Deconvolution Reporting Software 051409 - Agilent · MSD Deconvolution Reporting Software (MSD-DRS...

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

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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

20090514 e-Seminar

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

20090514 e-Seminar

Deconvolution Reporting Software 051409 - Agilent · MSD Deconvolution Reporting Software (MSD-DRS...

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