finding non-targeted (unknown) pesticides using … · finding non-targeted (unknown) pesticides...
TRANSCRIPT
July, 2008Page 1
Finding Non-targeted (Unknown) Pesticides
Using GC/MS and LC/Q-TOF
Chin-Kai Meng, Ph.D.Applications ChemistWilmington, Delaware
July, 2008Page 2
Outline - Workflow for Non-targeted Pesticides Analysis
• Business Issues and Challenges
• Sample Prep (QuEChERS) – Lehotay, Anastassiades and Mastovska
• GC/MSD
• (GC or LC)-QQQ
• LC/Q-TOF
July, 2008Page 3
Business Issues and Challenges
• Global trade increases the number of pesticides to monitor (> several hundreds)
• Regulation driving lower detection limits
• Speed: doing more analyses in a day
• Need for fast startup
Faster Data Review and Reporting
Total Solution Offering
Screen for Unknowns
July, 2008Page 4
GC-MSD Workflow
GC/MS (PTV) SIM/Scan– for known and unknown Final Report
Deconvolution
(+backflush)
Screen
Confirm
Quantify
July, 2008Page 5
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
TICs of Surface Water Extracts
How many pesticides (drugs, allergens etc.) are in these samples and how long does it take you to confirm?
July, 2008Page 6
17 Surface Water TICs (Scan): Pesticide Analysis Using DRS with Pesticide Database (927 entries)
*CDFA is the California Department of Food and Agriculture
CDFA* Agilent DRS
Targets Found (not counting
ISTD)37 Same 37
+99 more
False Positives 1 0
Processing Time ~8 hrs 32 min
Saving 7.5 hoursDRS: Deconvolution Reporting Software
July, 2008Page 7
What is Deconvolution?“…in the broad sense of extracting one signal from a complex mixture...” (From AMDIS)
There are four deconvoluted components under the white TIC.
July, 2008Page 8
AMDIS
Automatic Mass spectral Deconvolution and IdentificationSystem
• Developed by the National Institute of Standards and Technology (NIST)
• Developed to detect chemicals in violation of Chemical Weapons Convention (must minimize false positives and false negatives in reporting)
• Used to identify target compounds in complex matrices
How Does Deconvolution Work?
July, 2008Page 9
50
170 280
31075
185
160
Eliminate Ions Don’t Fit the Criteria
July, 2008Page 10
50
170 280
50170280
31075
75
185
185
310
160
160
Extracted Ion Chromatograms
(EIC)
Ion grouping criteria:
1. Same RetTime at apex
2. Same peak width
Spectrum is Deconvoluted/Cleaned
July, 2008Page 11
50
170 280
50170280
Related ions are grouped together as
a component.
A component in AMDIS
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Deconvolution
TIC of Spinach Extract
More than 370 peaks
found
Library Search
July, 2008Page 13
Carbaryl
PeachScan at 5.615 min
Deconvoluted/extracted spectrum
Library spectrum
A component in the scan above.
The power of deconvolution is appreciated while comparing the top two spectra (raw scan and the compound spectrum hidden in the raw scan).
July, 2008Page 14
Fenbuconazole
PeachScan at 10.776 min
Deconvoluted/extracted spectrum
Library spectrum
A component in the scan above.
July, 2008Page 15
DRS Report - Peach
July, 2008Page 16
DRS report from a SIM data file – for Additional Confirmation (4 ions for each target compound)
July, 2008Page 17
Can We Quant on Deconvoluted Ions?
• Yes!
• MSD ChemStation (G1701AA) E.02
• Deconvolution Reporting Software, DRS (G1716AA) A.04
• Pesticide RTL Library (G1672AA) – 927 entries
July, 2008Page 18
5 ion overlay
3 spectra
MSD & AMDIS areas & amounts
MSD ion AMDIS ion
“x” and “A”indicators
QEdit, p,p’-DDT selected
July, 2008Page 19
Graphics Report
MSD and AMDIS 5-ion overlay
Raw, deconvoluted and library spectra
MSD and AMDIS areas and amounts
July, 2008Page 20
Summary Quant Report optionally includes both MSD ChemStation and AMDIS amounts
partial report
July, 2008Page 21
DRS A.04 Report after importing AMDIS results QEdit reviewed with manual integrations
July, 2008Page 22
GC-MSD Workflow Summary
• GC/MS in full scan mode combined with deconvolution reporting software (DRS) enables unknown pesticide analysis at 10 µg/kg (ppb) in various food commodities from one injection
• Significant time saved on data analysis (screen and quant on 927pesticides), from hours to minutes
Application Note 5989-7670EN: Replacing Multiple 50-Minute GC and GC-MS/SIM Analyses with One 15-Minute Full-Scan GC-MS Analysis for Non-targeted Pesticides Screening and >10x Productivity Gain
SIM/ScanGC/MS (PTV)– for known/unknown
256
83
401 Final Report S
C
Q
Deconvolution
Library Search(+backflush)
July, 2008Page 23
LC/MS Workflow
LC/Q-TOF or TOF Full Spectrum – for unknown compounds Another
injection for MS/MS (QQQ or QTOF)
Confirm
Molecular Formula GenerationC16 H19 N3 P ClC15 H25 O P S ClC18 H21 O P Cl
Exact Mass Database Search
Screen
Quantify
July, 2008Page 24
Why Use (Q-)TOF?
QQQ• Routine target compound screening/quantitation in a single injection
• MRM detection limit meets all regulatory requirements
TOF/Q-TOF• Always full spectrum• Un-limited number of routine target and non-target compound screening
(using exact mass databases)
Q-TOF• Full scan accurate MS/MS spectra for added confidence for total
unknowns
All systems share SAME software platform, ion source, and collision cell – 100% workflow portability
July, 2008Page 25
Agilent Q-TOF Fundamentals
Ion opticsCommon with Q & QQQ
Flight tubeCommon with TOF
Octopole 1
DC Quad
Collision cellCommon with QQQ
Rough Pump
Turbo Turbo Turbo
Quad Mass Filter (Q1)
Collision CellLens 1 and 2
Octopole 2
Ion Pulser
Ion Mirror
Detector
Turbo
Exact Mass
Mass Accuracy (ppm)
July, 2008Page 26
What does “Exact Mass” mean?Element Atomic Number Exact Mass
H 1 1.007825C 6 12.000000N 7 14.003074O 8 15.994915
C6H6Cl6 287.8600665 LindaneC10H12N2O6S 288.0416000 CarbasulamC9H21O2PS3 288.0441285 TerbufosC13H21O3PS 288.0949000 IprobenfosC15H17N4Cl 288.1141743 MyclobutanilC12H21N2O4P 288.1238937 Diazoxon C11H20N4O3PS 288.1256000 EpronazC11H21N4O3P 288.1351000 PirimetaphosC16H20N2O3 288.1473925 Imazamethabenz
0.2874 amu
Uses accurate mass on TOF/Q-TOF to identify all of them.
July, 2008Page 27
v = d/t
E = ½ m v2 = ½ m (d/t)2
m = (2E/d2) t2
Energy (E) and Distance (d) are fixed
The measured mass is proportional to the flight time (time-of-flight).
The Key for getting useful TOF results is good mass accuracy.
Mass Analysis for TOF
July, 2008Page 28
Calculation of Exact Mass and Errorin Measured Mass (MH+) of Reserpine
Error if the electron was not omitted!
NH
N
O OO
O
OO
O OO
CH3
CH3
CH3
CH3
CH3
CH3
H
HH
Reserpine (C33H40N2O9)
Atom Mass of Atom # of Atoms SumHydrogen 1.00783 40 40.31300Carbon 12.00000 33 396.00000Nitrogen 14.00307 2 28.00615Oxygen 15.99492 9 143.95424Total 608.27338Plus H 1.00783 1 1.00783Total 609.28121Minus e- 0.00055 1 0.00055
609.28066
Calculated = exact
(Measured - Calculated)Calculated
0.9027038 ppm
X 1,000,000 = ppm
July, 2008Page 29
How Much Accuracy is Needed?
Reserpine (C33H40N2O9) has a protonated ion at 609.28066 (MH+)
Single quad reports mass to +/- 0.1 = 165 ppm
(0.1/609.28 x 10e6 = 164.128 ppm)
Number of possible formulas using only C, H, O & N:
Accuracy Mass Difference Number of Formulas165 ppm 0.1 amu (609.18 - 609.38) 20910 ppm 0.061 amu 135 ppm 0.030 amu (609.25 - 609.31) 73 ppm 0.0018 amu 42 ppm 0.0012 amu (609.2795 - 609.2819) 2
Measured mass accuracy can narrow the number of hits in a TOF database search, therefore reduce risk of investing effort on the wrong molecule.
July, 2008Page 30
Automatic Internal Referencing(Easy-to-use)
Reference Sprayer
Analytical Sprayer
Dual Spray ESI source andCalibration Delivery System (CDS) automates the introduction of reference masses
AutoTune
July, 2008Page 31
TOF/Q-TOF Unlimited Number of Compound Screening
5556.D (Grape)MS1 Full Spectrum
15 out of 510 compounds had hits from database search.
Molecular Feature Extractor
July, 2008Page 32
Run Another Analysis in MS/MS (Targeted) Mode to Confirm Hits Found in MS1 Mode
Compound RTPrecursor Mass
(MH)+
Spiroxamine 10.884 298.27406
Terbuconazole 14.139 308.15242
Tebufenoxide 15.492 353.22236
July, 2008Page 33
Next Step: Find
compounds by Targeted MS/MS
MS/MS data collected
July, 2008Page 34
4x10
0
1
2
3
4
5
6
7
Cpd 4:+ Scan (10.654-11.177 min, 25 scans) 5556.d
121.05092
298.27442C18H36NO2
3x10
0
1
2
3
4
5
Cpd 4:+ Product Ion (10.972 min) (298.27405[z=1] -> **) 5556.d
144.14176C8H18NO
298.27944100.11532C6H14N
Counts vs. Mass-to-Charge (m/z)80 100 120 140 160 180 200 220 240 260 280 300 320
O
O
NCH3
CH3
CH3 CH3
CH3
100.18206143.22668
Spiroxamine
MS1 Full Spectrum
MS/MS Full Spectrum
The compound name Spiroxamine came up in the MS1 mode.
Use formula results and MS/MS results to confirm the hit.
3x10
0
0.25
0.5
0.75
1
1.25
1.5
Cpd 4:+ Scan (10.654-11.177 min, 25 scans) 5556.d
298.27442C18H36NO2
299.27649
300.28402
Counts vs. Mass-to-Charge (m/z)97.5 298 298.5 299 299.5 300 300.5 301
Boxes represent theoretical isotope ratios
July, 2008Page 35
NN
N
OH
Cl
CH3
CH3
CH3
4x10
0
1
2
3
4
5
6
7
Cpd 11:+ Scan (13.974-14.248 min, 5 scans) 5556.d
121.05086
308.15216C16H23ClN3O
4x10
0
1
2
3
4
5
6
Cpd 11:+ Product Ion (14.121 min) (308.15240[z=1] -> **) 5556.d
70.04052C4H6O
125.01448C7H6Cl 308.15199
Counts vs. Mass-to-Charge (m/z)60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
125.57554Terbuconazole
MS1 Full Spectrum
MS/MS Full Spectrum
The compound name Terbuconazole came up in the MS1 mode.
Use formula results and MS/MS results to confirm the hit.
4x10
0
0.2
0.4
0.6
0.8
1
Cpd 11:+ Scan (13.974-14.248 min, 5 scans) 5556.d
308.15216C16H23ClN3O
310.14951
311.15253
Counts vs. Mass-to-Charge (m/z)307 308 309 310 311
Boxes represent theoretical isotope ratios
151.61282
July, 2008Page 36
Screen Pesticides with LC-TOF/Q-TOF
• Accurate Mass provides added compound selectivity
• Higher resolution provides added interference selectivity
• Always full spectral data
• Unlimited number of compounds can be screened (search exact mass compound database for identification)
• Sensitivity is the same regardless of number of compounds screened*
• MS/MS (Q-TOF) assists compound confirmation
*Triple quadrupole (QQQ) mass spectrometer can be more sensitive up to a limited number of compounds. That limit has not been definitively determined.
July, 2008Page 37
Representative Sample
Extraction
Pesticide Workflow: Screen , Confirm and Quantify
Clean-up
LC/QQQ MRM – for known targets S
C
Q
QuEChERS
S C Q
C16 H19 N3 P ClC15 H25 O P S ClC18 H21 O P Cl
LC/QTOF or TOF Full Spectrum– for unknown compounds
S
Exact Mass Database Search
Molecular Formula Generation
Q CAnother injection for MS/MS (QQQ or QTOF)
GC/MS (PTV) SIM/Scan– for known and unknown Final Report
S
C
Q
Deconvolution
(+backflush)
3 1
3 1
3 1
GC/QQQ MRM – for known targets S
C
Q
July, 2008Page 38
Summary
• GC/MS and Deconvolution to Screen, Confirm and Quantify
• QQQ for Targeted analysis in complex matrix – Sensitivity unsurpassed– Complex matrix with less clean-up
• TOF/Q-TOF for Targeted and Unknown Screening– Sensitive full scan analysis– Searching exact mass database leads to identification– Quantitative– Accurate mass MS/MS for identification of fragments and structure
elucidation
Page 39
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