presentación teórica ms-ms

7/27/2019 Presentacin terica MS-MS

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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


3/72

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


8/72

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


11/72



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


12/72

Liquid

Electrospray Plume

Stainless Steel CapillaryStainless Steel Tube

Electrospray Probe Tip


13/72

Liquid

Nebulizer Gas

Nebulizer Gas Electrospray Plume


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



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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.


20/72


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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


24/72

Quattro microTM

:Sample Cone and Cone Gas Nozzle


25/72

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


28/72

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


38/72

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



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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


50/72


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


51/72

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



52/72

Produc t Ion Scan

m1+

m2+

m2+

m2+

Product ion spectrum of a particular compound

m1+ set

m2+ 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



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




M+H

gEffect of Changing Collision Energy(Cont.)

MS/MS Spectra of

Chlorpheniramine(MW=274)Daughter Ions of m/z=275



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++CID

CID

++

DifferentNeutralFragments

Different CompoundsThat Are SomewhatSimilar In Structure

SameChargedFragment


+

+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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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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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 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 Scans can be used to detect those compounds whosemolecular ions produce the same neutral fragment.



62/72


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.



63/72


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


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



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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



68/72

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



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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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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



71/72


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



72/72

Precursor ionset

Product ionset

Fragmentation(CID)


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