Upload
vokien
View
219
Download
0
Embed Size (px)
Citation preview
Últimos Desarrollos en Tecnología de Alta Resolución: GCQTOF – IMS QTOF. Trabajo con Muestras Complejas
Jaume C. Morales
MS Product Specialist
February 12, 2014
1
Febrero 2014
GC/Q-TOF para Target, No-target y Desconocidos :
Las ventajas de la Alta Resolución, Masa Exacta y la alta
Velocidad de adquisición en MS y MS/MS
7200 GC-QTOF Una nueva herramienta para
solucionar complejos problemas
analíticos
Febrero 2014
Portfolio de Agilent en GC/MS
PRECIO
RE
ND
IMIE
NT
O
240 IT
7000C TQ
5977A SQ 7890BGC
5977E SQ 7820 GC
GC Q-TOF
5975T SQ
Full Scan/SIM
MS/MS
MS/MS masa exacta
Page 4
El reto en GCMS : problemas analíticos complejos
La identificación de compuestos en muestras complejas a nivel de trazas
(ng/mL o menor) es difícil y generalmente requieres estrategias analíticas
no-rutinarias o sistemas de configuración atípica y poco robustas como :
1- Potentes métodos de extracción/enriquecimiento
2- Sistemas GC de alto poder de separación (e.g.GCxGC)
3- Detectores selectivos y/o MS.
Sin embargo, para análisis a nivel rutinario de éste tipo de compuestos se
requieren enfoques con técnicas más simples, rápidas y robustas para
incrementar la productividad.
Desafortunadamente los sistemas GC/MS (SIM) o 1D GC en combinación
con detectores específicos como el PFPD o ECD no son a menudo
suficientes para conseguir la selectividad requerida.
Page 5
El reto en GCMS : problemas analíticos complejos
1a Estrategia :
La tecnología MS-TOF recoge y analiza simultáneamente todos los iones a
lo largo del rango de masas en contraposición a los instrumentos de barrido
convencional donde los iones son filtrados y detectados secuencialmente.
Consecuentemente, el GC-QTOF-MS en modo TOF no tiene parangón en
sensibilidad trabajando en “Full Scan”, comparable a la técnica GC-MS en
modo SIM, pero con el espectro completo y
Además ,el GC-QTOF-MS genera datos en masa exacta lo cual permite
obtener una alta selectividad y sensibilidad utilizando ventanas de
extracción del orden de 0.02-0.05Da.
PERO, ¿Qué pasa cuando la Resolución y la Δ Masa no son suficientes?
2a Estrategia: GC-QTOF-MS/MS
Page 6
= +
¿Que es el 7200? 7200 GC/Q-TOF = 7890 + 7000 + 6500
MS Cuadrupolo /Tiempo de vuelo
MS Tiempo de Vuelo
MS Triple Cuadrupolo
Page 7
La fusión de dos plataformas
Turbo 2
Ion Pulser
Turbo 3
Ion Source
Turbo 1b Turbo 1a
Quad Mass Filter (Q1)
Collision Cell
Transfer
optics
6500 LC/MS
Q-TOF based
7000 GC/MS
QQQ based
Ion Mirror
Ion Detector
Page 8
Nuevo . . . Pero totalmente probado Dual-stage ion mirror improves
second-order time focusing for
high mass resolution.
Hexapole collision cell accelerates
ion through the cell to enable faster
generation of high-quality MS/MS
spectra without cross-talk
Split-flow turbo differentially
pumps the ion source and
quadrupole analyzer compartments
4GHz ADC electronics enable a high
sampling rate (32 Gbit/s) which improves
the resolution, mass accuracy, and
sensitivity for low-abundance samples.
Dual gain amplifiers simultaneously
process detector signals through both low-
gain and high gain channels, extending the
dynamic range to 105.
Analog-to-digital (ADC) Detector:
Unlike time-to-digital (TDC) detectors
which record single ion events, ADC
detection records multiple ion events,
allowing very accurate mass
assignments over a wide mass range
and dynamic range of concentrations.
New Removable Ion Source
includes repeller, ion volume,
extraction lens and dual filaments
Proprietary INVAR flight tube
sealed in a vacuum-insulated
shell eliminates thermal mass
drift due to temperature changes
to maintain excellent mass
accuracy, 24/7. Added length
improves mass resolution.
Hot, quartz monolithic quadrupole
analyzer and collision cell identical
to the 7000 Quadrupole MS/MS
New Internal Reference Mass
can be delivered to the source at
a low and high concentration
Two 300L/s t urbos pump the
focusing optics and flight tube
Page 9
Removable Ion Source (RIS)
Automated
Retractable
Transfer Line
RIS
Automated
Gate Valve
Page 10
¿Que puede hacer el GC-QTOF por nosotros?
• En modo TOF
• Espectros “full scan” de alta resolución
• Medida de masa exacta
• Adquisición a alta velocidad de espectros “full scan”
• En modo MS/MS
• Espectros “full scan product ion” con alta resolución y
masa exacta
• La herramienta ideal para abordar complejos
problemas analíticos.
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
Page 12
Resolución y exactitud de masa
R = 614/0.68 = 903
Δmz = 0.1/614
= 160 ppm
Pw=0.68
Mz=614
SQ
TQ
IT
TOF
Q-TOF
R = 614/0.0423 = 14522
Δmz = 0.0004/613.96
= 0.7 ppm
Resolución :
R=mz/FWHM
Exactitud de masa:
Δmz=dm/mz*106, partes por millon
(ppm)
PFTBA mass 614
C12F24N=613.964203
1 Da.
1 Da.
Page 13
1. Bloqueo del eje de masas por Referencia Interna o
Calibración simultanea para exactitud sub 5ppm incluso
en muestras con alta carga de matriz
2. Fuente extraíble RIS para una limpieza, cambio de
filamentos o intercambio de fuentes EI/CI rápida y sin
romper vacío.
3. Q-TOF MS/MS:
• Reducción del ruido químico
• Selectividad
• Information estructural
• Desarrollo de métodos
4. Herramientas de software – Formula calculator / MSC (MS/MS Structural Correlation Tool)
Características Clave en el 7200
Page 14
El reto en GCMS : problemas analíticos complejos
1a Estrategia :
La tecnología MS-TOF recoge y analiza simultáneamente todos los iones a
lo largo del rango de masas en contraposición a los instrumentos de barrido
convencional donde los iones son filtrados y detectados secuencialmente.
Consecuentemente, el GC-QTOF-MS en modo TOF no tiene parangón en
sensibilidad trabajando en “Full Scan”, comparable a la técnica GC-MS en
modo SIM, pero con el espectro completo y
Además ,el GC-QTOF-MS genera datos en masa exacta lo cual permite
obtener una alta selectividad y sensibilidad utilizando ventanas de
extracción del orden de 0.02-0.05Da.
PERO, ¿Qué pasa cuando la Resolución y la Δ Masa no son suficientes?
2a Estrategia: GC-QTOF-MS/MS
Page 15
El reto en GCMS : problemas analíticos complejos
2a Estrategia :
El modo QTOF-MS puede :
- Reducir el ruido al seleccionar y filtrar un Precursor, suministrando así
mayor selectividad.
- Confirmar la identidad de un compuesto a través de su espectro MS/MS
de Alta Resolución.
- Elucidación Estructural.
Page 16
Elucidación estructural por MS/MS.
C16H14O4 (Anillos + Enlaces Dobles = 10)
(M – H)+
269.0802
Estructuras
candidatas m/z
(experimental)
Fórmula Error
(ppm)
Score
269.0802 C16H13O4 2.2 80.7
193.0494 C10H9O4 0.6 96.7
167.0334 C8H7O4 3.0 N/A
166.0259 C8H6O4 0.6 N/A
138.0310 C7H6O3 1.1 98.1
110.0359 C6H6O2 3.0 N/A
95.0127 C5H3O2 0.9 99.5
– CH2=CH–C6H5
– CO
– CH3
– CO
– H
– C6H5
– CH=CH–C6H5
Page 17
Formula Calculator: fórmulas consistentes con
la masa exacta y fórmula del Ión padre
C5H12O2PS3 m/z = 230.9732
Page 18
Aplicaciones 1. Metabolic profiling of yeast sterols using the Agilent 7200 Series
GC/Q-TOF system
2. Metabolomics of Carbon Fixing Mutants of Cyanobacteria by GC/Q-
TOF
3. Metabolomics of Opiate-Induced Changes in Murine Brain by GC/Q-
TOF
4. Untargeted Metabolomic Analysis of UV Stress Response in C.
reinhardtii by GC-QTOF
5. Simultaneous analysis of tryptophan, kynurenines and amino acids
using the GC/QTOF in Negative CI mode
6. Accurate mass retention time locked flavor database by GC/Q-TOF
7. Analysis of trace levels of sulfur compounds in coffee by the Agilent
7200 GC/Q-TOF system
8. Olive oil characterization using Agilent GC/Q-TOF MS and Mass
Profiler Professional software
9. Rapid simultaneous screening of multiple pesticide residues in Food
matrices
10. Simultaneous targeted and non-targeted screening for pesticides in
vegetables by GC/Q-TOF MS
11. Analysis of biomarkers in crude oil using the Agilent 7200 GC/Q-TOF
12. Characterization and classification of heroin from illicit heroin seizures
by GC/Q-TOF
13. Unknowns analysis of natural products using GC/Q-TOF and
GC/IonTrap in EI and PCI modes with MS/MS
14. Determination of odor compounds in surface water by solid phase
micro-extraction and GC/Q-TOF
15. The role of GC/QTOF in exposomics
Page 19
Food Testing and Flavors:
Olive Oil Characterization
UC Davis Olive Center
&
Stephan Baumann, Agilent Technologies
• MPP for statistical processing of GC/Q-TOF data
• MS library searching using GC/Q-TOF spectra
• CI data provide accurate mass information for molecular ions
Page 20
1. Olive oil samples had been subjected to sensory test and classified as passed or failed
2. GC/Q-TOF data then were acquired in both EI and PCI modes
3. Chromatographic deconvolution was performed with MassHunter Qual, and the data
were exported as CEF files to perform statistical analysis using Mass Profiler
Professional (MPP).
4. MPP was used for statistical evaluation of the data including construction of class
prediction model
5. The model was able to correctly predict whether the sample would pass or fail the
sensory test
Olive Oil Characterization: Workflow
Goals:
- to create a model that could predict whether olive oil sample would pass or
fail sensory test
- to recognize statistically significant olive oil components that are present at
distinct levels depending on whether they passed or failed sensory test
Page 21
Olive Oil Characterization: Data Filtering
442 unique compounds were
distinguished by
chromatographic
deconvolution, most of which
occur only once or twice and
are filtered out by MPP.
The table shows how many of these 442
compounds were actually found in each sample.
Page 22
Olive Oil Characterization: Visualization of Data Clustering
Principal Component Analysis (PCA) of MPP helps to visualize
clustering of the data
failed
passed
Page 23
Olive Oil Characterization: Fold Change Analysis
The Volcano Plot (on the right) shows fold-change for each entity on the x-axis and
significance on the y-axis.
Compounds accumulated
in the samples that failed
the sensory test.
Page 24
Olive Oil Characterization: Compound Identification
1. EI spectra were used to search NIST library to obtain tentative identification of
the compounds
2. PCI data were used to obtain molecular formula for the compounds
3. Further MS/MS experiments allowed to generate ‘clean’ spectra in the
presence of matrix interference and could possibly be used for structure
elucidation
Page 25
Olive Oil Characterization: Library Search
Commercial unit mass EI spectral libraries can be searched using accurate mass
EI GC/Q-TOF data to identify compounds
Compound spectrum
NIST library spectrum
Compound spectrum
(accurate mass)
EI
Page 26
Olive Oil Characterization: MS/MS Example
C12H17
5.11 ppm C9H11
-3.58 ppm
C8H9
-2.63 ppm
C10H13
0.93 ppm
α-Cubebene, full scan
C15H24
α-Cubebene: MS/MS
Precursor: 204
CE: 10 eV
(replib) α-Cubebene
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 2300
50
100
41
55
69 77
8191
105
119
133147
161
175 189
204
Accurate masses of ion fragments are consistent with molecular formula
Page 27
Olive Oil Characterization: Combining EI and PCI Data
Tentative NIST ID Formula EI , M*+ PCI, (M+H)+
Calculated Measured Mass error, ppm Calculated Measured Mass error, ppm Hexadecanoic acid C16H32O2 256.2397 256.2385 4.68 257.2475 257.247 1.94
Ethyl-octadecanoate C20H40O2 312.3023 312.3008 4.80 313.3101 313.3091 3.19
Squalene C30H50 410.3907 410.3904 0.73 411.3985 411.3987 0.49
α-Cubebene C15H24 204.1873 204.1883 4.90 205.1951 205.1945 2.92 Unknown C14H26O2 226.1927 N/A N/A 227.2006 227.1987 8.36
PCI spectral data provided accurate mass information for molecular ions of the
accumulated compounds in olive oils that fail the sensory test, including the
case where the EI spectrum showed no prominent molecular ion
Page 28
Olive Oil Characterization: MPP Results
• The model correctly predicted the pass or fail status of all samples, including
those not used to construct the model.
• The samples that were not used for building the prediction model are listed
with the Training parameter set as ‘None’
Page 29
Photodegradation Products of Beer
Page 30
Problem – identify degradation Products of beer
• Completely untargeted (initially) study of beer
photodegradation
• Method highlights
• 30 min extraction at 30 ˚C using manual
SPME holder and conditioned 50/30 µm
DVB/Carboxen/PDMS StableFlex SPME
fiber (Supelco), no agitation
• Desorption at 300 ˚C for 2 min in the SSL
injector; 1:10 split
• Agilent J&W column DB-5MS 30 m x 0.25
mm x 0.25 µm
Page 31
Changes in the Chromatogram
No exposure to direct sunlight
3 hours
6 hours
Appears following the exposure of the sample
to direct sunlight. Peak height is dependent on
the duration of exposure to the sun
Molecular ion
m/z=165.1120
C10H15NO
Page 32
Summary of MS/MS Experiments Accurate mass measurement of molecular ion and fragments
C4H5
C10H14N
C9H14N C7H8N
C9H11N
C6H7NO
C6H8N C6H6NO
C5H6N
C5H7N
C4H5
C4H6N
(mainlib) 1-Butanamine, N-(2-furanylmethylene)-3-methyl-
50 100 150 200 250 300 350 4000
50
100
39
53
67
81
95
109
122
136
164
O
N
C10H15NO
109 122
C7H8NO 136 C9H14N
148 C10H14N
133 C9H11N
80 C5H6N
94 C6H8N
108 C6H6NO
81 C5H7N
55 C3H5N
53 41 C3H5
66 C4H4N
78 C5H4N
C6H7NO MS
MS/MS
Page 33
Summary of MS/MS Experiments Calculate possible empirical formulas
C4H5
C10H14N
C9H14N C7H8N
C9H11N
C6H7NO
C6H8N C6H6NO
C5H6N
C5H7N
C4H5
C4H6N
(mainlib) 1-Butanamine, N-(2-furanylmethylene)-3-methyl-
50 100 150 200 250 300 350 4000
50
100
39
53
67
81
95
109
122
136
164
O
N
C10H15NO
109 C6H7NO
122 C7H8NO 136
C9H14N
148 C10H14N
133 C9H11N
80 C5H6N
94 C6H8N
108 C6H6NO
81 C5H7N
55 C3H5N
53 41 C3H5
66 C4H4N
78 C5H4N
MS
MS/MS
Page 34
Summary of MS/MS Experiments MS/MS on fragments + accurate mass to find empirical formulas
C4H5
C10H14N
C9H14N C7H8N
C9H11N
C6H7NO
C6H8N C6H6NO
C5H6N
C5H7N
C4H5
C4H6N
(mainlib) 1-Butanamine, N-(2-furanylmethylene)-3-methyl-
50 100 150 200 250 300 350 4000
50
100
39
53
67
81
95
109
122
136
164
O
N
C10H15NO
109 C6H7NO
122 C7H8NO 136
C9H14N
148 C10H14N
133 C9H11N
80 C5H6N
53 78
C5H4N
MS
MS/MS
-OH
-CH3 -C5H8
-CHN -H2
Page 35
Summary of MS/MS Experiments MS/MS on other fragments
C4H5
C10H14N
C9H14N C7H8N
C9H11N
C6H7NO
C6H8N C6H6NO
C5H6N
C5H7N
C4H5
C4H6N
(mainlib) 1-Butanamine, N-(2-furanylmethylene)-3-methyl-
50 100 150 200 250 300 350 4000
50
100
39
53
67
81
95
109
122
136
164
O
N
C10H15NO
109 C6H7NO
122 C7H8NO 136
C9H14N
148 C10H14N
133 C9H11N
80 C5H6N
94 C6H8N
108 C6H6NO
81 C5H7N
55 C3H5N
53 41 C3H5
66 C4H4N
78 C5H4N
MS
MS/MS
Page 36
• Los nuevos retos requieren en ocasiones nuevas
herramientas/soluciones.
• El GC Q-TOF ofrece la capacidad de solucionar problemas con
nuevas estrategias.
Resumen – Qué recordar
• La Alta Resolución (HR), Mejor Exactitud de
masa (MA) y Alta Velocidad de barrido mejora
los resultados analíticos.
• Los espectros MS/MS con HR y MA hacen
posible la Elucidación Estructural
• Agilent ofrece el portfolio más amplio en
herramientas GC/MS :
SQ, IT, TQ, & Q-TOF
Novel Ion Mobility Technology for QTOF LC/MS
April 23, 2014
37
IMS QTOF - Overview
HD QTOF
IMS Background
Ion Mobility Basics
Instrument & Software Overview
Applications
- Software tools
- Ω
- Lipids
- Carbohydrates
Summary
April 23, 2014
38
ASMS 2013 Ion Mobility
Abstracts
Aportación de los sistemas de Tiempo de Vuelo
43
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
Aportación de los sistemas de Tiempo de Vuelo
TOF
QTOF
TOF
Adquisición de todo el espectro (Full Scan)
QTOF
Adquisición de todo el espectro (Full Scan)
Adquisición del espectro MS/MS:
Tiapride 0.8 ppm
Tiapride 0.8 ppm
6550 iFunnel Q-TOF
LC/MS System
Sensitivity
• Dramatically improved quantitative capabilities
• New Qual/Quan Workflows
• Superior metabolite and protein detection
• Non-targeted compound screening
Comprehensive Performance Enhancements
• Mass Resolution >40,000
• 50 spectra /sec MS and 33 spectra/sec MS/MS
• 5 orders of linear dynamic range
• <1 ppm MS mass accuracy; <2 ppm MS/MS
• Unrivalled sensitivity
New 6550 iFunnel QTOF
10X Sensitivity Gain Enables Applications
QTOF Acquisition – MS and MS/MS Modes of Operation
• MS Only – “TOF only” mode
• MS/MS All Ions . MS & MS/MS info at the same time.
• Data Dependent MS/MS Experiments
• Precursor selection based on intensity of n-highest (with relative and absolute threshold)
• Excluded and Preferred mass lists
• Configurable charge-state selection preference
• Data Directed (Targeted) MS/MS experiments
• Import of target mass lists from Mass Profiler or Mass Profiler Professional software
• Import of mass lists from other applications
• Automatic dynamic creation of time segments
2. Instrument and Software Overview
Screening and identification workflow
Agilent’s approach
• Combination of UHPLC separation and accurate mass TOF technology
• Effective data mining algorithms to FIND compounds in a sample
• Optional software to COMPARE samples or sample sets to identify differences
• AMRT Databases and MS/MS Library Search to easily IDENTIFY targeted compounds
• Several algorithms to help IDENTIFYING unknown compounds (MFG, MSC)
• User Interface to easily NAVIGATE RESULTS
• Custom reporting to comprehensively REPORT results
• Full AUTOMATION of data acquisition, processing and reporting
TOF/
Q-TOF
Analysis
ID via
AMRT
DBs
Find
Compounds
MFG of
Compounds
w/ MSMS
MSMS
structural
correlation
ID via
MSMS
libraries
OPTION:
Differential
Analysis
custom
report
Automation
Screening and identification workflow
TOF/
Q-TOF
Analysis
ID via
AMRT
DBs
Molecular
Feature
Finding
MFG of
Compounds
w/ MSMS
MSMS
structural
correlation
ID via
MSMS
libraries
Automation
OPTION:
Differential
Analysis
MS/MS Structural Correlation (MSC)
custom
report
• Algorithm to correlate “proposed
structures” with accurate mass
MS/MS fragment ion spectrum.
• Favor systematic bond
dissociation approach over rule
based fragmentation prediction
approach.
• Proposed structures can be selected directly or searched in a PCDL or via ChemSpider
IMS QTOF - Overview
HD QTOF
IMS Background
Ion Mobility Basics
Instrument & Software Overview
Applications
- Software tools
- Ω
- Lipids
- Carbohydrates
Summary
April 23, 2014
50
ASMS 2013 Ion Mobility
Abstracts
Ion Mobility – A Brief History…
April 23, 2014
51
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
Drift Ion Mobility for LC-MS
Cross sectional areas
Complex Samples
Shape and Charge
Conformers
Isomers
Chromatography, Mass Resolution &
now Ion Mobility
2013 ASMS Scientific Presentations:
• Disease research
• Proteomics, Metabolomics, Lipidomics
• Natural Products
• Fundamental studies
Ion Mobility
MS
Pacific Northwest
Labs
Texas A&M
Vanderbilt University
Boston University
NIH
April 23, 2014
52
Solving Analytical Problems
April 23, 2014
53
Better IM resolution
Higher IM sensitivity
Resolve complex samples
Direct measurement
of Ω
Preserve molecular structures
• Enhance throughput, improve sensitivity and quantitation
• For large scale -omics studies PNNL
• Improving glycan analysis
• Disease research - Entamoeba Boston University
• Ion mobility fundamentals
• Study of metallo-protein structures Texas A&M
• Collisional cross section data (Ω)
• Mapping specific chemical classes – natural products
Vanderbilt University
• Separation of androgenic steroids not amenable to LC & MS
• Ω used to identify isobaric steroids NIH
• Characterization of trans membrane domains.
• Preservation of fragile protein folding structures Agilent
IMS QTOF - Overview
HD QTOF
IMS Background
Ion Mobility Basics
Instrument & Software Overview
Applications
- Software tools
- Ω
- Lipids
- Carbohydrates
Summary
April 23, 2014
54
ASMS 2013 Ion Mobility
Abstracts
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)
April 23, 2014
55
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.
April 23, 2014
56
Resolving Stereoisomers
α-glucose β-glucose
Ion mobility enables separation of glucose stereoisomers
Resolving Structural Sugar Isomers C18H32O16
Melezitose
Raffinose
Resolving two isobaric trisaccharides
Melezitose
Raffinose
α-glucose b-glucose
Resolving Different type of Isomers
Resolution Is Important!
Chromatographic Ion Mobility Mass
~seconds ~60 milli-seconds ~ 100 m seconds
April 23, 2014
60
It’s All About Separation
Chromatography Ion Mobility Mass
~seconds ~60 milli-seconds ~100 m seconds
April 23, 2014
61
Aldicarb-sulfone (C7H14N2O4S)
[M+Na]+ = 245.056649
Acetamiprid (C10H11ClN4)
[M+Na]+ = 245.056445
D mass is 0.2 mDa requires ~2,000,000 resolution
Separation of Isobaric Pesticides
4 x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
19.441
17 17.5 18 18.5 19 19.5 20 20.5 21 21.5
6 x10
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
18.297
Drift Time (ms) 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5
Aldicarb-sulfone
Acetamiprid
Drift Time (ms)
17 17.5 18 18.5 19 19.5 20 20.5 21 21.5
4x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
+IMS DriftSpec (m/z: 245.013827-245.177238) (rt: 0.026-1.987 min) Aldicarbsulfone_A…
* 18.297
* 19.441
Counts vs. Acquisition Time (min)17 17.5 18 18.5 19 19.5 20 20.5 21 21.5
Theoretical Plot
IMS Drift
Separation
April 23, 2014
62
IMS QTOF - Overview
UHPLC
HD QTOF
IMS Background
Ion Mobility Basics
Instrument & Software Overview
Applications
- Software tools
- Ω
- Lipids
- Carbohydrates
Summary
April 23, 2014
63
ASMS 2013 Ion Mobility
Abstracts
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
April 23, 2014
65
The Agilent Ion Mobility System
• Nitrogen buffer gas
• Funnels drive sensitivity
• Uniform Field Drift Tube allows direct determination of Ω
• Longer drift tube drives resolution approaching theoretical limit
• Fragmentation after IMS means parents and fragments have common drift times
enables an all ion experiment (with the precursor ions separated by drift times).
Mobility Resolution
∝ 𝐿𝐸𝑍𝑒
April 23, 2014
66
The Agilent Ion Mobility System
Mobility Resolution
∝ 𝐿𝐸𝑍𝑒
April 23, 2014
67
• Nitrogen buffer gas
• Funnels drive sensitivity
• Uniform Field Drift Tube allows direct determination of Ω
• Longer drift tube drives resolution approaching theoretical limit
• Fragmentation after IMS means parents and fragments have common drift times
enables an all ion experiment (with the precursor ions separated by drift times).
The Agilent Ion Mobility System
Electric Field
Mobility Resolution
∝ 𝐿𝐸𝑍𝑒
April 23, 2014
68
𝑣 = 𝐾 𝐸 ∝𝑒 𝐸
𝑃 𝑇 Ω
• Nitrogen buffer gas
• Funnels drive sensitivity
• Uniform Field Drift Tube allows direct determination of Ω
• Longer drift tube drives resolution approaching theoretical limit
• Fragmentation after IMS means parents and fragments have common drift times
enables an all ion experiment (with the precursor ions separated by drift times).
Front-end Instrumentation
Ion funnel technology drives sensitivity gain
April 23, 2014
69
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
April 23, 2014
70
• Frame Navigation tool
• Frame viewer
• Heat map
IM Drift TIme
MS
New Agilent MassHunter IM-MS Browser Visualizing Ion Mobility LC/MS Data
IMS/MS Frame
Selection
Software
Solutions for
Improving your
Productivity
April 23, 2014
71
IM Drift TIme
MS
Chromatogram View
Ion Mobility Resolution. How much?
536.5960 536.9300
537.2644
Resolution = 84!
Zipper peptide
April 23, 2014
72
IMS QTOF - Overview
HD QTOF
IMS Background
Ion Mobility Basics
Instrument & Software Overview
Applications
- Software tools
- Ω
- Lipids
- Carbohydrates
Summary
April 23, 2014
74
ASMS 2013 Ion Mobility
Abstracts
Published Collisional Cross Sections
April 23, 2014
75
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
Reveal Greater Detail All Ions: Ondansetron, Colchicine, Reserpine
Reserpine
Colchicine
Ondansetron
609.2800
400.1749
294.1597
IM Drift TIme
MS
All Ion MS using 20 Volt Fragmentation Energy
Collective drift spectrum includes all ions generated from 3 compounds
Reserpine
Colchicine Ondansetron
100 m/z 600
12
dri
ft t
ime (
ms)
4
0
Drift Time Separated Fragmentation
Simultaneous separation and fragmentation for ondansetron
Ondansetron
[M+H]+
100 m/z 600
12
dri
ft t
ime (
ms)
4
0
Simultaneous separation and fragmentation for colchicine
Drift Time Separated Fragmentation
Colchicine
[M+H]+
100 m/z 600
12
dri
ft t
ime (
ms)
4
0
Simultaneous separation and fragmentation for reserpine
Drift Time Separated Fragmentation
Reserpine
[M+H]+
100 m/z 600
12
dri
ft t
ime (
ms)
4
0
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
April 23, 2014
83
Tetraalkylammonium Salts CCS Values Compared to Literature
April 23, 2014
84
Analyte
Measured
Cross-Section
[Å2]
TAA-4 166.61 ± 0.5%
TAA-5 189.21 ± 0.6%
TAA-6 212.71 ± 0.3%
TAA-7 236.34 ± 0.2%
TAA-8 257.19 ± 0.1%
TAA-10 294.53 ± 0.1%
TAA-12 323.62 ± 0.2%
TAA-16 362.03 ± 0.2%
TAA-18 381.58 ± 0.3%
Literature
Cross-Section
[Å2]
166.00 ± 0.3%
190.10 ± 0.1%
214.00 ± 0.3%
236.80 ± 0.2%
258.30 ± 0.4%
Relative Standard
Deviation
[%]
0.56
0.28
0.41
0.01
0.24
• High experimental precision
(< 0.5% relative deviation)
• Agreement with literature
(most < 0.5% deviation)
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
April 23, 2014
86
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
April 23, 2014
87
Lipid classes
Source: Wikipedia
Fatty acids
Glycerolipids
Glycerophospholipids
Sphingolipids
Sterol lipids
Prenol lipids
Saccharolipids
Polyketides
Main classes Examples of Glycerophospholipids
April 23, 2014
88
Cerebrosides
Cerebrosides are glycosphingolipids called
monoglycosylceramides which are important components in
animal muscle and nerve cell membranes.
April 23, 2014
89
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
April 23, 2014
90
Lipid Analysis
Tetraalkylammonium Salts
+2 ions
+3 ions
+1 ions
+4 ions
Ion
Mo
bilit
y D
rift
Tim
e (
ms)
Mass (Da)
0
0
20
40
50
500 1000 1500 2000
10
30
60
70
L-α-phosphotidylethanolamines (PE)
TAA-3
TAA-16
TAA-12
TAA-10
TAA-8 TAA-7
TAA-6
TAA-5
TAA-4
April 23, 2014
91
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)
April 23, 2014
92
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)
April 23, 2014
93
Carbohydrates; Great complexity by linkage
Source: Blixt et al., PNAS, 2004
Current dominant strategies: MS(n) or Library searches
April 23, 2014
94
Carbohydrates Analysis
maltodextrins (1 to 8) cyclodextrins (α, β, γ)
human milk oligosaccharides (7)
Tetraalkylammonium Salts
+2 ions
+3 ions
+1 ions
+4 ions
Ion
Mo
bilit
y D
rift
Tim
e (
ms)
Mass (Da)
0
0
20
40
50
500 1000 1500 2000
10
30
60
April 23, 2014
95
Carbohydrates IM-MS 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
Mixture of Lacto-N-difucohexaose I & II
Mass (Da)
1018 1022 1024 1026 1028 1020
Drift Time (ms)
37 39 40 41 42 38 36 35
Lacto-N-
difucohexaose II
Drift Time (ms)
37 39 40 41 42 38 36 35
Lacto-N-
difucohexaose I
Lacto-N-difucohexaose I
Lacto-N-difucohexaose II
Gal Glc Gal GlcNAc
Fuc Fuc
Gal Glc Gal GlcNAc
Fuc Fuc
April 23, 2014
96
• Next generation of IMS QTOF Technology
• Added dimension of separation based on size, charge and
molecular conformation
• Resolve and characterize the complex samples
- Increased peak capacity
• Direct determination collision cross sections
• Preservation of molecular structures via lower thermal
excitation
Summary
April 23, 2014
97
MUCHAS GRACIAS
Jaume C. Morales Especialista de Producto
AGILENT TECHNOLOGIES
901.11.68.90
C L E A R LY B E T T E R M S S O L U T I O N S
NEW
NEW
Questions?
C L E A R LY B E T T E R M S S O L U T I O N S
Mass Spectrometry
Technology
Products
Solutions
NEW