1. Orbitrap Technology: New Frontiers in Pesticide Residue Analysis
Michal Godula
Special Solutions Center Europe
Thermo Scientific

2. We Are The World Leader in Serving Science
50,000 employees in 50 countries
$17 billion in annual revenues
Unparalleled commercial reach
Global Scale
Premier Brands
I’d like to be the first to welcome to you Thermo Fisher Scientific and I want to give you some background on this great company that you’re now a part of.
Thermo Fisher is the world leader in serving science. We’re a $17 billion company with almost 50,000 employees, creating products and services that enable our customers to make the world healthier, cleaner and safer.
With our leadership position comes the advantages of scale and depth of capabilities, which differentiates us in the marketplace. There really is no other company that has the range of customer touch points we have – technologically, geographically or commercially.
We leverage our premier brands to create the most value for our customers:. I’ll talk more about the areas those brands represent in a moment.
Unmatched Depth
Leading innovative technologies
Applications expertise
Lab productivity partner 2 2

3. Orbitrap: The Performance Leader since 2006
What Orbitrap provides
No compromise MS, MS/MS and MSn
Speed, sensitivity, ultrahigh resolution, precise mass accuracy, and dynamic range - at the same time
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
Ian Jardine reviews “state of the art” in the portfolio – the LTQ Orbitrap Velos
Unmatched ultrahigh resolution, accurate mass performance 3

4. Orbitrap FTMS
Makarov A. Anal. Chem. 2000, 72,

5. 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

6. How accurate is your mass?
Mass accuracy
Quadrupole MS
Orbitrap MS TOF MS

7. 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
10.6
Time (min) 10 20 30 40 50 60 70 80 90
100
Relative Abundance
RT: 8.45
9.29
8.72
8.67
8.99
8.78
9.11
8.21
7.85
8.83
8.13
7.62
7.59
7.81
8.10
8.35
7.38
7.33
7.27
9.46
10.61
10.50
9.50
10.34
10.16
9.64
10.09
NL: 1.25E5
m/z=
F: FTMS
{1,0} + p APCI Full
ms [ ]
MS pivo_5_UH
100 ppm
20 ppm
2 ppm
8.25
7.40
7.37
7.92
7.99
8.61
7.30
7.26
8.88
10.31
NL: 1.20E5
F: FTMS
8.55
7.49
7.91
7.76
7.41
7.67
F: FTMS
*Zachariasova M et al, Anal. Chim. Acta

8. Resolution (resolving power)
m (FWHM)
m 10%
Quadrupole MS
Orbitrap MS

9. 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.

10. Orbitrap Mass Analyzer
Q Exactive MS - a 3D view
Hyperbolic Quadrupole
Mass filter
HCD Cell
Orbitrap Mass Analyzer
C-Trap
RF-Lens

11. Q Exactive FS-ddMS2 screening process
Full Scan MS Data: Screening/Quantitation
Ion Accumulation
Full Scan
Orbitrap Analysis
Inclusion List
Precursor Selection
Precursor accumulation
Full Scan MS/MS Data:
Confirmation
Fragmentation in HCD
MS2 Orbitrap Analysis

12. 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
In today’s presentation we aim to demonstrate a complete workflow solution for quantitative analysis of pesticide residues taking into account both relevant legislative and high throughput sample analysis requirements and challenges.
We are going to demonstrate how easily and quickly a complete and ready-to-use workflow can be implemented and applied for routine multi-residue analysis with the latest GC-MS/MS technology.
For this reason we have selected 140 GC amenable pesticide compounds covering different physico-chemical characteristics including known problematic compounds and three matrices which are typically measured in routine analytical laboratories. These were strawberry referring to high water content and acidic character matrix, leek as high water and chlorophyl containing and wheat flour as low water, moderate fat containing matrix with high starch/protein content.
A complete in-house validation has been conducted for the implemented method and method perfomance parameters were established for all target compounds and matrices. These values were then compared to the current legislative requirements as proof of fit for purpose criteria.
strawberry
leek
wheat flour

13. Quechers extraction Extraction Analysis
10 g of homogenized sample is weight into Quechers extraction tube
+ 20 mL of water
+ 10 mL of ACN
Extraction
shaking 10 min
Centrifugation rpm
Analysis

14. 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
The complete analytical method was directly imported as ready to use acquisition and process file including settings for more than 600 target compounds from Thermo Fisher source directly into the TraceFinder control software.
For sample injection splitless PTV injection with 75°C inlet temperature, 3 min transfer time, 300°C transfer temperature and 1.2 ml/min flow rate and 1 ul injection volume was applied.
On the GC site 1.2 ml/min He flow was applied on the TG-5 SilMs GC column; the relevant temperature gradient setting is shown in the figure in left bottom of the slide – the total run time was 35 min excluding the time needed for cooling down and equilibrating the instrument which took only few min.

15. 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 m/z
ddMS2: resolution 35,000 FWHM
Inclusion list on
isolation 1 m/z
exclusion 5 s
List of 556 target analytes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 …
556

16. TraceFinderTM Screening Data Review
Isotopes
Target analytes
Fragments
Chromatogram
Library
Spectrum

17. Thermo EFS Screening Database: 1729 substances
Targeted screening in “known RT” compounds
Untargeted in the rest of database (RT not known)

18. Screening Example – Acetamiprid in honey sample

19. Acetamiprid confirmation
Isotopic Pattern
+ Main fragment

20. Acetamiprid details Sample – determined concentration 5.3 ug/kg
Standard 50 ug/kg
Sample
MS2 spectra
Standard MS2 spectra

21. Isotopic pattern fitting is influenced by resolution

22. 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
In contrast to well known and widely used data dependent MS2 acquisition (ddMS2), data independent acquisition techniques will provide a complete record of data. This way unknown or suspected components can be confirmed by fragmentation data in a retrospective way even after years. This is possible already by application of full scan / all ion fragmentation data acquisition (FS / AIF), but here the sensitivity of significant fragments can be significantly reduced compared to the full scan. DIA basically slices the AIF scan range into smaller chunks, so the dynamic range of every chunk is improved. This enhances the sensitivity of these fragments to a level comparable to the preceding full scan. Additionally, the selectivity of specific fragments improves at the same time, getting close to the selectivity of ddMS2 scans. On the other hand, DIA scanning does not need any specific setup with respect to the components analyzed, so one generic method serves for the analysis of a very wide range compounds.
So no changes at all have to be made with respect of the individual analysis run, no time lost.

23. Variable DIA – alternative to All Ions Fragmentation
Traditional AIF
Variable Data Independent Analysis
Presented by P Zomer, RIKILT at EPRW 2014:

24. AIF vs. vDIA Oxacillin in Pig Kidney @ 2 ppb AIF vDIA background
Here AIF fails completely because of even stronger complexity, while with DIA still clear confirmation with three fragments is achieved. Even ddMS2 doesn’t do better.
vDIA

25. Running 380 Compounds with the Q Exactive Focus
Data Dependent-MS2
vDIA
Quan/Qual
Quantitation
Targeted Screening
# of targeted compounds
Total: 380
345 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
Goal of the project: to compare
Solvent and black tea
35 – mycotoxins and alcalides

26. vDIA in targeted screening
5 parameters for identification or confirmation
(HR m/z, RT, isotopic pattern, fragments, library search)
Search results in different libraries
# of compound ppb
84%
16%

27. Peaks in honey sample spiked to 1ppb
vDIA sensitivity in honey
80%
20%
Number of compounds with <1ppb sensitivity
Peaks in honey sample spiked to 1ppb

28. 345 target pesticides in tea matrix
Data still
being processed
% of positive responses (green=yes)
Recovery checked at 2 concentration levels for 5 replicates

29. 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
Put simply, these limitations in GC-MS analysis do not apply any more with the arrival of Orbitrap technology to GC
The Q Exactive GC provides unique view of your samples, a view not possible with other technologies
The system provides hugely powerful high resolution-accurate mass in full scan MS and MS/MS analyses
As well as the quantitative power up there with the best triple quadrupoles, in terms of sensitivity, precision and linear range
It is also, very easy to use!

30. Bringing GC and Orbitrap Technology Together
AQT
Quadrupole
Bent
Flatapole
C-TRAP
HCD Cell
ExtractaBrite™
Ion Source
Thermo Scientific™ ExtractaBrite™ ION SOURCE
Robust, rugged electron impact (EI) and chemical ionization (CI) performance – proven in routine applications in the Thermo Scientific™ ISQ™ and TSQ 8000 series GC-MS systems. Fully removable without breaking vacuum for maintenance or switching to chemical ionization (CI). Also exchangeable for a unique source plug, making GC columns easily exchanged, without venting the MS system
BENT FLATAPOLE
For lowest possible noise and maximum robustness
ADVANCED QUADRUPOLE TECHNOLOGY (AQT)
High transmission of selected masses for low-level detection and quantitation of low abundance compounds in highly complex matrices
C-TRAP
Curved linear trap for precise ion injection—delivers excellent in-spectrum dynamic range and ensures outstanding HR/AM performance across a wide range of concentrations through automatic gain control (AGC)
HCD COLLISION CELL
Higher-energy collisional dissociation for MS/MS characterization of ions—used in combination with chemical ionization to help elucidate chemical structures
ORBITRAP MASS ANALYZER
Incredible resolving power up to 120,000 FWHM at m/z 200, sub-ppm mass accuracy with a high acquisition rate, driving unrivalled spectral quality
Orbitrap
Mass Analyzer

31. Breakthrough in GC-MS Performance
Highest selectivity and confidence with high resolving power
Unrivalled Resolving Power
Up to 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
Slide

32. 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 m/z 200) provides interference free detection
Excellent mass accuracy for confident identification
Chlorphropham in leak at 10ng/g extracted using QuEChERS XIC shows resolution
values of 60k and 120k, providing interference-free, confident detection with
excellent mass accuracy.

33. High resolving power crucial to avoid false negatives
Pesticides screening
High resolving power crucial to avoid false negatives
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 m/z 200)
False negatives at 15,000 RP
Graph showing the lowest detected standard for 55 pesticides in horse feed. Identification based on accurate mass <2ppm and retention time ±20 seconds. Half MRL limit (5 ppb) and MRL (10 ppb) displayed.

34. Breakthrough in GC-MS Performance
Highest selectivity and confidence with high resolving power
Fast acquisitions
Important for accurate profiling of narrow GC peaks
Full scan with resolving power of 60 m/z 200) generates scans
Fast enough for GC!
XIC of dieldrin in baby food (m/z ) 100ppb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
60,000 RP (m/z 200)
2.4 s peak
17 scans
7 Hz acquisition
The peak width is representative of GC peaks that are typically 2-4 s

35. 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
Scan to-scan
XIC of dieldrin in baby food (m/z ) 100ppb
60,000 RP (m/z 200)
2.4 s peak
0.7
0.6
0.5
0.3
0.5 ppm RMS
SLIDE – consistently excellent mass accuracy – from all angles…
Scan to scan, matrix, mass range, concentration
CLICK
This capability allows data to be confidently extracted with small extraction widows of 2ppm and less which ensures high selectivity.
16 scans/peak, peak width 2.4 sec

36. 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
Accurate mass is also very good over a wide range of concentrations
Data courtesy of Hans Mol and Marc Tienstra, RIKILT

37. Is it sensitive enough? Q Exactive GC vs. Triple Q

38. Breakthrough in GC-MS Performance
Unprecedented quantitation power for HR/AM GC-MS
SRM
Full Scan
Summary of instrument detection limit for 150 pesticides when using the Q Exactive GC(in full scan – 60k) and the TSQ 8000 Eco (in SRM)analyzing a mixed vegetable matrix
Detection levels for both well below the MRL levels
Comparable detection limits between instruments.
Achieved with a fast GC separation all compounds eluting <10 mins

39. Compound Discovery and Identification
Tracefinder: automated compound detection and identification
Peak detection
Spectral deconvolution
Library searching (e.g. NIST, Wiley, custom)
% TIC explained using elemental composition and accurate mass filtering
Within Tracefinder the Q Exactive GC takes advantage of a novel software tool that fully automates the discovery and identification of compounds. First it performs:
Peak detection….

40. Automated Workflow for Accurate Mass Confirmation
Unknown Identification Workflow
Spectral Deconvolution on EI HRAM Data
Spectral Library
Search
Scoring
Additional
High Resolution Scoring
m/z =
m/z =
This accurate mass confirmation of the unit mass library search can be automated so that the process is not time intensive to the analyst. Note that adding spectral deconvolution to the beginning of the process can insure a clean spectra is used to perform the library search.
In the case shown on this slide, the first hit of the unit mass spectral library cannot produce an ion that is within the mass tolerance of the acquired ion at m/z After rescoring based on this accurate mass consideration, bis (TBDMS) malonate is determined to be the correct hit, as it can produce fragments that match the acquired accurate mass data within the mass tolerance of the instrument at all masses including m/z
C11H23O4Si2
m/z =

41. Horse feed detection limit
Pesticides screening
Horse feed detection limit
Graph showing the lowest detected standard for 55 pesticides in horse feed. Identification based on accurate mass <2ppm and retention time ±20 seconds. Half MRL limit (5 ppb) and MRL (10 ppb) displayed.
100 % detected at MRL level
Majority at ½ MRL

42. (triplicate at each level)
Pesticides screening
Quantitation
Curve from 0.5 to 50 ng/g
(triplicate at each level)
R2=0.9999
TraceFinder software view of the extracted ion chromatograms and calibration curve for fenpropimorph in leek. Triplicate injections of the calibration series
were performed with excellent linearity.
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

43. Complete solution for total pesticide analysis
Q Exactive Focus
Hybrid Quadrupole-Orbitrap LC-MS/MS System
Q Exactive GC
Hybrid Quadrupole-Orbitrap GC-MS/MS System