1 J. Prasain 01/19/11 Ion fragmentation of small molecules in mass spectrometry Jeevan Prasain [email protected]6-2612 J. Prasain 01/19/11 Nomenclature: the main names and acronyms used in mass spectrometry • Molecular ion: Ion formed by addition or the removal of one or several electrons to or from the sample molecules- Electron Impact (EI-MS). M + e - M +• + 2e - • Adduct Ion: Ion formed through interaction of two species and containing all the atoms of one of them plus one or several atoms of them (e.g. alkali, ammonium).
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Ion fragmentation of small molecules in mass · PDF file1 J. Prasain 01/19/11 Ion fragmentation of small molecules in mass spectrometry Jeevan Prasain [email protected] 6-2612 J. Prasain
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• Product ion/daughter ion: Ions resulting from parent/precursor ions.
• Neutral loss: Fragments lost as neutral molecules
• In positive ionization mode, a trace of formic acid is often added to
aid protonation of the sample molecules; in negative ionization mode a trace of ammonia solution or a volatile amine is added to aid deprotonation of the sample molecules. Proteins and peptides are usually analysed under positive ionization conditions and polyphenols and acids under negative ionization conditions. In all cases, the m/z scale must be calibrated.
J. Prasain 01/19/11
Terminology..
• Ionspray denotes pneumatically assisted ESI
operating at aflow rate of approximately 5 to 50
uL/min.
• Turboionspray is ionspray with additional heated
gas for flow rate of 0.1 to 2 ml/min.
• Heated nebulizer is the trade mark of AB Sciex for
APCI.
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Precursor ion
or parent ion
Activated ion
Fragmenting ion
Neutral loss
Product ions
Schematic of CID fragmentation
What is Collision Induced Dissociation (CID)
or Collisionally Activated Dissociation (CAD) ?
Other activation processes:
PSD (post source-decay)
ECD (electron capture dissociation)
SID (surface-induced dissociation
o
o
o
o
o
o
o
o
o
oo
oo
Collision gas
Collision cell
J. Prasain 01/19/11
Various types of MS/MS experiments
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Steps involved in fragmentation
Step # 1: Creation of ions
Step # 2: Add energy of activation
Step # 3: Charge directed fragmentation
C N H
OCH2CH3
C N H
OCH2CH3
CID
C N H
OCH2CH3
Fragment ion + neutral molecule
ESI
J. Prasain 01/19/11
Applications of MS/MS
• Identification and characterization drug metabolites
• Authentification and profiling of chemical
components in a crude mixture
• Substructure analysis of unknown components
• Quantification of analytes in biological samples
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Ways to approach predicting MS/MS
• Likely sites of protonation or deprotonation.
• Likely leaving group.
• Mobility of protons
• Literature study
Fragmentation always follows the basic
rules of chemistry
Where are the sites of
Deprotonation/protonation?
What is the most likely leaving
Group in this molecule?
OHO
O
O
O
CH2OHHO
HO
HO
OCOOH
OH
OHOH
J. Prasain 01/19/11
Ion fragmentation for identification of phase
II drug metabolites (glucuronide/sulfate
conjugates)
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100 200 300 400
m/z
100
50
0
Rela
tiv
e I
nte
nsit
y (
%)
59
85
113
133 181 224
269
Neutral loss (176 Da)
O
OHHO
OH
COOH
445
Product ion spectrum of genistein glucuronide in ESI-MS/MS
Glucosides/glucuronides conjugates are easily
cleaved off by higher potential at orifice
Characteristic fragments
Aglycone
genistein
Parent
ion
What fragment ions are characteristics
for glucuronide conjugates?
J. Prasain 01/19/11
The loss of 80 Da from the parent ion and the presence of m/z 80
in the product ion spectra are the indicative of sulfate conjugates of
phenolic compounds like daidzein [A] and equol [B]
20 60 100 140 180 220 260 300
5.0e5
1.5e6
2.5e6
3.5e6
4.5e6
5.5e6
Inte
ns
ity, c
ps
116.9
253.0
134.9
252.0132.991.0224.0
207.980.0
96.9 225.0197.0160.0
[A] OHO
O
O
SO
O O-
m/z, amu
20 60 100 140 180 220 260 300 340
2.0e6
4.0e6
6.0e6
8.0e6
1.0e7
1.2e7
1.4e7
Inte
ns
ity, c
ps
121.0
119.0
135.0
79.9
93.0 241.0147.0
91.0 320.9
[B]
OO
OH
SO
O
O-
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What happens with aliphatic sulfates in MS/MS?
Aliphatic and aromatic sulfate conjugates behave differently in
MS/MS aliphatic typically show m/z 97 (HSO4-) and m/z 80 (SO3-.)
J. Prasain 01/19/11
Source: Weidolf et al. Biomed. and Environ. Mass Spec. 15, 283-289, 1988
The absence of the m/z 97 fragment with the base peak