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New Screening strategies with Exact Mass QTOF-MS LC & GC
New tools and workflows for more efficiency and confidence
Jaume C. Morales
LCMS Product Specialist
08/04/2015
AccMass_Screening_Pesticides
1
Accurate Mass Screening and Quantitation Different approaches and challenges
• Target screening and quantitation
- Limited list of target compounds
- Reference compounds are available in the lab
- Databases and libraries are available
- Challenge: Efficient data review and quantitation
• Qualitative target screening with or without reference standards
- Affordable and Comprehensive list of target compounds
- Reference standards might not be available or not at hand
- Databases and libraries with spectral data are available
- Challenge: Elimination of false positives
08/04/2015
AccMass_Screening_Pesticides
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Accurate Mass Screening and Quantitation Different approaches and challenges • Target screening and quantitation
- Limited list of target compounds
- Reference compounds are available in the lab
- Databases and libraries are available
- Challenge: Efficient data review and quantitation
• Qualitative target screening with or without reference standards
- Comprehensive list of target compounds
- Reference standards might not be available or not at hand
- Databases and libraries with spectral data are available
- Challenge: Elimination of false positives
• Non-target screening
- You do not know what you are looking for
- No databases and libraries available
- Requires efficient feature finding algorithms, profiling tools and
in-silico fragmentation programs
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Dos enfoques de Screening
TARGET
QUALITATIVE
Introduction Current regulations and residue situation
• EU regulation
- Maximum residue levels regulated in commision regulation (EC) 396/2005
• appendix II – IV regulates >170 000 MRLs (matrix-pesticide combinations)
- European and national monitoring programs, regulated by (EC) 396/2005
- 173 alert notifications related to pesticide residues entered in the Rapid Alert
System for Food and Feed (RASFF) in 2009
• US regulation
- 40 CFR Part 180:
Regulates tolerances and exemptions for pesticides in food
Specifies allowed methodologies for analysis
- Special regulations are in place for:
• Organic food
• Baby food
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SANCO guidelines for pesticides screening Implementations for qualitative and quantitative methods
• Qualitative multi-residue methods
- To extend the analytical scope in a cost-effective way to analytes which have low
probability to be present in samples
- Screening Detection Level (SDL) for individual compounds must be established and
confidence of detection must be validated for different commodities
- Method performance must be shown for every batch with ≥ 10 indicator analytes at
the SDL
• Quantitative multi-residue methods
- Initial method validation (intra-laboratory)
- Recovery determination for 10% of analytes in each batch in a rolling program
- Strict compliance with identification criteria (e.g. accurate mass for > 2 ions and RTs)
- Usually calibration with all targeted analytes for each batch, but at least for a number
of representative analytes (minimum 25 + 10% of total number of analytes)
- Re-calibration for positive compounds possible, re-analyze in case of MRL violation
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Introduction Pesticide screening – Status today • More than 1000 pesticides and pesticide metabolites need consideration
• QuEChERS as „universal“ extraction procedure is widely accepted
• Detection method of choice is chromatographic seperation and mainly QQQ
detection
• Current situation
- 600 to 800 compounds included in routine monitoring programs
- Only about 150 pesticides are typically detected in food commodities
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Introduction Pesticide screening – • More than 1000 pesticides and pesticide metabolites need consideration
• QuEChERS as „universal“ extraction procedure is widely accepted
• Detection method of choice is chromatographic seperation and mainly QQQ
detection
• Current situation
- 600 to 800 compounds included in routine monitoring programs
- Only about 150 pesticides are typically detected in food commodities
• Wouldn‘t it be nice ...
- to measure everything ?
- to only calibrate and quantify what is likely ?
- to find what else is in the sample ?
- to retrospectively look into the sample ?
- to use profiling tools to find the unexpected ?
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QTOF Acquisition – MS and MS/MS Modes of Operation
• MS Only – “TOF only” mode (RETROSPECTIVE but just MS [*])
• Data Directed (Targeted) MS/MS experiments (NON RETROSPECTIVE)
• Import of Target mass lists from Target compounds List or Mass Profiler Professional software
• Import of mass lists from other applications
• Automatic dynamic creation of time segments (like DMRM on QQQ)
• All Ions - MS & non selective MS/MS (RETROSPECTIVE)
• Low/High alternate Collision Energy
• Data processing with FBF & PCDL integration
• Data Dependent MS/MS Experiments (NON RETROSPECTIVE)
• Precursor selection based on intensity of n-highest (with relative and absolute threshold)
• Excluded and Preferred mass lists
• Configurable charge-state selection preference
HRMS QTOF ACQUISITION MODES
Aportación de los sistemas de Tiempo de Vuelo
11
El TOF es un cronómetro que mide el
tiempo que tardan los diferentes iones
en llegar al detector desde que se
disparan en el PULSER.
Los iones más ligeros llegan antes y
los más pesados, más tarde.
Ese tiempo se contrasta con una
calibración del equipo t <-> m/z y
sabemos con exactitud la m/z del ión.
Genéricamente se entiende por masa exacta cuando el error en la medida es menor de 5 ppm.
Los sistemas basados en SQ/QQQ suelen mostrar un error de masa > 150ppm.
(Masa Medida - Masa Calculada)
Masa Calculada
= ppm Error en la medida = X 1.000.000
HRMS TOF/QTOF
TOF
QTOF
TOF
Full Scan acquisition
QTOF
Full Scan acquisition
MS/MS spectrum
Tiapride 0.8 ppm
Tiapride 0.8 ppm
HRMS TOF/QTOF – MS SCORING
13
Compounds Identification at MS level is based
at least on three parameters :
• monoisotopic mass accuracy
• isotopes rel. abundance
• isotopic spacing
Matched with theoretical distribution (formula)
At MS/MS level we can match also
fragmentation spectrum.
Monoisotopic mass
(change in ppm)
Isotopoc spacing
(change in ppm)
Isotopopic Distribution
(Change in %)
Scoring based on
Is there a way to avoid the limitations of MS/MS ? Acquisition of all ions all the time using new All Ions MS/MS
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All Ions MS/MS CID fragmentation without precursor selection
Simultaneous acquisition of a low energy trace
and one or more high energy traces (CID)
The Implementation of All Ions MS/MS in LC/MS Acquisition with low & high collision energy channels
Acquisition
- Special method required
- Chromatography is monitored
in 2 channels (or more if desired)
Low energy channel (CE = 0V)
- Spectrum dominated by molecular ions
and clusters
High energy channel (CE > 0V)
- Spectrum dominated by fragment ions
(not only of target compound)
Fragments
Pesticide screening
GC-Q/TOF
Example for all ions spectra Carbofuran in multi-residue solvent standard
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Low energy
spectrum
Example for all ions spectra Carbofuran in multi-residue solvent standard
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Low energy
spectrum
High energy
spectrum
Example for all ions spectra Carbofuran in multi-residue solvent standard
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Low energy
spectrum
High energy
spectrum
Screening workflow using LC-(Q)TOF-MS Personal compound database and library
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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
20
Pesticides
-Test Mix: 253 compounds
-DB: 1680+ compounds
-Library: 750+ compounds
Veterinary Drugs
-Test Mix: 146 compounds
-DB: 1150+ compounds
-Library: 850+ compounds
Forensic Toxicology
-Test Mix: 139 compounds
-DB: 8900+ compounds
-Library: 3400+ compounds
All Ions MS/MS Features: Targeted Identification Using Agilent’s PCDLs Personal Compound Database and Libraries (PCDLs) contain library
searchable MS/MS spectra. All Ions MS/MS uses the product ions in the
spectra to confirm the identification of hits in the screen—the result is
fewer false positives!
.
Application PCD PCDL
Forensics / Tox 8.998 compounds Adds spectra for 3.497
compounds
Pesticides 1.685 compounds Adds spectra for 770
compounds
Veterinary Drugs 1.191 compounds Adds spectra for 866
compounds
Metabolomics METLIN rev. B.07.00
(317.245 compounds)
METLIN rev. B.07.00
(adds spectra for 11.897
compounds)
21
Qualitative screening using All Ions MS/MS What is new about All Ions MS/MS ? • All Ions MS/MS offers a complete workflow for screening, verification and setup of a
quantitation method
• It features:
- Support of TOF as well as QTOF
- Easy setup of the acquisition method
- Unique identification of product ions using Agilent’s MS/MS libraries
- Chromatographic confirmation of product ions using Agilent’s unique
coelution score
- Autodynamic setup of Quant method based on the identification results
• Additional possibility to:
- Re-interrogate data for unknowns without reacquiring
- Quickly add unknowns to the MS/MS library to include
them in the screening
08/04/2015
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Workflow of All Ions MS/MS acquisition Experimental setup of validation study
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Data analysis
using
„Find-by-Formula“
Verified by
fragment coelution
profile Quant with verification by
accurate mass metrics
and qualifier fragments
Yes
LC-QTOF
All Ions MS/MS
acquisition
List of Suspects
No
Experimental UHPLC and QTOF method parameters
08/04/2015
AccMass_Screening_Pesticides
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UHPLC column Agilent ZORBAX Eclipse Plus C18 RRHD
2.1 x 150 mm, 1.8 µm @ 30°C
Mobile phase A: 5 mM NH4 formate + 0.1% formic acid
B: 5 mM NH4 formate + 0.1% formic acid in methanol
Gradient program
Min % B
0 5
0.2 5
2.2 30
10.5 100
13.0 100
13.5 5
Stop time 15 min
Flow rate 0.50 ml/min
• G6540 UHD QTOF operated in 2GHz High extended dynamic range mode
• Acquisition rate 3 scans/sec in MS with 2 CEs for
All Ions MS/MS
All Ions MS/MS Targeted Screening Workflow
Acquisition PCDL Qual Quant
MassHunter 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
Targets
m/z, ion species
Qualitative screening using All Ions MS/MS All Ions MS/MS workflow • Modified “Find by Formula Algorithm” searches compound based on database entry in the
low energy domain
08/04/2015
AccMass_Screening_Pesticides
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All Ions MS/MS Targeted Screening Workflow
Acquisition PCDL Qual Quant
MassHunter 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
Targets
m/z, ion species
EICs for
fragments on MS
high channel
Find
Extract
Align EICs of
fragments with
parent EIC
Qualitative screening using All Ions MS/MS All Ions MS/MS workflow • Modified “Find by Formula Algorithm” searches compound based on database entry in the
low energy domain
• Uses PCDL spectra as source of fragments to look for in the high energy domain
08/04/2015
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Qualitative screening using All Ions MS/MS All Ions MS/MS workflow • Modified “Find by Formula Algorithm” searches compound based on database entry in the
low energy domain
• Uses PCDL spectra as source of fragments to look for in the high energy domain and
compares elution profiles
08/04/2015
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All Ions MS/MS Targeted Screening Workflow
Acquisition PCDL Qual Quant
MassHunter 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
Targets
m/z, ion species
EICs for
fragments on MS
high channel
Find
Extract
Align EICs of
fragments with
parent EIC
Correlate qualified
fragments with
target
Qualify and confirm
fragments
Qual
Report
Qualitative screening using All Ions MS/MS Results overview
• Carbendazim was found
with 5 valid qualifier
fragments from the PCDL
spectrum
• Overlay of the EICs with the
Coelution Plot shows
excellent agreement
between the products and
precursor ion
08/04/2015
AccMass_Screening_Pesticides
31
Qualitative screening using All Ions MS/MS Results overview
08/04/2015
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32
Pesticides screening with accurate mass Validation data for 3 different matrices
08/04/2015
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33
• Most compounds found in lowest
spiking level in all matrices
• Even at lowest spiking level reliable
fragment detection
• Missing qualification for compounds
with low abundant fragments or only
low mass fragments
found by FBF algorithm and verified by
fragment coelution
found by FBF algorithm
Blank 0.005 0.01 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Acetamiprid qualified qualified qualified qualified qualified qualified
Aldicarb qualified qualified qualified not qualified not qualified qualified qualified qualified
Azoxystrobin qualified qualified qualified qualified qualified qualified qualified qualified qualified
Bifenazate (D 2341) qualified qualified qualified qualified qualified qualified qualified qualified qualified
Buprofezin qualified qualified qualified qualified qualified qualified not qualified not qualified not qualified
Carbaryl qualified qualified qualified not qualified not qualified qualified qualified qualified qualified
Carbendazim (Azole) qualified qualified qualified qualified qualified qualified qualified qualified
Chlorfenvinphos(I) qualified qualified qualified qualified qualified qualified qualified qualified qualified
Chloroxuron qualified qualified qualified qualified qualified qualified qualified qualified qualified
Chlorpyrifos qualified qualified qualified qualified qualified qualified qualified qualified qualified
Chlorpyrifos-methyl not qualified not qualified not qualified
Cyprodinil qualified qualified qualified qualified qualified qualified not qualified qualified qualified
Difenconazole(I) qualified qualified qualified qualified qualified qualified qualified qualified qualified
Dimethoate qualified qualified qualified qualified qualified qualified qualified qualified qualified
Dimethomorph(E) qualified qualified qualified qualified qualified qualified qualified qualified qualified
Dimoxystrobin qualified qualified qualified qualified qualified qualified qualified qualified qualified
Dinotefuran qualified qualified qualified qualified qualified qualified qualified qualified qualified
Dioxacarb qualified qualified qualified not qualified qualified qualified not qualified not qualified qualified
Ethoxyquin qualified qualified qualified not qualified qualified qualified qualified qualified qualified
Fenamiphos qualified qualified qualified qualified qualified qualified qualified qualified qualified
Fenhexamid not qualified qualified qualified not qualified not qualified qualified not qualified not qualified qualified
Fluquinconazole(I) not qualified qualified qualified not qualified qualified not qualified not qualified qualified qualified
Flutriafol qualified qualified qualified not qualified not qualified qualified qualified qualified qualified
Imazalil(II) (Enilconazole) qualified qualified qualified qualified qualified qualified qualified qualified
Imidacloprid qualified qualified qualified qualified qualified qualified qualified qualified qualified
Metalaxyl qualified qualified qualified qualified qualified qualified qualified qualified qualified
Methidathion qualified qualified qualified qualified qualified qualified qualified qualified qualified
Myclobutanil not qualified not qualified qualified not qualified not qualified not qualified qualified
Penconazole(I) not qualified qualified qualified not qualified qualified not qualified not qualified qualified
Pendimethalin (Penoxalin) not qualified not qualified qualified not qualified not qualified qualified qualified
Phosmet (Imidan) qualified qualified qualified qualified qualified qualified qualified qualified qualified
Pirimicarb qualified qualified qualified qualified qualified qualified qualified qualified qualified
Propamocarb qualified qualified qualified qualified qualified qualified qualified qualified qualified
Propiconazole(II) not qualified not qualified qualified not qualified not qualified not qualified not qualified not qualified not qualified
Pyraclostrobin qualified qualified qualified qualified qualified qualified qualified qualified qualified
Pyridaben qualified qualified qualified not qualified not qualified not qualified qualified qualified
Quinalphos (Diethquinalphione) qualified qualified qualified not qualified qualified qualified qualified qualified qualified
Spinosyn A qualified qualified qualified qualified qualified qualified qualified qualified qualified qualified
Spiroxamine qualified qualified qualified qualified qualified qualified qualified qualified qualified
Sulfentrazone not qualified not qualified qualified not qualified qualified qualified not qualified not qualified qualified
Tebuconazole(II) (Terbuconazole) not qualified not qualified not qualified not qualified not qualified not qualified not qualified not qualified not qualified
Tebufenpyrad qualified qualified qualified not qualified not qualified qualified not qualified qualified qualified
Thiabendazole qualified qualified qualified qualified qualified qualified qualified qualified
Thiacloprid qualified qualified qualified qualified qualified qualified qualified qualified qualified
Thiamethoxam qualified qualified qualified qualified qualified qualified qualified qualified qualified
Triadimefon qualified qualified qualified not qualified qualified qualified not qualified qualified not qualified
Triazophos qualified qualified qualified qualified qualified qualified qualified qualified qualified
Trifloxystrobin not qualified qualified qualified qualified qualified qualified qualified qualified
Uniconazole-P(I) not qualified not qualified not qualified not qualified not qualified not qualified not qualified not qualified not qualified
Vamidothion qualified qualified qualified qualified qualified qualified qualified qualified qualified
Zoxamide qualified qualified qualified qualified qualified qualified qualified qualified qualified
Tomato Lemon AvocadoCompound
All Ions MS/MS Targeted Screening Workflow
Acquisition PCDL Qual Quant
MassHunter 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
Targets
m/z, ion species
EICs for
fragments on MS
high channel
Find
Extract
Align EICs of
fragments with
parent EIC
Correlate qualified
fragments with
target
Qualify and confirm
fragments
Qual
Report
Export
Batch review for All Ions MS/MS results Method setup from screening results
08/04/2015
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35
Batch review for All Ions MS/MS results Method setup from screening results
08/04/2015
AccMass_Screening_Pesticides
36
Fragment ions with different CEs
are used as qualifiers.
Quantify target with
additional fragment
ion confirmation
All Ions MS/MS Targeted Screening Workflow
Acquisition PCDL Qual Quant
MassHunter 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
Quantitation of pesticides using accurate mass Batch review process including accurate mass metrics
08/04/2015
AccMass_Screening_Pesticides
38
Quantitation of pesticides using accurate mass Batch review process using Compounds at a Glance
08/04/2015
AccMass_Screening_Pesticides
39
Cal
sample
Pesticides screening and quantitation Apple sample from local market
08/04/2015
AccMass_Screening_Pesticides
40
Pirimicarb
Conc.: 120 µg/kgMass error: -0.54 ppmTOF score: 99.2Library score: 97.0
Pesticides screening and quantitation Apple sample from local market
08/04/2015
AccMass_Screening_Pesticides
41
Boscalid
Conc.: 20 µg/kgMass error: -0.15 ppmTOF score: 97.5Library score: 93.0
O
N
NH
Cl
Cl
Pesticides screening and quantitation Apple sample from local market
08/04/2015
AccMass_Screening_Pesticides
42
Trifloxystrobin
Conc.: 10 µg/kgMass error: -1.00 ppmTOF score: 99.3Library score: 87.8
O
ON
OCH3
ON
CH3 F
F
F
CH3
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Fast and Fully Automated Multi-Residue Pesticide Screening in Fruit / Vegetable
Extracts using a GC-Q/TOF
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
The Agilent 7200 Series GC-Q/TOF
= +
Quadrupole Time of Flight MS
Time of Flight MS
Triple Quadrupole MS
7890 + 7000 + 6500 = 7200 GC-Q/TOF
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
7200 Q/TOF Components
7000B TQ 6500 Q/TOF
NEW Optics
NEW Removable Ion Source
Two 300 L/s Turbos
Four stages of pumping
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
7200 GC/Q-TOF Performance Specifications
• Resolving Power: > 12.5K at m/z 272
– 13K to 15K typical
• Mass Accuracy: < 5 ppm at m/z 272
– < 2 ppm typical
• MS Sensitivity >400:1 at m/z=272
• MS/MS Sensitivity is between SRM and Product Ion scan of a Tandem Quad MS
• Dynamic Range: 3-5 orders of magnitude
• Quad Mass range: 20-1050 Da (0.7-4.0 Da FWHM)
• TOF Mass range: 20-1700 Da
• Spectral Rate: up to 50 Spectra/sec
The Implementation of All Ions MS/MS in LC/MS Acquisition with low & high collision energy channels
Acquisition
– Special method required
– Chromatography is monitored
in 2 channels (or more if desired)
Low energy channel (CE = 0V)
– Spectrum dominated by molecular ions
and clusters
High energy channel (CE > 0V)
– Spectrum dominated by fragment ions
(not only of target compound)
Fragments
Pesticide screening
GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Screening Pesticides LC-Q/TOF vs GC-Q/TOF
Pesticide screening GC-Q/TOF
LC/MS ESI+ GC/MS EI+
Malathion
MWt = 330.0361
CE = OV
Spectrum dominated by [M]+ ion is the exception rather than the rule
Fragments
[M+H]+ Low energy CE = OV
High energy CE > OV
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Screening Pesticides with a GC-Q/TOF
• Accurate Mass provides added compound selectivity
- Higher resolution provides added interference selectivity
• TOF mode : Always full spectral data, Non-targeted acquisition
• ‘Unlimited’ number of compounds can be screened (search accurate mass compound database for identification)
• Sensitivity is the same regardless of number of compounds screened
• MS/MS (Q/TOF Mode) further assists compound identification
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Screening Pesticides with a GC-Q/TOF Objectives
• Similar functionality, look and feel as All Ions for LC/MS
- Adapt MH Qual All Ions to process GC/MS EI mass spectra
• Customizable UI, fast data processing, filtering result data, export found compounds for quantitation in MH Quant SW
• Enable user creation of accurate mass EI Personal Compound Database Libraries (PCDL) for GC-Q/TOF mass spectra by modifying PCDL Manager
• Create accurate mass EI mass spectral database of pesticides including locked retention times
- Collaborate with 2 European Labs to build pesticide library
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
All Ions : Targeted Screening Workflow
1. Acquisition 2. PCDL 3. MH Qual SW 4. MH Quant SW
Compound library (compounds with
spectra and RTs)
Quantify target with additional fragment ion confirmation
GC-Q/TOF MH Acquisition
Constant Collision Energy on Q-TOF
(CE=0)
Find by Formula on MS
EICs for fragments
Correlate qualified fragments with target
Find
Extract
Qualify and confirm fragments
Export
Align EICs of fragments
Targets
m/z, ion species
Quant Report
Qual Report
Retention Time Locked Method (optional)
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
(1)
7200 GC-Q/TOF RTL Pesticide Analyzer CF Method
7890B GC
PCM
Column (1) : 15m x 0.25mm ID x 0.25um HP-5MSUI (19091S-431UI)
Column (2) : 15m x 0.25mm ID x 0.25um HP-5MSUI (19091S-431UI)
Same column configuration and flow rate for 40 minute and 20 minute
methods, only difference is Oven Temperature program
7200 Q/TOF
MMI Inlet
Purged Union
ALS
(2)
Purged ultimate union
1. Acquisition
Mid-Column Back Flush
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
PCDL : Personal Compound Database Library
1. Acquisition 2. PCDL
Pesticide screening GC-Q/TOF
Accurate mass and Retention time Database + EI Accurate Mass Spectra
• Name • Mol. Formula • Mono-isotopic exact mass • Locked RT • CAS # • Accurate mass spectrum
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
PCDL Manager - view / edit entries
User created GC/MS Accurate Mass PCDLs
Pesticide screening GC-Q/TOF
1. Run standard (RTL Method)
2. Extract compound spectra (Find by integration or
by deconvolution)
3. Add compound information
4. Send spectra to PCDL
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
User Created PCDLs
Pesticide screening GC-Q/TOF
Find compounds by integration or by deconvolution Optional search of
unit mass MS database
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
1. Select Spectra 2. Right Mouse click
3. Specify PCDL 4. Press OK
Steps to transfer the spectra to PCDL
Pesticide screening GC-Q/TOF
PCDL Manager - view / edit entries
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
1. Acquisition 2. PCDL 3. MH Qual SW
Qualitative screening using All Ions MS
Pesticide screening GC-Q/TOF
Gun Powder Tea
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
All Ions data processing - Method setup (1) Formula Source
Pesticide screening GC-Q/TOF
• Use locked Retention time (If available)
• Specify PCDL
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
All Ions data processing - Method setup (2) Formula matching
Pesticide screening GC-Q/TOF
• Set acceptable mass tolerance for identifying molecular ion
• If molecular ion found measure :
Mass accuracy
Isotope ratios
Isotope spacing
• Set mass window for fragment EICs
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
All Ions data processing - Method setup (3) Fragment Identification
Pesticide screening GC-Q/TOF
• Select minimum number of qualified fragments required to identify compound
• Co-elution score is the major function to qualify compounds based on similarity of extracted ion peak shapes
• Select number of fragment ions to be evaluated
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Pesticide screening GC-Q/TOF
All Ions Results Overview GC-Q/TOF Tea extract
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
All Ions Results Overview GC-Q/TOF Tea extract
1 2
3 4
5
Pesticide screening GC-Q/TOF
Pesticide screening GC-Q/TOF
Target Compound List and Results
Compound Identification Results
EICs and Co-Elution Plots Compound MS Spectrum Results
Compound Fragment Spectrum Results
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Window 1 Target Compound list and Results
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Window 2 Compound identification results
Pesticide screening GC-Q/TOF
• EIC Co-elution Scores based on : − Retention time − Peak shape (symmetry) − Peak width − Abundance
• Mass accuracy of [M]+
(if present)
• RT difference
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Window 3 Compound Chromatogram Results and EIC Co-Elution Plots
Overlayed EICs
Co-elution plot
Pesticide screening GC-Q/TOF Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Window 4 Compound MS Spectrum results
Observed isotope pattern for molecular ion cluster + Theoretical overlay from FBF spectrum
Observed
• If molecular ion is detected, otherwise window is blank
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Window 5 Compound Fragment Spectrum results
Averaged mass spectrum across peak
Qualifier fragments
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Import and build MH Quant Method from MH Qual
MH Qual
.cef file Quant method - Accurate mass
EICs for quantifier and qualifier
- Relative responses - Retention times
Mass-to-Charge (m/z)
741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756
C o u n
t s
x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6 746.4838 (M+H)+
747.4871 (M+H)+
748.4901 (M+H)+
749.4929 (M+H)+ 751.4984
(M+H)+
Compound results
MH Quant
Pesticide screening GC-Q/TOF
1. Acquisition 2. PCDL 3. MH Qual SW 4. MH Quant SW
Qualified targets
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
All Ions Summary
Pesticide screening GC-Q/TOF
• Continuing trend towards comprehensive screening methods using accurate mass instruments
• Innovative data mining tools for identification of trace contaminants in complex matrices
• All Ions integrated workflow for enhanced qualification of suspected compounds by additional fragment ions in qualitative screening
- Similar Workflow as on LC- QTOF • Easy customizable and generation of user PCDL Libraries • Personal compound database library containing > 700 pesticides
with GC/MS accurate mass MS spectra and locked retention times under is construction
• Seamless integration of compound identification and MH Quantitation for easy and quick batch review
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Acknowledgements
Klaus Wilmers, Thorsten Bernsmann, Peter Fürst CVUA-MEL, Munster, Germany
Noelia Belmonte, Milagros Mezcua, Amadeo R. Fernández-Alba EURL for Pesticides in Fruits and Vegetables Almeria, Spain
Pesticide screening GC-Q/TOF
CSEMP
Contaminants in mussel
GC-QQQ
March 2011
Acquisition
• Easy method setup
• All Ions is faster than MS/MS
• Chromatography is monitored in 2 channels (or more if desired)
– A low energy channel ensures optimum molecular ion generation
– A high enery channel produces fragments
• support of TOF (fragmentor) as well as Q-TOF(collison cell)
Finding?
• As with TOF screening compounds are found using the same find by formula algorithm
already well proven
Verifying
• verification of results by using PCDL MS/MS library
• Chromatographic verification of fragment ions uses a unique co-elution score
• Allows retrospective data interogation, growing libraries can be applied
Qualitative Screening using All Ions LC-MS/MS
Pesticide screening GC-Q/TOF
Novel Ion Mobility Technology for LC/MS
Jaume C. Morales
MS Product Specialist
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Ion Mobility – A Brief History…
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1905
Ion mobility theory
Paul
Langevin
1969
Transport of Ions in Gases
McDaniel & Mason
1997
Applications to clusters & biomolecules
Clemmer & Jarrold
2006
Synapt Triwave
G2 in 2009
G2S in 2011
2013
Agilent IMS QTOF
1872 - 1946
Mass Spectrograph
Aston & Thomson
1919
Mass Accuracy Does Not Equal Compound Identification: Seven Golden Rules - Oliver Fiehn
Empirical formula is not unique above
mass m/z 100 (searching PubChem)
Number of formula: ChemSpider mass
search at m/z 400.3787
• 1 ppm mass error → 1742 entries
• 0 ppm mass error → 340 entries
Need additional physical information to
identify
• MS/MS spectra
• Physical properties such as:
• Chromatographic retention time
• Ion mobility cross section (size,
charge)
Number of Database Entries
(Assuming Zero Mass Error)
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What Does Ion Mobility Bring to Mass Spectrometry?
Separation
• Ion Mobility resolves of many isomeric analytes otherwise impossible to
determine by mass spectrometry alone.
Improves Detection Limits
• Ion Mobility dramatically reduces interference from other analytes and
background.
Confirmation
• Collision Cross Section data gives additional information supporting
compound characterization and identification.
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Resolution Is Important!
Chromatographic Ion Mobility Mass
~seconds ~60 milli-seconds ~ 100 m seconds
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It’s All About Separation
Chromatography Ion Mobility Mass
~seconds ~60 milli-seconds ~100 m seconds
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tdrift
Detector
Analyte
Ions
Gating
Optics
Ion Mobility Cell
t0
VH VL
Electric Field
Stacked ring ion guide gives linear field
𝑣 = 𝐾 𝐸 ∝𝑒 𝐸
𝑃 𝑇 Ω
Basic Operational Principle of Ion Mobility For Conventional DC Uniform Field IMS
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Resolving Stereoisomers
α-glucose β-glucose
Ion mobility enables separation of glucose stereoisomers
IM-QTOF Instrument Overview Novel Aspects Achieve New Performance Capabilities
DC only drift tube to achieve lowest ion temperatures
System sensitivity optimized using electrodynamic ion funnels
Designed for both high Ion Mobility and Mass Resolution
Ion Fragmentation can be selected using standard QTOF collision cell (CID)
10x increase in data acquisition system to support the additional data rates of adding the 3rd Ion Mobility Dimension
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IM-MS Operational Modes
83
• QTOF Only Mode
• Mobility Separated Precursor Ion Mode
• Mobility Separated All Ions Fragmentation Mode
• Mobility Separated Targeted MS/MS Mode
m/z selection ON/OFF
Fragmentation ON/OFF
Mobility
separation
IM-QTOF workshop 01/28/14
New Agilent MassHunter IM-MS Browser Visualizing Ion Mobility LC/MS Data
322.0481
622.0294
922.0098 1221.9906 1521.9711
1821.9521 2121.9332
2421.9138
2721.8941
IMS/MS Frame
Selection
Chromatogram View
Software
Solutions for
Improving your
Productivity
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• Frame Navigation tool
• Frame viewer
• Heat map
IM Drift TIme
MS
New Agilent MassHunter IM-MS Browser Visualizing Ion Mobility LC/MS Data
Ion mobility Frame Viewer provides linked views for easier navigation
2721.8941
2722.8974
2723.9003
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HEAT MAP
IMS QTOF - Overview
Applications
-Software tools
-Ω
-Lipids
-Carbohydrates
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ASMS 2013 Ion Mobility
Abstracts
Published Collisional Cross Sections
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90
Analyte Mass
[Da]
CCS
Literature
[Å2]
CCS
This Work
[Å2]
%
Deviation
from Lit.
Colchicine1 399.4 196.2 196.2 ± 0.54 Å2 0%
Odansetron2 293.4 172.7 173.8 ± 0.36 Å2 0.6%
Threonine 119.1 130.1 ±0.45 Å2 <2%
Phenylalanine 165.2 140.9 ±0.5 Å2 <2%
Tyrosine 181.2 148.4 ±0.6 Å2 <2%
Fructose 180.2 143.4 ±0.6 Å2 <2%
Sorbitol 182.2 142.7 ±0.5 Å2 <2%
1. Anal.Chem. 2012;84:1026.
2. Int. J. MS. 2010;298:78
3. JASMS.2007;18:1163
New Analyte Ion
CCS
IMS QTOF
[Å2]
5α-dihydrotestosterone (M+H)+ 181.6 ± 0.
5α-dihydrotestosterone (M+Na)+ 201.5±1.0
5β-dihydrotestosterone (M+H)+ 179.8±0.8
5β-dihydrotestosterone (M+Na)+ 199.5±0.8
androsterone (M+Na)+ 200.0±0.7
etiocholanolone (M+Na)+ 196.3±1.1
5-androstenediol (M+Na)+ 174.0±1.5
epiandrosterone (M+Na)+ 197.0±0.8
Excellent agreement between published
and measured cross sections
Collaboration with NIH
Collision Cross Section Correlates with Structure
91 IM-QTOF workshop 01/28/14
92
Structural studies of proteins possible with CCS calculation
[M+5H]5+ [M+6H]6+
[M+7H]7+
[M+8H]8+
[M+9H]9+
[M+10H]10+
[M+11H]11+
[M+12H]12+
[M+13H]13+
[M+14H]14+
5+ 6+
7+
8+
9+
13+
14+
10+ 11+ 12+
m/z
drift t
ime (
ms)
Structural Analysis of Ubiquitin Protein
IM-QTOF workshop 01/28/14
93
Ion Charge State CCS
experimental (Å2)
CCS
literature (Å2)
[M+5H]5+ 5 1196
[M+6H]6+ 6 1431, 1658
[M+7H]7+ 7 1755, 1886 1910
[M+8H]8+ 8 1966 1990
[M+9H]9+ 9 2008 2090
[M+10H]10+ 10 2114, 2197 2200
[M+11H]11+ 11 2239, 2348 2340
[M+12H]12+ 12 2412, 2511 2480
[M+13H]13+ 13 2556, 2620 2600
[M+14H]14+ 14 2680, 2726
Reference: Bush et al., Anal Chem. 2010, 82, 9557-9565.
Automated collision cross section calculation without the use of calibration curves
Cross Section Calculation of Ubiquitin Charge States
IM-QTOF workshop 01/28/14
Reveal Greater Detail All Ions: Ondansetron, Colchicine, Reserpine
Reserpine
Colchicine
Ondansetron
609.2800
400.1749
294.1597
IM Drift TIme
MS
Reveal Greater Detail All Ions: Ondansetron
June 2013
95
294.1597
184.1117
170.0960
212.1064
294.1597
Collision Cross Section Benchmark Vanderbilt University
• Tetraalkylammonium salts (TAA)
• Proposed as an “ideal” ion mobility standard
• Wide CCS range (TAA-4 to TAA-18; 100 to 400 Å2)
• TAA salts do not form clusters
• Literature CCS values exist N2 drift gas
+2 ions
+3 ions
+1 ions TAA-16
TAA-18
TAA-12
TAA-10
TAA-8 TAA-7
TAA-6 TAA-5
TAA-4
0
10
30
40
200 400 600 800
0
20
Mo
bilit
y D
rift
Tim
e (
ms)
Mass-to-Charge (m/z) 1000 1200
50
TAA-5 N-(CH2CH2CH2CH2CH3)4
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Conformational Space Occupancy of Biomolecules: Class Association by Trend Curves
Size Shape
Charge
Using a Synapt does NOT allow compound class association
Drift tube IMS allows
Class association
Conformational Space Occupancy of Biomolecules
Co
llis
ion
Cro
ss
Se
cti
on
(Å
2)
Mass (Da)
Hypothetical Ordering of
Biomolecular Classes
lipids
carbohydrates
peptides
oligonucleotides
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Lipid nomenclature
Trivial nomenclature Palmitoleic acid Trivial names (or common names) are non-systematic historical names.
Systematic
nomenclature (9Z)-octadecenoic
acid
Systematic names (or IUPAC names) derive from the standard IUPAC Rules for
the Nomenclature of Organic Chemistry, published in 1979,[1] along with a
recommendation published specifically for lipids in 1977.[2] Counting begins from
the carboxylic acid end. Double bonds are labelled with cis-trans isomerism-
/trans- notation or E-/Z- notation, where appropriate.
Δx nomenclature cis,cis-Δ9,Δ12
octadecadienoic acid
In Δx (or delta-x) nomenclature, each double bond is indicated by Δx, where the
double bond is located on the xth carbon–carbon bond, counting from the
carboxylic acid end. Each double bond is preceded by a cis- or trans- prefix,
indicating the conformation of the molecule around the bond.
n−x nomenclature n−3
n−x (n minus x; also ω−x or omega-x) nomenclature both provides names for
individual compounds and classifies them by their likely biosynthetic properties in
animals. A double bond is located on the xth carbon–carbon bond, counting from
the terminal methyl carbon (designated as n or ω) toward the carbonyl carbon.
Lipid numbers 18:3; or 18:3, n-6; or
18:3, cis,cis,cis-
Δ9,Δ12,Δ15
Lipid numbers take the form C:D, where C is the number of carbon atoms in the
fatty acid and D is the number of double bonds in the fatty acid. This notation can
be ambiguous, as some different fatty acids can have the same numbers.
Source: Wikipedia
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Lipid classes
Source: Wikipedia
Fatty acids
Glycerolipids
Glycerophospholipids
Sphingolipids
Sterol lipids
Prenol lipids
Saccharolipids
Polyketides
Main classes Examples of Glycerophospholipids
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Cerebrosides
Cerebrosides are glycosphingolipids called
monoglycosylceramides which are important components in
animal muscle and nerve cell membranes.
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Diseases Based on Sphingolipids
Disease Deficient enzyme Accumulated products
Niemann-Pick disease Sphingomyelinase Sphingomyelin in brain and RBCs
Fabry disease α-galactosidase A Glycolipids in brain, heart, kidney
Krabbe disease Galactocerebrosidase Glycolipids in oligodendrocytes
Gaucher disease Glucocerebrosidase Glucocerebrosides in RBCs, liver and
spleen
Tay-Sachs disease Hexosaminidase A GM2 gangliosides in neurons
Metachromatic
leukodystrophy Arylsulfatase A or
prosaposin Sulfatide compounds in neural tissue
Source: Wikipedia
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Lipid Analysis Io
n M
ob
ilit
y D
rift
Tim
e (
ms)
Mass (Da)
0
0
20
40
50
500 1000 1500 2000
10
30
60
70
PE 60:N PE 62:N
PE 64:N
PE 33:N PE 35:N
PE 37:N PE 39:N
PE 41:N
PE 23:N PE 21:N
PE 19:N
PE oligomers (+1)
PE oligomers (+2)
L-α-phosphotidylethanolamines (PE)
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Lipid Analysis Io
n M
ob
ilit
y D
rift
Tim
e (
ms)
Mass (Da)
0
0
20
40
50
500 1000 1500 2000
10
30
60
70
PE 35:(6-2) PE 37:(8-4) PE 39:(10-6) PE 33:(4-2)
+Na +K
Mass (Da)
740 760 770 780 790 750 800 810 820
PE 60:N PE 62:N
PE 64:N
PE 33:N PE 35:N
PE 37:N PE 39:N
PE 41:N
PE 23:N PE 21:N
PE 19:N
PE oligomers (+1)
PE oligomers (+2)
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Summary Separation
Chromatography
Ion Mobility
Size & Charge
Mass Resolution
Peak Capacity (~8-fold)
Isobaric Separation Pesticides,…
Compound Class
Natural Products
Finding outliers
Shape & Biological Activity
Class Association
Mapping conformational space
Vanderbilt work
Boston University
CCS (Ω)
Direct determination
Excellent Precision
Accuracy vs. Literature
<2%
Easier than Calibration Tables
Texas A&M
Isomers
Small molecules
Stereoisomers
Structural Isomers
Charge state separation
Chiral Compounds?
NIH – steroids
mAb IgG2 conformers
Better IM resolution
Higher IM sensitivity
Resolve complex samples
Directly obtain Ω
Preserve molecular structures
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New Screening strategies with Exact Mass QTOF-MS LC & GC
08/04/2015
AccMass_Screening_Pesticides
106
¿Preguntas?
