Introducción: Proceso de Ionización en ESI-MS: - Iones ...
Transcript of Introducción: Proceso de Ionización en ESI-MS: - Iones ...
➢ Introducción: Proceso de Ionización en ESI-MS:
- Generación de iones: - Ión pseudomolecular
- Iones multicarga, dímeros y aductos
➢ Interpretación de Espectros MS y MS/MS.
➢ Reglas de Fragmentación en ESI-MS:
- Fragmentación con Migración de carga
- Fragmentación con Retención de carga
➢ Bases de Datos y herramientas informáticas para Predicción de Fragmentación in silico.
➢ Casos Prácticos: - Metabolitos primarios: Aminoácidos
- Fármacos: Haloperidol
Indice
- Metabolitos secundarios (plantas): Policétidos aromáticos: Flavonoides
- Metabolitos secundarios (plantas): Triterpenos
- Metabolitos secundarios: Glycerofosfolípidos y Lisofosfolípidos
Case Studies
3. Gas-phase Fragmentation reactions
2. Mass Spectrum interpretation
4. Rationalization of the Fragmentation process
5. Structural identification and charaterization
In order to interpret the Mass Spectrum,
one should attain an understanding the:
1. Ionisation process
✓ Databases✓ In-silico fragmentation prediction: software tools
Structural information for a compound can be obtained in different ways:
• by exploiting the in-source fragmentation (IS-CID)
• by performing targeted collision-induced dissociation (CID) MS/MS experiments
➢ Negative ions usually have less internal energy and poor in-source fragmentation is observed. To
fragment them it is necessary higher voltages.
➢ Positive ions usually have more internal energy and more in-source fragmentation is observed. [M+H]+
are preferable for fragmentation, because they result in more informative spectra for many compounds.
Rationalization of the Fragmentation process
In-Source Collision-Induced Dissociation (IS-CID)
in-sourceCID region
Fragmentorvoltage
MS
Capillary
QTOF
QQQ
MS/MS
IT
MSn
high vacuumatmospheric pressure medium vacuum
ESI
In source-CID region
MS/MS CID
medium vacuum
Skimmer
ESI QTOF-MS
Capillary
Fragmentor
ISF
fragmentor voltage collision energy
General Steps in Mass Spectral interpretation
Obtain a “good” spectrum
Evaluate the general appearance
Find the “Molecular Ion” if posible
Determine de Elemental Composition
Approach an approximate Molecular Formula
Study the Isotopic Peaks set, adducts, dimers…
Considering Fragmentation reaction rulesStudy of the most significant Fragments
Identify structural features
Propose possible structures
Be aware of the ionization mechanismm/z
inte
nsi
ty
+ ESI Scan
General Steps in Mass Spectral interpretation
(i) EIC(+) of LysH+ (147.1128) (ii) Average spectrum of LysH+ peak top
m/zMigration time (min)
Rel
. In
t(%
)
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Migration time (min)
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m/z
Rel
. In
t(%
)
(b) Fragmentor voltage 200 V
m/z= 84.0811 (In-source fragment )
m/z= 130.0862 (In-source fragment )
m/z=147.1126 Lysine [M+H]+
LysH+
LysH+
m/z= 130.0862 (In-source fragment )
m/z= 293.2186 (Dimer [2M+H]+)
m/z= 84.0812 (In-source fragment )
m/z= 315.2003 (Dimer and adduct [2M+Na]+)
m/z=147.1126 Lysine [M+H]+
(i) EIC(+) of LysH+ (147.1128) (ii) Average spectrum of LysH+ peak top
(iii) EIC’s of related ions with same shape
(iii) EIC’s LysH+ increased fragment ions (iv) Scale to large in all EIC’s
(iv) Scale to large in all EIC’s
(a) Fragmentor voltage 100 V
General Steps in Mass Spectral interpretation
[fragment+HCOONa]-
[fragment]-
[M-H]-
[M+H]+ [M+Na]+
[M+K]+ [2M+H]+
[2M-H]-
[M+H-H2O]+
[fragment]+
[fragment]-
[fragment]-
[fragment]-
[fragment]-
[fragment]-
General Steps in Mass Spectral interpretation
CHARGE MIGRATION FRAGMENTATIONS (CMF)
A.1. A.2.
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Fragmentation Reactions in ESI-MS
Chemistry is confusing enough with IUPAC nomenclature procedures to know, and the difference between E/Z and cis/trans alkene descriptions, and so many other new terms, models, units, and symbols.
… But the most common and important symbol in chemistry is the arrow.
Many students learn their arrows incorrectly and never manage to correct themselves.
Basic Organic Chemistry concepts
➢ Knowing the conventions for chemistry arrows is important. You should make sure that you know the arrows used in general chemistry.
Guide to common arrows in chemistry:
Basic Organic Chemistry concepts
[fragment+HCOONa]-
[fragment]-
[M-H]-
[M+H]+ [M+Na]+
[M+K]+ [2M+H]+
[2M-H]-
[M+H-H2O]+
[fragment]+
General Steps in Mass Spectral interpretation
pathway bpathway a
pathway b
Charge Migration Fragmentation (CMF):
CASE STUDY: N6-Methyl-L-Lysine In-source fragmentation (Positive ionization mode)
Fragments observed in the MS spectrum:
144.1021, 130.0864, 126.0922, 112.0767, 98.0962, 84.0816.
M
(C) MS spectrum of chlorogenic acid (FRAGMENTOR LOW ENERGY 90 V):
Inte
nsi
ty
m/z
CASE STUDY: Chlorogenic acid In-source fragmentation (Negative ionization mode)
Inte
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m/z
(D) MS spectrum of chlorogenic acid (FRAGMENTOR HIGH ENERGY 175 V): increased fragment ions
M(A) MS spectrum obtained by averaging the spectra over the chromatographic peak of chlorogenic acid:
Inte
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m/z
m/z = 503.1420
m/z = 569.1145
m/z = 455.1768
m/z = 353.0886
m/z = 353.0886
m/z = 353.0886
(B) Extracted ion chromatograms (EICs) of the individual ions in the spectrum:
Inte
nsi
ty
Rt (min)
(C) which ions are aligned in the retention time and peak shape and are analyte related:
m/z = 353.0886 [M-H]-
m/z = 191.0571[M-H-caffeoyl moiety]-
m/z = 707.1823 [2M-H]-
m/z = 503.1420co-elution
Inte
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Rt (min)
Rt (min)
Full Scan (-ESI) BPC
CASE STUDY: Chlorogenic acid In-source fragmentation (Negative ionization mode)
CFR: Retro-heteroene reactionsMcLafferty-type rearrangements
CMF: elimination assisted by hydrogen removal
Competition between a remote Hydrogen rearrangement and McLafferty-type rearrangement in caffeoylquinic acids
Charge Migration Fragmentation (CMF):
CASE STUDY: Chlorogenic acid In-source fragmentation (Negative ionization mode)
Terminology used:CS* = charge site, which can be positive or negativeABCDE+ or ABCDE- = atoms, a functional group or even several groups such as an alkyl chain
- Remote b-hydrogen rearrangements:
- Retro Diels-Alder (RDA):
- Retro-ene reactions:
(McLafferty rearrangement)
- Retro-heteroene reactions
- Charge remote fragmentation
- Carbon monoxide elimination from cyclic carbonil compounds:
CHARGE RETENTION FRAGMENTATIONS (CRF)p
eric
yclic
reac
tio
ns
(typically occurs in compounds possesing saturated and unsaturated long-chains and, such fatty acids)
Fragmentation Reactions in ESI-MS
Figure 16. Proposed haloperidol fragmentation scheme.Figure 4. Proposed fragmentation scheme of haloperidol.
Software for the prediction of mass spectral fragmentation
Journal Impact factor : 0.90 Journal Impact factor: 9.526
CASE STUDY: Haloperidol MS/MS Experiment (Positive ionization mode)
CASE STUDY: Haloperidol MS/MS experiment (positive ionization mode)
[M+H]+
[M+H]+
Charge Migration Fragmentation (CMF):
CASE STUDY: Haloperidol MS/MS experiment (positive ionization mode)
[M+H]+
CASE STUDY: Haloperidol MS/MS experiment(positive ionization mode)
A)
B)
RT= 1.19 min
277.1 → 202.9
277.1 → 258.9
CASE STUDY: Clembuterol MS/MS experiment (positive ionization mode) (QQQ)
CASE STUDY: Clembuterol MS/MS experiment (positive ionization mode)
MS/MS experiment (QTOF)CASE STUDY: Lysophosphocholine (LysoPC 16:0)
MS/MS spectra resulted from native standard state LPC(16:0) at 20 eV. acquired in positive ion mode (A) and in negative ion mode (B)
CASE STUDY: Lysophosphocholine (LysoPC 16:0) MS/MS experiment (negative ionization mode) (QTOF)
Proposed Major Fragmentation Pathway 1 of negative ions of LPC(16:0). Competition between aMcLafferty-type rearrangement (route a) ad a displacement reaction (route b) to yield characteristicMolecular Lipid species-specific Fragments (MLFs).
CASE STUDY: Lysophosphocholine (LysoPC 16:0) MS/MS experiment (negative ionization mode) (QTOF)
Proposed Minor Fragmentation Pathways 2 and 3 of negative ions of LysoPC(16:0). Competition between a
McLafferty-type rearrangement (route a) and a displacement reaction or elimination assisted by hydrogen removal
or (route b) to yield common Lipid Class-selective Fragments (LCFs).
MS/MS experiment (QTOF)CASE STUDY: Lysophosphocholine (LysoPC 16:0)
CASE STUDY: Lysophosphocholine (LysoPC 16:0) MS/MS experiment (positive ionization mode) (QTOF)
Proposed Fragmentation Pathways of positive ions for LysoPC(16:0)
[M+H]+
[M+Na]+
-H2O
-H2O-H2O
-H2O
-HCOOH
-H2O
[2M+Na]+
m/z
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-H2O
ESI+ - MS spectra
enlarged spectra
Analysis under ESI+ MS conditions gave abundant products ions generating typical fragmentation patterns:
CASE STUDY: Terpenoids: Coreanogenoic acid In-source fragmentation (positive ionization mode)
CMF: simple inductive clavage
CRF: remote hydrogen rearrangement
CRF: remote hydrogen rearrangement
[carbocation rearrangement]
CRF: Retro-Diels-Alder reaction
CASE STUDY: Terpenoids: Coreanogenoic acid In-source fragmentation (positive ionization mode)
Intensity vs. m/z
RT=12.5 min
m/z=299.0572
m/z=284.0331
m/z=367.0441
[M-H]-
[M-H-CH3]-
[M-H+HCOONa]-
Pseudo MS3(negative ionization mode) (QTOF)CASE STUDY: Chrysoeriol (flavone)