scan
XIC of dieldrin in baby food (m/z 262.85642) 100ppb
60,000 RP (m/z 200) 2.4 s peak
Breakthrough in GC-MS Performance
Consistently excellent mass accuracy
< 1ppm (internal)
< 3ppm (external)
• Scan-to-scan
• Low level in matrix
• Over full mass range
• Over full concentration range
• No need for continuously calibrating in sequence
0.70.7
0.6
0.5
0.3
0.6
0.5
0.3
0.3
0.3
0.5
0.6
0.6
0.70.5
0.5 ppm RMS
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Breakthrough in GC-MS Performance
Consistently excellent mass accuracy
Mass accuracy over >6 orders
• Hexachlorobenzene
• 0.2 – 250,000 ng/mL
• Mass accuracy within 1 ppm
• Orbitrap spectral fidelity independent of concentration
Over full concentration range
Data courtesy of Hans Mol and Marc Tienstra, RIKILT
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Is it sensitive enough? Q Exactive GC vs. Triple Q
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Breakthrough in GC-MS Performance
Unprecedented quantitation power for HR/AM GC-MS
SRMFull Scan
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Compound Discovery and Identification
Tracefinder: automated compound detection and identification
1. Peak detection
2. Spectral deconvolution
3. Library searching (e.g. NIST, Wiley, custom)
4. % TIC explained using elemental composition and accurate mass filtering
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Automated Workflow for Accurate Mass Confirmation
Unknown Identification Workflow
Spectral Deconvolution on EI HRAM
Data
Spectral Library SearchScoring
AdditionalHigh
Resolution Scoring
m/z = 275.11298
C11H23O4Si2m/z = 275.11294
m/z = 275.11298
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Pesticides screening
Horse feed detection limit
100 % detected at MRL levelMajority at ½ MRL
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Pesticides screening
Quantitation
Fenpropimorph calibration curve in leek matrix.
• For all pesticides, the coefficient of determination (R2) was >0.99 with an average value of R2 = 0.997
Curve from 0.5 to 50 ng/g (triplicate at each level)
R2=0.9999
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Complete solution for total pesticide analysis
Q Exactive GC Hybrid Quadrupole-Orbitrap GC-MS/MS System
Q Exactive FocusHybrid Quadrupole-Orbitrap LC-MS/MS System