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Quattro microTM
Operator Training Course
MS/MS Theory Presentation
(Neonatal application focus)
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Topics
Introduction to Mass Spectrometry
Electrospray Ionization (ESI)
Ion travel through the Mass Spectrometer
Quadrupole Theory
Mass Spectra and MS/MS Data Acquisition Mode
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What is Mass Spectrometry?
An analytical technique in which:
Gaseous Ions are produced from neutral molecules
Ions are separated according to their mass-to-charge
(m/z) ratio
Ions are detected and recorded as a plot of ion
abundance vs. m/z (mass spectrum)
m1m3m4 m2
m3
m1
m4
m2
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An analytical technique in which: Several mass spectrometers are serially linked
Conventionally, MS/MS consists of two massanalyzers separated by a collision cell
Ion
Source
MS 1
CollisionCell
MS 2
What is MS/MS or
Tandem Mass Spectrometry?
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How Does MS/MS Work?
Sample must be first ionized
Ionized sample is sent to first mass analyzer
Ionized sample mixture is sorted by m/z A single ion can be selected
Selected ions are sent to a collision cell Fragmentation of selected ions takes place
Fragment ions are sent to second mass analyzer Fragment ions are sorted by m/z
A single fragment ion can be selected
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What is an Ion?
Phe : Molecular Weight = 165.08
PheH+ : m/z = 166.08
MassAnalysis9 of C (12.000) = 108.000
1 of N (14.003) = 14.003
11 of H (1.008) = 11.088
2 of O (15.995) = 31.990
165.081
Protonated (Ionized) Phe
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Gas Phase Ion Fragmentation
AB+ A + B+
Precursor or
molecular
Ion
Neutral
Loss
CAD
Product
Ion
This fragmentation behavior allows forseveral types of scanning modes
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Tandem Quadrupole Instruments
MS1 Collision Cell MS2
MS1 is used to scan a
range of precursor ionmasses or to select aparticular precursorion mass and pass theions to the collision
cell
In the collision cell,
the ions from MS1collide with Argonatoms and fragmentinto daughter(product) ions
MS2 is used to scan
a range of daughterion masses or toselect a particulardaughter ion massand pass the ion(s)
on to the detector
In a triple quadrupole or tandem mass spectrometer,
MS1 and MS2 are mass analyzers that filter ions.
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Atmos-
pheric
Pressure
Create
GasPhase
Ions
Low
Vacuum
Region
Get Ions
into theMass
Spec
A Generic LC/MS Configuration
Ion
Source
Ion
TransportMass
AnalyzerDetector
High Vacuum
Region
Mass Analyze Ions
228m/z
162m/z
Minutes
1 . 00 2 . 00 3 . 00 4 . 00 5 . 00 6 . 00 7 . 00 8 . 00 9 . 00 1 0 .0 0
198m/z
Data
Liquid
from LC
SampleIntroduction
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Schematic Overview of the Quattro
microTM
10
-1
mbarRotary Pump
10-3 to 10-4 mbar
Turbo Pump 10-5
mbarTurbo Pump
RemovableSample
Cone
ESI
Probe
Transfer
OpticsRF Lens
Pre Filter
Quadrupole
MS1
Quadrupole
MS2
Hexapole
CollisionCell
Phosphor
PMT
ConversionDynode
Post Filter
Pre Filter
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Electrospray Ionization (ESI)
Electrospray Ionization (ESI)
Liquid is sprayed out of a capillary tube to which ahigh voltage is applied to form a spray of charged
droplets.
ESI is a type of Atmospheric Pressure Ionization (API)
since the ions are formed at atmospheric pressure
4H082004 Waters Corporation
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Liquid
Electrospray Plume
Stainless Steel CapillaryStainless Steel Tube
Electrospray Probe Tip
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Liquid
Nebulizer Gas
Nebulizer Gas Electrospray Plume
Electrospray Probe Tip
When a nebilizer gas (usually nitrogen) is used,
the electrospray process is often referred to as
Pneumatically Assisted Electrospray
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Liquid
Nebulizer Gas
Nebulizer Gas
Desolvation Gas
Desolvation Gas
Electrospray Plume
Electrospray Probe Tip
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Nitrogen
Nitrogen
Heater Wires
Heater Wires
Desolvation Gas Flow
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API Probes for Z SPRAYTMSource
Electrospray Probe
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High Voltage PowerSupply
+
2.5-4.0 kV
Counter Electrode
+-
Example of Positive Electrospray
Electrospray Ionisation
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-
--Liquid
Electrospray
Probe Tip
+ + + +
High Voltage
+ + + +
+
+ +
+
+-
+
++
+
++
+
+
+ -+
+
++
+
-
-+
+ +
+
+
--+
+ +
+
+ --+
+ +
+
+ --+
++--
-+- -
--+
--
-+
-+
-- -+-
-+ -+
-
--
+-
-+
- -+
--
-
+
Droplet Formation in Positive Ion
Electrospray
Taylor Cone
More Negative Ions
than Positive Ions
More Positive Ions
than Negative Ions
Positively
Charged
Droplets
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+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
SolventEvaporation
++
+
+
+
+
CoulombicFission
+
+
+
Electrospray Droplet Undergoing Fission
Charge resides on the surface of the droplet.
Solvent evaporates from the droplet and the droplet shrinks until
the charge density on the surface reaches a point where the
repulsive force between charges exceeds the liquid surface
tension that holds the drop together.
At that point, the drop fissions and a set of small droplets are
expelled from the main droplet.
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Electrospray Ions
Positive Electrospray Ions are produced by the addition of a
positively charged ion (e.g H+, NH4+, Na+) to a molecule.These positively charged ions that are added are oftenreferred to as adducts.
N N C H3
OC H 3
H
C H 3
O
OH
C H 3
CH 3
C H 3
O
O
C H3
CH 3
Negative Electrospray Ions are most often produced by theremoval of a proton (hydrogen ion) from a molecule.
+ H+
+ H+
Lidocaine
Ibuprofen
N N C H3
OC H 3
H
+
H
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ESI
Probe
Ions Enter the Z SPRAYTMSource
Ions
Ions created by theElectrospray probe
are drawn in through
the sample cone
along with nitrogengas (and some other
gases from the
mobile phase).
QuadsRF Lens
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Cone Gas
Plume of Ions,
Clusters, Neutralsand Stuff
The cone gas helps
create ions with fewer
clusters and helps
keep out neutrals
which yields better
S/N. Thus, less stuff
collects on the inner
Orifice Cone
ESI Probe
N2 N2
Cone Gas Cone Function
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Quattro microTM
:Sample Cone and Cone Gas Nozzle
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Z SPRAYTMSource - Electrospray
ExtractionCone
Nebulizer
Gas
Desolvation
Gas
Sample
Cone Gas
Exhaust
Sample Cone
Isolation
Valve
RotaryPump
Turbomolecular
Pump
RF Lens
Quads
Z SprayTM
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Spray = Neutral solvent
Evaporating (cone-shaped
region from initial spray)
Solution inlet
Sample
Cone
Skimmer
To analyzer
To analyzer
of mass
spectrometer
Extraction cone
Ion beam
Ion beam
Spray
Spray
Solution inlet
Why ZSpray?
In ZSpray
In a Z-Spray source,
more ions make it into
the mass spec
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Quattro microTM Z-Spray Source
Desolvation Heater Electrospray Probe
Isolation
Valve
Sample
Cone
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Ions Enter the RF Lens Region
As the ions pass by the
entrance to the RF Lens
region, the ions are
extracted from the gas
flow and accelerated by
the voltage difference andpressure difference
between the source
region and the RF Lens
region.
The source region is both
at a higher potential (cone
voltage) and pressure
than the RF Lens region.
ESIProbe
Sample Cone
and SourceBlock at Cone
Voltage
Extraction Cone and RF
Lens at Lower Voltages
and Lower Pressure
Ions at
AtmosphericPressure
QuadsRF Lens
To RoughPump
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RF Lens
Ions Pass Through the
Quadrupoles
As the ions pass from the source region, through the RF Lens and
quadrupoles, a series of potential differences and pressure drops (better
vacuum) help propel the ions through the middle of the RF Lens andquadrupoles and on to the detector.
Quads, Collision Cell
and Detector
ES or APcI Probe Sample Cone and Source
Block at Cone Voltage
Extraction Cone and RFLens at Lower Voltages
and Lower Pressure
Ions Analyzer Section (at Lowest
Pressure (Best Vacuum))
To Rough
Pump
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Quadrupole Theory
Quadrupole Theory
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Quadrupole Theory
A quadrupole mass analyzer is an assembly of four parallel rodsarranged equidistantly from a central (imaginary) axis.
Through the application of DC and RF (radio frequency) voltages,ions can be filtered along the central axis and their mass measuredto yield a mass spectrum.
Depending upon the exact potential applied to the quadrupole, ionswith masses too large or too small will not pass through thequadrupole. These ions will strike the rods and be lost.
End View
Q tt i TM RF T f
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Quattro microTM- RF Transfer
Lens
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RF Lens - Hexapole
End View
Note Voltage
Polarities
Hexapole Assembly
Radio frequency plus
a small bias voltage
transports all masses.
Designed to insure ion
focusing in a relatively
poor vacuum.
Delivers the ions in atightly focused beam
to the quadrupole
where they can be
analyzed.
RF Lens +
++
- -
-
Ions
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Trajectories of the Ions
As the ions pass down the middle of the quadrupleassembly, the ions are either pulled towards a rod orpushed away from the rod depending on the voltagesapplied to the rod
The trajectories of ions as they pass through thequadrupole assembly can be calculated
Since voltage applied to a quadrupole rod is a
combination of a constant voltage and an oscillating RFvoltage and there are two pairs of these rods, thiscalculation is very complicated so only a qualitativedescription of the calculation will be given
Q d l M A l
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Quadrupole Mass Analyzer:
RF Voltage
0
+V
-V
0
+V
-V
Each rod in a quadrupole is connected to the rod on the
opposite side. The RF voltage is applied 180 degrees
out of phase to each pair of rods.
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Quadrupole Analyzer
Pre-filter Quadrupole Mass FilterRejected Ions Ion with
Stable Trajectory
-1
0
If the correct voltages (DC and RF) are applied to the rods,
ions with the desired mass can pass through the rodassembly down the middle of the quadrupoles and reach
the detector. All other ions will spiral out and be lost. By
changing the voltages, different masses can be filtered
through the system to produce a mass spectrum.
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Quattro microTM- Ion Optical Rail
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Applied Potential to Rods
The voltage applied to an opposing pair of rods is given by:
f = U - V cos wT
DC Voltage RF Voltage
The voltage applied to the other pair of opposing rods is:
f = - U + V cos wT
Typically: DC Voltages (U) are in the range of 1000 V
RF Voltages (V) range from 1000 to 6000 V
RF frequencies (w) are around 1 MHz and fixed
Q d l O t d M Filt
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0
0.2
0.4
0.6
0.8
1
1.2
0 0.2 0.4 0.6 0.8 1 1.2
m3
m2
m1
0
1
200 250 300 350 400 450 500 550 600
m1 m2 m3
m/z
m/z: m3 > m2 > m1
U
(DC)
V (RF)
Quadrupole Operated as a Mass Filter
Optimum Quadrupole Operational Line
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Derivatization
A chemical modification during sample preparation to aid indata acquisition
The addition of a butyl group to the carboxylate functionality
of the analyte
The butyl ester that is formed aids in sample analysis by
forcing a permanent positive charge (acylcarnitines) or by
making the charging process more efficient (amino acids)
Butylation increases the non-polar character of the analytes
and lower polarity =better desolvation and better sampleintroduction to the vacuum environment
D i ti ti f A i A id d
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Derivatization of Amino Acids and
Acylcarnitines
H2N CH C
R
OH
O
CH3
H2C
CH2
H2C
HO
H
O
H
H2N CH C
R
O
O H2C
C
H2
H2C
CH3
+
+
H C l,H e a t
Aminoacid
(Freeacid)
A minoacidbutylester
R =AminoacidSideChain
Amino acids
1-Butanol CH3
H2C
C
H2
H2C
H3C N+
H3C
H3C
CH2
CH
CH2
O
C
O
O
H2C
CH2
H2C
CH3
CR
O
+ H O H
HC l,
He at
Acylcarnitine(Freeacid)
A cylcarnitinebutylester
HO+H3C N
+H3C
H3C
CH2
CH
CH2
O
C
O
OH
CR
O R =AcylChain
Acylcarnitines
1-Butanol
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Mass Spectra
and
MS/MS Data Acquisition Modes
2004 Waters Corporation4H08
MS/MS Data Acquisition Modes
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MS/MS Data Acquisition Modes
MS Mode MS1 Scan
MS/MS Modes
Daughter/Product Ion Scan
Parent/Precursor Ion Scan
MRM
Constant Neutral Loss
4H08
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How Does MS/MS Work?
Sample ionizationand introduction
Ion sorting
and selection
Ionfragmentation
Fragment Ion
sorting and selection
Iondetection
IonSource
MS 1
CollisionCell
MS 2
MS1 Scan
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MS1 Scan
MS1 Collision
Cell (No Argon)
MS2
m1m2
m3
RF
10 -100V
ScanningRF (+ DC)
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MS1 Scan
MS/MS Modes
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MS/MS Modes
MS1Collision
Cell (w/Argon)MS2
MS1 is used as amass selectorand allows ionsof a particular
mass to passinto the collisioncell
In the collisioncell, the ionsfrom MS1 collidewith Ar atoms
and fragmentinto daughter(product) ions.
MS2 is used as amass selectorand allowsdaughter ions of
a particularmass to pass onto the detector
In the collision cell, a potential is applied (typically 5-40 eV) to
control the energy of the collisions between the ions and Ar atoms.
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MS/MS Modes
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Quattro microTM MS/MS
Low energy collisions (simple fragmentationpathways)
Collision gas of choice is Argon
Collision gas pressure is normally fixed while the
collision energy is used to alter the degree of
fragmentation
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Daughter/Product Ion Scan
MS1 Collision
Cell (w/Argon)MS2
m1
5-40 eV Scanning
-5V
Fixed
1V m2
m1
m3Determines Collision Induced Dissociation (CID) produced daughter ions of a
particular parent ion
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Daughter ion scan- common MS/MS mode Select one mass in MS1 and send into the
collision cell and fragment, MS1 is fixed
MS2 scans the fragments for a given massrange
Used for structural information gathering
and identification of product ions
First step to developing quantitative
method
Daughter/Product Ion Scan
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Produc t Ion Scan
m1+
m2+
m2+
m2+
Product ion spectrum of a particular compound
m1+ set
m2+ scan
Daughter/Product Ion Scan
Daughter Ion Scan
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150 160 170 180 190 200 210 220 230 240 250 260 270 280 290m/z0
100
%
0
100
%
0
100
%
0
100
%
275
275230
230
275
230
MS/MS Spectra of Chlorpheniramine (MW=274)
Daughter Ions of m/z=275Collision Energy = 5 eV
Collision Energy = 10 eV
Collision Energy = 12 eV
Collision Energy = 17 eV
M+H
gEffect of Changing Collision Energy
Daughter Ion Scan
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150 160 170 180 190 200 210 220 230 240 250 260 270 280 290m/z0
100
%
0
100
%
0
100
%
230
167230
201180 202
167
201180 194 230
Collision Energy = 17 eV
Collision Energy = 30 eV
Collision Energy = 38 eV
M+H
gEffect of Changing Collision Energy(Cont.)
MS/MS Spectra of
Chlorpheniramine(MW=274)Daughter Ions of m/z=275
Parent/Precursor Ion Scan
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++CID
CID
++
DifferentNeutralFragments
Different CompoundsThat Are SomewhatSimilar In Structure
SameChargedFragment
Parent/Precursor Ion Scan
+
+Parent Ion Scans can be used to detect those compounds whosemolecular ions produce the same charge fragment.
Consider a class of compounds that are similar in structure:
P t/P I S
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Parent/Precursor Ion Scan
MS1 CollisionCell (w/Argon)
MS2
m2
5-40 eV Fixed
5V
Scanning
5V
m3m1
Find ions that will produce via CID, daughter ions with a particular m/z
P t/P I S
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Parent/Precursor Ion Scan
Select fragment at m/z x on MS2 (fixed) Scan MS1 for a given mass range
Observe signal from ions giving fragment of m/z x
(selected in MS2)
Used to determine the origin of particular production(s) created in the collision cell
Seen in newborn screening literature for
acylcarnitines
Parent/Prec rsor Ion Scan
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Precu rso r Ion Scan
m1+
m2+
m1+
m1+
A set of compounds with a common product ion
m1+ scan
m2+ set
Parent/Precursor Ion Scan
Parent Ion of 85 scan: Acylcarnitines
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CAD
H3C N+
H3C
CH3
CH2
HC
HC
C
O
OH
H3C N+CH3
CH3
CH2
CH
CH2
O
C
O
OH
CR
O
H3C N+
H3C
CH3
CH2
CH CH
O
C
O
OH
RC
O
H
OH
RC
O
H3C N
CH3
CH3
H2C CH
H2C
CH3
H2C+
CH
HC
C
O
OH
H3C N+
CH3
CH3
CH2
CH
CH2
O
C
O
O
H2C
CH CH2
CH3
CR
O
H
H3C N+
CH3
CH3
CH2
CH
CH2
O
C
O
O
H2C
CH2
H2C
CH3
CR
O
H2C
HC CH
C
+O
O
H
Acylcarnitine butyl ester
derivative
Loss of 1-butene by
1,4 Hydrogen
rearrangement
Loss of faty acid
functionalityby
1,4 Hydrogenrearrangement
Loss of trimethyl amineby-cleavage
(heterolytic cleavage)
Underivatized
Acylcarnitine free acid
Faty acid1-Butene
Trimethyl amineCommo n Fragment Ion
m /z 85
Oxonium Ion
Carbonium Ion
R = Acyl Chain
CAD
Parent Ion of 85 scan: Acylcarnitines
Analysis of Acylcarnitines by MS/MS
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Analysis of Acylcarnitines by MS/MS
- Parent Scan
Neutral Loss (NL) Scan
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++
CID
CID+
SameNeutralFragment
Different CompoundsThat Are SomewhatSimilar In Structure
DifferentChargedFragments
+
Neutral Loss (NL) Scan
+
+
Neutral Loss Scans can be used to detect those compounds whosemolecular ions produce the same neutral fragment.
Neutral Loss (NL) Scan
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Neutral Loss (NL) Scan
MS1 CollisionCell (w/Argon)
MS2
m2
5-40 eV Scanning
-5V
Scanning
1Vm1 - offsetm
1
m2 - offset
Q1 and Q2 scan together. m/z of Q2 is m/z of Q1 minus an offset.
Neutral Loss (NL) Scan
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Neutral Loss (NL) Scan
Neutral loss scan (Example NL102) MS1 & MS2 both scan a given mass range but with
a constant offset (difference) between rangesscanned
Spectra indicate which ions lose a neutral speciesequal to MS1 MS2 difference
Seen in newborn screening literature for aminoacids
Complement to Parent/Precursor Ion Scan
N t l L (NL) S
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Cons tant Neutral Loss Scan
m1+
m2+
m2+ m1
+
A set of compounds with a common neutral fragment
m1+ scan
m2+ scan
m-m
-m
Neutral Loss (NL) Scan
NL of 102 Scan: Amino Acids
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NL of 102 Scan: Amino Acids
H2N
CHC
R
O
O
CH2
H2C
CH2
CH3
H2N+
CH
R
CO
O
CH2
H2C
CH2
CH3
H
H3N+
CHC
R
O
O
CH2
H2C
CH2
CH3
H2N+
CHC
RO
O
CH2
H2C
CH2
CH3
H
AA-Butyl esterDelivered to MS by LC System
+ H+
Protonation,Takes place
in Ion Source
Protonated Precursor Ion
Mass Selected by Q1
Collisional
Activation
with N2
Collisionally ActivatedPrecursor Ion
(transition State)
Fragmentation
Take place inside
collision cell
+
Neutral loss of28 + 74 = 102
+
Fragment IonMass selected
by Q3
Analysis of Amino Acids by
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OHN
O
ON
O
OH
Phenylalanine
Tyrosine
y y
MS/MS NL
Analysis of Amino Acids by
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ON
O
OH
ON
O
OHN
O
ON
O
OH
Deriv
Phenylalanine
Tyrosine
Deriv
m/z = 222
m/z = 238
y y
MS/MS NL
Analysis of Amino Acids by
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ON
O
OH
ON
O
N
OH
NOH
N
O
ON
O
OH
Deriv C.I.D.
Phenylalanine
Tyrosine
DerivC.I.D.
CID Results in the Loss of 102 Da
m/z = 222
m/z = 120
m/z = 238
m/z = 136
H
y y
MS/MS NL
Analysis of Amino Acids by
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Derivitzed Amino Acid Mix Infused 10 L/min Quattro micro
180 185 190 195 200 205 210 215 220 225 230 235 240 245 250m/z0
100
%
NeoLynxTestMix_CNL 1 (0.510) Neutral Loss 102ES+
4.28e7191.1188.0
222.1
209.0
206.1
227.2
238.1240.1
Phenylalanine
Tyrosine
Methionine
Leucine
Other peaks are from deuterated forms of these amino acids
MS/MS NL
Multiple Reaction Monitoring (MRM)
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Multiple Reaction Monitoring (MRM)
MS1 Collision
Cell (w/Argon)MS2
m1
5-40 eV Fixed
-5V
Fixed
1V
mx
MRMs are used to monitor selected analyte(s) via their daughter ions
Multiple Reaction Monitoring (MRM)
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Multiple Reaction Monitoring (MRM)
Select a particular mass transition, both MS1 and MS2are fixed
Measure ion intensity from that single mass transition
Can cycle through many transitions during course of
measurement
Measure only what you set up to see
Time is spent measuring only desired signals
More signal per transition, best way to maximize
signal intensity of product ions
Multiple Reaction Monitoring (MRM)
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Precursor ionset
Product ionset
Fragmentation(CID)
Multiple Reaction Monitoring (MRM)
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