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1
J. Prasain 01/19/11
Ion fragmentation of small
molecules in mass
spectrometry
Jeevan Prasain
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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Contd..• Pseudomolecular ion: Ion originating from the analyte
molecule by abstraction of a proton [M-H]- or addition of proton [M+H]+
• Tandem mass spectrometry (Cooks, 1976): MS/MS (McLafferty, 1978), tandem in space or time
• Precursor ion/parent ion: Ions undergoing fragmentation.
• 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
m/z 80 makes the distinction between aromatic and
aliphatic sulfates
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Change in mass
Methylation 14
Demethylation -14
Hydroxylation 16
Acetylation 42
Epoxidation 16
Desulfuration -32
Decarboxylation -44
Hydration 18
Dehydration -18
Metabolic rxn
Change in mass is associated with possible
metabolic reaction
J. Prasain 01/19/11
Ionization mode Scan
Glucuronides pos/neg NL 176 amu
Hexose sugar pos/neg NL 162 amu
Pentose sugar pos/neg NL 132 amu
Phenolic sulphate pos NL 80 amu
Phosphate neg Precursor of m/z 79
Aryl-GSH pos NL 275 amu
Aliphatic-GSH pos NL 129
taurines neg Precursor of m/z 124
N-acetylcysteins neg NL 129 amu
NL = neutral loss.
Conjugate
Characteristic fragmentation of drug
conjugates by MS/MS
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20 60 100 140 180 220 2605.0e5
1.5e6
2.5e6
3.5e6
4.5e6
5.5e6
6.5e6
Inte
ns
ity, c
ps
107.1
135.1
159.0
133.0147.1
109.1
173.1
121.2
145.1
93.181.1
How steroids get fragmented in MS/MS?
+OH2
HO
HO
+
m/z 159
+HO
m/z 107
20 40 80 120 160 200 240
5.0e5
2.5e6
4.5e6
6.5e6
8.4e6
Inte
nsity
, cps
133.1
159.1
157.1
197.1
107.1 145.181.1 213.1 253.2183.0131.1 198.193.1 121.2
171.1
141.1105.1 179.1147.179.1
HO
O
OH+
+OH2
m/z 135
Estradiol
m/z 273
Estrone m/z 271
J. Prasain 01/19/11
Estradiol Standard Curve 0.05 – 25 µM
r = 0.9959Untitled 1 (Estradiol 273.2 / 107.1): "Quadratic" Regression ("1 / (x * x)" weighting): y = -215 x̂ 2 + 1.7e+004 x + -104 (r = 0.9959)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Concentration, uM
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
Are
a,
cou
nts
Sensitivity is an issue in quantification of steroids
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Derivatization of estradiol with dansyl chloride
leads to the formation of E2-dansyl (m/z 506)
Source: Nelson et al. Clinical Chemistry, 2004
Easy
protonation
J. Prasain 01/19/11
Time, min
1 3 5 7 90
40
100
160
220
280
340
400
460
Inte
nsity
, cps
5.62
5.806.26
6.62 7.807.095.50 7.53 8.005.22 8.494.884.37 8.63
Representative MRM chromatogram (mass transition 506/171)
obtained from 50 picomole concentration of dansylated E2
Does derivatization help increase sensitivity?
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Calibration curve for dansylated E2 showing linearity
from 50 picomole to 100 nanomole concentration range
(r = 0.999)
0 10 20 30 40 50 60 70 80 90 100
Concentration, nM
5.0e5
2.0e6
3.5e6
5.0e6
6.5e6
8.0e6
Are
a, c
ounts
J. Prasain 01/19/11
O
OH
O
O
O
OHOH
CH2OH
OH
O
OH
HO
O
OHO
CH2OH
Puerarin; mol. wt. 416 Daidzin; mol. wt. 416
OH
HO
Can MS/MS analysis help distinguish isoflavone
glucosides?
The Kudzu as a
source of isoflavones
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O
OH
HO
O
OHO
HO
CH2OHOH
O
OH
O
O
O
OHOH
CH2OH
OH
Puerarin
m/z 415
O- and C-glucosides fragment
differently in ESI-MS/MS
Prasain et al. J. Agric. Food Chem. 2003
220 280 300 320 360 380m/z
0
%
0
100
%
255.050
256.057
297.043
267.037
268.041281.051
307.065321.046
363.046335.061
351.044381.055
100
240 260 340
[A]
[B]
-162 Da
Yo+
-120 Da
Daidzin m/z 415
J. Prasain 01/19/11
Possible product ions of puerarin in ESI-MS/MS
in negative ion mode
O
O-
HO
O
OH
O
O-
HO
O
O
O-
HO
O
HO
HO
O
O-
HO
O
OHO
CH2OH
OH
HO
m/z 415
m/z 325
-90 Da
m/z 295
-120 Da
m/z 267
-28 Da
Prasain et al. J Agric Food Chem. 51, 4213, 2003.
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J. Prasain 01/19/11
m/z
100
50
0
100 200 300 400
m/z
100
50
0.0
Rel. I
nt.
(%
)253
109
161
224
253
271
433
433
180 Da
162 Da
Neutral losses (162 and 180) is useful in deciding
whether sugar is attached to an aromatic ring or not
O
O-
HO
O
O
OHO
OH
HO
CH2OH
O
O-
O
O
OH
OHO
HO
HO CH2OH
162 Da
Possiblestructure
Hexose attached
To the ring B
162 loss is an indicative
of aromatic attachment
J. Prasain 01/19/11
m/z
100
50
0
50 100 150 200 250m/z
100
50
0
Rel. I
nt.
(%
) 65107
117
151
225
269
63
91107
133
159 180196
224
240
269O
OH
HO
OOH
Genistein
O
OH
HO
OOH
Apigenin
O
O-
O-
149
Isomers like genistein and apigenin are readily
separated by tandem mass spectrometry
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J. Prasain 01/19/11
M/z
100
50
0
50 100 150 200 250 300
100
50
0
63 107 139
167
183
195211 239
267
303
139
167
195
223
251
287-36
-28
-28
-28
-28
-36
-28
-28
O
OO_
HO
Cl
O
OO_
HO
Cl
OH
Neutral loss of HCl (36 Da) is diagnostic for 3’-chloro
derivative of genistein and daidzein in ESI-MS/MS
but not in 8-chloro derivatives
J. Prasain 01/19/11
Comparison of product ions help
elucidate the unknown structures
O
OOH
HO
O
HO
OH
OH
H2C
OH
+OH
150 200 250 300 350 400
100
%
255268
282
311
-120431
Monohydroxylated
puerarin
200 250 300 350 400
100
50
0
267 295
\
415
-120
Puerarin
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Fragmentation of taxoids in ESI-MS/MS
J. Prasain 01/19/11
Fragmentation of basic taxoids from T.
Wallichiana extract
Prasain et al. Anal Chem, 2001
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ESI-MS/MS spectra of taxoids (1-3). Peaks m/z 194
and 210 represent the intact alkaloid side chain.
Alkaloid
Side chain
m/z 210
Diterpenoid
Scaffold
Loss of 60 or
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J. Prasain 01/19/11
References
1. Electrospray Ionization Mass Spectrometry by Richard B.
Cole.
2. Stefanowicz P, Prasain JK, Yeboah KF, Konishi Y. Detection
and partial structure elucidation of basic taxoids from
Taxus wallichiana by electrospray ionization tandem mass
spectrometry. Anal Chem. 2001;73:3583-9.
3. Prasain JK, Patel R, Kirk M, Wilson L, Botting N, Darley-
Usmar VM, Barnes S. Mass spectrometric methods for the
analysis of chlorinated and nitrated isoflavonoids: a novel
class of biological metabolites. J Mass Spectrom.
2003;38:764-71.
4. William Griffiths. Tandem mass spectrometry in the study of
fatty acids, bile acids and steroids. Mass Spectrometry
Reviews, 2003;22:81-152.