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Introduction to Mass Spectrometry
BioAnalytical Technologies (I) Pvt. Ltd.
Navnath Jaybhaye Manager- Product development & Application
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Agenda
Introduction Overview of MS Ionization Techniques Mass Analyzers Quadrupole Operations Applications Of LCMS
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Analytical Assays used in PharmaceuticalIndustry Labs for New Chemical Entities
0
98%
0
2%
2006
10%10%10%Immunoassay(ELISA/FPIA etc.)
60-75%40-50%3%LC/MS/MS
2%3%12%GC/MS
20%50-60%75%HPLC(UV &Fluorescence)
200019981990Method
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Mass Spectrometers
Separate and measures ions based on their mass-to-charge (m/z) ratio.
Operate under high vacuum (keeps ions from bumping
into gas molecules) Key specifications are resolution , mass measurement
accuracy , and sensitivity .
Several kinds exist: for analysis, quadrupole, time-of-flight (TOF) and ion traps are most used.
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InletInlet DetectDetectMass
AnalyzeMass
AnalyzeIonizeIonize
MSMS
InletInlet FragmentFragmentMass
Analyze
Mass
AnalyzeIonizeIonize
MassAnalyze
MassAnalyze
DetectDetect
MS1MS1 CollisionCell
CollisionCell
MS2MS2
MS/MSMS/MS
MS vs. MS/MS
GCHPLCCE
Separation Identification
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CH3COCH3CH3COCH3
SampleInlet
SampleInlet
CH 3+COCH 3CH3+COCH 3
Ionization& Adsorption
of Excess Energy
Ionization& Adsorption
of Excess Energy
Mass AnalysisMass Analysis
CH 3C+OCH 3CH3C+OCH 3
+COCH 3+COCH 3
+CH 3+CH3+COH+COH
Fragmentation(Dissociation)Fragmentation(Dissociation)
DetectionDetection
Mass Spectrometry
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Components of Tandem Mass Spectrometer
CollisionCell
MassSpectrometer
MassSpectrometer
Detector
Ionization Source
ESIAPPIAPCIMALDI
ArgonXenon
QuatrupoleMagnetic Sector
Quatrupole
Magnetic SectorTime-of-flight
Collisioncell
MS1MS2
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Sample introduction
Ion Souce Transforms sample molecules to ions Soft ionization
Places positive or negative charge on the analyte withoutsignificantly fragmenting the analyte M+1 ion (or M-1 ion) No need to volatilize Down to fmol detection limits
Atmospheric Pressure Ionization (API) Electrospray MALDI APCI APPI
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The Abb Nolletexperimentedwith electrified liquids in the 18thcentury !
He observed that when a personwas connected to a high-voltagegenerator he/she would not bleednormally after cutting ...blood
sprayed from the wound !
F. Lemire, LCGC Europe LC-MS Supplement,December 2001, p29-35
The Macabre History of Electrospray
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How Sample is IntroducedSample Inlet
Ion Spray Probe
Ion Spray Heater
Heater
Ion Spray Inlet
The sample is heldon the surface of a
capillary tube, afine wire, or a smallcup.
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11J. Zelene, Phys. Rev ., 10, 1-6 (1917)
The Electrospray Phenomenon
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Ionization Source
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Sample ConeOrifice = 400m
Sample ConeOrifice = 400m
Spraying NeedleSpraying Needle
VacuumIsolation Valve
VacuumIsolation Valve
Ionization Source
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Ionization Methods .
Gas Phase Ionization Electron Impact (EI) Chemical Ionization (CI)
Spray Ionization / Atmospheric Pressure Ionization (API) Electrospray (ESI) / API Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure photo Ionization (APPI)
Desorption Ionization
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II. Spray Ionization/APIThe compound of interest in a volatile buffer mobilephase, is passed through heated, narrow bore tubingdirectly into the ion source of a mass spectrometer. Thesolution is vaporized in the tubing, and analyte ionsdesorb into the gas phase and pass into the massanalyzer.
Electrospray (ESI) / API Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure photo Ionization (APPI)
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Electrospray Ionization(ESI)
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High voltage appliedto metal sheath (~4 kV)
Sample Inlet Nozzle(Lower Voltage)
Charged droplets
++
++
+
+
+
+
+ ++
+
+
+
+ ++
++
+ +
++ +
++ +
++
++
+
+
+
+
+
+
++ +
++ +
++
+
MH +
MH 3+
MH 2+
Pressure = 1 atmInner tube diam. = 100 um
Sample in solution
N2
N2 gas
Partialvacuum
Electrospray ionization:
Ion Sources make ions from sample molecules
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TURBOIONSPRAY (TIS)
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API 4000TM
Turbo Ion Spray
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ESI Spectrum of Trypsinogen (MW 23983)
1599.8
1499.9
1714.1
1845.91411.9
1999.62181.6
M + 15 H +
M + 13 H +
M + 14 H +M + 16 H +
m/z Mass-to-charge ratio
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A P C I
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A P P I
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hLaser
1. Sample is mixed with matrix (X)and dried on plate.
2. Laser flash ionizes matrixmolecules.
3. Sample molecules (M) are
ionized by proton transfer:XH+ + M MH+ + X.
MH+
+/- 20 kV Grid (0 V)
Sample plate
M A L D I : M a
t r i x
A s s
i s t e d L a s e r
D e s o r p
t i o n
I o n
i z a
t i o n
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The mass spectrum shows the results
R e
l a t i v e
A b u n
d a n c e
Mass (m/z)
0
10000
20000
30000
40000
50000 100000 150000 200000
MH +
(M+2H) 2+
(M+3H) 3+
MALDI TOF spectrum of IgG
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Differential vacuum in MS
OR
RNG
QO
IQ1
ST RO1IQ2 RO2 IQ3
RO3
DF
DET
1x10 -5 torr
Collision Cell (N 2)
1-5 x 10 -4 torr
1 torr
8 x 10 -3 torr
Q1 Analyzer Q3 AnalyzerCollisionalFocussing
DP-FP
Turbo
Roughing pump
1-2 x 10 -5 torr
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Significance of vacuum
A vacuum is necessary to permit ions to reach thedetector.
It reduces or eliminates the chances of ion collisions
with mass analyzer. The major reason for maintaining high vacuum is to
increase the mean-free path of ions. Increases sensitivity and resolution of Mass
Spectrum .
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Types Of Analyzers
Quadrupole Ion Trap Time-of-flight
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Analytical Quadrupole
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Schematic Diagram of Triple quad
Instrument
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Quadrupole TheoryPre-filter Quadrupole Mass Filter Stable Trajectory
Unstable Trajectories
Only ions with the correct m/z values have stabletrajectories within an RF/DC Quadrupole field.Ions with unstable trajectories collide with the rods, orthe walls of the vacuum chamber, and are neutralised.
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1. Quadrupole
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Advantages Relatively small and low-cost systems Low-energy collision-induced dissociation (CID) MS/MS Triple quadrupole and hybrid mass spectrometers.
LimitationsLimited resolutionPeak height vs. mass response must be 'tuned'.Not well suited for pulsed ionization methods
Applications Majority of benchtop GC/MS and LC/MS systems
Triple quadrupole MS/MS systemsquadrupole hybrid MS/MS systems
Quadrupole: Pros & Cons
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Tandem Quadrupole
CollisioncellMS1 MS2
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Components of Tandem Mass Spectrometer
CollisionCell
MassSpectrometer
MassSpectrometer
Detector
Ionization Source
ESIAPPIAPCIMALDI
ArgonXenon
QuatrupoleMagnetic Sector
Quatrupole
Magnetic SectorTime-of-flight
Collisioncell
MS1MS2
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Operation Modes
Product Ion Scanning Analyzes all products of a single precursor
Precursor Ion Scanning Analyzes all precursors of a single charged product
Neutral Loss Scanning Analyzes all precursors of a single uncharged product
Multiple Reaction Monitoring Analyzes for specific precursors producing specificproducts.
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SCANNING MODE: The first quadrupole massanalyzer is Scanning over a mass range. Thecollision cell and the second quadrupole massanalyzer allow all ions to pass to the detector.
SCANNING MODE: The first quadrupole massanalyzer is Scanning over a mass range. Thecollision cell and the second quadrupole massanalyzer allow all ions to pass to the detector.
MS1 MS2Collision
Cell
Scanning Rf only, pass all masses
CollisioncellMS1 MS2
F u
l l S c a n
A c q u
i s i t i o n
M o d e
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Mass Spectrum: Progesterone
200 220 240 260 280 300 320 340 360 380 400m/z0
100
%
315.1
316.1
[M+H]+
O
O
CH3
CH3CH3
F u
l l S c a n
A c q u
i s i t i o n
M o
d e
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Static (m/z 315.1) Scanning
The first quadrupole mass analyzer is fixed at the mass-to-chargeratio ( m/z ) of the precursor ion to be interrogated while the secondquadrupole is Scanning over a user-defined mass range.
The first quadrupole mass analyzer is fixed at the mass-to-chargeratio ( m/z ) of the precursor ion to be interrogated while the secondquadrupole is Scanning over a user-defined mass range.
Argon gasArgon gas
PrecursorPrecursorProductsProducts
CollisioncellMS1 MS2
P r o
d u c
t i o n s c a n n
i n g
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Product Ion Spectrum: Progesterone
300 305 310 315 320 325 330m/z0
100
%
315.1
316.1
Mass Spectrum fromMS1
100 125 150 175 200 225 250 275 300 325m/z0
100
%
109.097.0
Product ion spectrum from MS2 P r o
d u c
t i o n s c a n n
i n g
Product ions
OCH
2
CH2
CH3
O
CH3
CH3
O
O
CH 3
CH3CH3
Precursor ion
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Static Scanning
Precursor Ion Scan
The first quadrupole mass analyzer is Scanning a mass range while thesecond quadrupole is fixed, or Static , at the mass-to-charge ratio ( m/z )of a product ion known to be common to the analytes in a mixture.
The first quadrupole mass analyzer is Scanning a mass range while thesecond quadrupole is fixed, or Static , at the mass-to-charge ratio ( m/z )of a product ion known to be common to the analytes in a mixture.
Argon gasArgon gas
Pr ecur sor sPr ecur sor s
Pr oductPr oduct
CollisioncellMS1 MS2
P r e c u r s o r
i o n
s c a n n i n g
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- RCOOH
-(CH3)3N-C4H8
- RCOOH
-(CH3)3N-C4H8
CIDCID
ButylationButylation
CH2CH2 CHCH CHCH
RCOORCOO HH
COOHCOOH(CH3)3N(CH3)3N
CH2CH2 CHCH CHCH
RCOORCOO HH
COOC4H8COOC4H8(CH3)3N(CH3)3N
CH2CH2 CHCH CHCH COOHCOOH[[ ]+]+
(m/z 85)(m/z 85)
AcylcarnitinesDerivatization and Fragmentation
All compounds of this typefragment to produce the 85ion.
P r e c u r s o r
i o n
s c a n n i n g
R=0 to 18 carbon alkyl chain.
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42225 250 275 300 325 350 375 400 425 450 475 500
m/ 0
100
%
d3-free carnitin ed3-free carnitin e
C2 carnitineC2 carnitine
C16 carnitineC16 carnitine
d3-C3 carnitined3-C3 carnitine
d3-C8 carnitined3-C8 carnitine
d3-C16 carnitined3-C16 carnitine
Normal Acylcarnitine Profile
P r e c u r s o r
i o n
s c a n n i n g
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Scanning (M-102) Scanning (M)
In a neutral loss scan the two quadrupole mass filters are Scanning synchronously at a user-defined offset. The neutral loss is known to becommon to the analytes in a mixture.
Argon gas
Pr ecur sor s
Pr oduct s
CollisioncellMS1 MS2
N e u
t r a
l l o s s s c a n n
i n g
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Neutral and Acidic Amino Acids
Derivatization and Fragmentation (Generic )
+
Butanol
CH3
OH
O
OHCH3
Butyl formateNeutral loss of
102Da
+
O
NH2
OHR
Neutral or Acidic AA
HCl
Amino acid butyl ester
O
NH2
OR
CH3
Neutral or Acidic AA
O
NH3+
OR
CH3Fragmentation
Fragment
NH2+
R
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45140 160 180 200 220 240 260 280
m/z0
100
%
d3-Leu
d4-Ala
d3-Met
d5-Phe d6-Tyr
d8-Val
Gly
Ser
Pro
Glu
Deuterated internal standards for quantification
Normal Amino Acid Profile
N e u
t r a
l l o s s s c a n n
i n g
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Both the first and second quadrupole mass analyzers are held Static atthe mass-to-charge ratios ( m/z ) of the precursor ion and the mostintense product ion, respectively.
Both the first and second quadrupole mass analyzers are held Static atthe mass-to-charge ratios ( m/z ) of the precursor ion and the mostintense product ion, respectively.
Static (m/z 315.1) Static (m/z 109.0)
Argon gasArgon gas
Precursor(s)Precursor(s)Product(s)Product(s)
CollisioncellMS1 MS2
M u
l t i p l e
R e a c
t i o n
M o n
i t o r i n g
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Collision induced dissociation
Collision conditions (FRAGMENTATION) is controlled by altering:
The collision energy (speed of the ions as they enter the cell) Number of collisions undertaken (collision gas pressure)
Argon gas
O
O
CH 3
CH3CH3
Precursor ion Product ions
OCH
2
CH2
CH3
O
CH3
CH 3
In the collision cell, the TRANSLATIONAL ENERGY ofthe ions is converted to INTERNAL ENERGY .
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2. Quadrupole Ion Trap
In an Ion trap the ions are trapped in a radio-frequency quadrupolefield.
The ions are then ejected detected as the radio frequency field isscanned.
Ions are dynamically stored in a three-dimensional quadrupole ionstorage device.
The RF and DC potentials can be scanned to eject successivemass-to-charge ratios from the trap into the detector.
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Q0 Q1 Q2 Q3
QTRAP Linear ion trap
N2 CAD Gas
linear ion trap 3x10 -5 Torr
ion selection
Ion accumulation
Fragmentation
Exit lens
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3-D Ion Trap Schematics
Heated quartzcapillary
Ion TrapID ~ 10 mm
MS ScanProduct Ion ScanMS n
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Q-Ion Trap- Pros & Cons
Benefits
High sensitivityMulti-stage MS
Limitations
Poor quantitation.Subject to space charge effects and ion moleculereactions.Collision energy not well-defined in CID MS/MS.
Applications
Compact mass analyzer
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3. Time-of-Flight (TOF)
Time of flight mass spectrometer measures the mass-dependent time
It takes ions of different masses to move from the ion
source to the detector.
Ions are either formed by a pulsed ionization method(usually MALDI), or various kinds of rapid electric fieldswitching are used as a 'gate' to release the ions fromthe ion source in a very short time.
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TOF - Pros & Cons
BenefitsFastest MS analyzerWell suited for pulsed ionization methodsHigh ion transmissionHighest practical mass range of all MS analyzers
LimitationsRequires pulsed ionization method or ion beam switchingFast digitizers used in TOF can have limited dynamic rangeLimited precursor-ion selectivity for most MS/MS experiments
Applications
MALDI systemsVery fast GC/MS systems
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Mass analysis is the separation of bunches orstreams of ions according to their individual
mass-to-charge (m/z) ratio
The mass analyzer sorts the ions according tom/z and the detector records the abundance ofeach m/z.
DETECTORS
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Detectors are eyes of the Instrument
Once the ion passes through the mass analyzerit is then detected by the ion detector,
The final element of the mass spectrometer.
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METHODS OF I ON DETECTI ON
The detector generates a signal from incidentions by two ways:
1. Inducing current generated by a moving charge
2. Generating secondary electrons, which are furtheramplified.
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Current generated by a moving charge .
Flow of electrons in the wire isdetected as an electric currentwhich can be amplified andrecorded.The more ions arriving, the greaterthe current.Variation in the magnetic field,changes the flow of ion stream tothe detector which produce acurrent proportional to the numberof ions arriving.Timing mechanisms, whichintegrate those signals with thescanning voltages, allow theinstrument to report which m/zstrikes the detector
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Detector operates by producing a signal current fromincident ions by generating secondary electrons, whichare further amplified
Key part of such type of detectors is a dynode. Dynode is electron-multiplying electrode.
Current generated by secondary electrons
Incoming Ion
Secondary Electrons
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Contd
Process of Secondary electron emission. Electrons accelerated, and strike the surface of
electrode (dynode) Energy deposited by the incident electrons result in
re- emission
Secondaryelectrons
Electrodesurface(dynode) Dynodes
ForAmplification
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Certain of thesecharacteristics are common,like :
high sensitivity
linear, quantitative response
Some detectors aredesigned for specificfunctions or applications.
CHARECTERISTICS
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Electron multiplier is made up of a series of dynodesmaintained at ever increasing potentials.
Typical amplification or current gain of an electronmultiplier is one million.
Electron Multipliers contd
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Two major modifications:
Multiple-dynode type
Continuous-dynode type
Electron Multipliers contd
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1. Multiple-dynode type EM
Incoming ion reaches the first dynode
Ejects several other electrons by secondary emission.
This process repeated at each succeeding dynode having
a higher potential than the preceding dynode. When it arrives at the anode, the electron flow is
significantly amplified
Incident ions
Cathode Anode
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2. Continuous Electron Multiplier
Contains a glass pipe with a coating on its inner surface
The electron flow moves along the pipe, reflecting fromthe inner wall and progressively gaining electrons
The electrical field accelerating the flow is formed bythe high voltage applied across the two ends
resistive-material
inner coating
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Advantages: Very high current gain Sensitive Fast
Disadvantage: Short lifetime Requires good vacuum to operate
Electron multipliers are widely used in Quadrupole andIon trap Instruments.
Features of Electron Multipliers
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Channel Electron Multiplier
A modified continuousdynode electron multiplier
Comprised of "the channel,"a hollow, cornucopia-shapedtube made of semi conductiveglass.
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Channel Electron Mult iplier contd ..
IONS
The primary incoming ions passes through the inlet and strikes thesurface of the CEM
The collision energy eject an electron from CEM wall Ejected electrons accelerated into interior of CEM
Trigger secondary emission and the process continues to produce anoutput electron
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RECORDER
Analogue signal is produced by the detector. Analogue Digital Converter sends the output to the
computer.
Detector ADC Recorder
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IDENTIFICATION
Single MS
Specificity
Precursor Ion
Neutral Loss
Enh.Multi Charged
QUANTITATION
SIM or MRM
AUTOMATION
I nfo. D ep. Acquisition
Met ID
CHARACTERIZATION
High Sensitivity Full Scan MSMS
MS 3 Capabilities
Plug & Play Sources
TurboIonspray Heated Nebulizer (APCI)
Nanospray
Integrated Syringe pump
Built-in divert valve
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Where are Mass Spectrometer Used
Biotechnology: the analysis of Proteins,Peptides, Oligonucleotides
Pharmaceutical: Drug Discovery, Combinatorial
Chemistry, Pharmacokinetics, Drug Metabolism
Clinical: neonatal Screening, Hemoglobinanalysis, drug testing
Environmental: PAHs, PCBs, water quality.Food contamination
Geological: Oil composition
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How can Mass Spectrometry helpBiochemists
Accurate molecular weight measurements : sample confirmation, todetermine the purity of a sample, to verify amino acid substitutions, todetect post-transnational modifications, to calculate the number ofdisulfide bridges
Reaction monitoring: to monitor enzyme reactions, chemicalmodification, protein digestionAmino acid sequencing :sequence confirmation, de novocharacterization of peptides, identification of proteins by databasesearching with a sequence tag from a proteolytic fragment
Oligonucleotide sequencing :the characterization or quality control ofOligonucleotides
Protein structure :protein folding monitored by H/D exchange, protein-ligand complex formation under physiological conditions,macromolecular structure determination
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LC-MS/MS
Selectivity and Sensitivity comparisons
Application Area: Environmental
Application Example 1
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LC/MS or LC/MS/MS?Selectivity & Sensitivity
Quantitation of 3 pesticides in a surfacewater extract Simazine; Atrazine and
Metabenzthiazuron
Chromatographic and Mass Specconditions: Ionization technique : APCI
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Product Ion Spectra
Simazine (202-132)
Metabenzthiazuron (222 - 165)
Atrazine (216-174 )
MH+
MH+
MH+
N2NO2 NO2
MSMS Product Ion Scan= NO2
Q1 fixed, = CAD Collision Q3 scanningNO2
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Simazine in surface water extract(50 pg injected on column)
(SIM MODE; m/z 202) (MRM MODE; m/z 202-174)
API 150 EX TM LC/MS System API 2000 TM LC/MS/MS System
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Application Example 2
LC-MS/MS
Impurity profiling
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Impurity/Degradation Product Profiling
Experimental Conditions: 4 Commercially available OTC Melatonin Tablet
preparations
LC Column: 4.6 x 50 mm Chromolith SpeedRodRP-18 Targeted analysis using MRM as survey for
known impurities and EMS for profiling in IDAfollowed by EPI
Confirmation of impurity/degradation product byMS/MS using EPI
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Multiple MRMs (1)
Second DependentScan (s)
IDA CriteriaLevel 1
EMS (2)
Dependent Scan (s)
Enhanced Resolution
Add toExclusion ListIDA CriteriaLevel 2
Dependent Scan (s)Dependent Scan (s)Dependent Scan (s)
Second DependentScan (s)
Second DependentScan (s)
Impurity/Degradation Product Profiling
For known impurities,targeted MRM analysiscan be used. At the sametime an EMS survey scan
can search for unknownimpurities
for unknown impurities,IDA triggered MS/MS and
MS3 information can becollected to aid inidentification.
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H 3C O NH
O
HN
OH
H OOH
HN
NH
O
H 3C O
oxidation Impurities249 m/z
di-oxidation Impurity265 m/z
Targeted MRMKnown Impurities
EMSUnknown Impurities
Impurity/Degradation Product Profiling
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+E P I (477.00) C h arge (+ 2) C E (3 0): E xp 3 , 5 .568 m in from S am ple 1 (S tre ssed 10 ng/uL) o f S tre ss ... M ax. 1 .6e5 cps .
60 80 100 120 140 160 180 200 220 240 260 28 0 30 0 320 340 360 380 400 420 4 40 4 60 4 80 500m/z, amu
5 %
10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%100% 245.0
186.0202.9
228.0 477.2405.2160.0
273.0 418.3267.1233.0 329.0
H3COHN
ON
H 3CO NH
ON
245
Melatonin-FormaldehydeImpurity477 m/z
EPI
Impurity/Degradation Product Profiling
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X IC o f + E M S : E x p 1 , 2 4 9 .0 a m u fr o m S a m p le 9 ( M e la to n in T a b le t (1 0 0 n g /u L ) ID A E M S_ E R _ E P I w i. . . M a x . 8 .3 e 6 c p s .
1 2 3 4 5 6 7 8 9Ti m e , m i n
0 .0
5 . 0 e 5
1 . 0 e 6
1 . 5 e 6
2 . 0 e 6
2 . 5 e 6
3 . 0 e 6
3 . 5 e 6
4 . 0 e 6
4 . 5 e 6
5 . 0 e 6
5 . 5 e 6
6 . 0 e 6
6 . 5 e 6
7 . 0 e 6
7 . 5 e 6
8 . 0 e 68 . 3 e 6
2 . 8 6
7 . 2 1
6 . 9 1
3 . 8 1
XICs of peaks present in Sample,but not Control.
MS/MS information collected through IDA
software can help with data analysis through sampleand control comparison for degradation products
Impurity/Degradation Product Profiling
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Specificity of Detection for LC
UV chromophore all compounds with a chromophore responding at the
selected wavelength will interfere
MS molecular mass interference from isobaric compounds chemical noise
MS/MS molecular mass and structuralinformation interference from structural isomers only
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1. Wash all glassware in methanol x2 and tert-butyl methyl ether (TBME) x2.2. Place 50 L of internal standard (in methanol) into each screw-cap glass
tube.3. Add 200 L Sirolimus calibrator (5x concentrated in methanol) or 200 L
methanol for patient samples.4. Add 1.0mL blank whole blood to calibrators or 1.0mL patient whole blood .
5. Add 2.0mL 0.1M ammonium carbonate buffer.6. Mix thoroughly.7. Add 7.0mL TBME and extract for 15min.8. Transfer upper layer to clean tube and re-extract lower layer with 7.0mL
TBME.9. Combine TBME extracts and evaporate to dryness .10 . Redissolve residue in 5.0mL ethanol and evaporate to dryness .
11 . Redissolve residue in 1.0mL ethanol, transfer to Eppendorf tube andevaporate to dryness .
12 . Redissolve residue in 100 L 85% methanol.13 . Inject 80 L (equivalent to 800 L whole blood ) and analyse using two
4.6mm x 250mm C18 columns connected in series (30min run time) .
HPLC-UV Analysis of Sirolimus inWhole Blood
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Sirolimus: HPLC - UV Example
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Add ZnSO4
Soln.
Whole Blood(10 L - 40L)
Add 2 volumes MeCNwith IS, Seal & Vortex Mix
Centrifuge,Inject 5 - 20 L
Immunosuppressant Sample PreparationLC-MS/MS Analysis
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Sirolimus: MS Spectrum
790 795 800 805 810 815 820 825 830 835 840 845 850m/z0
100
%
821.5
810.5
822.5
826.5
827.5[M+H]+
[M+NH4]+
[M+Li]+
[M+Na ]+
[M+K]+
F u
l l S c a n
A c q u
i s i t i o n
M o
d e
Sirolimus:
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Sirolimus:LC-MS (SIM) vs LC-UV
0
100
%SIR m/z 821
30g / L
1.5 min
HPLC-UV
HPLC-MS
S i n g l e
i o n m o n
i t o r i n g
( M
S )
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Sirolimus: MS Spectrum
790 795 800 805 810 815 820 825 830 835 840 845 850m/z0
100
%
821.5
810.5
822.5
826.5
827.5[M+H]+
[M+NH4]+
[M+Li]+
[M+Na ]+
[M+K]+
F u
l l S c a n
A c q u
i s i t i o n
M o
d e
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MS1 MS2Collision
Cell
Static (m/z 821.5) Scanning
The first quadrupole mass analyzer is fixed, orStatic , at the mass-to-charge ratio ( m/z ) of theprecursor ion to be interrogated while the secondquadrupole is Scanning over a user-defined massrange.
The first quadrupole mass analyzer is fixed, orStatic , at the mass-to-charge ratio ( m/z ) of theprecursor ion to be interrogated while the secondquadrupole is Scanning over a user-defined massrange.
Ar (2.5 3.0e -3mBar)Ar (2.5 3.0e -3mBar)
PrecursorPrecursorProductsProducts
P r o d u c
t i o n s c a n n
i n g
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790 795 800 805 810 815 820 825 830 835 840 845 850m/z0
100
%
821.5
810.5
822.5
826.5827.5
Mass spectrum from MS1Mass spectrum from MS1
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z0
100
%
768
576
558548718 750
786821
Product ion spectrum from MS2Product ion spectrum from MS2
P r o d u c
t i o n s c a n n
i n g
NH4+
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MS1 MS2Collision
Cell
Static (m/z 821.5) Static (m/z 768.5)
Ar (2.5 3.0e -3mBar)Ar (2.5 3.0e -3mBar)
Precursor(s)Precursor(s) Product(s)Product(s)
MS/MS : Compound-Specific Monitoring
M u
l t i p l e
R e a c t i o
n M o n i t o r i n g
Sirolimus
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SirolimusLC-MS(SIM) vs LC-MS/MS (MRM )
SIR m/z 821
0.50 1.00 1.50Time0
100
%
0
100
%
0.50 1.00 1.50Time0
100
%
0
100
%
MRM m/z 821>768
3g / L 30g / L
M u
l t i p l e
R e a c t i o
n M o n i t o r i n g
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