intro to ms nrao
TRANSCRIPT
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Introduction
toQuadrupole Mass
Spectrometry
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Components of a Mass
Spectrometer
SampleIntroductionMethod to vaporize sample
Inlet
Ionization/ desorption
Analyzer
Mass Sorting
Ion Detection
Detection
Data Analysis
Source
+
1330 1340 1350
100
75
50
25
0
Solid
Liquid
Vapor
Detect
ions
Form ions(charged
molecules)Sort Ions by Weight
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LC-MS Components
Sample introduction
e.g., FIA, HPLC
Sample ionization Nebulizer System
Sample transfere to high vacuum regionAPI Interface
Ion mass-to-charge filtering Mass Analyzer
Ion detection Detector
Data acquisition and analysis
Data System
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Ionization
Techniques
ElectronImpact
(EI)
ChemicalIonization
(CI)
APIMALDI
Hard, Fragments
Fast AtomBombardment
(FAB)
Soft, Intact
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LC-MS Sample Inlets
Themost common sample inlets for LC-MS are
Atmospheric Pressure Ionization (API)
Sources
Two distinct types of API sources:
Heated Nebulizer Source
ElectroSpray Source
IonSpray
TurboIonSpray
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Heated Nebulizer (APCI)
Heated Nebulizer:
Atmospheric Pressure Chemical Ionisation (APCI)
corona discharge needle
polar to unpolar and thermally stable compounds
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Heated Nebulizer (APCI)
Heated Nebulizer:
Atmospheric Pressure Chemical Ionisation (APCI)
corona discharge needle
polar to unpolar and thermally stable compounds
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Heated Nebulizer (APCI)
Heated Nebulizer:
Atmospheric Pressure Chemical Ionisation (APCI)
corona discharge needle
polar to unpolar and thermally stable compounds
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Heated Nebulizer (APCI)
Heated Nebulizer:
Atmospheric Pressure Chemical Ionisation (APCI)
corona discharge needle
polar to unpolar and thermally stable compounds
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Heated Nebulizer (APCI)
Heated Nebulizer:
Atmospheric Pressure Chemical Ionisation (APCI)
corona discharge needle
polar to unpolar and thermally stable compounds
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Heated Nebulizer
1st picture inside of APCI/HN, with corona needle offset between orifice andquartz tube
2nd picture back of APCI/HN unit with articulations for the HN on back and
the corona needle on top.
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Heated Nebulizer (APCI) Inlet
Suitable for polar, thermally stablecompounds
Usually, Molecular Weight < 1000 amu
Probe is heated to facilitate vaporization
Requires nebulizer and auxiliary gas
Requires corona discharge needle to produce
ionization (APCI)
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ElectroSpray Inlet
(Ion Evaporation)
++++
+ +++ +
+++
++
Liquid
5 kv
++ +++++++++++
+
+
+
+++
++
++
Droplet
Formation
Droplet
Evaporation
Coulomb
Explosion
Ion Evaporation
Liquid Phase Ionization
MolecularIon
H+
++ +++
++++++
++++++
+++
+++
+++
+++
+++
+++
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TurboIonSpray Inlet
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TurboIonspray Source
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TurboIonSpray Inlet
Pros:
Hot drying gas improves evaporation of charged
droplets
no thermal degradationbetter sensitivity at higher flow rates
tolerates higher aqueous solvent compositions
Improved performance over a wide range of LC flow
rates without splitting (5L/min to 1mL/min)
Detection limits are very good
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Photo Ionization Source
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Operating Principle
Samples are sprayed with the aid of a nebulizing gasinto a heated probe, as in APCI.
A dopantcompound is vaporized in the auxiliary gaswithin the heated nebulizer probe.
In the ionization region, dopant molecules arepredominantly ionized by UV radiationand formphotoions.
The photo ionsinitiate a cascade of ion-moleculereactions leading to the formation of the ionizedanalytes in the form [MH]+ (by proton transfer) or [M]+(by charge transfer).
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The role of the Dopant
The dopant acts as an intermediate in the
ionization process
Opens up an alternative reaction channel
Increases analyte ionization efficiency
Two dopants were extensively tested
Toluene
Acetone
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PhotoIonization lamp
Krypton lamp
10 eV
CompoundIonizationPotential(eV)
Nitrogen15.58Water12.62
Acetonitrile12.20Oxygen12.07Methanol10.84
MethylPentanoate10.40Hexane10.13Heptane9.93Acetone9.70Pyridine9.26Benzene9.24
Amphetamine8.99Toluene8.83
Naphtalene8.14Reserpine7.88
Triethylamine7.53
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Ion Source Schematics
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PhotoSprayTMSource Block
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PhotoSpray Source
Pros: Entire mobile phase and sample vaporized into the gas phase (with
heat), then ionized
Uses Dopant (Toluene/Acetone) to promote ionization
Accommodates high LC flow from (0.2 - 2 mL/min)
Uses heat (400500 C) & nebulizer gas to vaporizeHPLC eluent and steam distill sample into the gasphase for photoionization
Detection limits are very good
Improvements over ESI and APCI compound dependant
Wider application range
Cons: Thermally labile analytes may degrade when vaporized
Low mol weights only (
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PhotoSpraysource vs APCI vs
TurboIonspray
Sensitivity
Polar compounds: TIS > APCI > PhotoSpray
Non Polar compounds: PhotoSpray > APCI > TIS
Chemical Background
TIS > APCI > PhotoSpray
Suceptibility to the MP compositionPhotoSpray > TIS > APCI
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Ion Sources Sensitivity
SampleCharacteristics
ESI APCI PhotoSpray
Ionic
Ionizable
Polar Non-ionic
Nonpolar
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Ion Sources Characteristics Molecular
Weight Consideration
ChemicalProperties
ESI APCI PhotoSpray
Ionic Ionizable Polar Non-ionic Nonpolar - - molecular weight >100000
>2000 >1000 >500
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PhotoSprayTMPotential Application
Areas
Pharmaceutical Companies/CROs
Steroids/Hormones/Apolar Vitamins
Relative Apolar drugs
Alternative Markets
Petroleum/Oil Industry
Polymer Analysis
Food Analysis
Sugars
Carotenoids
Essential Oils
Environmental Analysis
PAHs; PCBs; Dioxines
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PhotoSprayTMPotential Application
Areas
Clinical
Vitamins
Food Analysis
Sugars, Carotenoids. Essential Oils, Flavonoids
Environmental Analysis
PAHs; PCBs; Dioxines, Pesticides
Petroleum/Oil Industry
Polymer Analysis
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PhotoSprayTMPotential Application
Areas
Prohibited classes of substances
Stimulants
Narcotics
Anabolic Agents
Anabolic androgenic steroids
Beta-2-agonists
Diuretics
Peptide hormones, mimetics and analogues
Masking agents
Beta-Blockers
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API Inlets
Heated Nebulizer, TurboIonSpray and Photo
Ionization Inlets are soft ionization sources
They produce abundant molecular or protonated ions
with little or no fragmentation
Ideally suited for high sensitivity (quantitative)
analyses
Qualitative analyses can be enhanced with the
presence of characteristic fragment ions
I T f f At h t
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Ion Transfer from Atmosphere to
a High Vacuum Environment
2 Tor Declust. RegionProblem of all conventionalinterface designs:
Matrix, e.g., salt praticles, may
cause clogging of the orifice
and/or form deposition inside the
interface or further downstream
How to overcome this problem:
Off-axis spraying
Z spray
Orhtogonal sprayingStill not good enough
Clustered
I ons
Declustering
on
Off -axis spray
direction
Th API P t t d C t i G
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The API Patented Curtain Gas
Interface
2 Tor Declust. RegionAll AB Sciex LC/MS use thepatented Curtain Gas Interface
Combination of simple off-axisspraying with active gas protection
Simple design
Easy to optimize
Curtain gas protects MS fromcontamination
dirt forms deposition off-axis oncurtain gas plate
Matrix particles (neutrals)can not pass N2pressure barrier
reduces matrix induced drift to aminimum
very infrequent cleaning
CurtainGas
Clustered
I ons
Declustering
on
Off -axis spraydirection
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1. Curtain Gas assists in droplet evaporation
ATMOSPHERIC PRESSURE VACUUMCUR DP SK
FP
N2
+ ++
+
+
+
How the curtain gas works
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1. Curtain Gas assists in droplet evaporation
ATMOSPHERIC PRESSURE VACUUMCUR DP SK
FP
N2
+
+ +
+
++
How the curtain gas works
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1. Curtain Gas assists in droplet evaporation
2. Curtain Gas declusters ions
ATMOSPHERIC PRESSURE VACUUMCUR DP SK
FP
N2
+
How the curtain gas works
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1. Curtain Gas assists in droplet evaporation
2. Curtain Gas declusters ions
ATMOSPHERIC PRESSURE VACUUMCUR DP SK
FP
N2
+
How the curtain gas works
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1. Curtain Gas assists in droplet evaporation
2. Curtain Gas declusters ions
ATMOSPHERIC PRESSURE VACUUMCUR DP SK
FP
N2
+
How the curtain gas works
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State-Of-The-Art API Interface
N2gas curtain and orificevoltage assist in
declustering ions
Increasing orifice voltage can
cause analyte fragmentation(compound dependant)
advantageous with Single
Quad MS for achieving
structural information
A Vi B hi d th API I t f
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A View Behind the API InterfaceThe RF0 Ion Guide
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Ion Sampling Region
Expansion of gas and ions into vacuum
random
motion
of
ions andgas
760 torr 1 torr
250 micronorifice
atmosphere vacuum
barrel shock
silent zone
-mach disk-
gas velocity breaks
sound barrier
ion motion
alligned and
confined to
gas
4.5 mm
Different Ion Sampling
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Different Ion Sampling
Approaches
ions
gas
opticsRf onlyquadrupole
ions
highly charged
clusters
ions
opticsRf only quad
gas
skimmer
ring
electrode
ions
highly charged
clusters
ions
gas
ions
highly charged
clusters
focusing
ring
A. On Axis Sampling
B. Off Axis Sampling
C. L (dog leg) Sampling
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Patented RF Ion Guide (Q0)
Ion beam expands behind Skimmerdifferential pressure difference
space charge RF Ion Guide prevents loss of ions and focuses them into and
through Ion Guide
8 mTorr Ion Guide region promotes collisional focussing
+
+
SkimmerOrifice
Patented Collisional Focussing
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Patented Collisional Focussing
vs. Conventional Ion Guides
Collisional Focussing
dampens beam
amplitudeFocussed ion beam
improved transmision
Conventional Ion
Guides
poorer ion transmisson(ions are lost)
Sensitivity of Ion Transmission vs
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Sensitivity of Ion Transmission vs.
Q0 Pressure
M A l
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Mass Analyzers
All mass spectrometers sort ions based on their mass-to-
charge ratios (m/z) in a vacuum
Common analyzer types:
Quadrupole
single quadrupole
triple quadrupole
Time of flight
Ion trap
Magnetic and electric sector
Fourier transform ion cyclotron resonance
Quadrupole Fundamentals
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Quadrupole Fundamentals
Mass
Voltage
-DC RF amplitude
Voltage
Mass
+DC RF amplitude
r0
+
+
A quadrupole is a tunable band-pass filter
RF/DC ratio is constant
filter is tuned by raising
voltage amplitude
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Selecting Quadrupole Resolution
DC
RF
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+
+
+
1. Ion enters the quadrupole system
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+
+
+
2. Electrical repulsion and attraction,
respectively, between quadrupole rods and ion
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+
+
+
3. Movement of the ion into direction of the
nearest quadrupole rod with the opposite charge
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+
+
+
4. RF-voltage changes polarity and electrical repulsion
and attraction, respectively, between quadrupole rods
and ion
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+
+
+
5. Movement of the ion into direction of the nearest
quadrupole rod with the opposite charge
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+
+
+
6. RF-voltage changes polarity and electrical repulsion
and attraction, respectively, between quadrupole rods
and ion
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+
+
+
7. Movement of the ion into direction of the nearest
quadrupole rod with the opposite charge
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+
+
+
8. RF-voltage changes polarity and electrical repulsion
and attraction, respectively, between quadrupole rods
and ion
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+
+
+
9. Movement of the ion into direction of the nearest
quadrupole rod with the opposite charge
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Start of the animation:How is the oscillating RF voltage working?
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Quadrupole Fundamentals
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Stable & Unstable Ion Trajectories
At any RF/DC potential setting only ions of one particular
mass/charge ratio have a stable trajectory
Scanning Using a Quadrupole
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Mass Filter
3 7 4 . 0
4 8 2 .5
263.3
347.3
459.4
518.5300 360 420 480 540
m/z, amu
10
20
30
40
5060
70
80
90
%I
ntensity
2 29 1 .2 7 .24
Pi t f Q d l
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Picture of a Quadrupole
++ +
-
-
Single Quadrupole MS
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(generic API 150 schematics)
- Patents numbers 4,963,736 and 5,179,278
RF-only Ion Guide Filt. QuadStubbies
8 mT Ion-Guide Region 2x10-5 Tor MS Region2 Tor Declust. Region
Curtain Gas
Clustered
Ions
on
Declustering MS-filtrationFocusing Extraction
Patented Curtain Gas interface
Patented collisional focussing quadrupole ion guide
Schematics of a Triple
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Quadrupole MS
RF Ion Guide: Focusses ion beam into Q1
Q1: Analyzer Quadrupole 1can be scanned or set to pass one specific m/z
Q2: Collision Cell
can be pressurized with N2for fragmentation of ionscan be at low pressure for bandpassing all ions
Q3: Analyzer Quadrupole 3
can be scanned or set to pass one specific m/z
N2 Inlet
RF
Ion Guide
Q1 Q2
Coll is ion Cel l
Q3 Detector
Fragmentation in a Tripple
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Quadrupole MS
precursor ion
molecular weight
information
possible fragment ions
structural information
Cross Talk in a Conventional High
P C lli i C ll
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Pressure Collision Cell
Collision cell transit time: 50-100 msec
(conventional design)
Cross-talk between consecutivelymeasured MRMs with same Q3mass
Q1 mass Q3 mass Time
( msec )
136.1 43.1 45.0
136.1 51.1 45.0
189.2 56.1 45.0232.2 56.1 45.0
204.1 56.1 45.0
246.1 56.1 45.0
218.1 56.1 45.0
152.1 59.1 45.0
Cross Talk in a Conventional High
P C lli i C ll
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Pressure Collision Cell
The Problem:potential systematic errors
fragment ions having identical mass but differentprecursors can not be differentiated
sensitivity loss when using very short dwell times forMRM
Scan speed and number of analytes/sample(throughput) must be reduced
Solutions:
Software or firmware (clean pulse)work-arounds
LINAC (AB Sciex Patent)
API Linac Collision Cell
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(Linear Accelerator, Patented)
y
x
y
x
X < y X > y
+5.75 v +4.25 v+1.5v field gradient
entrance exit
Q2 Linac (linear accelerator) eliminates cross-talk
allows faster MS/MS scanning withoutsensitivity loss
API Linac Collision Cell
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(Linear Accelerator, Patented)
Q2 Linac (linear accelerator) eliminates cross-talk
allows faster MS/MS scanning withoutsensitivity loss
Q2 rods are tilted and separate DC potentials are appliedto each pair of rods to create an axial electric field
gradient
Ions exit collision cell much faster
potentialgradie
nt
ionvelocity collisions with N2
reduce ion ion velocity
Cross-Talk without Linac
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Cross Talk without Linac
3.67e4 cpsXIC of +MRM (4 pairs): for 136.1 / 91.1 amu from Seleg 2500pg No-LINAC
Dwell 100msec
0.5 1.0 1.5 2.0Time, min
20
40
60
80
%I
ntensity
1.04e6 cpsXIC of +MRM (4 pairs): for 188.2 / 91.1 amu from Seleg 2500pg No-LINAC
0.5 1.0 1.5 2.0Time, min
20
40
60
80
%I
ntensity
25 ng Selegiline
Cross-talkfor amphetamine
100
100
Injection of selegiline to determine the level of cross-talk in
the amphetamine MRM transition
Different precursor masses, same fragment mass
No Cross-Talk with Linac
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No Cross Talk with Linac
Injection of selegiline to determine the level of cross-talk in the
amphetamine MRM transition with a Linac Q2
8.66e3 cpsXIC of +MRM (4 pairs): for 136.1 / 91.1 amu from Seleg 2500pg w LINAC
0.5 1.0 1.5 2.0Time, min
2000
4000
6000
8000
Intensity,cps
3.76e6 cpsXIC of +MRM (4 pairs): for 188.2 / 91.1 amu from Seleg 2500pg w LINAC
0.5 1.0 1.5 2.0Time, min
1e6
2e6
3e6
Intensity,cp
s
25 ng Selegiline
Cross-talk for amphetamine
No Cross-Talk with Linac
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No Cross Talk with Linac
0.5 1.0 1.5 2.0 2.5 3.0Time, min
10
20
30
40
50
60
70
80
90
%I
ntensity
10025
msec
50mse
c
100msec
250msec
Selegiline
Selegiline response using different MRM dwell times
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MSMS scanning in an
Quadrupole LCMSinstrument
Q1/Q3 Scanning Modes
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Q1/Q3 Scanning Modes
Q1 Full Scan(StartStop):Q1 always used as single MS analyzer
Used primarily for identification of precursor ion
Q3 operates in RF-only mode
Q3 transmits all ions to detectorQ3 acts as ion guide for product ions
In all MS/MSmodes, Q3 is a mass filter
Ions are separated based on mass/charge ratio
Q2 is source of product ions entering Q3
N2 Inlet
RFIon Guide
Q1 Q2Coll ision Cell
Q3 Detector
Single MS Operation
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Selected Ion Monitoring (SIM) (Width = 0):Used to optimizeanalyzer for specific ions
SIM used for quantitative analyses
Q1 SIM used to optimize precursor ion
Maximize signal in preparation for MS/MS
MS/MS - Product Ion Scan
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After identification, the precursor ion is sent into the
collision cell and fragmented
Q1 is fixed, Q3 scans a defined mass range
Provides structural information and identification of product
ions
Product Ion Scan
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Select
Precursor Ion
Scan ProductsCAD
m1
+
m2+
m2+
m2+
Product ion spectrum
of a particular compoundm1+ set
m2+ scan
Quadrupole 1 Coll is io n Cell (Q2) Quadrupole 3
MS/MS - Product Ion Scan
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After identification, the precursor ion is sent into the
collision cell and fragmentedQ1 is fixed, Q3 scans a defined mass range
Provides structural information and identification of
product ions
MS/MS - Precursor Ion Scan
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Precursor ion scan
Q1 scans a defined mass range, Q3 is fixed
Used to determine the origin of particular
product ion(s) created in the collision cellFrequently used for drug metabolite identification
(common product ion observed in the
metabolites)
MS/MS - Precursor Ion Scan (cont.)
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MS/MS Precursor Ion Scan (cont.)
MS/MS Constant Neutral Loss
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Neutral loss scan:
Q1 & Q3 both scan a given mass range but with a
constant difference between the ranges scanned
Spectrum indicates which ions lose a neutral species
equal to Q1 - Q3 difference
Complement to Precursor Ion Scan
Neutral gain indicates a multiply charged precursor
ion was fragmented
MS/MS Constant Neutral Loss (cont.)
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MS/MS Constant Neutral Loss (cont.)
MS/MS - Multiple reaction Monitoring (MRM)
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MS/MS Multiple reaction Monitoring (MRM)
If Q1 and Q3 width=0, then MRM
Many precursor to product ion pairs can be
monitored
MRM analysis is the best way to maximizesignal/noise ratio of compounds
MRM used primarily for quantitation studies
Multiple Reaction Monitoring (MRM)
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p g ( )
Precursor
ion set
Product
ion setFragmentation
(CAD)
Ion Detection
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Detectors Channel Electron Multiplier (CEM)
Discrete Dynode Detector
Data Acquisition
& Post Analysis Evaluation
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& Post Analysis Evaluation
Two computer and software platforms are offered
Windows NT and MAC
Automatic Resolution
Optimization
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Optimization
Ability to pre-define Unit,High and Low Resolution
Auto Resolution
optimization and Auto-Calibration
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