Tandem MS for Drug Analysis

8/2/2019 Tandem MS for Drug Analysis

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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


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


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


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



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


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)


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)




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



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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



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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



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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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89

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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90

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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91

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


93/93

93

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Tandem MS for Drug Analysis

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