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

(i) EIC(+) of LysH+ (147.1128) (ii) Average spectrum of LysH+ peak top

m/zMigration time (min)

Rel

. In

t(%

)

Inte

nsi

ty

Migration time (min)

Inte

nsi

ty

m/z

Rel

. In

t(%

)

(b) Fragmentor voltage 200 V

m/z= 84.0811 (In-source fragment )


m/z=147.1126 Lysine [M+H]+

LysH+

LysH+


m/z= 293.2186 (Dimer [2M+H]+)


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


[fragment+HCOONa]-

[fragment]-

[M-H]-

[M+H]+ [M+Na]+

[M+K]+ [2M+H]+

[2M-H]-

[M+H-H2O]+

[fragment]+

[fragment]-

[fragment]-

[fragment]-

[fragment]-

[fragment]-


CHARGE MIGRATION FRAGMENTATIONS (CMF)

A.1. A.2.

-

-

-

-

-

-

-

-

-

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]+


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

nsi

ty

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

nsi

ty

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

nsi

ty

Rt (min)

Rt (min)

Full Scan (-ESI) BPC


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



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]+


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

Inte

nsi

ty

-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)

Introducción: Proceso de Ionización en ESI-MS: - Iones ...

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