Orbitrap Technology:
New Frontiers in Pesticide Residue
Analysis
Michal Godula
Special Solutions Center Europe
Thermo Scientific
We Are The World Leader in Serving Science
Global Scale
• 50,000 employees in 50 countries
• $17 billion in annual revenues
• Unparalleled commercial reach
Unmatched Depth
• Leading innovative technologies
• Applications expertise
• Lab productivity partner
2
Premier Brands
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
Unmatched ultrahigh resolution,
accurate mass performance
3
• Proteomics, drug discovery, toxicology,
forensics, environmental, food safety and
many more
Orbitrap FTMS
Makarov A. Anal. Chem. 2000, 72, 1156-1162.
4
Orbitrap Mass Analyzer Features
•
•
•
•
•
•
•
•
5
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
mmeas  mtrue 6
m / z 
10
mtrue
• Quadrupole MS
• Orbitrap MS
TOF MS
500.1  500.0 6
m / z 
10  200 ppm
500
m / z 
500.10314  500.10214 6
10  2 ppm
500.10314
Selectivity increases with higher mass accuracy
RT: 8.45
100
N
m
4
4
{
m
M
90
80
70
60
Relative
Abundance
2 ppm
50
40
30
20
10
7.26
7.41
7.49
7.67
0
7.2
7.4
7.6
7.91
7.76
7.8
8.55
7.99
8.0
8.2
8.4
8.8
Time (min)
9.0
9.2
9.4
10.31
10.16
9.46
8.6
9.6
9.8
10.0
10.2
10.50
10.4
10.61
10.6
RT: 8.45
100
90
80
70
Relative
Abundance
60
50
40
20 ppm
30
20
10
7.37
7.30
7.26
7.40
7.59
7.62
7.85
7.81
7.92
7.99
8.21
8.25
8.61
0
7.2
7.4
7.6
7.8
8.0
8.2
8.4
8.67
8.6
8.88
9.11
8.8
9.0
Time (min)
9.46
9.2
9.4
10.09
9.6
9.8
10.0
10.16
10.31
10.2
10.50
10.4
10.61
10.6
RT: 8.45
100
90
80
70
Relative
Abundance
60
50
40
8.67
20
10
9.29
8.72
8.78
30
7.27
7.33
7.38
7.59
7.62
7.85
7.81
8.10
8.13
8.21
8.99
9.11
8.83
9.46
9.50
0
7.2
7.4
7.6
7.8
8.0
*Zachariasova M et al, Anal. Chim. Acta
100 ppm
8.35
8.2
8.4
8.6
8.8
Time (min)
9.0
9.2
9.4
9.6
9.64
10.09
9.8
10.0
10.16
10.2
10.50
10.34
10.4
10.61
10.6
Resolution (resolving power)
• Resolution
m
R
m
• Quadrupole MS
• Orbitrap MS
m (FWHM)
m 10%
m 500
R

 833
m 0.6
m
500
R

 100000
m 0.005
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: amadeo@ual.es
Q Exactive MS - a 3D view
Hyperbolic Quadrupole
Mass filter
RF-Lens
C-Trap
HCD
Cell
Orbitrap Mass
Analyzer
Q Exactive FS-ddMS2 screening process
Ion Accumulation
Full Scan MS Data:
Screening/Quantitation
Full Scan
Orbitrap Analysis
Inclusion List
Precursor Selection
Precursor accumulation
Full Scan MS/MS Data:
Confirmation
Fragmentation in HCD
MS2 Orbitrap Analysis
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
Extraction
+ 20 mL of water
+ 10 mL of ACN
shaking 10 min
Centrifugation 5 min @ 5000 rpm
Analysis
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
scan range
• ddMS2: resolution
Inclusion list
isolation
exclusion
70,000 FWHM
120-1200 m/z
35,000 FWHM
on
1 m/z
5s
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
TraceFinderTM Screening Data Review
Isotopes
Target analytes
Fragments
Chromatogram
Spectrum
Library
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
Sample – determined
concentration 5.3 ug/kg
Standard 50 ug/kg
Sample
MS2 spectra
Standard
MS2 spectra
Isotopic pattern fitting is influenced by resolution
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
22
Variable DIA – alternative to All Ions Fragmentation
Traditional AIF
Variable Data Independent Analysis
Presented by P Zomer, RIKILT at EPRW 2014:
23
AIF vs. vDIA
Oxacillin in Pig Kidney @ 2 ppb
AIF
vDIA
24
background
Running 380 Compounds with the Q Exactive Focus
Quan/Qual
# of targeted
compounds
Selectivity
Linearity – working
range [ppb]
Matrix
Sample Prep
25
Data Dependent-MS2
vDIA
Quantitation
Targeted Screening
Total: 380
345 Pesticides, 35 Mycotoxins and Alkaloids
Retention time, fragment ions, ion ratios, isotope
ratio, library match
1-500
(6 levels and 2 replicates)
Black Tea/Honey
Buffered QuEChERS method
clean-up made by MgSO4, PSA and C18
vDIA sensitivity in honey
Number of compounds with <1ppb sensitivity
Peaks in honey sample spiked to 1ppb
20%
27
80%
345 target pesticides in tea matrix
Data still
being processed
28
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
29
Bringing GC and Orbitrap Technology Together
C-TRAP
AQT
Quadrupole
Bent
Flatapole
HCD Cell
ExtractaBrite™
Ion Source
Orbitrap
Mass Analyzer
30
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
31
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
32
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)
33
Breakthrough in GC-MS Performance
Fast acquisitions
XIC of dieldrin in baby food (m/z 262.85642) 100ppb
9
• Important for
accurate profiling of
narrow GC peaks
8
60,000 RP (m/z 200)
2.4 s peak
7
11
• Full scan with
resolving power of
60 (FWHM @ m/z
200) generates
17 scans
6
5
• Fast enough for
GC!
4
3
1
34
10
2
17 scans
12
13
14
15
16
17
Breakthrough in GC-MS Performance
< 1ppm (internal)
XIC of dieldrin in baby food (m/z 262.85642) 100ppb
Scan to-scan
0.3
< 3ppm (external)
• Scan-to-scan
• Low level in matrix
0.5
60,000 RP (m/z 200)
2.4 s peak
• Over full mass
range
0.3
0.5 ppm RMS
0.5
0.5
0.6
35
0.6
0.3
• Over full
concentration range
• No need for
continuously
calibrating in
sequence
0.3
0.7
0.7
0.6
0.6
0.7
0.5
Breakthrough in GC-MS Performance
Over full concentration range
Mass accuracy over >6
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
36
Is it sensitive enough? Q Exactive GC vs. Triple Q
37
Breakthrough in GC-MS Performance
Full Scan
38
SRM
Compound Discovery 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
39
Automated Workflow for Accurate Mass Confirmation
Unknown Identification Workflow
Spectral
Deconvolution
on EI HRAM
Data
Spectral
Library
Search
Scoring
m/z = 275.11298
Additional
High
Resolution
Scoring
m/z = 275.11298
C11H23O4Si2
m/z = 275.11294
40
Pesticides screening
100 % detected at MRL level
Majority at ½ MRL
41
Pesticides screening
Curve from 0.5 to 50 ng/g
(triplicate at each level)
R2=0.9999
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
42
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
43