Post on 14-Aug-2018
MSD DeconvolutionReporting Software
(MSD-DRS or DRS)(MSD-DRS or DRS)A tool to Aid in the Identification and Quantitationof Target Compoundsof Target Compounds
Mike SzelewskiAgilent TechnologiesM 14 2009May 14, 2009
20090514 e-Seminar
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TICs of surface water extracts
How many ypesticides are in these samples ?
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
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5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Seventeen (17) surface water samples
CDFA* Agilent DRS Agilent DRS (N
567 927
CDFA* Agilent DRS (Original) (New
Database)Targets Found (not counting 37 Same 37 Same 37(not counting
ISTD)37 + 34 more +99 more
False Positives 1 0 0
Processing Time
~8 hrs (ChemStation
Only)20 minutes 34 min
*CDFA is the California Department of Food and Agriculture
y)
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Data files courtesy of Dr. Mark Lee and Steve Siegel
DRS Report from 3 Integrated Processes
Total ion chromatogram
Targets are identified by comparison to locked R T s and 3 qualifier ion
Deconvoluted Target spectra confirmed by AMDIS searched
AMDIS deconvolutescomponent spectra and
searches target MS database using locked RT as a qualifier
Quant Results
R.T.s and 3 qualifier ion ratios, then quantified using target ion area vsISTD cal table
AMDIS, searched against NIST08 MS database
Confirmed NIST08 hits
g q
Confirmed AMDIS hits
AMDIS Quant Result
Combined quantitative and qualitative HTML S t
Quant Results Confirmed NIST08 hits
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Summary report
Report-Spinach Example 1CSpinach Report, results from 3 DRS processesNIST 08 Match and Hit number of the top 100 hits from 191,000+ compounds
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ISTD, used for targets amount Match against
AMDIS library
RT diff in secs vs expected locked RT
• A t ti M t l D l ti d
DRS is Based on Industry Standard AMDIS
• Automatic Mass spectral Deconvolution and Identification Software
– Developed to automatically detect chemicals in– Developed to automatically detect chemicals in violation of Chemical Weapons Convention
– Identify compounds at low concentration levels in complex matrices
– Developed by National Institute of Standards and TechnologyTechnology
– Retention Time can be used as a qualifier, so RTL is a big advantage
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• A t ti M t l D l ti d
True Deconvolution using AMDIS
• Automatic Mass spectral Deconvolution and Identification Software
– All spectra are deconvoluted not just those selected– All spectra are deconvoluted, not just those selected by the user
– Identification is based on full spectral matching
– User does not have to “pick” ions
– More confidence in result using all ions
– Published algorithims, not proprietary
– Accepted for world-wide use
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AMDIS Deconvolution Pulls Out Individual Components and their Spectra p p
Deconvoluted components and spectra
t iTIC
Components and Mixed Spectra
matrixTIC
Component 1C t 2
interference
Component 2
Component 3
Deconvolution
target
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17 280
How Does Deconvolution Work?
50
170 0
310
75
18516
0 00
Extracted Ion Ch t
160 shapeChromatograms
(EIC)
Aft d
50170
p
same shape and same retention time
After de-skewing
0280
75 late retention time 185 shape & early retention time
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310 early retention time
Eliminate Ions that Do Not Fit the Criteria 17 28
050
170 0
310
75
18516
0 00
Extracted IonOnly the ions in black have the
5017
Extracted Ion Chromatograms
(EIC)
black have the same shape
and retention time as shown028
0
time as shown by 50, 170, 280 -
plus others
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Combine Ions That Do Fit the Criteria 17 28
050
170 0
These bi dExtracted Ion
Chromatograms (EIC)
combined deconvoluted
ions are a 50
( C)component
170
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280
What can you do with a full spectrum component ?
• Identification by matching against an AMDIS user library- Locked RT or RI can also be part of the library entryp y y
• Send to NIST Search for additional confirmation– All components can be NIST searched, not just target compounds
• Add to an existing library
• Build a user library of deconvoluted components, in NIST, AMDIS or Agilent formatsg
• Component spectra are flat files that can easily be shared or edited
• Full spectrum graphics output confirms compound for reports– Often required by government agencies and courts (drugs)
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A portion of RTLdemo.D, clean standards
3000000
3500000
4000000
4500000
5000000
5500000
6000000
6500000
7000000
Abundance
TIC: RTLdemo.d
16.604
Quant results show NOidentified compounds at 16.604
16.10 16.20 16.30 16.40 16.50 16.60 16.70 16.80 16.90 17.00 17.10 17.20 17.300
500000
1000000
1500000
2000000
2500000
3000000
Time-->
min, using a target and 3 qualifier ions
Abundance
1000
2000
3000
4000
5000
6000
7000
8000
9000
Scan 2530 (16.604 min): RTLdemo.d286
125
263
79
47 197168 233 323100 146
50 100 150 200 250 300 350 400 450 5000
m/z-->
3000
4000
5000
6000
7000
8000
9000
Abundance
#218: Chlorpyrifos Methyl286
125
A single hit is hard to believe, especially with a 99 match quality – the danger
50 100 150 200 250 300 350 400 450 5000
1000
2000
3000
m/z-->
7947
170 197 321228100 146 260 405343 430375 476498 of reverse search
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Deconvoluted Peaks from AMDIS
There are 2 small “T” symbols indicating Targets found by AMDIS
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Match First of 2 Peaks from AMDIS
Black is uncleanedBlack is uncleaned spectrum at 16.599 min White is deconvoluted spectrum at 16.6000 min
Chlorpyrifos methyl AMDIS match = 94 Δ r.t. = 0.4 sec NIST05 r-match = 89NIST05 r-match = 89 NIST05 f-match = 87 Probability = 97.5
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Match Second of 2 Peaks from AMDIS
Black is uncleanedBlack is uncleaned spectrum at 16.599 min White is deconvoluted spectrum at 16.6000 min
Methyl parathion AMDIS match = 94 Δ r.t. = 0.4 sec NIST05 r-match = 91NIST05 r match 91 NIST05 f-match = 90 Probability = 97.9
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Can I ?
Add Compounds to Existing AMDIS databases ?• Yes, detailed instructions are in the Help files, you start with a
d t fil th t t i th d( ) t dd A t ldatafile that contains the compound(s) to add. App notes also available.– remember to add these to your Chemstation method also
B ild AMDIS D t b ?Build my own AMDIS Databases ?• Yes, detailed instructions are in the Help files, you start with a
user Library, in Agilent format, of your compounds. App notes also available.– If you haven’t already created a Library in Agilent format, try
this. Collect all your data in one or more files using RTL conditions Use Excel and the rtl import command toconditions. Use Excel and the rtl_import command to simultaneously create a Library in Agilent format and a quant database.
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Agilent Databases - # Compounds Part #
Hazardous Industrial Chemicals – 731 G1671AA
Pesticides 927 G1672AAPesticides - 927 G1672AA
Indoor Air Toxics – 171 G1673AA
Forensic Toxicology – 723 G1674AAForensic Toxicology 723 G1674AA
Japanese Positive List – 430 G1675AA
Fiehn Metabolomics - > 1000 G1676AA
Environmental Semivolatiles – 343 G1677AA
DRS Base Product, rev A.04 G1716AA
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Report-Spinach Example 1CToxicology Sample using G1674AA
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DRS Results from Environmental Soil and Water Samples using G1677AASamples using G1677AA
Total for8 samples
Total Real Positives based on manual review 183
Chemstation False Positives at 20% Absolute 35Qualifier Ion Criteria
Chemstation False Negatives at 20% Absolute 56Qualifier Ion CriteriaQualifier Ion Criteria
AMDIS False Positives 11
AMDIS False Negatives 11g
Both AMDIS and Chemstation Same False Negatives 0
Both AMDIS and Chemstation Same False Positives 2
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DRS A.04 – Quantitation on the Deconvoluted Ion
AMDIS Information fully integrated into QEdit
No need to switch between programs
R l d lib t di l d f i lRaw, clean and library spectra displayed for visual confirmation
Deconvoluted target ion for easier integration
5000
6000
Raw MSD 70 Prochloraz 70
g g
2000
3000
4000
Raw MSD 70 Prochloraz 70
Interfering 70
0
1000
2000
1645 1646 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660
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Scan
Neat Orange Oil Spiked with 20 pesticides at 200 pg each on column – 1162 total components found
2.7x10e7 max
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
5x10e55x10e5DRS finds 2,4-D ethyl ester
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3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80
MSD Chemstation alone does not find 2,4-D ethyl ester based on 3 Qualifier Ions
3.992 minscan 503sca 503
Expected Actual 20% 50%Ion Ratio Ratio absolute relative
175 100 100248 76 9 no no
|
185 75 94 yes yes177 64 15 no no ||
|
DRS shows it present and manual integration yields MSD AMDIS AMDIS AMDIS NIST
pg pg match RT diff matchpg pg atc d atcQuant ion
175 727 186 71 -1.1 sec 74
Where did this come from ? later
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Where did this come from ? - later
MSD Apex Spectrum does not match 2,4-D-ee but the AMDIS component spectrum does match
60008000
Abundance
Scan 503 (3.992 min): 2_100cp_2^2_25_F.D\data.ms93MSD Raw Spectrum
0 20 40 60 80 100 120 140 160 180 200 220 240 260
200040006000
m/z-->
55 1337967 120105 147161 190175 203218 248231 262277
Abundance
EIC 175
MSD
|
02000400060008000
[_MS_PT]*AMDIS Extracted Spectrum
175248
111 213187200751
||
|
EIC 175
AMDIS Component Spectrum
0 20 40 60 80 100 120 140 160 180 200 220 240 2600
m/z-->Abundance
[_MS_PT]*AMDIS Library Spectrum
EIC 175
AMDISAMDIS Library Spectrum, 2,4-D Ethyl Ester
||
|
0 20 40 60 80 100 120 140 160 180 200 220 240 26002000400060008000
m/z-->
175 248145111 162 2131877559 13387 99471
RT difference of 2 apices = 0.011 min
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RT difference of 2 apices 0.011 min
MSD TIC (scaled 1/100), MSD 175 EIC and the MSD raw spectrum
30000
Raw MSD TIC (0.01x)
20000
25000
15000
Raw MSD 175Shaded area quant amount = 727 pg
5000
10000Raw MSD 175amount = 727 pg
0496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
Scan #
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Scan #
There are actually 2 separate components found by AMDIS under the MSD TIC
30000
Raw MSD TIC (0.01x)
20000
25000
Component
Component 2 TIC
15000
p1 TIC (0.01x)
5000
10000
0496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
Scan #
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Scan #
Each of the two components has a 175 ion and AMDIS separates the ion contribution from each
30000
20000
25000
15000
Raw MSD 175
Component 1 175
5000
10000Raw MSD 175
Component 2, 175
Quant amount = 186 pg From component 2 175 EIC
0496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
Scan #
From component 2, 175 EIC
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Scan #
Component TICs and 175 EICs with raw, deconvoluted and2,4-D Ethyl ester library spectra
30000 Raw MSD TIC (0.01x)
20000
25000 TIC - 2,4-D ee Component
15000
Component 1 TIC (0.01x)
Raw MSD 175
5000
10000Component 1 175
Raw MSD 175
175 - 2,4-D-ee Component
0496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
Scan #
Component
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Scan #
DRS A.04 Report from the original Spinach example, before importing AMDIS results
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DRS A.04 QEdit Menu Items1
23
1
4
56
2- Displays Text Report with MSD and AMDIS Results
6
1- Import spectra and extracted ion from AMDIS
p y p
3- Reloads existing results from AMDIS results file
4- Displays DRS report with updated amounts
5- Opens AMDIS standalone
6- Link to DRS Help
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QEdit, p,p’-DDT selected
5 ionMSD ion AMDIS ion
5 ion overlay
MSD & AMDIS areas & amounts
3 t“x” and “A”
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3 spectra indicators
DRS A.04 Report after importing AMDIS results, before review and manual integration
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DRS A.04 Report after importing AMDIS results, QEdit reviewed with manual integrations
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Text report comparing MSD and AMDIS RTs and amounts available in QEditand amounts available in QEdit
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Graphics Report
MSD and AMDIS 5-ion overlay
Raw, deconvoluted and library spectra
MSD and AMDIS areasMSD and AMDIS areas and amounts
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Summary Quant Report optionally includes both MSD Chemstation and AMDIS amountsboth MSD Chemstation and AMDIS amounts
partial reportp p
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Enhanced Data Analysis DRS Menu:New A.04 Items
1
2
3
1) Include quant results from AMDIS on the Summary Quant Report
2) Automatically create AMDIS libraries, quant and screener databases from a user selected PBM library, with a few mouse clicks
3) Automatically update retention times in user selected AMDIS libraries with retention times from the currently loaded quant database. A backup of the initial libraries is made.
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DRS Tools Available
“Comprehensive Pesticide Screening by GC/MSD using Deconvolution Reporting Software” pub # 5989-1157
“New Tools for Rapid Pesticide Analysis in High Matrix Samples” pub # 5989-1716
“A Bli d St d f P ti id R id i S ik d d U ik d F it“A Blind Study of Pesticide Residues in Spiked and Unspiked Fruit Extracts Using Deconvolution Reporting Software” pub # 5989-1654
“Building and Editing RTL Screener/Quant Databases and Libraries” pub # 5989-0916
“Building Agilent GC/MSD Deconvolution Reporting Libraries for Any Application” pub # 5989-2249Application pub # 5989 2249
“Fast and Accurate EPA 8270 Quantitation Using Deconvolution” pub# 5990-3253
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DRS Tools Available
“Screening for Hazardous Chemicals in Homeland Security and Environmental Samples Using GC/MS/ECD/FPD with a 731 Compound DRS Database” pub # 5989-4834p p
“New Features of Deconvolution Reporting Software Revision A.02”, pub # 5989-4159
“Improved Forensic Toxicology Screening Using A GC/MS/NPD System with a 725-Compound DRS Database” pub # 5989-8582
“Semivolatiles Retention Time Locked (RTL) Deconvolution Databases ( )for Agilent GC/MSD Systems “ pub # 5989-7875
“Screening for Pesticides in Food Using the Japanese Positive List Pesticide Method: Benefits of Using GC/MS with DeconvolutionPesticide Method: Benefits of Using GC/MS with Deconvolution Reporting Software and a Retention Time Locked Mass Spectral Database” pub # 5989-7436
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DRS User Benefits, Time = $$$
Ease of use – no need to learn deconvolution software
Automation – part of a method or part of a sequence, analyst can be doing other things
Quality lt t bj t t h iQuality – program results are not subject to changes inmood/attention” of analyst
Sensitivity – AMDIS will find answers that an analyst y ymight miss
Confidence – DRS will report the fewest false positiveand false negatives in the shortest timeand false negatives in the shortest time
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