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Thursday 19th September
Workshop B LC/MS Libraries and Personal Compound Databases for TOF and QTOF
Gordon A Ross Ph.D.
Agilent Technologies
Agenda
• Databases and Libraries
• Excel Based Databases (*.csv)
• Personal Compound Database (PCD)
• Personal Compound Databases and Library (PCDL)
• PCDL at the heart of Screening and Identification Workflows
• Find by Formula
• All Ions and MS/MS Libraries
Databases and Libraries
Database (*.csv, PCD)
Consists of (*.csv, PCD)
• Name
• Empirical formula
• Accurate monoisotopic mass
Optional (PCD)
• Structure
• CAS_Number
• Chemspider Number
Library (PCDL)
QTOF - PCD plus L
• MS/MS Spectra from
– Different collision energies
– Different ion sources
– Different polarities
Databases and Libraries
Database *.csv files
Consists of
• Name
• Empirical formula
• Accurate Mass
• Retention time (optional)
Simple to construct in Excel
Personal Compound Database and Library (PCDL)
Personal Compound and
Database Libraries
(PCDL)
Pesticides:
- 1600 Entries / >500 MS/MS
Forensic and Toxicology:
Broecker, Herre and Pragst Library
- 7500 Entries / 2500 MS/MS
Veterinary Drugs:
- 900 Entries / 500 MS/MS
All PCDLs can be customised with
addition of retention times, structures
and MS/MS spectra. Also by adding
compounds or removing irrelevant
compounds for a particular
application.
Personal Compound Database and Libraries (PCDL)
Personal Compound and Database Libraries (PCDL)
Accurate Mass and Retention Time Database (MS) and Libraries (MS/MS)
MS/MS Spectra
Multiple Collision Energies
Different Polarities
Accurate Mass LC/MS Application Kits Untargeted Screening with TOF or QTOF
LC/MS Application
Kits
Method
Standards
On Site Training
LC Column
Accurate Mass
MS/MS Library
Pesticides
-Test Mix: 253 compounds
-DB: 1600+ compounds
-Library: 500+ compounds
Veterinary Drugs
-Test Mix: 148 compounds
-DB: 900+ compounds
-Library: 500+ compounds
Forensic Toxicology
-Test Mix: 139 compounds
-DB: 7500+ compounds
-Library: 2500+ compounds
Creating a PCD
• A PCD is a Personal Compound Database
• PCDs contain compound information e.g. name, formula, mass, retention time,
external IDs (CAS and ChemSpider IDs), IUPAC name, structural information (in
the form of mol file text), and notes
• PCDs are created by the user in PCDL Manager by manually entering each
compound or by importing from a .csv file and then adding .mol file information
• PCD masters are created by an internal Agilent tool.
Creating a PCD- Useful resources
General
• ChemSpider: http://www.chemspider.com/
• PubChem: http://pubchem.ncbi.nlm.nih.gov/
• NIST Chemistry WebBook: http://webbook.nist.gov/chemistry/
• Sigma-Aldrich: https://www.sigmaaldrich.com/
• Cayman Chemical: http://www.caymanchem.com/
Metabolite and drug based
• METLIN: http://metlin.scripps.edu/metabo_advanced.php
• KEGG: http://www.genome.jp/kegg/
• Human Metabolome Database (HMD or HMP): http://www.hmdb.ca/
• Lipid Maps: http://www.lipidmaps.org/resources/resources.html
• ChEBI: http://www.ebi.ac.uk/chebi/
• BioCyc: http://biocyc.org/
Forensics and Toxicology
• Veterinary Substances DataBase: http://sitem.herts.ac.uk/aeru/vsdb/index.htm
• Cheminformatics Database: https://www.forensicdb.org/quickstart.aspx
Pesticides
• EURL Datapool: http://www.eurl-pesticides-datapool.eu/
• Pesticide Properties DataBase (PPDB): http://sitem.herts.ac.uk/aeru/footprint/en/index.htm
• IUPAC Agrochemical Inofrmation: http://sitem.herts.ac.uk/aeru/iupac/
• Compendium of Pesticide Common Names: http://www.alanwood.net/pesticides/
Note: Wikipedia too! –
but always confirm data
with multiple sources.
Public databases
contain errors!
Creating a PCD
1. Open PCDL Manager and choose the Empty.cdb
2. Click File and create a New PCDL
3. Either Create an Empty PCDL specifying PCDL type or
Create a PCDL from an existing PDCL and delete unwanted entries.
Creating a PCD
4. Enable editing by marking Allow Editing on the
PCDL menu.
5. Click the Edit Compounds tab.
6. Click Add New. Insert structure information in
either of the following ways:
• Click open file in the Structures area to open a
.mol file, or
• Click on the MOL Text tab and paste in MOL
file text.
Note: Chemspider has 4 CAS ID’s listed for DEET but only 1
is correct. It also has multiple names and not all are correct.
This is common for all public (free) databases.
Creating a PCD
7. Type information on the left side of the Edit Compounds tab, such as
Name, Mass etc.
8. Click Update Selected.
Creating a PCDL
• A PCDL is a Personal Compound Database and Library
• PCDLs contain the same information as a PCD plus MS/MS spectra for
one or more compounds
• PCDLs can be created in PCDL Manager by manually adding MS/MS
spectra to compounds
• PCDL masters are created by an internal Agilent tool – a great deal of
processing and mass correction is done in the import tool that is not
available in the PCDL Manager
• The MS/MS spectra which are imported for the Master PCDLs are
accurate mass (precursor and fragments).
• The spectra in the Master PCDLs are curated to ensure that the following
issues which produce bad spectra are not present;
• Low intensity data files
• Noisy/Impure data files
• Saturation
• Bad calibration
• Low m/z calibration problems
• Chromatography problems
Creating a PCDL
1. Open the data files in MassHunter Qualitative Analysis.
2. Create a new method using the default method as a template.
3. Set Extract (MS/MS) parameter:
a. In Method Explorer > Spectrum, select Extract (MS/MS).
b. In the Peak Filters tab, set:
• Absolute height to 10 counts
• Relative height to 1.000 % of largest peak
• Maximum number of peaks to no limit (clear the check box)
Note: These parameters are the
default fort PCDL importation to
ensure library match is not
affected by spectral noise.
Parameters can be adjusted to
vary the number of ions imported
into the PCDL as required.
Creating a PCDL
4. In MassHunter Qualitative Analysis click File > Export > as CEF
5. In PCDL Manager click Load Spectra.
6. Select the .cef file of interest and click Open.
Note: Profile spectra or non-MS/MS spectra will be skipped!
Creating a PCDL
7. Select each spectrum and click Add Spectra
8. Or you can Copy to Clipboard and paste into the Acquired spectra
table. But this will bring over noise peaks- NOT the preferred method
Creating a PCDL
• Copied spectra will contain all noise peaks.
Imported from cef
Copied from clipboard
Agilent Master and created User PCDLs
• Agilent Master PCDL will only contain the monoisotopic precursor
• Agilent Master m/z all adjusted to correct accurate mass
Master PCDL
User: Imported .cef
Original DEET data compared to Master PCDL
• 10 eV spectrum has a forward score of 93 due to medium abundance of
high mass isotopes.
• These peaks disappear in 20 and 40 eV spectra (forward scores ≥ 98).
• Reverse score is very high for all
Accurate Mass Library Search Scoring
• The score calculation is a dot-product algorithm which uses the abundance.
• A score is high if the bulk of the ion signal is assigned.
Forward
• Peaks in Target spectrum are compared to peaks in Library spectrum.
• Forward search penalizes peaks that are in Target but not in Library AND the
peaks that are in Library but not in Target.
• A low forward search indicates noise and/or impurities.
Reverse
• Peaks in Library spectrum are compared to peaks in Target spectrum.
• Reverse search only penalizes peaks that are in Library but not in Target.
• A reverse search will work well for weak or noisy signals if all library ions are
included at the approx. correct abundance.
• A low reverse search indicates a bad match.
Note: The forward search can never be greater than the reverse search
Search Scoring- Consequences
• Reverse search only penalizes peaks that are in Library but not in Target.
• Library spectra which only include one ion will have a good reverse
search score for many compounds.
• Rich fragmentation patterns – Identify with confidence
• Poor fragmentation patterns – Do not belong in a PCDL
Solutions:
• Ensure CID energy is sufficient to create fragmentation but not blow
up molecule.
• Measure spectra at more than 1 CID energy
Note: This is similar to the QQQ approach of measuring at least 3 MRM
transitions to ensure specificity.
Why is the low m/z range so important? For/Tox
Calibration for low mass analysis
Page 23
Anatomy and Physiology of Q-TOF
C2H3N + Na+
Calibration for low mass analysis (cont.)
Calibration for low mass analysis (cont.)
CF3-
Negative Ion Calibration 25:1 Dilution 4% water
Screening for TOF/QTOF
Increasing Confidence in Identification
Accurate Mass
(AM)
AM
+
Isotope Pattern
(IP)
AM
+
Retention Time
(RT)
+
IP
AMRT
+
MS/MS
Or
All Ions
TOF/QTOF
TOF/QTOF
(PCD)
TOF/QTOF
(PCD)
TOF/QTOF
Personal
Compound
Database and
Library
(PCDL)
PCDL at the Heart of Screening and Identification
Find by
Formula
All Ions
Molecular
Structure
Correlator
Library
Searches
Finding Compounds (TOF/QTOF)
• Find by Formula – Extracts given empirical formula from data file (Post acquisition decision)
– Also used for MS data from All Ions, Auto MS/MS and targeted MS/MS
• Molecular Feature Extraction (MFE)
– Removes background
– Extract compounds
– Used in MS and Auto MSMS experiment
• Find by Auto MSMS / Targeted MSMS
– Extracts Auto MSMS Spectra from data file (Post acquisition decision)
– Extracts Targeted MSMS Spectra from data file (Pre acquisition decision)
Finding Compounds (TOF/QTOF)
• Find by Formula – Extracts given empirical formula from data file (Post acquisition decision)
– Also used for MS data from All Ions, Auto MS/MS and targeted MS/MS
• Molecular Feature Extraction (MFE)
– Removes background
– Extract compounds
– Used in MS and Auto MSMS experiment
• Find by Auto MSMS / Targeted MSMS
– Extracts Auto MSMS Spectra from data file (Post acquisition decision)
– Extracts Targeted MSMS Spectra from data file (Pre acquisition decision)
Screening for TOF/QTOF
TOF/
QTOF
MS Run Find by
Formula I.D. via AMRT
Target
Acquisition
MS/MS Run
Find by Target
MS/MS I.D. via Library
Searching
Target List from FBF cmpds with No MS/MS data
Auto MS/MS
Run
Find by
Formula
(extracts MS/MS data)
I.D. via AMRT
and Library
Search
Target List from FBF cmpds
Screening for TOF/QTOF
TOF/
QTOF Find by Formula All Ions
I.D. via AMRT
and Fragment
Co-Elution
ALL Ions MSMS Workflow
33
Modified Find by Formula Algorithm
for All ions
Use PCDL Lib as source of fragments
Export modified CEF file for Quant
method building
Quant requirement : Ability to convert
new CEF file in Quant method and
Quantitate on All Ions data file.
PCDL Requirement : PCDL library
content for pesticide available.
Acquisition PCDL FindbyFormula on
MS low channel
EICs for fragments
on MS high channel
Correlate qualified
fragments with target
Qual
.d data file Targets
Find
m/z, ion
species
Extract
Qualify and confirm
fragments
Export
Align EICs of
fragments with
parent EIC
Targets
fragment ions
December 2012 China NPT
Find Compounds by Formula
Find by Formula (MS)
Compound information from formula or Database
Ion Information
Ion chromatogram Spectral comparison
Spectral performance
Find Compounds by Formula
Find by Formula (MS/MS)
Compound information from formula or Database.
MS and MS/MS Spectra extracted
Ion Information
Ion chromatogram
MS/MS Spectra extracted
from matched peak
Spectral performance
Spectral Matching
Monoisotopic mass
(varies in ppm)
Isotope spacing
(varies in ppm)
Isotope abundance
(varies in %)
Scoring based on Mass Match +
Abund. Match +
Spacing
Match =
Overall Score
Find By Formula: Preliminaries Purpose
“Does this LC/MS data file contain any evidence for the presence
of specified compound(s)?”
FBF: Preliminaries Nomenclature
• Search Target – one of the compounds whose presence we are trying to determine.
• Hit Candidate – an EIC peak, or a spectrum from an EIC peak, which is being tested to see
whether it meets all of our search criteria.
• Hit – an EIC peak and associated spectra that has been found to meet all of our
criteria as evidence for the presence of a specific search target.
FBF: Search Targets
Formula + possible ion species
(M+H)+ (M+Na)+
Expected isotope distribution(s)
FBF: Search Targets
Formula + possible ion species
Expected isotope distribution(s)
Optionally with expected retention time
(M+H)+ (M+Na)+
FBF: Search Targets
Observed isotope distribution
Taken directly from CEF file that
does not have a formula.
Optionally with expected retention time
FBF: The Basic Search Strategy
1. Presume any spectrum in the file might contain evidence for the search
target
2. Filter candidate spectra on the basis of chromatographic behavior (and
expected RT, if available)
3. Filter remaining candidate spectra based on m/z match to expected ions
4. Grade (score) remaining candidate spectra
5. Apply some final “quality” filters
6. If any candidates survive, they are hits – report the best one(s)
7. Optionally, report the candidates that failed (and why)
FBF Walkthrough: Step 1
Determine “most significant ions” for each expected ion species
• Use most abundant predicted ion
• If less than 75% of the total abundance, include the
next most abundant ion
• Use no more than 5 ions per ion species.
(parameters are not visible in UI)
(M+H)+ of 7-aminoclonazepam
FBF Walkthrough: Step 2 RT Filtering
Extract and integrate EIC of all significant ions for the target
Method parameters control how many peaks are
reported by the integrator.
FBF Walkthrough: Step 2 RT Filtering
Eliminate spectra not belonging to an EIC peak.
FBF Walkthrough: Step 2 RT Filtering
If the expected RT is known …
FBF Walkthrough: Step 2 RT Filtering
… remove peaks falling outside the RT range.
Each surviving peak becomes a hit candidate – in this case, there’s only one!
For the purposes of the walk-through, however, assume that there are still multiple EIC
peaks that have survived the RT filters.
FBF Walkthrough: Step 3 m/z Filtering
Extract a spectrum for each candidate peak in the EIC
The default saturation and
background parameters are
highly recommended!
FBF Walkthrough: Step 3 m/z Filtering
Find the ions (if any) matching the expected ion distribution for each species
FBF Walkthrough: Step 3 m/z Filtering
Compute the (average) mass error for each species
(M+H)+ -15.7 ppm
(M+Na)+ -35.6 ppm
(M+H)+ -2.4 ppm
(M+Na)+ not found
(M+H)+ -11.2 ppm
(M+Na)+ -29.4 ppm
FBF Walkthrough: Step 3 m/z Filtering
Discard any species outside the mass tolerance.
(M+H)+ -15.7 ppm X
(M+Na)+ -35.6 ppm X
(M+H)+ -2.4 ppm OK
(M+Na)+ not found
(M+H)+ -11.2 ppm OK
(M+Na)+ -29.4 ppm X
FBF Walkthrough: Step 3 m/z Filtering
… and discard any spectrum with no “good” ion species.
(M+H)+ -15.7 ppm X
(M+Na)+ -35.6 ppm X
(M+H)+ -2.4 ppm OK
(M+Na)+ not found
(M+H)+ -11.2 ppm OK
(M+Na)+ -29.4 ppm X
X
FBF Walkthrough: Step 4 Scoring
For all remaining ion species in remaining spectra, calculate match scores
Mass Abundance Spacing
96.27 96.68 91.99
45.93 0 59.44
FBF Walkthrough: Step 4 Scoring
The overall score for an ion species is the weighted average of the individual
mass/abundance/spacing/RT scores.
Overall Mass Abundance Spacing
95.37 96.27 96.68 91.99
36.03 45.93 0 59.44
FBF Walkthrough: Step 4 Scoring
The overall score for a hit candidate (EIC peak + spectrum) is
taken from its best-scoring ion species.
FBF Walkthrough: Step 5 Final Filters
(a) Minimum required score for a candidate to qualify
Overall Mass Abundance Spacing
95.37 96.27 96.68 91.99
36.03 45.93 0 59.44X
FBF Walkthrough: Step 5 Final Filters
(b) Finding only a single ion (per species) may disqualify a candidate
Two ions of same
species - ok
FBF Walkthrough: Step 5 Final Filters
Second ion not seen.
It should have been 480 counts.
Candidate is disqualified.
480 counts
FBF Walkthrough: Step 5 Final Filters
Second ion not seen.
Is only expected to be 280 counts.
Still qualifies (with warning)
280 counts
FBF Walkthrough: Step 6 Report Hit(s)
Rank all remaining hits for the search target
Hits are ranked by overall score, with abundance as a tie-breaker
Report the top N hits:
Special Situations: Missing (or “wrong”) Hits
A new “Flags” column in Compound List shows why some targets were not
reported and warns about poor results with others.
Note: “red” flags only appear when “only generate” is unchecked
Special Situations: Isomeric Search Targets
• Results for isomers depend on whether the compounds can
be distinguished by retention time:
– The method enables matching based on retention times
– All isomers have a known retention time
– The retention times differ by more than the match tolerance.
• When all three conditions are met, the isomers are treated as
separate compounds and are correctly identified.
Special Situations: Isomeric Search Targets (Distinguished by RT)
Correctly identified:
Special Situations: Isomeric Search Targets (not Distinguishable by RT)
When isomers can not be distinguished by RT, the same peak is usually
reported as the hit for all isomers
Special Situations: Isomeric Search Targets (not Distinguishable by RT)
A new option explicitly deals with this situation
Special Situations: Isomeric Search Targets (not Distinguishable by RT)
Now there is one compound reported for each EIC peak, with a new “multiple
IDs” flag.
Pesticides screening with accurate mass TOF screening and confirmation
• 55 most common pesticides spiked
in Rucola extract to 10 µg/kg
Pesticides screening with accurate mass Preliminary results
Blank 0.005 0.01 0.05 0.1 Blank 0.005 0.01 0.05 0.1 Blank 0.005 0.01 0.05 0.1
2.4-D FALSE
Acetamiprid FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Azoxystrobin TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Bifenazat FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Boscalid FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Buprofenzin TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Carbaryl FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Carbendazim FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Chlopyrifos FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Chlorpyrifos-methyl FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Cyhexatin FALSE TRUE TRUE TRUE TRUE
Cyprodinil FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
DEET FALSE TRUE TRUE TRUE TRUE TRUE TRUE
Dichlorvos FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Difenoconazole FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Diflubenzuron FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Dimethoat TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Dimethomorph FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Famoxadon FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Fenhexamid FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Fluazifop free acid FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Fludioxonil FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Fluquinconazole FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Flutriafol FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Imazalil TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Imidacloprid FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Iprodion TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Kresoxim-methyl FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Linuron FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Mandipropamid FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
MCPA FALSE
Metalaxyl FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Methidathion FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Myclobutanil FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Penconazole FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Pendimethalin FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Phosmet FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Phoxim FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Piperonylbutoxid FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Pirimicarb FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Pirimicarb-desmethyl FALSE TRUE TRUE TRUE TRUE TRUE TRUE
Propamocarb TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Propargite FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Propiconazole FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Prosulfocarb FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Pyraclostrobin FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Pyridaben FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Teflubenzuron FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Thiabendazole FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Thiacloprid TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Thiamethoxam FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Thiophanat-methyl TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Triadimefon FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Triazophos FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Trifloxystrobin FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
Ergebnis 45/55 50/55 53/55 53/55 33/55 43/55 50/55 51/55 40/55 46/55 51/55 53/55
Mit RT 0 2 2 4 4 2 2 4 4 3 0 1 3 3 3
Zusätzlich Gefunden
CompoundGurke Zitrone Rucola
Pesticides screening with accurate mass Pesticide PCDL - preliminary
Pesticides screening with accurate mass Library matching of MS/MS spectra (e.g. Dimethoate in rucola)
• Library match score: 82.73
Contents
• What is All Ions MS/MS
• How does it work
• Software implementation
• Acquisition
• Qualitative Analysis
• Quantitative Analysis
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All Ions MS/MS is enabled on all high-res
instrument
• 6200 Series TOF Instruments
• 6500 Series QTOF Instruments
• 6550 Series iFunnel Q-TOF
5/22/2012 ASMS 12
72
Ion Source Region
Collision Cell
Quantify target with
additional fragment
ion confirmation
All Ions MS/MS Targeted Screening Workflow
Acquisition PCDL Qual Quant
MH Acquisition
High/Low
Fragmentor
Voltage on TOF
and Q-TOF
Pesticide
library (~450-
500
compounds
with spectra)
Forensics
/Tox library
(2720
compounds
with spectra )
High/Low
Collision Energy
on Q-TOF
Find by Formula
on MS low
channel
EICs for
fragments on MS
high channel
Correlate qualified
fragments with
target
Find
Extract
Qualify and confirm
fragments
Export
Align EICs of
fragments with
parent EIC
Targets
m/z, ion species
Quant
Report
Qual
Report
All Ions MS/MS Features: Easy Acq Method Setup
The MassHunter Acquisition method only needs to be set up
with a “low channel” (e.g. no Collision Energy) and “high
channels” (e.g. CE = 20). There is no need to enter every
compound like in MRM. The result is much faster method
setup!
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New Experiment
# field allows set
up of up to four
low and high CE
or Fragmentor
channels per
Time Segment
All Ions Method setup
• Extension of Find by Formula
• Works with or without Library
• Coelution score as the major function
to qualify compounds based similarity
of peakshapes between low energy
and high energy spectra
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All Ions MSMS Results Overview
76 September 19, 2013 LGC Meeting UK
1
3
2
4 5
All Ions MSMS Results Overview
77 September 19, 2013 LGC Meeting UK
Score calculation based on
Isotopic pattern
Mass accuracies of the individual ions
Isotopic spacing
Calculated resolution 18500 Structure from Library
Fragment Identification – Origin of fragments
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All Ions MSMS Results Overview
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Fragments confirmed in
green 5 x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
Cpd 16: Alprazolam: +ESI HighE Scan (7.991-8.228 min, 38 Scans) 3CEs_001.d
309.0905
226.9516
255.0682 281.0715
205.0762 90.9768
158.9638 59.0494
288.9217 149.0231 105.0336 274.1212 77.0387
65.0388 121.0509 165.0210 242.9252 193.0883 138.0103 220.9344 177.0212
Counts vs. Mass-to-Charge (m/z)
50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300 305 310 315 320
All Ions example Cycloate
One fragment found
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Phospamidon
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Example for a low coelution score
Normally ruled out
Can All Ions MS/MS help to distinguish between
isomers?
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Can All Ions MS/MS help to distinguish between
isomers?
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Codeine
Hydrocodone
Can All Ions MS/MS help to distinguish between
isomers?
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Codeine
Hydrocodone
Can All Ions MS/MS help to distinguish between
isomers?
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Codeine
Hydrocodone
Yes, we can!
• These are perfect examples of
• Turning a new function into a perfect productivity tool
• Using and combining PCDL libraries with All Ions MS/MS
• Exporting information and automatically creating quant methods in
seconds
• Reducing processing time from
one day to hours
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0
200
400
600
800
1000
1200
1400
Without All Ions With Allons
min
ute
s
Processing time
Quant
Qual
