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Pesticides e-Seminar Series -
Pesticide Analysis Workflow Using Hyphenated Mass Spectrometry Techniques
Date: 30 April 2009
Time : 10am Singapore Time
* Phone line needs to be IDD enabled. Not accessible from Mobile Phones
International Telephone Number for Singapore: +65-6622 1044 or Toll Free 8008 523 396International Telephone Number for Hong Kong: +852 3006 8101Toll Free International Numbers:Australia 1800 999 130 New Zealand 0800 450 755China 800 876 5011 Malaysia 1800 807 180Indonesia 0018 038 526 350 Taiwan 0080 185 5735India* 0008 008 521 136 South Korea 0079 885 214 717India (Backup)* 0008 001 006 542 Philippines 1800 185 50065Thailand* 0018 008 526 361 Vietnam 120 650 065
E-seminar, April 2009
Page 2
Pesticide Analysis Workflow Using
Hyphenated Mass Spectrometry
Techniques
E-Seminar, April 30, 2009
Chinkai (Kai) Meng, Ph.D.Senior Applications ChemistWilmington, Delaware USA
E-seminar, April 2009
Page 3
MS Techniques Outline
• MS Analyzers Selection
• GC/MS – deconvolution
• QQQ (MS/MS)
• LC/Q-TOF
• Summary
E-seminar, April 2009
Page 4
Analytical Instrumentations
Chromatography (Separation – the best friend of any detector)• GC – volatile and semi-volatile non-polar compounds• LC – polar or moderately polar or thermally labile compounds
Detectors• GC (element selective, FPD, NPD, ECD) – high sensitivity, poor specificity • LC (UV, FLD) – general purpose, limited sensitivity• MS –
– SQ: high confidence from spectral confirmation, limited MDL– QQQ: low detection limits of target compounds in dirty matrices (MRM)– TOF/Q-TOF: always full spectrum and accurate mass for screening– TOF-TOF, Q-Trap, …and others (generally not for ROUTINE use)
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Guiding Principles in MS Analyzer Selection
The correct choice of analyzer depends on whether one is “LOOKING for UNKNOWNS” or whether one is “CONFIRMING & MEASURING KNOWNS”.
The correct choice of analyzer depends on the COMPLEXITY of the MATRIX.
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Basic Questions – Which MS Solution
Target analysis only?
Analysis of unknowns?
How much chemical noise from the matrix?
How much sample prep?
Scan or SIM or MS/MS
Scan MS with Quad, IT or TOF
Deconvolution or MS/MS
MS/MS
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Page 7
Representative Sample
Extraction
General Workflow: Screen , Confirm and Quantify
Clean-up
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)3
1
LC/QQQ MRM – for known targets S
C
Q
3 1
GC/QQQ MRM – for known targets S
C
Q
3 1
GC/MS (PTV) SIM/Scan– for known and unknown Final Report
S
C
Q
Deconvolution
(+backflush)
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Page 8
MS Techniques Outline
• MS Analyzers Selection
• GC/MS – deconvolution
• QQQ (MS/MS)
• LC/Q-TOF
• Summary
E-seminar, April 2009
Page 9
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?
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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
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50
170 280
50170280
31075
75
185
185
310
160
160
Eliminate Ions Don’t Fit the Criteria
Extracted Ion Chromatograms
(EIC)
Ion grouping criteria:
1. Same RetTime at apex
2. Same peak width
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50
170 280
50170280
Spectrum is Deconvoluted/Cleaned
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 SearchPage 14
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Page 15
5 ion overlay
3 spectra
MSD & AMDIS areas & amounts
MSD ion AMDIS ion
“x” and “A”indicators
QEdit, p,p’-DDT selected
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Pesticides in Red Wine - Fenpropathrin
250 ppb
1 ppm
5 ppm MF = 877Fenpropathrin
MF = 783Fenpropathrin
Not found
MF = 83
250 ppbRaw scan
Deconvoluted
Library entry
Found by AMDIS
MF = 65
100 ppbRaw scan
Deconvoluted
Library entry
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MS Techniques Outline
• MS Analyzers Selection
• GC/MS – deconvolution
• QQQ (MS/MS)
• LC/Q-TOF
• Summary
E-seminar, April 2009
Page 18
Analyzers for Trace Target Compounds
LC/QQQ MRM – for known targets S
C
Q
GC/QQQ MRM – for known targets S
C
Q
Who should use QQQ (MS/MS)?
User doing Selected Ion Monitoring (SIM) for target compound analysis in traditional markets, needing additional sensitivity and selectivity with less sample prep, to meet more demanding analytical requirements.
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Advantages of a QQQ as a Chromatographic Detector - Multiple Reaction Monitoring (MRM)
170 210 250 290
210
222
268 280165
Quad Mass Filter (Q3)Quad Mass Filter (Q1) Collision Cell
Spectrum with backgroundions (from EI)
Q1 lets onlytarget ion 210 pass through
190 210
210
Collision cell breaks ion 210 apart
150 170 190 210
210158
191
Q3 monitors onlycharacteristic fragments 158 and 191 from ion 210 for quant and qual.
160
158
190
191
no chemical background
Source
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Page 20
Why MS/MS?Greater Selectivity Than SIM
PrecursorIon
Product 1Product 3
Product 2
EI-SIMselectivity proportional to
spectral resolutionno selectivity against ions
with same m/z
EI-MS/MSPrecursor selectivity same as SIM
High probability that at least one product ion will be a unique dissociation product
of the precursor BUT not the interferenceinterference
analyte
interference
The precursor ion should NOT be used for ion ratios or quantitation since the interferences will
be the same as the SIM ion
unit mass resolution
Consistent MS/MS Results, Regardless of Matrices– Analysis of spiked p,p’-DDE at 10 ppb (All injections = 1 µL)
Apple
Orange
Spinach
Ginseng
Cabbage
SIM - EIC (246) MRM (2 transitions)
A c q u is it io n T im e (m in )1 1 .8 1 1 .9 1 2 1 2 .1 1 2 .2 1 2 .3
Cou
nts 4x1 0
-0 .1
0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .8
0 .9
1
1 .1
2 4 6 .0 -> 1 7 6 .1 , 2 4 6 .0 -> 1 7 5 .1
R a t io = 2 3 .5
A c q u isitio n T im e (m in )1 1 .8 1 1 .9 1 2 1 2 .1 1 2 .2 1 2 .3
Cou
nts 3x1 0
0
1
2
3
4
5
6
7
8
2 4 6 .0 -> 1 7 6 .1 , 2 4 6 .0 -> 1 7 5 .1
R a t io = 2 3 .6
A c q u is it io n T im e (m in )1 1 .8 1 1 .9 1 2 1 2 .1 1 2 .2 1 2 .3
Cou
nts 4x 1 0
-0 .1
0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .8
0 .9
1
1 .1
1 .2
2 4 6 .0 -> 1 7 6 .1 , 2 4 6 .0 -> 1 7 5 .1
R a t io = 2 4 .0
A c q u is it io n T im e (m in )1 1 .8 1 1 .9 1 2 1 2 .1 1 2 .2 1 2 .3
Cou
nts 3x1 0
0
1
2
3
4
5
6
7
8
2 4 6 .0 -> 1 7 6 .1 , 2 4 6 .0 -> 1 7 5 .1
R a t io = 2 2 .9
Acquisition Time (min)11.8 11.9 12 12.1 12.2 12.3
Cou
nts 4x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
246.0 -> 176.1 , 246.0 -> 175.1
Ratio=23.3
S/N = 448
S/N = 456
S/N = 260
S/N = 446
S/N = 241
AppNote: 5990-3578; 175 pesticidesPage 21
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Page 22
Carrot Extract - 1 µL with GC/QQQ is Much Better than 5 µL GC/Q
GC/Q GC/QQQ5 µL (Multi-mode Inlet) 1 µL
Pesticide Cold SL Scan + DRS Cold SL SIM Hot SL (ppb)
Diclobenil 0.38*Pentachlorobenzene 0.75*Trifluralin 2.3*Tefluthrin 0.53*4,4'-Dichlorobenzophenone 1.2*Chlorpyrifos 24.7o,p'-DDE 3.7p,p'-DDE X X 240o,p'-DDD 9p,p'-DDD X Sum = 45o,p'-DDT Xp,p'-DDT X X 130Fenazaquin X Not in Method
* = Below Calibration Level
E-seminar, April 2009
5 µLColdSL ScanEIC
5 µLColdSL SIMEIC
Acquisition Time (min)11.8 11.9 12 12.1 12.2 12.3
Cou
nts 5x10
-0.10
0.10.2
0.30.4
0.50.6
0.70.8
0.91
1.11.2
1.3
246.0 -> 176.1 , 246.0 -> 175.1
Ratio=23.8
1 µLHotSL QQQMRM
S/N = 35
S/N = 375 S/N = 434
p,p’-DDE in Carrot Extract (240 ppb) – 5 µL GC/Q vs. 1 µL GC/QQQ
Same as left, after deconvolution
Page 23
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Page 24
Why a GC/MS/MS System?
• Allows for the selective quantitation of target compounds in high chemical background samples
• Better S/N in complex matrices than can be achieved by single quadrupole scan or SIM approaches.
• Newer regulations in some markets specify analytical power commensurate with GC/MS/MS
The Limitations of Converting an LC/MS/MS to GC/MS/MSESI, APCI and APPI - Atmospheric pressure sources
– Designed to separate ions from molecules before entering the vacuum manifold– Higher m/z metal rods are typically ‘temperature compensated’ for 100ºC
operation (some only 50ºC) – no problem since only ions enter the analyzer
EI, PCI and NCI - Vacuum sources
– All neutral molecules (carrier gas, analytes and matrix) enter the vacuummanifold BEFORE ionization
– Most molecules are NOT ionized and must be pumped away– High boiling molecules common to GC will contaminate all cooler surfaces
• Contamination causes loss of sensitivity• Cool (< 100ºC) metal quadrupoles are easily contaminated under GC/MS
Page 25
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Page 26
Why Heated Quartz “Gold” Quads?
• Higher temperature reduces potential for contamination– 200 C max – Virtually eliminates the need to clean quads
• Low coefficient for thermal expansion for quartz– Stable structure during maintenance cycles (hot-cool-hot) for source
or detector
More stable tunes and methods over a longer period of time in real world sample environments
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Page 27
MS Technique Outline
• MS Analyzers Selection
• GC/MS – deconvolution
• QQQ (MS/MS)
• LC/Q-TOF
• Summary
E-seminar, April 2009
Page 28 September, 2008Page 28
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
Two key concepts for TOF:
1. Exact Mass
2. Mass Error
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Page 29
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).
Mass Analysis for TOF
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Page 30 September, 2008Page 30
What does “Exact Mass” mean?Element Atomic Number Exact Mass
H 1 1.007825
C 6 12.000000
N 7 14.003074
O 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.
The Key for getting useful TOF results is good mass accuracy.
E-seminar, April 2009
Page 31 September, 2008Page 31
Calculation of Error in Measured Mass (MH+) of Reserpine
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
Error if the electron was not omitted!0.9027038 ppm
X 1,000,000 = ppm
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Page 32 September, 2008Page 32
TOF/Q-TOF Unlimited Number of Compound Screening
5556.D (Grape) MS1 Full Spectrum
• Molecular Feature Extractor found 510 compounds in the TIC.
• 15 out of 510 compounds had hits from EXACT MASS database search, i.e., these 15 matched entries in the database within 3 ppm mass accuracy.
The three highlighted compounds were further confirmed by MS/MS.
Terbuconazole
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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
125.0158
MS1 Full Spectrum
MS/MS Full Spectrum
The compound name Terbuconazole came up in the MS1 mode search.
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
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Page 34
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.
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MS Technique Outline
• MS Analyzers Selection
• GC/MS – deconvolution
• QQQ (MS/MS)
• LC/Q-TOF
• Summary
E-seminar, April 2009
Page 36
Summary
• GC/MS and Deconvolution to Screen, Confirm and Quantify • QQQ for routine Targeted trace analysis in complex matrix
– MRM Sensitivity unsurpassed (up to a few hundred compounds)– 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
• Unlimited number of compounds• Sensitivity is the same regardless of number of compounds screened
– Quantitative– Accurate mass MS/MS for identification of fragments and structure
elucidation (hotlink available to search on-line databases)
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Page 37
References
Application Note 5989-7670: 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
Pesticide Brochure 5989-8652
GC-QQQ Brochure 5990-3152
Current Trends in Mass Spectrometry, pp 33-38, Nov. 2008 (Supplement to LC/GC North America): Comprehensive Screening, Confirmation, and Quantification of Organic Pesticides in Foods by GC-MS and LC-MS
The Applications Book, LC/GC Europe, pp 9-11, Dec. 2008: The Benefits of Incorporating GC/QQQ into Pesticide Analysis Methods
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Page 39
Technology
Agilent took the best technologies from their industry leading 597X Series GC/MSD:
– Heated monolithic gold plated quartz quadrupole– Proven reliable high performance source design– AUTOTUNE
and the 6410A LC/QQQ:
– Linear acceleration enhanced Collision Cell– Wide Mass-Bandwidth QQQ ion optics– MassHunter software
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Page 40
Identify Dichlofenthion in MSD ChemStation- Red wine
MF = 630/670Dichlofenthion
No match
No match250 ppb
500 ppb
1 ppm
hidden
E-seminar, April 2009
Page 41
Identify Dichlofenthion in AMDIS
MF = 630/670Dichlofenthion
No match
No match250 ppb
500 ppb
1 ppm
MF = 82
250 ppbRaw scan
Deconvoluted
Library entry
hidden
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Page 42
Agilent Has the TOTAL Portfolio for PesticideAnalysis
(semi)-volatile or non-polar
(moderate)-polar or thermally labile
GCFPD/ECD
LCUV, FLD
GC/QDRS
LC/QLC/QQQMRM
GC/QQQMRM
Q-TOFTOF
GAS
LIQUID
Routine Analysis Identify Unknowns
phase
phase
Exact Mass Database Search
hidden