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Finding a Needle in a Haystack:
Using High Resolution Mass Spectrometry in Targeted and Non Targeted Searching for Food Contaminants
Erik VerschuurenStrategic Account Manager, Food Safety and Environmental
Thermo Fisher Scientific
• Melamine in milk powder• Illegal dyes in chilli powder• Acrylamide in crisp bread & French fries• BADGE in canned fish• 2-Isopropylthioxanthone (ITX) in packaged foods• Illegal addition of proteins to milk• Industrial chemicals & pharmaceuticals in environment
Where Target Analysis has Failed?
Different Modes of Analysis
Pure QualitativePure Quantitative
Detect & Quantify
Targeted Analysis
Non-Target Analysis
Non-Target Analysis
Determine structure
• Structural ID• Compound Confirmation• Reaction Monitoring• Process Monitoring• Metabolism• Proteomics• Metabolomics
Screening
QuantificationConfirmation
ProfilingFingerprinting, Statistical
Database searching
Identification of unknown Find your needles
Triple Quadrupoles Used for Targeted Analysis
TSQ Quantum Access TSQ Quantum Ultra TSQ VantageTSQ Quantum Access Max
Ultimate Value Ultimate Performance
However….
Technology is evolving…
….direction glaring in the mist
Shift Towards HRAM : Crossover Point
Official Food Control Authority of the Canton of
Zurich
Shift Towards HRAM : Crossover Point - cont
High Resolution Analyzers Are Claiming Their Share
Bench-top HRMS Ultimate Performance
QExactiveExactive LTQ Orbitrap
[M+H]+ 381.07828
Mass accuracy Number of hits
100 ppm 34
50 ppm 17
30 ppm 11
10 ppm 4
5 ppm 2
3 ppm 2
•Compounds containing •C[5-15]N[0-10]O[0-15]H[0-40]
340 350 360 370 380 390 400 410m/z
0
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Rel
ativ
e A
bu
nd
ance
381.07828
C27 H11 O2 N
How Much Mass Accuracy is Needed?
Selectivity Increases With Higher Mass AccuracyRT: 0.00 - 18.59 SM: 5B
0 2 4 6 8 10 12 14 16 18Time (min)
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1000
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Re
lative
Ab
un
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nce 0
20
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1005.465.40 5.99 6.69
8.094.934.36
8.543.75 8.74 10.10
10.4411.25
12.0712.73
13.550.82 14.371.85
15.0416.27
8.09
9.6510.59 12.04 17.858.462.31 3.50 5.644.971.14 7.82 17.0616.2715.00
8.09
NL: 2.14E6m/z= 207.0489-207.2489 F: FTMS {1,1} + p ESI Full ms [100.00-1000.00] MS STL_Cal_02
NL: 3.29E5m/z= 207.0989-207.1989 F: FTMS {1,1} + p ESI Full ms [100.00-1000.00] MS STL_Cal_02
NL: 3.26E5m/z= 207.1439-207.1539 F: FTMS {1,1} + p ESI Full ms [100.00-1000.00] MS STL_Cal_02
207.1489± 0.1µ
207.1489± 0.05µ
207.1489± 0.005µ
288.0441 C9H21O2P1S3 Terbufos
288.0949 C13H21O3P1S1 Iprobenfos
288.1142 C15H17N4Cl1 Myclobutanil
288.1256 C11H20N4O3S1 Epronaz
288.1351 C11H21N4O3P1 Pirimethaphos
288.1474 C16H20N2O3 Imazamethabenz
Is a simultaneous measurement possible?
Element Exact Mass H 1.007825 C 12.000000 N 14.003074 O 15.994915
Detection of Isobaric Compounds in Mixtures
289.00 289.05 289.10 289.15 289.20m/z
0
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Rel
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e A
bund
ance
289.1314
289.0513 289.1022
289.1215
289.1329 289.1424
R = 10,000
289.00 289.05 289.10 289.15 289.20m/z
0
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Rel
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bund
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289.1390
289.1021289.0514
289.1215
289.1329289.1424
R = 20,000
289.00 289.05 289.10 289.15 289.20m/z
0
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Rel
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bund
ance
289.1424289.1547289.1329289.1022
289.0514
289.1215
R = 100,000
0.1033µ
239.00 239.05 239.10 239.15 239.20 239.25m/z
0
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Rel
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bund
ance
239.15181C11 H 19 O2 N4
6.50 ppm
R = 15,000Error = 6.50 ppm
Detection of Analytes in Heavy Matrix
Pesticide Mixture in Horse Feed Matrix
R = 80,000Error = 0.32 ppm
239.00 239.05 239.10 239.15 239.20 239.25m/z
0
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Rel
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e A
bund
ance
239.15033C 11 H 19 O2 N 4
0.32 ppm
Target Screen Approach
Raw Data
Library of “Knowns”
Targeted Screen Report
Non Targeted Analysis – What’s the challenge?
• High resolution MS data acquisition- Unknown is unknown…- Matrix presence- Variability of samples- Small concentrations of potential threats- Extraction procedures: polarity, pH
• Data processing?
MS Detectors for Unknowns
Unknowns
Ion Trap MS High Resolution
Orbitrap MS
• Full scan mode acquisition
• Confirmation by MSn
• Exact mass and high resolution data
• Confirmation by isotopic pattern and MS/MS
• Confirmation and Quantitation in 1 run
10052_520 #157 RT: 2.23 AV: 1 NL: 1.62E5T: FTMS {1,1} + p ESI Full ms [100.00-1500.00]
127.0 127.2 127.4 127.6 127.8 128.0
m/z
0
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Re
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Ab
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127.07263
128.07596127.10697 127.54161127.41898
10052_520 #118 RT: 1.67 AV: 1 NL: 1.91E6T: FTMS {1,1} + p ESI Full ms [100.00-1500.00]
650 700 750 800
m/z
0
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Re
lativ
e A
bu
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an
ce
707.21625
C30 H31 O 11 N10
723.19019
C30 H29 O 13 N9
785.23486
C30 H45 O 15 N10
695.20386
C27 H33 O 15 N7649.21008
C27 H33 O 13 N6
Key Requirements: Mass Accuracy + Resolution
ppm error Number of hits
150 11
50 4
10 1
5 1
2 1
m/z 127.07263C, N, O, H
m/z 707.21625C, N, O, H
ppm error Number of hits
150 38
50 12
10 3
5 3
2 2
Milk sample 2g
Add 20 ml Acetonitrile/Water70:30 v/v
Extraction 20 min
Centrifugation 14000 rpm 10 minutes
Filtration through 0.2 µm PTFE into LC vial
UHPLC/Exactive MS analysis
SIEVE data processing
Poster: NEW STRATEGIES FOR FOOD PROFILING AND MONITORING USING HIGH RESOLUTION MASS SPECTROMETRY APPROACH, M. GODULA1, A. CHARLTON2, D. ROBERTS2 AND K. MITTENDORF1
Example: Analysis of Milk Powder
RT: 0.39 - 60.03 SM: 7B
5 10 15 20 25 30 35 40 45 50 55 60Time (min)
0
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Re
lative
Ab
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44.4638.18 43.82
46.56
14.81 41.80 48.62
10.2641.27 48.98
13.829.82 33.20 49.7219.818.5637.765.47 16.73
50.4021.12 22.71 27.95 34.9031.41 51.0727.60
51.6953.57
55.96
44.46
38.20
10.25 46.56
14.81 41.80
33.1446.81
48.8541.462.03
50.189.30 37.76 50.8319.84
8.322.59 12.78 37.52 51.2215.18 21.10 52.3622.62 30.21
54.95
NL: 1.58E8TIC F: FTMS {1,1} + p ESI Full ms [50.00-1000.00] MS Non_Target_Milk_PosC18ESI_250809Sample
NL: 3.70E7Base Peak F: FTMS {1,1} + p ESI Full ms [50.00-1000.00] MS Non_Target_Milk_PosC18ESI_250809Sample
TIC (Total Ion Current)
BPC (Base Peak Chromatogram)
TIC and BPC of Milk Extract - No Difference…
Background subtracted file
Components extracted
Automated Component Detection - ExactFinder
RT: 8.71 - 60.03 SM: 7B
10 15 20 25 30 35 40 45 50 55 60Time (min)
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Re
lative
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44.46
38.1843.82
46.56
14.8141.80 48.62
10.26
41.2748.98
40.53 49.2813.82
33.20 49.7219.81
50.1837.7616.73
50.4021.12
22.71 50.7927.9534.9022.95
31.4151.07
51.2227.6026.12
51.6953.57
54.9555.96 58.20
46.5814.81
38.23
41.80
10.29
37.5333.16
12.79
49.48
35.9625.0119.87
52.9826.32 27.8916.7122.58 28.1417.75
54.09
NL: 4.47E7TIC F: FTMS {1,1} + p ESI Full ms [50.00-1000.00] MS Non_Target_Milk_PosC18ESI_250809SampleNL: 4.41E7TIC F: FTMS {1,1} + p ESI Full ms [50.00-1000.00] MS non_target_milk_posc18esi_250810blank
The only visually identified difference
FenthionSudan dyesMelamineCyanuric acidUrea
Comparing Contaminated Sample (10ppm) With Blank
Non_Target_Milk_PosC18ESI_250809Sample 8/26/2010 1:52:43 AM S5 spike 10ppm B
RT: 42.64 - 46.16 SM: 7B
42.8 43.0 43.2 43.4 43.6 43.8 44.0 44.2 44.4 44.6 44.8 45.0 45.2 45.4 45.6 45.8 46.0Time (min)
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Rel
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RT: 43.82
44.46
45.22
45.6945.3642.94 45.0342.91 44.22 44.69
43.5943.24 43.27 46.0945.7645.91
NL: 4.47E7TIC F: FTMS {1,1} + p ESI Full ms [50.00-1000.00] MS Non_Target_Milk_PosC18ESI_250809Sample
Non_Target_Milk_PosC18ESI_250809Sample #3077 RT: 43.84 AV: 1 SB: 31 43.35-43.61 , 44.09-44.25 NL: 3.52E6T: FTMS {1,1} + p ESI Full ms [50.00-1000.00]
60 80 100 120 140 160 180 200 220 240 260 280 300m/z
0
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Rel
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unda
nce
93.05714
249.10082
128.04893
156.04360
188.06968116.98526 232.09821
133.03367149.01094
184.0724684.95951
160.07487
101.00794 177.0056770.98044 195.08568 279.64330214.91702 297.00241253.32847243.59590225.36233
20 hits < 10 ppmCHNOSP
Blank Milk vs. Spiked Milk – Visual Comparison Only
Blank sample
Real sample
Alignment Framing ChemSpider Search
Compensation of RT changes
Table of identified frames
Table of positive hits
SieveTM –Data Comparison Tool
Sieve™ – Framing Process
•ChemSpider search
•Web based
• Number of databases
available
•Targeted search MZ Lookup
•Custom build library
•Adducts searched
Sieve™ – Database Search Option
Advanced Algorithms – Elemental Composition Calculation
MH+
ASMS 2010 Poster-T501: High-Resolution LC-MS Analysis of Compounds for Early Drug Discovery: Quantitative and Qualitative Screening in One Pass, Caroline Ding1, Jim Shofstahl1, Karen Salomon1, Hans Pfaff2, and Thomas McClure1
ExactFinder considers isotopic pattern as a confirmation criteria
•Isotopic patterns compared
• Resolution is needed to separate overlapping
peaks
Table of candidates
Isotopic Pattern Fitting is Influenced by Resolution
Conclusions – Future View
• Minimize Sample preparation• Simplified sample preparation strategies• Direct analysis where possible
• Advanced data processing algorithms must be used• Data extraction and background subtraction• Comparative approaches• Libraries of accurate masses and fragments required• Isotopic pattern fitting
• Validation• Validation for diverse matrices required
