2lcms Handouts

8/8/2019 2lcms Handouts

1/22


2/22

Dr. Shulamit Levin, Medtechnica1

The LC in LC/MSThe LC in LC/MS

HHighigh PPerformanceerformance LLiquidiquid CChromatographyhromatography

Separates compounds based on their chemical

characteristics (e.g. polar, nonpolar, acidic, basic,

etc.)

The MS in LC/MSThe MS in LC/MS

MMassass SSpectrometrypectrometry

A process in which ions are generated and analyzed

according to their mass-to-charge ratio (m/z) and in

which the number of ions is determined electrically.

The /The /The Transition in LC/MSThe Transition in LC/MS

Fact:Fact: Compounds in liquid phase eluting from theHPLC at atmospheric pressure

Fact:Fact: Mass spectrometer accepts only gas phase ions

at high vacuum

Therefore the interfaceinterface must: Remove the solvent

Leave the analyte

Charge the analyte

+

+

SAMPLE DESOLVATION

AND IONIZATION

EI

Electrospray

APCILC/MSLC/MS

INTERFACEINTERFACE

SOURCESOURCE

ANALYSERANALYSERION DETECTORION DETECTOR

HPLCHPLCMASS SPECTRUM

SORTING OF IONS

Quadrapole

Time of Flight Paul Trap

+

++

+

+ ++ +

+

-

+

+

++

++

-

+

+

+

Typical LC/MS System ProgressionTypical LC/MS System Progression

DATA PROCESSINGDATA PROCESSING


3/22

Dr. Shulamit Levin, Medtechnica

2

Transition from LC to MSTransition from LC to MS

State of Matter: LiquidLiquid to GasGas

Charge State: NeutralNeutral to IonIon

Pressure: 760 torr760 torr to 1010--55 to 10to 10--88 torrtorr

From Raw Data to Mass Spectrum

Raw Data

Instrument Basic

Threshold Parameters

Instrument Advanced

Threshold Parameters

Mass Range

Data Format

Scan Time

Inter-Scan Delay

Mass Spectrum

102,1

103,1

Scan N

Centroid

100 101 102 103

Intensity

m/z

Scan N

ContinuumN Scans

MCA

100 101 102 103

Intensity

m/z100 101 102 103

Intensity

m/z

Defining Data Description of Data FormatDefining Data Description of Data Format

(Spectrum Format)(Spectrum Format)

Defining Data Description of Data FormatDefining Data Description of Data Format

CentroidCentroid-- andand ContinousContinous --SpectraSpectra

15085 15090 15095 15100 15105 15110 15115 15120 15125 15130 15135 15140 15145 15150 15155 15160 15165mass0

100

%

0

100

%

HFN1 1 (2.958) Cn (Top,5, Ar); ME [Ev-43833,It17] (Gs,0.830,980:1400,0.50,L33,R33) Scan ES+1.77e815127

HFN1 1 (2.958) ME [Ev-43833,It17] (Gs,0.830,980:1400,0.50,L33,R33) Scan ES+6.33e715127

Continous

Centroid

Converted

All MassAll Mass Spectrometers Must:Spectrometers Must:

Generate ions

Separate ions

Detect ions

Compute ion intensities

Interpret Data

GenerateGenerate ionsions

SeparateSeparate ionsions

DetectDetect ionsions

ComputeCompute ionion intensitiesintensities

InterpretInterpret DataData


4/22


Diode Array 3D RunDiode Array 3D Run

Mixture

2.00 4.00 6.00 8.00 10.00Time0

Abs

1: Diode Array5.054.65

3.82

0.74

8.62

5.65

8.02

10.62

200 210 220 230 240 250 260 270 280 290 300 310nm0

Int. 210.00

246.00

294.00

Diode Array Chromatogram

with poor resolution

Mass Spectrometer 3D RunMass Spectrometer 3D Run

Mixture

2.00 4.00 6.00 8.00 10.00Time0

Int.

1: Mass Chromatogram5.054.65

3.82

0.74

8.62

5.65

8.02

10.62

Total-Ion-Current Chromatogram

with poor resolution

Mix

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z0

100

%

(10.696) 1: Scan ES+4.34e5262.87

59.99

213.90

195.98

120.8068.9298.85

76.87128.82

170.92

222.87

235.87

240.88

263.87

264.85

267.91 287.01 309.02

333.84

Mix

2.00 4.00 6.00 8.00 10.00 12.00 14.00Time0

100

%

1:ScanES+262.874.59e5

10.60

Mixture

2.00 4.00 6.00 8.00 10.00 Time0

Int. 5.054.65

3.820.74

8.625.65

8.02

10.62

SelectivitySelectivity of Mass Spectrometer Detectorof Mass Spectrometer Detector

Extracted Ion Chromatogram

of a single Component from

a mixture of Components

Ionization ModesIonization Modes

I. Electron Impact-Ionization (EI)/Chemical Ionization

(CI)

II. Atmospheric Pressure Ionization (API)

Atmospheric Pressure Chemical Ionization (APCI)

Electrospray (ESI)


5/22


MS Configuration for IonizationMS Configuration for Ionization

High Vacuum

IonSource

Interfaceto

VacuumMass

AnalyzerDetector

Sample

Introduction

API

High Vacuum

IonSource

MassAnalyzer

Detector

Sample

Introduction

EIInterface

to Vacuum

Electron Ionization (EI)Electron Ionization (EI)

MSample Inlet

Filament

Collector Ion FocusingLenses

Ions

ToAnalyzer

-

-Repeller

+

- + -M + e M + 2e.

F F F1 2 3

A beam of solute particles (Particle Beam) enter the MS source and

hits a heated wall where they sublime. Once in vapor phase, the moleculesare ionized by Electron Impact (EI) or Chemical Ionization (CI).

EI SpectrumEI Spectrum


I. Electron Impact-Ionization (EI)/Chemical Ionization

(CI)



Electrospray (ESI)


6/22


APCIAPCI

Ion formation (Aerosol/Evaporation/Ionization/Evacuation)

Heat rapidly evaporates both solvent and solutes.

APCI creates gas-phase ions at atmospheric pressure with the Corona

discharge.

Small Gas-phase ions interact with neutrals.

Volatile salt buffer are recommended.

APCI MechanismAPCI Mechanism

Ionization produces

solvent ions

The solvent ions react

w ith analyte m olecules

form ing clusters

Corona

Needle

X = Solvent M olecules e.g.H 2O, M eCNM = Sam ple M olecule

Heated Nebulizer

xx

xH+ M

xx

xH+

M

x

x

M

M H+

xx

xx

XH +x

xH+

M H+

APCIAPCI

Ionization Process

After evaporations of neutral species ions are formed by the Corona

discharge process

All species in the gas phase undergo significant collisions with surrounding

gases

SH+ + M ----> MH+ + S (S= solvent, M= molecule)

APCIAPCI

General applicability

Molecule < 2000 amu

Ionizable, polar and mid-polar molecules

Available on all MS types

Extremely sensitive technique for compounds

with high proton affinity like amines

Positive and negative ionization modes


7/22


APCIAPCI

Advantages Molecular weight information

Easy to use

Rugged

Very efficient ionization

Accommodates LC flows

up to 2.0 ml/min

Good sensitivity

Good complement to ESI

(same hardware)

Disadvantages Thermal degradation may occur

High chemical noise at low mass

Not appropriate for compounds

with MW above m/z 2000

Volatile buffers required

I. Electron Impact-Ionization (EI)/Chemical Ionization(CI)



Electrospray (ESI)


ElectrosprayElectrospray IonizationIonizationPositive or Negative?Positive or Negative?

Basic Compounds (-NH2) (M+H)+

Acidic Compounds (-CO2H, -OH) (M-H)-

Basic Compounds (-NH2) (M+H)+

Acidic Compounds (-CO2H, -OH) (M-H)-


8/22


Recognizing Multiply Charged IonsRecognizing Multiply Charged Ions

Mass spectrometers operate on the basis of mass-to-charge ratio (m/z).

Mass assignments are normally made assuming a single charge per ion

(i.e. m/z = m)

Single charge Mass = (M+H)

Double charge Mass = 1/2

(M+2H)

n charge Mass = 1/n (M+nH)

Isotopes of doubly charged ions are separated by 0.5 Da

Mass spectrometers operate on the basis of mass-to-charge ratio (m/z).

Mass assignments are normally made assuming a single charge per ion

(i.e. m/z = m)

Single charge Mass = (M+H)

Double charge Mass = 1/2

(M+2H)

n charge Mass = 1/n (M+nH)

Isotopes of doubly charged ions are separated by 0.5 Da

n = 20

n = 22

n = 18

n = 16, m/z = 1060

n = 23, m/z = 738

n = 21

n = 19

n = 17

Horse Heart Myoglobin

Mass RangeMass RangeMultiply Charged MoleculesMultiply Charged Molecules

Calculated MassAcquired Mass range

Hemoglobin SpectrumHemoglobin Spectrum

Presence of More Than One Charged EnvelopePresence of More Than One Charged Envelope

1000 1050 1100 1150 1200 1250 1300 1350 1400

m/z0

100

%

1081.60

1009.36

1058.83

1164.52

1133.92

1261.64

1221.181376.08

1323.14

Deconvolution by MaxEntDeconvolution by MaxEnt

HemoglobinHemoglobin

15000 15100 15200 15300 15400 15500 15600 15700 15800 15900 16000 16100mass0

100

%

15125.0

15857.0

15149.0

15866.0


9/22


Multiply Charged IonsMultiply Charged Ions

(M+H)+

(M+H)+

(M+2H)2+(M+2H)2+

1 Da1 Da

0.5 Da0.5 Da

ESI GeneralESI General applicabilityapplicability

Small singly charged ions.

Large ionic molecules.

Virtually any ion in solution is a candidate for ESI

(+ or - ions).

The production of multiple charge ions (proteins) extendsthe mass range detection to no limit.

Available on all MS types

Most sensitive LC/MS technique Molecular weight

Structure with MS/MS or CID

ElectrosprayElectrospray

Advantages MW confirmation

High MW determination

Volatile & non-volatile solutes

No limit in MW

Ionic/polar analytes

low temperature, no degradation

Good sensitivity

Quantitative method

Suitable for CapLC

Disadvantages Requires low LC flow rates for best aerosol

Must form ions in solution

Ion suppression with high salt condition

Adduct ions may result

Limited structural information

Users must understand chemistry of aqueous

solution (acid-base equilibrium, redox chemistry..)

Unknowns should be analyzed in both positive and

negative ion modes.

1. The Quadruple

2. Time of Flight

3. Ion Trap4. Magnetic sector

5. Fourier Transform Ion Cyclotron

Ion Separation AnalyzersIon Separation Analyzers

T im e Of F l ig h t Mass A n a lyz ersT im e Of F l ig h t Mass A n a lyz ers

T im e Of F l ig h t Mass A n a lyz ersT im e Of F l ig h t Mass A n a lyz ers


10/22


190

ElectronMultiplier

Inlet

Ring Electrode,Rf

End Cap Electrode

AxialModulation

IonIon TrapsTraps

+ + ++++ ++

Types ofTypes of

Mass SpectrometersMass Spectrometers

AnalyzersAnalyzers

S O U R C E

DETECTOR

REFLECTRON MODE

R EF L EC T R ON ONDETECTOR

LINEAR MODEDRIFT TUBE

SOURCE

DETECTOR

REFLECTRON MODE

R EF L EC T R ON OF FDETECTOR


S O U R C E

DETECTOR

REFLECTRON MODE

R EF L EC T R ON ONDETECTOR


SOURCE

DETECTOR

REFLECTRON MODE

R EF L EC T R ON OF FDETECTOR


220

IonSource

Slit

Magnetic sector

Electrostatic Sector

(ESA)

Detector

SlitNier-Johnson-Geometry (EB)

SectorSector Mass SpectrometersMass Spectrometers

199

FTFT--ICRICR--SpectrometerSpectrometer

DCDC

DC

Source

Filament

Transferoptic

TrappingPlates

TransmitterPlates

Receiver Plates

Sender

Elektroden Electrodes

Y

ZX

Magnetic Fielt B

77

Ion Source

Detector

nonres onant Ion

resonantIon

dcandRf Voltages

TheThe Quadrupole AnalysatorQuadrupole Analysator

Starting with theStarting with the quadrupolequadrupole

Source

DetectorNonresonant Ion

Resonant Ion

dc and Rf voltages

V(t) = -V dc -Vrfcost

V(t) = V dc + Vrfcost

Fragmentation of Molecules forFragmentation of Molecules forIdentificationIdentification Triple Quadrupole (MS/MS)Triple Quadrupole (MS/MS)

Precursor ions and product ions are created and analyzed indifferent physical spaces.

Ions must be moved from "source" to analyzer (differentphysical regions) where different functions take place.

Q1 Q3Q2

CID

Region

IonSource

Tandem-in-Space

T i lT i l S ifi tiS ifi ti


11/22


TypicalTypical SpecificationsSpecifications

Mass Range: 2-4000 u

Resolution: Unit Resolution

Mass Accuracy: ca 0.1 u

Scan Speed: 4000 u/s (in practice

500 - 1000 u/s)

Vakuum: 10-4 up to 10-5 Torr

Quantification:best choice for solving

quantification problems.

Variations: single QuadSIR/scanning

Triple Quad-SRM/scanning Hybrids, Q-

TOF

Positive and negative Ions

References: Miller, P.E., Denton, M.B.,

J.Chem Educ. 7, 617, (1986).

TimeTime ofofFlight Mass AnalyzersFlight Mass Analyzers

SOURCE

SOURCE

DETECTOR

REFLECTRON MODE

DETECTOR

REFLECTRON MODE

REFLECTRON OFF

REFLECTRON ONDETECTOR

LINEAR MODE

DETECTOR

LINEAR MODE

DRIFT TUBE

DRIFT TUBE

TimeTime ofofFlightFlight

A package of ions is accelerated by a potential Vinto a field free flight tube.

The time tfight needed for ions to reach a detector placed at distance dismeasure:

m/z = [k V/d2] t

2fight

Small ions have higher velocity than large mass ions therefore their time of flightin the tube are different and related to m/z ratio.

A high percentage of the generated ions is detected thereforethis technology provides very high sensitivity.

Fast scanning capabilities (>106 dm/dt) and no limit in mass range.

However it requires very high vacuum (10-7 torr).

TypicalTypical Specifications TOFSpecifications TOF

Mass Range: 2-15000, high

sensitivity

Resolution: 10000 (Reflectron)

Mass Accuracy: 5 ppm

Scan Speed: very high 106 u/s,

sampling rates 300-500 MHz.

Vakuum: 10-7 Torr

Quantification: low dynamic range

Variations: Linear and Reflectron, Tandem

with quads and sectors

Positive and negative Ions : ESI, APCI,MALDI

References: Cotter, R.J., Time-of-Flight:

Instrumentation and Applications in

Biological Research, ACS: Washington, D.C.

1994.


12/22


WhereWhere Exact MassExact Mass becomes reallybecomes really

interesting...interesting...

2 organic molecules may have the same nominal mass but differentelemental composition

An exact mass measurement may be able to show this

This becomes useful in

metabolism studies

impurity studies

environmental analysis

synthetic chemistry industrial applications

...

CO = 27.9949

N2 = 28.0061

C2H4 = 28.0313

These have the same nominal mass but

different exact mass

5ppm5ppm -- So What?So What?

Differentiation of nominally isobaric metabolites

For example

Dehydroxylation followed by methylation...

R-CH2-OH R-CH3(-O)

R-OH R-OCH3(+CH2) (-O+CH2 = -1.9793 Da)

Alcohol to Aldehyde .

R-CH2-OH R-CH=O (-H2 = -2.0157 Da)

Difference in m/z between the 2 metabolites is 0.0364 Da

At m/z 500 this is a 73ppm difference

5ppm will allow you to correctly ID the metabolite

Component Calculated

m/z

Measured

m/z

+/- mDa +/- ppm

Parent 360.1382 360.1366 1.6 4.4

Sulphide 344.1433 344.1424 0.9 2.6

Sulphone 376.1331 376.1330 0.1 0.3

Desmethyl 346.1225 346.1218 0.7 2.0

S-Desmethyl 330.1276 330.1265 1.1 3.3

Aldehyde 344.1069 344.1074 0.5 1.5

S-Pyridone 272.0858 272.0867 0.9 3.3

Mass Measurement AccuracyMass Measurement Accuracy Power and Limitations of Exact MassPower and Limitations of Exact Mass

For C0-100 H3-74 O0-4N0-4

Mass 118 : closest neighbour at 34ppm

Mass 500 : 5 compounds within 5ppm Mass 750.4 : 626 compounds within 5ppm

Note 5ppm at m/z 500 means m/z 500 0.0025

(reference J. Am. Soc. Mass Spectrom.)

Ion TrapIon Trap


13/22


QQ--TOF HybridTOF Hybrid Mass SpectrometerMass Spectrometer

PUSHER DETECTOR

ESI PROBE

SAMPLING CONESKIMMER

QUADRUPOLE

RF HEXAPOL

REFLECTRON

HEXAPOLE

COLLISION CELL

QTofQTofQTofQTof:::: Quadrupole-TOF Tandem"

Ion TrapIon Trap

Accumulation

Isolation

Excitation

Fragmentation

Fragment

Accumulation

Detection

Ion TrapIon Trap MS/MS/MSMSnn TypicalTypical Specifications ITSpecifications IT

Mass Range: 20-2000 u

Resolution: Unit Resolution

Mass Accuracy: ca 0.1 u

Scan Speed: 4000 u/s

Vakuum: 10-3 Torr

Quantification: possible, but not very

accurate

Variations: MS/MS, Sources- Internal and

External, Resonance Excitations


References: March, R.E., Huges, R.J.

Quadrupole Storage MS, John Wiley and

Sons: New York, 1989.

S tSector M S t tMass Spectrometers

TypicalTypical SpecificationsSpecifications SectorSector


14/22


IonSource

Slit

Magnetic sector

Electrostatic Sector

(ESA)

Detector

SlitNier-Johnson-Geometry (EB)

SectorSectorMass SpectrometersMass Spectrometers

Magnet

Ion beam

Incr

ease

d m/z

1234

The radius con be obtained from:

r = m (2 V/z B2)

V = accelerating potential applied to ions leaving the source

B = magnetic field strength

TypicalTypical SpecificationsSpecifications -- SectorSector

Mass Range: 20000u Resolution:

with double focusing: 100000

Mass Accuracy: 1 ppm

Scan Speed: slow

Vakuum: 10-7 Torr

Variations: 1-, 2,-, 3-, 4-Sectors

Tandem with quads and TOF

Positive and negative Ions : ESI, APCI, EI,

CI, PB, Continuos flow FAB

References: Dass, Chhabil Chapter 1

Instrumentation and Techniques, IN: Mass

Spectrometry: Clinical and Biomedical

Applications; Desidero, D.M. (Ed.), Plenum,

New York (1994).

FTFT--ICRICR--SpectrometerSpectrometer

DCDC

DCSource

Filament

Transferoptic

TrappingPlates

Transmitter Plates

Receiver Plates

Sender

Elektroden Electrodes

Y

ZX

Magnetic Field B

TypicalTypical SpecificationsSpecificationsFTFT--ICRICR

Mass Range: > 15000

Resolution: High, 106

Mass Accuracy: up to 100 ppb

Scan Speed: fast ms Vakuum: 10-

7 - 10-9 Torr

Quantification: possible, but not very

accurate

Variations: MS/MS, Sources- Internal and

External, SWIFT: Stored Waveform Inverse

FT


References: Buchanan, M.V. (Ed.), Fourier

Transform MS: American Chemical

Society: Washington D.C, 1987.

Ph l i liPh t lti li


15/22


Electron MultiplierElectron Multiplier

+

+ ++--

-Electron Multiplier

(voltage setting lower thanDynode)

Conversion Dynode

(Voltage 1- 20 kV)

Current is measured

++

Mass Analyser

A conversion dynode is used to convert either negative or

positive ions into electrons. Higher potentials on the conversion

dynodes are used to accelerate high mass ions and thereby

enhance sensitivity.

PhotomultiplierPhotomultiplier

high voltage conversion dynode (converts either positive or negative

ions into electrons)

electrons impinge onto light emmitting phosphor that is optically

coupled to a photomultiplier

photomultiplier permanently sealed in its own glass envelope. The

detector is protected from contamination and thus longer lifetimes are

achieved.

The photomultiplier has a 10 year maintenance-free lifetime.

dynodedynode

phosphorphosphorphotomultiplierphotomultiplier

MassMass

Nominal Mass

The mass of an ion with a given empirical formula calculated using the integer

mass numbers of the most abundant isotope of each element

Ex : M=249 C20H9+ or C19H7 N+ or C13H19N3O2+

Exact Mass

The mass of an ion with a given empirical formula calculated using the exact

mass of the most abundant isotope of each element

Ex : M=249 C20H9+ 249.070

C19H7N+ 249.0580

C13H19N3O2+ 249.1479

ElectrosprayElectrospray Produces Multiple ChargingProduces Multiple Charging

(consequently high(consequently high MWsMWs can be measured)can be measured)

Ion SeriesIon m/zM+ X/1M2+ X/2

M3+ X/3M/4+ X/4M5+ X/5

M+M2+M3+

M4+

M5+

XX/2X/3X/4X/5

Mass/ChargeMultiply charged ion series is the key to determining molecular mass with Electrospray MS

Resolution


16/22


Mass RangeMass RangeMultiply Charged MoleculesMultiply Charged Molecules

650 700 750 800 850 900 950 1,0000

2,000

4,000

6,000

8,000

m/z

Intensity

[M+18H]18+

[M+17H]17+

[M+16H]16+

[M+15H]15+

[M+14H]14+

[M+13H]13+

Positive ion mode,ESI, 2 ul/min,50% MeOH:49%H2O:1% HOAc

5 uM Cytochrome c (horse heart)

MW = 12,360.9

Resolution

Resolution, equally called Resolving Power, of a massspectrometer is a measure of its ability to separate

adjacent ions.

At higher resolution, small may differences may be detected.

249 249.0700 249.0580 249.1479

3 different compounds

Same nominal mass

Low resolution

3 different compounds

3 different exact masses

High resolution

C20H9+

C19H7N+

C13H19N3O2+ C20H9+ C19H7N+ C13H19N3O2+

Determining Resolution

2 adjacent ion peakswith a 10% valley max

Double Ion method

R =mave

mr

Full Width at Half Maximum(FWHM)

or at 5% of the peak height

Single Ion method

R =m

m

mr

mavePPG 2000 Resolut ion

2007 2008 2009 2010 2011 2012 2013 2014 2015 2016mass0

10 0

%

0

10 0

%

ppg (0.517) Cu (0.25); Is (1 .00,1.00) C 102H210O 35N Scan ES +

3.40e122010.47

2009.47

2011.47

2012.47

2013.472014.47

ppg (0.517) Cu (0.50); Is (1 .00,1.00) C 102H210O 35N Scan ES +

3.42e122010.47

2009.47

2011.47

2012.47

2013.47

50%

Defining Mass ResolutionDefining Mass Resolution

FWHM Resolution ExampleFWHM Resolution Example

50%

m=0.5 amu

m=0.25 amu

FWHM~8040

FWHM~4020

Mass Accuracy M A R l ti


17/22

Dr. Shulamit Levin, Medtechnica

1

Mass Accuracy

Ability of a mass analyzer to assign the mass

of an ion close to its true value (exact mass)

m accuracy = mreal - mmeasured

ppm = 106 * m accuracy / mmeasured

High mass accuracy (exact mass measurement)

is usually associated to high resolution analyzers

Goals :

- Unknown compound determination

Exact mass helps to define its atomic composition

- Target analysisExact mass proves the presence of a particular

ion in a mixture

maccuracy

Mass Accuracy vs Resolution

SensitivitySensitivity

Term used to describe the ability of the MS to respond to a given amount ofsample analyte at a given mass to charge ratio (compound dependent)

Mass Spectrometers are mass-flow sensitive device.Sensitivity = Area( or height)

Cmax x flow rate

The sensitivity of your LC/MS system is directly relatedto the efficiencies of all the processes of the entire system :

- LC separation and flow

- Interface

- Ionization

- Mass transmission

- Detection

Cmax = 4 M (N)1/2

d2 L (1+k) (2)1/2

Scan Speed (or rate)Scan Speed (or rate)

The rate at which we can acquire a mass spectrum, (mass units/sec).

Is an essential acquisition parameter for MS

Will affect the amount of information (qualitative and quantitative) thatcan reasonably be attained with a given mass analyzer.

Minimum Scan Rate:

dm/dt 10 points = 10macquisition / WLC peak

Triple QuadrupoleTriple Quadrupole


18/22


Triple QuadrupoleTriple Quadrupole

MS/MSSRM

MS/MS

ProductIon Mode

SIM

Full ScanMS

Q1 Q2 Q3

[email protected]

A Typical BioA Typical Bio--Analytical Quantitative Study Using TripleAnalytical Quantitative Study Using Triple

QuadrupolesQuadrupoles::

Measurement of Amphetamines in Human SalivaMeasurement of Amphetamines in Human Saliva

by LCby LC--MS/MSMS/MS

HO

ONH

CH3

C3

M/Z

MS Scan of Ecstasy (MDMA)MS Scan of Ecstasy (MDMA)

O

O

CH3

Product ion Scan ofm/z194

Collision energy = 12eV, Argon = 2.5x10-3

mbar

NH

CH3

Specificity = MRM

Parent 194 Daughter 163

Signal:Noise forSignal:Noise for CarbosulfanCarbosulfan Calibration Graph forCalibration Graph for CarbosulfanCarbosulfan


19/22


g

(0.25pg/L std)Compound name: Carbosulfan

Coefficient o f Determination: 0.999380

Calibration curve: 2736 92 * x + 6294.89

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

0.0 5.0 10.0 15.0 20.0 25.0pg/l0

6926597

Response

pp

QQ--TOF HybridTOF Hybrid Mass SpectrometerMass Spectrometer--

Peptide & Protein SequencingPeptide & Protein Sequencing

PUSHER DETECTOR

ESI PROBE

SAMPLING CONESKIMMER

QUADRUPOLE

RF HEXAPOL

REFLECTRON

HEXAPOLE

COLLISION CELL

QTofQTofQTofQTof:::: Quadrupole-TOF Tandem"

Nominally isobaric metabolitesNominally isobaric metabolites --

rabeprazolerabeprazole

N

N

SN

O

O

O

H

Aldehyde

[M+H]+

344.1069

N

N

SN

O

O

H

O

H

Sulphide

[M+H]+ 344.1433

N

N

SN

O

O

H

Difference = 36 mDa

Will MS/MS help?Will MS/MS help?

Exact mass chromatogramsExact mass chromatograms


20/22


m/z 119.0609

( 7.5 ppm)

m/z 226.0902

( 4.1 ppm)

H

N

N

H+

S+

N

O

O

m/z 226.0538

( 2.7 ppm)

m/z 119.0609

( 5.9 ppm)

H

N

N

H+

S

+

N

O

O

H

O

Aldehydem/z 344.1069

N

N

SN

O

O

H

O

H

Sulphide

m/z 344.1433

N

N

SN

O

O

H

1 Da window

0.03 Da window

0.03 Da window

Sulphide

Aldehyde

SulphideSulphide ++ AldehydeAldehyde MetabolitesMetabolites

N

NS

N

O

O

H

N

NS

N

O

O

H

O

H

Sulphide

m/z 344.1433 ( 2.6 ppm)

Sulphide

m/z 344.1433 ( 2.6 ppm)

Aldehyde

m/z 344.1069 ( 2.0 ppm)

Aldehyde

m/z 344.1069 ( 2.0 ppm)

Exact Mass is important to Multiply Charged IonsExact Mass is important to Multiply Charged Ions

(M+H)+(M+H)+

(M+2H)2+(M+2H)2+

+/- 1 Da+/- 1 Da

+/- 0.5 Da+/- 0.5 Da

Monoisotopic M/ZMonoisotopic M/Z

Monoisotopic M/ZMonoisotopic M/Z

Peptide and Protein Sequencing Strategy Followed in Protein Identification using MassStrategy Followed in Protein Identification using Mass


21/22


Nomenclature of PeptideNomenclature of Peptide

Fragment IonsFragment Ions

R1R

1 R2R

2 R3R3 R4R4

COOCOOHCHCH NHNHCOCOH2NH2N CHCH CHCHCHCHNHNHNHNH COCOCOCO

y1y1

+2H+2H

b3b3

y2y2

+2H+2H

b2b2

y3y3

+2H+2H

b1b1

a2a2 a3a3

Peptide and Protein Sequencing gy ggy gSpectrometrySpectrometry

COMPLEX

PROTEIN

MIXTURE

UNKNOWN

INTACT

PROTEIN

PEPTIDE ESI-MS

ANALYSIS

e.g. nanoflow

IDENTIFICATION OF

PROTEIN

FRACTIONATION

OF PROTEIN

MIXTURE

e.g. by gel

electrophoresis

CHEMICAL OR

ENZYMATIC

DIGESTION

e.g. tryptic

digest

PEPTIDE

SAMPLECLEAN-UP

e.g. by LC

Computer search of

the experimentally

determinedmolecular weights

against a database of

peptide fragments

Drug Discovery ProcessDrug Discovery Process

Identification of TargetIdentification of Target

ADH 250fmoles inj.

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00Time0

100

%

HV001 1: TOF MS ES+BPI779

28.38

13.81

27.1118.92

17.41

15.24

16.51

17.50

24.66

19.32

21.63

20.10

21.09

22.17

22.8726.30

31.73

31.02

ADH 250fmoles inj.

100 200 300 400 500 600 700 800 900 1000 1100 1200m/z0

100

%

HV001 78 (22.825) Cm (78:81) 2: TOF MSMS 536.34ES+112487

240

228

222

201147129

355260

341

327

456

440

373385

428

412

815586

555527

510

699587

695667

701

794713

816

816

943926

824

Nanoscale LCNanoscale LC--ESIESI--MSMS--MSMS

ChromatogramChromatogram

MS-MS spectrumMS-MS spectrum

D t Di t d A l i (DDA)D t Di t d A l i (DDA)

Unit mass MS/MSUnit mass MS/MS


22/22


Data Directed Analysis (DDA)Data Directed Analysis (DDA)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z0

100

%

0

100

%

DG020118_03 7 (0.616) TOF MSMS 1465.80LD+311465.84

355.19

200.11159.10

158.10

325.20

315.16

298.13

211.15

483.25

386.20

424.26

581.36

554.30

535.29

598.39

868.43

652.30 691.40 740.38

1448.821266.721080.60912.52

DG020118_02 94 (8.118) TOF MSMS 1464.70LD+261464.79

323.16

305.15

175.13

159.10

110.08

225.11

296.17

780.41667.31649.32

509.24

355.19 481.25371.22

458.25

424.25

572.30

596.28 685.38

763.38 956.58798.48 869.51 1446.79

1279.651142.621080.641057.85 1265.25

1465.84

1464.81464.8 -- MSMSMSMSSearching over the InternetSearching over the Internet

Documents

2lcms Handouts