uhplc-ms basic principles and applications · srm fixed m/z pass all (+ ce) neutral loss scanning...

UHPLCUHPLC--MS Basic PrinciplesMS Basic Principlesand Applicationsand Applications

Jitnapa Voranitikul

December, 2017 Product Specialist LC/MS

UHPLCUHPLC--MS Basic PrinciplesMS Basic Principlesand Applicationsand Applications

Jitnapa Voranitikul

December, 2017 Product Specialist LC/MS

• It measures mass betterthan any other technique

• It can give informationabout chemicalstructures.

What does a Mass Spectrometer do?What does a Mass Spectrometer do?

• It measures mass betterthan any other technique

• It can give informationabout chemicalstructures.

What does a Mass Spectrometer do?What does a Mass Spectrometer do?

Applications of Mass SpectrometryApplications of Mass Spectrometry

• Pharmaceutical analysis– Bioavailability studies– Drug metabolism studies,

pharmacokinetics– Characterization of potential drugs– Drug degradation product analysis– Screening of drug candidates– Identifying drug targets

• Biomoleculecharacterization– Proteins and peptides– Oligonucleotides

• Environmental analysis– Pesticides on foods– Soil and groundwater contamination

• Forensic analysis/clinical

• Pharmaceutical analysis– Bioavailability studies– Drug metabolism studies,

pharmacokinetics– Characterization of potential drugs– Drug degradation product analysis– Screening of drug candidates– Identifying drug targets




Applications of Mass SpectrometryApplications of Mass Spectrometry







Fundamental of LiquidFundamental of LiquidChromatographyChromatography

https://www.thermofisher.com/order/catalog/product/IQLAAAGABHFAPUMZZZ?SID=srch-srp-IQLAAAGABHFAPUMZZZ

Fundamental of LiquidFundamental of LiquidChromatographyChromatography

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IntroductionIntroduction –– HPLCHPLC--System: General DesignSystem: General Design

• Pump with Degasser• Autosampler• Column (installed in a Column Compartment)• Detector• Computer with control software


Autosampler

IntroductionIntroduction –– HPLCHPLC--System: General DesignSystem: General Design



Column

ColumnRelated

Terminologies

ColumnRelated

Terminologies

ColumnRelated

Terminologies

ColumnRelated

Terminologies

Sample The Original Representative Material Which Is To Be Analyzed Also

Called The Sample Matrix (Coffee)

Analyte(s)• A Specific Compound(s) Contained In The Sample Which Is(Are) To Be

Separated And Analyzed (Caffeine)

Compound• Pure Chemical Component In A Sample, Also Called An Analyte Or

Solute

Chromatography TerminologyChromatography Terminology






Solute






Solute







Solute

Stationary Phase The Chromatographic Packing Material Which Is Held In A Fixed

Position Usually Packed Into A Column, Or Coated Onto A Surface. ItPerforms The Chemical Separation (Also Called The Packing Material, TheChromatographic Material, Or The Adsorbent)

Mobile Phase• Carrier Of The Sample, Moving It Through The Stationary

Chromatographic Packing Material. The Mobile Phase Can Be A Liquid(HPLC), Or A Gas (GC)















Remember!

The Stationary Phase and The Mobile Phasewill have OPPOSITE PROPERTIES to Set-Up Competition

For Sample Matrix and Analytes

Remember!



Remember!



Remember!



IntroductionIntroduction –– The ChromatographicThe Chromatographic ProcessProcess

Continous stream of mobile phase

Column Stationary Phase

• The stationary phase retains analytes due to various interactions.• When different chemical components pass through the column at

different rates they become separated in single zones.

Column Stationary Phase

IntroductionIntroduction –– The ChromatographicThe Chromatographic ProcessProcess

Continous stream of mobile phase

Stationary Phase Mobile Phase

• The stationary phase retains analytes due to various interactions.• When different chemical components pass through the column at

different rates they become separated in single zones.

Stationary Phase Mobile Phase

Characteristics of a ChromatogramCharacteristics of a Chromatogram

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0-50

100

200

300

400

500

600

90,0

100,0

110,0

120,0

130,0

140,0

1 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 UV_VIS_12 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 Pump_PressuremAU bar

min

1

1 -

5,3

63

2 -

6,5

73

3 -

8,8

63

4 -

12

,93

0

2

Flow: 0,50 ml/minMethanol: 70,0 %%C: 0,0 %%D: 0,0 %

Detectorsignal

Peak Height

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0-50

100

200

300

400

500

600

90,0

100,0

110,0

120,0

130,0

140,0


min

1

1 -

5,3

63

2 -

6,5

73

3 -

8,8

63

4 -

12

,93

0

2


Peak area

Characteristics of a ChromatogramCharacteristics of a Chromatogram

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0-50

100

200

300

400

500

600

90,0

100,0

110,0

120,0

130,0

140,0


min

1

1 -

5,3

63

2 -

6,5

73

3 -

8,8

63

4 -

12

,93

0

2


Pressuresignal and

ripple

Retention time

Peak Height

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0-50

100

200

300

400

500

600

90,0

100,0

110,0

120,0

130,0

140,0


min

1

1 -

5,3

63

2 -

6,5

73

3 -

8,8

63

4 -

12

,93

0

2


Baseline

Pressuresignal and

ripple

Theory of ChromatographyTheory of Chromatography

tR2

Resolution R of two peaks: Goal of every chromatographic method!

Time, s

tR1

Sig

nal

Peak 1

Peak 2

w1 w2

Theory of ChromatographyTheory of Chromatography

• Distance between the peakcenters of two peaks divided bythe average base width of thepeaks.

• From theory R > 1.50 indicatesbaseline separation.

• In real life R ≥ 2 is usually thegoal (requested in regulatedenvironment).

• Much more resolution than 2does not improve separationquality but increases analysistime.

Resolution R of two peaks: Goal of every chromatographic method!• Distance between the peak

centers of two peaks divided bythe average base width of thepeaks.

• From theory R > 1.50 indicatesbaseline separation.

• In real life R ≥ 2 is usually thegoal (requested in regulatedenvironment).

• Much more resolution than 2does not improve separationquality but increases analysistime.

uCu

BAH

Pla

te h

eig

ht

(HE

TP)

VanVan DeemterDeemter PlotPlot

Eddy dispersion A

Longitudinal diffusion B/u

Resistance to mass

transfer C u

Pla

te h

eig

ht

(HE

TP)

Mobile phase velocity (u)

AA

u

BB

VanVan DeemterDeemter PlotPlot

Eddy dispersion A

Longitudinal diffusion B/u

Resistance to mass

transfer C u

Mobile phase velocity (u)

u

BB

CC

A TermA Term –– Eddy DiffusionEddy Diffusion

http://www.chromacademy.com/lms/sco3/Theory_Of_HPLC_Band_Broadening.pdf

Band broadening due to EddyDiffusion (A Term) in columns withlarge and small particleslarge and small particles – effects onchromatographic peak shape(Efficiency (N))

A TermA Term –– Eddy DiffusionEddy Diffusion

Band broadening due to EddyDiffusion (A Term) in columns withlarge and small particleslarge and small particles – effects onchromatographic peak shape(Efficiency (N))

B TermB Term –– Longitudinal DiffusionLongitudinal Diffusion


Band broadening due to LongitudinalDiffusion (B Term) in columns withlow and high mobile phase linearlow and high mobile phase linearvelocityvelocity – effects on chromatographicpeak shape (Efficiency (N))

B TermB Term –– Longitudinal DiffusionLongitudinal Diffusion

Band broadening due to LongitudinalDiffusion (B Term) in columns withlow and high mobile phase linearlow and high mobile phase linearvelocityvelocity – effects on chromatographicpeak shape (Efficiency (N))

C TermC Term –– Mass TransferMass Transfer

Cs – term, describing thecontributions to peakbroadening in stationary phase

https://chem.libretexts.org/Core/Analytical_Chemistry/Chromedia/01Gas_Chromotography_(GC)/Gas_Chromotography%3A_Basic_Theory/13The_Van_Deemter_equation


Cm – term, describing thecontributions to peakbroadening in mobile phase

Cs – term, describing thecontributions to peakbroadening in stationary phase

https://chem.libretexts.org/Core/Analytical_Chemistry/Chromedia/01Gas_Chromotography_(GC)/Gas_Chromotography%3A_Basic_Theory/13The_Van_Deemter_equation




Band broadening due to MassTransfer (C Term) in columns withmobile phase linear velocity andmobile phase linear velocity andstationary phase particle sizestationary phase particle size –effects on chromatographic peakshape (Efficiency (N))

Band broadening due to MassTransfer (C Term) in columns withmobile phase linear velocity andmobile phase linear velocity andstationary phase particle sizestationary phase particle size –effects on chromatographic peakshape (Efficiency (N))

Particle Size EvolutionParticle Size Evolution

Early 1970’s10µm Irregular micro-porous1000-2500 psi (70-180 bar)40,000 plates/meter

10 min.

Late 1960’s40µm pellicular non-porous coated100-500 psi (7-40 bar)5,000 plates/meter

10 min.


10 min.

Particle Size EvolutionParticle Size Evolution


Late 1960’s40µm pellicular non-porous coated100-500 psi (7-40 bar)5,000 plates/meter

10 min.


1980’s to present day3.5 - 5µm spherical micro-porous1500-4000 psi (110-280 bar)80,000 - 115,000 plates/meter

Thermo Analytical LC SystemsThermo Analytical LC Systems

UltimateTH 3000Max Pressure 1000 bar

Thermo Analytical LC SystemsThermo Analytical LC Systems

VanquishTH

Max Pressure 1517 bar

System ContributionSystem Contribution –– What Do We Want?What Do We Want?

• Qualitative analysis– Resolved analytes

• Quantitative Analyis– Reproducible peak areas

We want a reliable method working on a reliable system!




System ContributionSystem Contribution –– What Do We Want?What Do We Want?







• Accuracy is the degree of closeness of a measured quantity to its true value relevance for method transfer

• Precision is the degree of further measurements show the same results(reproducibility) deviation of repeated measurements

• The target analogy:

• A valid method or system is accurate and precise!

System ContributionSystem Contribution –– Accuracy and PrecisionAccuracy and Precision





accurate and precise(ideal result)

accurate, but not precise(random errors)





System ContributionSystem Contribution –– Accuracy and PrecisionAccuracy and Precision





accurate, but not precise(random errors)

precise, but not accurate(systematic error)

Neither accurate nor precise(useless)

TheThe UltiMateUltiMate™™ 30003000 LC SystemsLC Systems

Pumps

Autosampler

Binary

StandardBasic Automated

Isocratic

Column Compartments

Detectors

Standard

VWD MWD/DAD

TheThe UltiMateUltiMate™™ 30003000 LC SystemsLC Systems

Quaternary Dual-Gradient

Thermostatted + Fractionation

With ValvesStandard

Fluorescence Corona Coulochem

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Fundamental of Mass SpectrometryFundamental of Mass Spectrometry

“The basis in mass spectrometry (MS) is the productionof ions, that are subsequently separated or filteredaccording to their mass-to-charge (m/z) ratio, anddetected. The resulting mass spectrum is a plot of the(relative) abundance of the produced ions as a functionof the m/z ratio.”

What is Mass Spectrometry?What is Mass Spectrometry?


Niessen, W. M. A.; Van der Greef, J., Liquid Chromatography–Mass Spectrometry:Principles and Applications, 1992, Marcel Dekker, Inc., New York, p. 29.


What is Mass Spectrometry?What is Mass Spectrometry?


Niessen, W. M. A.; Van der Greef, J., Liquid Chromatography–Mass Spectrometry:Principles and Applications, 1992, Marcel Dekker, Inc., New York, p. 29.

Mass spectrometry CharacteristicsMass spectrometry Characteristics

•Operate at very low pressure (10-5 to 10-7 torr)

(Atmosphere = 760 torr)

•Mass spectrometer work with IONSIONS

•Measure gas-phase ions

•Determine the mass are separated according to their mass-to-charge

(m/z) ratio






(m/z) ratio

Mass spectrometry CharacteristicsMass spectrometry Characteristics






(m/z) ratio






(m/z) ratio

Mass Spectrometry “Simplified”Mass Spectrometry “Simplified”

GenerateMoveSelectMoveSelectDetect

The lifetime of an ion from the point of formationto detection is approximately 50 to 100 microseconds

Mass Spectrometry “Simplified”Mass Spectrometry “Simplified”

Ion Production

Ion OpticsIon Optics

Analyser (Quadrupole, Orbitrap)

Electron Multiplier

The lifetime of an ion from the point of formationto detection is approximately 50 to 100 microseconds

Mass SpectrometryMass Spectrometry –– Block DiagramBlock Diagram

LiquidChromatography

Ionization

• ESI• APCI• APPI

Very important!- Many columns- Many solvent systems

Detector/Data

Collection


Ionization Mass AnalyserDetector/

DataCollection

•Triple Quadrapoles•Ion-Traps•Orbitrap

Q1Hyperquad

Ion SourceInterface

TSQ Quantum ComponentsTSQ Quantum Components

Q00 Q0Q1

Hyperquad

Ion SourceInterface

Ion Transfer Tube

Q1Hyperquad

Q3Hyperquad

DetectionSystem

TSQ Quantum ComponentsTSQ Quantum Components

Q1Hyperquad

Q3Hyperquad

Q2Collision Cell

Mass Analyzer

ION SOURCEION SOURCE

IONIZATION TECHNIQUESIONIZATION TECHNIQUES

ION SOURCEION SOURCE

IONIZATION TECHNIQUESIONIZATION TECHNIQUES

Type of Ionization techniquesType of Ionization techniques

•Electron impact (EI)•Chemical Ionization (CI)

•Atmospheric Pressure Ionization (API)•Electrospray Ionization (ESI)•Atmospheric Pressure Chemical Ionization (APCI)•Atmospheric Pressure Photo-Ionization (APPI)

•Matrix Assisted Laser Desorption/Ionization (MALDI)




Type of Ionization techniquesType of Ionization techniques







Electrospray Ionization (ESI)Electrospray Ionization (ESI)

Three Fundamental Processes:

1. Production of charged droplets.2. Droplet size reduction, and fission.3. Gas phase ion formation.








ESI Needle

+/- 3-5 kV

Solvent evaporation andIon desolvation


Taylor Cone

Ion Transfer TubeSolvent evaporation andIon desolvation


ESIESI -- Ion Max SourceIon Max SourceESIESI -- Ion Max SourceIon Max Source

• Gas phase ionization via corona discharge• APCI is a three-step process:

1. High voltage (via corona needle) interacts with both the nitrogen carrier gas and the vaporizedHPLC solvent to produce primary ions:

O2 + e- → O2+. + 2e-

N2 + e- → N2+. + 2e-

2. Through a complex series of reactions primary ions react with solvent molecules formingreagent ions, H3O+ and CH3OH2

+

3. Reagent ions react with analyte molecules forming (M+H)+ in positive ion mode or (M-H)- innegative ion mode:

H3O+ + Analyte → (Analyte + H)+ + H2OOH- + Analyte → (Analyte – H)- + H2O

Atmospheric Pressure Chemical Ionization (APCI)Atmospheric Pressure Chemical Ionization (APCI)



O2 + e- → O2+. + 2e-

N2 + e- → N2+. + 2e-


+





O2 + e- → O2+. + 2e-

N2 + e- → N2+. + 2e-


+






O2 + e- → O2+. + 2e-

N2 + e- → N2+. + 2e-


+




© 2004 Dr. Paul Gates

University of Bristol


Ion Max Source DesignIon Max Source Design -- APCI ProbeAPCI ProbeIon Max Source DesignIon Max Source Design -- APCI ProbeAPCI Probe

• It depends on the exact application.

• Increasing polarity and molecular weight and thermal instabilityfavors electrospray.

– Most drugs of abuse are highly polar and are easilyanalyzed using electrospray.

– High molecular weight proteins also require electrospray

• Lower polarity and molecular weight favors APCI or APPI.• Lower background, but compounds must be more

thermally stable.

Which is Best?Which is Best?






thermally stable.






thermally stable.

Which is Best?Which is Best?






thermally stable.


LiquidChromatography

IonizationDetector/

DataCollection


Ionization Mass AnalyserDetector/

DataCollection

•Triple Quadrapoles•Ion-Traps•Orbitrap

MASSMASS ANALYSERANALYSER

QUADRUPLEQUADRUPLE


QUADRUPLEQUADRUPLE


QUADRUPLEQUADRUPLE


QUADRUPLEQUADRUPLE

• Operate under high vacuum (keeps ions from bumping into gasmolecules)

• Actually measure mass-to-charge ratio of ions (m/z)

• Key specifications are resolution, mass measurementaccuracy, and sensitivity.

• Several kinds exist: for ion traps, quadrupole, time-of-flight andorbitrap are most used.

Mass analyzers separateMass analyzers separate ionsions









Mass analyzers separateMass analyzers separate ionsions





TSQTSQ Quantiva MSQuantiva MS——Powered by AIM TechnologyPowered by AIM Technology

Systematic optimization of all electric fields,in concert, to produce breakthroughperformance.

Active Ion Management (AIM)

TSQTSQ Quantiva MSQuantiva MS——Powered by AIM TechnologyPowered by AIM Technology

Systematic optimization of all electric fields,in concert, to produce breakthroughperformance.

Active Ion Management (AIM)

TSQTSQ QuantivaQuantiva Animation (available on YouTube)Animation (available on YouTube)

http://www.youtube.com/watch?v=LFB14D8pkoc

TSQTSQ QuantivaQuantiva Animation (available on YouTube)Animation (available on YouTube)

http://www.youtube.com/watch?v=LFB14D8pkoc

Scan Modes

Scan Mode Q1 Q2

Full Scan Scanning Pass All

SIM Fixed m/z Pass All

Product Fixed m/z Pass All (+ CE)

SRM Fixed m/z Pass All (+ CE)

Neutral Loss Scanning Pass All (+ CE)

Precursor Scanning Pass All (+ CE)

Scan Modes

Q2 Q3 Purpose

Pass All Pass All MW Info.

Pass All Pass All Quantitation

Pass All (+ CE) Scanning Structural Info.

Pass All (+ CE) Fixed m/z TargetedQuantitation

Pass All (+ CE) Scanning Analyte Screening

Pass All (+ CE) Fixed m/z Analyte Screening

R T : 0 . 0 0 - 2 0 . 0 2

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0T i m e ( m i n )

0

51 0

1 5

2 0

2 5

3 0

3 5

4 04 5

5 0

5 5

6 0

6 5

7 0

7 58 0

8 5

9 0

9 5

1 0 0

Relative

Abundan

ce

1 . 2 4

4 . 6 0

2 . 6 2

3 . 7 9

3 . 1 05 . 3 8

7 . 7 87 . 5 8

1 8 . 3 41 6 . 9 3 1 8 . 9 41 5 . 9 58 . 2 3 1 5 . 0 91 4 . 0 58 . 9 0 1 2 . 7 39 . 8 1

N L :7 . 3 5 E 7T I C M SH S - h e l i n -1 0 2 4 - 1

One Click

Chromatogram

Full Scan ModeFull Scan Mode

One Click

Spectrum

H O

H O

O H

6060 8080 100100 120120 140140 16016000

100100

%%

R T : 0 . 0 0 - 2 0 . 0 2

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0T i m e ( m i n )

0

51 0

1 5

2 0

2 5

3 0

3 5

4 04 5

5 0

5 5

6 0

6 5

7 0

7 58 0

8 5

9 0

9 5

1 0 0

Relative

Abundan

ce

1 . 2 4

4 . 6 0

2 . 6 2

3 . 7 9

3 . 1 05 . 3 8

7 . 7 87 . 5 8

1 8 . 3 41 6 . 9 3 1 8 . 9 41 5 . 9 58 . 2 3 1 5 . 0 91 4 . 0 58 . 9 0 1 2 . 7 39 . 8 1

N L :7 . 3 5 E 7T I C M SH S - h e l i n -1 0 2 4 - 1

Chromatogram

Full Scan ModeFull Scan Mode

H +Base peak

at m/z 240 (MH+)N H tB uH

180180 200200 220220 240240 260260 280280 300300

240

241

Full Scan ModePurpose: Survey scan of a chromatographic peak

Q1 Scanning RF Only Q3 RF Only

Full scan Q1:

Full Scan (QFull Scan (Q11 or Qor Q33))

Q1 RF Only RF Only Q3 Scanning

Full scan Q1:

Full Scan Q3:

Full Scan ModePurpose: Survey scan of a chromatographic peak

Q1 Scanning RF Only Q3 RF Only

Full Scan (QFull Scan (Q11 or Qor Q33))

Q1 RF Only RF Only Q3 Scanning

SIM ModePurpose: Quantitation on a specific m/z range of ions

Selected Ion MonitoringSelected Ion Monitoring –– SIMSIM

Q1 Set RF Only + CE Q3 RF Only

SIM Q1:

Q1 RF Only RF Only + CE Q3 Set

SIM Q1:

SIM Q3:

SIM ModePurpose: Quantitation on a specific m/z range of ions


Q1 Set RF Only + CE Q3 RF Only

Q1 RF Only RF Only + CE Q3 Set

SIM is in essence a full scan acquisition on a relatively narrow masswindow (defined as center mass / scan width)

Fixed m/z Pass All


Advantages Targeted analyte monitoring High duty cycle

Disadvantages Can suffer from interferences Not as sensitive or selective as SRM

SIM is in essence a full scan acquisition on a relatively narrow masswindow (defined as center mass / scan width)

Pass All Pass All


Disadvantages Can suffer from interferences Not as sensitive or selective as SRM

RT: 0.00 - 75.04 SM: 7G

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

52.33

47.88

31.3055.14

34.47 50.24

39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03

31.30

39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03

NL: 2.91E8Base Peak F: + cNSI Full ms [400.00-1800.00]MS data14

NL: 7.97E7Base Peak m/z=1030.90-1031.90 F:+ c NSI Full ms [400.00-1800.00]MS data14

Full Scan

Full Scan versus SIMFull Scan versus SIMRT: 0.00 - 75.04 SM: 7G

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

52.33

47.88

31.3055.14

34.47 50.24

39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03

31.30

39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03



SIM

RT: 0.00 - 75.04 SM: 7G

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

52.33

47.88

31.3055.14

34.47 50.24

39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03

31.30

39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03



Full Scan versus SIMFull Scan versus SIMRT: 0.00 - 75.04 SM: 7G

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

52.33

47.88

31.3055.14

34.47 50.24

39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03

31.30

39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03



Pass AllFixed m/z

Q1 Q2

Selected Reaction Monitoring (SRM)Selected Reaction Monitoring (SRM)

Advantages Targeted analyte monitoring High duty cycle “Simultaneous” monitoring of

multiple transitions

Fixed m/zPass All

Q2 Q3

Selected Reaction Monitoring (SRM)Selected Reaction Monitoring (SRM)

Advantages Targeted analyte monitoring High duty cycle “Simultaneous” monitoring of

multiple transitions

Disadvantages No structural information

The Need for True MS/MSThe Need for True MS/MSThe Need for True MS/MSThe Need for True MS/MS

RT: 2.28 - 5 .89 S M: 15G

2.5 3.0 3.5 4.0 4.5 5.0 5.5Time (m in)

0

20

40

60

80

100

0

20

40

60

80

100

Rela

tive

Abun

danc

e

0

20

40

60

80

100

RT: 5 .37S N: 1093

RT: 5 .37S N: 27528

RT: 5 .37S N: 201353020

NL: 8 .14E7m/z= 191 .50-192 .50MS G enesis F u ll-MS2

NL: 1 .38E8m/z= 191 .50-192 .50 F :+ c EI Q 1MS[191 .945-191 .995 ] MSG enesisSIM-1_111128173605

NL: 3 .27E7T IC F : + c EI SRM ms2191 .950[126 .935-126 .985 ] MSG enesisF u ll-SRM-Survey-1

x300

Full MS Scan

Noise Level

SRM Selectivity in Complex MatricesSRM Selectivity in Complex Matrices

RT: 2.28 - 5 .89 S M: 15G

2.5 3.0 3.5 4.0 4.5 5.0 5.5Time (m in)

0

20

40

60

80

100

0

20

40

60

80

100

Rela

tive

Abun

danc

e

0

20

40

60

80

100

RT: 5 .37S N: 1093

RT: 5 .37S N: 27528

RT: 5 .37S N: 201353020




x300

SIM Scan

SRM Scan

Noise Level

RT: 2.28 - 5 .89 S M: 15G

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RT: 5 .37S N: 27528

RT: 5 .37S N: 201353020




x300

Noise Level

SRM Selectivity in Complex MatricesSRM Selectivity in Complex Matrices

RT: 2.28 - 5 .89 S M: 15G

2.5 3.0 3.5 4.0 4.5 5.0 5.5Time (m in)

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RT: 5 .37S N: 27528

RT: 5 .37S N: 201353020




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RT: 17.13 - 27.39

18 19 20 21 22 23 24 25 26 27Time (min)

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RT: 23.76

24.01

25.5223.55 24.11 24.55 25.3523.2822.9722.52 25.6522.17 25.87 26.45 26.7821.6721.3021.0920.4020.27

RT: 23.76

25.37

23.5327.3522.93

26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61

RT: 23.76

24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37

21.89 22.5221.6721.1520.5719.65 20.15

RT: 23.77

RT: 23.77

NL: 6.37E4m/z= 236.50-237.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim



NL: 6.10E5m/z= 207.50-208.50 F: + c EISRM ms2 237.000[207.999-208.001] MSGenesis Probe20F


SIM

Comparison of SIM and SRMComparison of SIM and SRMRT: 17.13 - 27.39

18 19 20 21 22 23 24 25 26 27Time (min)

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RT: 23.76

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25.5223.55 24.11 24.55 25.3523.2822.9722.52 25.6522.17 25.87 26.45 26.7821.6721.3021.0920.4020.27

RT: 23.76

25.37

23.5327.3522.93

26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61

RT: 23.76

24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37

21.89 22.5221.6721.1520.5719.65 20.15

RT: 23.77

RT: 23.77






SRM

RT: 17.13 - 27.39

18 19 20 21 22 23 24 25 26 27Time (min)

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RT: 23.76

24.01

25.5223.55 24.11 24.55 25.3523.2822.9722.52 25.6522.17 25.87 26.45 26.7821.6721.3021.0920.4020.27

RT: 23.76

25.37

23.5327.3522.93

26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61

RT: 23.76

24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37

21.89 22.5221.6721.1520.5719.65 20.15

RT: 23.77

RT: 23.77






Comparison of SIM and SRMComparison of SIM and SRMRT: 17.13 - 27.39

18 19 20 21 22 23 24 25 26 27Time (min)

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RT: 23.76

24.01

25.5223.55 24.11 24.55 25.3523.2822.9722.52 25.6522.17 25.87 26.45 26.7821.6721.3021.0920.4020.27

RT: 23.76

25.37

23.5327.3522.93

26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61

RT: 23.76

24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37

21.89 22.5221.6721.1520.5719.65 20.15

RT: 23.77

RT: 23.77






Superior Selectivity

Free from sample matrix

Applications of TripleApplications of TripleQuadrupole LCQuadrupole LC--MS/MSMS/MS




Why HighWhy High--throughput LCthroughput LC--MS/MS for Drugs of Abuse Analyses?MS/MS for Drugs of Abuse Analyses?Why HighWhy High--throughput LCthroughput LC--MS/MS for Drugs of Abuse Analyses?MS/MS for Drugs of Abuse Analyses?

Thermo Scientific Vanquish Horizon UHPLCThermo Scientific Vanquish Horizon UHPLCThermo Scientific Vanquish Horizon UHPLCThermo Scientific Vanquish Horizon UHPLC

Experimental DesignExperimental Design –– Liquid ChromatographyLiquid ChromatographyExperimental DesignExperimental Design –– Liquid ChromatographyLiquid Chromatography

Experimental DesignExperimental Design –– Mass SpectrometryMass SpectrometryExperimental DesignExperimental Design –– Mass SpectrometryMass Spectrometry

ApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMSApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMS

HIGH RESOLUTION MASSHIGH RESOLUTION MASS ANALYSERANALYSER

ORBITRAPORBITRAP


ORBITRAPORBITRAP


ORBITRAPORBITRAP


ORBITRAPORBITRAP

Mass Analyzers ParametersMass Analyzers Parameters

•Nominal Mass

The mass of an ion with a given empirical formula calculated using theinteger mass numbers of the most abundant isotope of each element

Ex : M=249 C20H9+ or C19H7N+ or C13H19N3O2

+

•Exact Mass

The mass of an ion with a given empirical formula calculated using theexact mass of the most abundant isotope of each element

Ex : M=249 C20H9+ 249.0070C19H7N+ 249.0580C13H19N3O2+ 249.1479

•Nominal Mass



+

•Exact Mass


Ex : M=249 C20H9+ 249.0070C19H7N+ 249.0580C13H19N3O2+ 249.1479

Mass Analyzers ParametersMass Analyzers Parameters

•Nominal Mass



+

•Exact Mass


Ex : M=249 C20H9+ 249.0070C19H7N+ 249.0580C13H19N3O2+ 249.1479

•Nominal Mass



+

•Exact Mass


Ex : M=249 C20H9+ 249.0070C19H7N+ 249.0580C13H19N3O2+ 249.1479

MASS RESOLUTIONMASS RESOLUTIONMASS RESOLUTIONMASS RESOLUTION

• Ability of a mass spectrometer to distinguish between ions ofnearly equal m/z ratios (isobars).

Mass Resolution: What is it?Mass Resolution: What is it?

• m - measured mass• Δm - peak width measured at

50% peak intensity (Full WidthHalf Maximum)

- or the mass differencebetween two adjacent peaksof equal intensity, in this casepw @ 10% valley definition isused.




• Ability of a mass spectrometer to distinguish between ions ofnearly equal m/z ratios (isobars).

Mass Resolution: What is it?Mass Resolution: What is it?







Resolution & Peak WidthResolution & Peak WidthResolution & Peak WidthResolution & Peak Width

• At minimum the resolution of the mass analyzer should besufficient to separate two ions differing by one mass unitanywhere in the mass range scanned (unit mass resolution).

• Typical values of resolution for low resolution mass analyzers(e.g. quadrupoles and ion traps) are below 5000.

• High resolution instruments have a resolution exceeding 15000.

Mass ResolutionMass Resolution: What is it?: What is it?












Mass spectrometerFT-ICR-MS

OrbitrapHR-ToFHR-ToF

Magnetic SectorsQuadrupole / IonTrap in UltraZoom mode

Quadrupole / IonTrap


Resolving Power (FWHM)1,000,000500,00060,00060,00010,000

Quadrupole / IonTrap in UltraZoom mode 5,0001,000

Commercial High Resolution MS Technology RaceCommercial High Resolution MS Technology RaceM

ass re

solu

tion (

FW

HM

)

300000

350000

400000

450000

500000

Orbitrap Tof / QTof

ORBITRAP’s spectacular climbin performance in a decade!

Time progression (year)

Mass re

solu

tion (

FW

HM

)

Bendix Tof0

50000

100000

150000

200000

250000

1955 1965 1975 1985

Commercial High Resolution MS Technology RaceCommercial High Resolution MS Technology Race

Tribrid Orbitrap

ORBITRAP’s spectacular climbin performance in a decade!

Q-Orbitrap*

New Tribrid Orbitrap

LIT-Orbitrap ETD

Time progression (year)

1995 2005 2015

Ion Trap-Orbitrap

Quad Orbitrap

First Q-Tof

New Q-Orbitrap

Entry Q-Orbitrap

MASS ACCURACYMASS ACCURACYMASS ACCURACYMASS ACCURACY

• Mass accuracy is the precision of which the mass is measured bythe mass spectrometer.

• Typical way of reporting mass error in ppm (relative mass error):

• Absolute mass error can be used (mDa).

• Main advantage: the possibility to determine the elementalcomposition of individual molecular or fragment ions, a powerfultool for the structural elucidation or confirmation.

Mass Accuracy: What is it?Mass Accuracy: What is it?









Mass Accuracy: What is it?Mass Accuracy: What is it?





• Accurate mass measurements take advantage of the fact that the combination ofelements contained in a molecule have a very specific, non-nominal molecularweight:

– Carbon has a mass of 12.0000– Hydrogen has a mass of 1.0078– Oxygen has a mass of 15.9949– Nitrogen has a mass of 14.0031

Mass accuracy depends on resolutionHigher resolution allows for better mass accuracy

Mass AccuracyMass Accuracy• Accurate mass measurements take advantage of the fact that the combination of

elements contained in a molecule have a very specific, non-nominal molecularweight:



• Accurate mass measurements take advantage of the fact that the combination ofelements contained in a molecule have a very specific, non-nominal molecularweight:



Mass AccuracyMass Accuracy• Accurate mass measurements take advantage of the fact that the combination of

elements contained in a molecule have a very specific, non-nominal molecularweight:



• Typical mass accuracy capability for various MS types:

Mass AccuracyMass Accuracy

• Typical mass accuracy capability for various MS types:

Mass AccuracyMass Accuracy

Source: Metabolomics Fiehn’s lab

Major accurateMajor accurate--massmass analyzersanalyzers for Life Sciencefor Life ScienceMajor accurateMajor accurate--massmass analyzersanalyzers for Life Sciencefor Life Science

OrbitrapOrbitrap Mass Analyzer:Mass Analyzer: Principle of OperationPrinciple of Operation

qm

kz /

Makarov A. Anal. Chem. 2000, 72, 1156-1162.

OrbitrapOrbitrap Mass Analyzer:Mass Analyzer: Principle of OperationPrinciple of Operation

)/ln(2/2

),( 222mm RrRrz

kzrU

z

φHyper-logarithmic potential distribution:

“ideal Kingdon trap”

r

12

2

R

Rmz 2

2

R

Rmzr

Makarov A. Anal. Chem. 2000, 72, 1156-1162.

Characteristic frequencies:• Frequency of rotation ωφ

• Frequency of radial oscillations ωr• Frequency of axial oscillations ωz

Many Ions Generate a Complex “Transient”Many Ions Generate a Complex “Transient”

FourierTransform

Time Domain

FourierTransform

Mass Spectrum

Many Ions Generate a Complex “Transient”Many Ions Generate a Complex “Transient”

FourierTransform

Frequency Domain

FourierTransform

Mass Spectrum

• Accurate mass measurement is the experimentally determined massmeasured to an appropriate degree of accuracy and precision ( Gross, J.Am. Soc. Mass Spectrom.,1994 )

• Accurate mass measurements narrow down the list of possibleformulae for a particular molecular weight

• Mass spectrum and analyst complete the picture:– Isotope distributions indicate/eliminate elements ( e.g. Cl, Br, Cu )

– User-supplied info eliminates others ( e.g. no F, Co )

– Suggested formula has to make chemical sense: (C6H28O2 is not reasonable noris Cl3H2Co4 )

Mass Accuracy: I.D. of unknown compoundsMass Accuracy: I.D. of unknown compounds











Mass Accuracy: I.D. of unknown compoundsMass Accuracy: I.D. of unknown compounds






• The effect of mass accuracy and molecular weight on thenumber of potential chemical formulae.

Mass accuracy for chemical formulae IDMass accuracy for chemical formulae ID

• The effect of mass accuracy and molecular weight on thenumber of potential chemical formulae.

Mass accuracy for chemical formulae IDMass accuracy for chemical formulae ID

Generate Formula fromGenerate Formula from MonoisotopicMonoisotopic MassMass

Right click on massThen select

Generate formula form mass

0.549 ppm Mass Accuracy

Generate Formula fromGenerate Formula from MonoisotopicMonoisotopic MassMass

10 ppb

100 ppb

1000 ppb

Elemental Composition Generates by Monoisotopic MassElemental Composition Generates by Monoisotopic MassElemental Composition Generates by Monoisotopic MassElemental Composition Generates by Monoisotopic Mass

80

100

120

140

160

No o

f For

mul

ae

Chemical element NoC 50H 100O 10N 10Cl 10

Elemental Composition StatisticsElemental Composition Statistics

0

20

40

60

80

1 1.5 2

No o

f For

mul

ae

Mass Accuracy (ppm)

22 55 77

147147

Elemental Composition StatisticsElemental Composition Statistics

5 10 50Mass Accuracy (ppm)

1616

3030

Compound ID confirmation through Isotopic Pattern MatchCompound ID confirmation through Isotopic Pattern MatchCompound ID confirmation through Isotopic Pattern MatchCompound ID confirmation through Isotopic Pattern Match

UHPLC with QUHPLC with Q ExactiveExactive Mass SpectrometerMass Spectrometer

http://planetorbitrap.com/

UHPLC with QUHPLC with Q ExactiveExactive Mass SpectrometerMass Spectrometer

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