ms/ms scan modes - elte

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MS/MS Scan Modes

Árpád Somogyi

• Eötvös University, BudapestApril 16, 2012

MS/MS Scan Modes

Select

Select

Select Select

Scan

Scan

Scan Scan

Dissociate

Dissociate

Dissociate

Dissociate

Product Ion Scan

Neutral Loss Scan

Precursor Ion Scan

Selected ReactionMonitoring (SRM)

Δ

2

Scan modes in a triple quadrupole (QqQ)(one quadrupole shown here)

http://www-methods.ch.cam.ac.uk/meth/ms/theory/quadrupole.html

100200

300

Vm1

RF

DC

Vm3Vm2

m/zm2m/zm1 m/zm3

Voltage

mass spectrum

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200 300

AnalyteMixture

Scan

3

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Vm1

RF

DC

Vm3Vm2

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200 300

AnalyteMixture

Scan

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300100

200 300

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200 300

Voltage

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Vm1

RF

DC

Vm3Vm2

100

200 300

AnalyteMixture

Scan

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300100

200 300

100

200 300

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200 300

Voltage

4

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Vm1

RF

DC

Vm3Vm2

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200 300

AnalyteMixture

Scan

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200 300

Voltage

200

RFDC

Vm2

m/zm2 mass spectrumDesiredAnalyte

100

200 300

AnalyteMixture

SelectVoltage

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Modes of scanning in a Triple Quadrupole (QQQ)

Q1 Q3q2 (gas)

• Quadrupole is a mass filter• QQQ used in this tutorial to describe scan modes

– Q1 and Q3 = analyzers– q2 (middle quadrupole) used for CID (dissociation)

• Ways to set quadrupoles: Scan, Select & rf only• Other instruments are used

scan orselect

scan orselect

rf only

A variety of instruments are used for MS/MS

To name a few…

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QQQ

Benefits:Simple, ion filterGood for quantification

Q1 q2

Q3

Q-TOF

Benefits:Higher resolution & mass accuracyAll ions recorded in parallel

Ref: Chemushevich, 2001

Q1 q2 TOF

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Q-Linear Ion Trap (Q-trap)

Benefits:Quadrupole-like CID spectra with ion trap sensitivityNo ion trap low mass cutoff

Ref: Hopfgartner, 2003

Q1 q2 LIT

API ion sourcelinear

Ion trap

quadrupoleMassfilter

C-trap

HCD collision

cell

reagentIon

source

reagent reagentorbitrap

LT-Orbitrap (pictured with ETD source)

Benefits:LTQ: Ion trap sensitivityOrbi: High dynamic range& high resolution & mass accuracy

Q1q2

Orbi

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Trapping Instruments

Benefits:SensitivityMS^n (most)

Q1

q2

Q3

MS/MS Scan Modes

Select

Select

Select Select

Scan

Scan

Scan Scan

Dissociate

Dissociate

Dissociate

Dissociate

Product Ion Scan

Neutral Loss Scan

Precursor Ion Scan


Δ

9

Product Ion Scan

• Qualitative structural information• Q1 is used to select one m/z• This “parent” ion is dissociated in Q2 (Rf only)

– Q2 in “Rf only” mode is high transmission device• Fragments (product ions) are formed by collisions• Product ions are scanned through Q3• Prerequisite: Produce an MS spectrum for selection• Output = MS/MS spectrum

Q1 Q3q2 (gas)

Select ScanDissociate

Tandem in Space (QQQ) – Product Ion Scan

Q1 Q3(gas)Source Detector

Select one m/z

(fixed Vac/Vdc)

10

Tandem in Space (QQQ) – Product Ion Scan


(collide with gas)

DissociateScan Products

(scan Vac/Vdc)

MS … select … MS/MS

MS select

MS/MS

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MS/MS of a Peptide (YGGFL, m/z = 556.2)

200 300 400 500 600

0

20

40

60

80

100R

elat

ive

Inte

nsity

m/z

556.2

-H2O

b4

a4

y3y2

Y G G F L

a/b4

y3y2

425

397

279 336

538

Multiple stages of MS in a trapping instrumentMSn of trocade (a drug metabolism study)

MS

MS2

MS3

MS4

Ref: Hopfgartner, 2003

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Product Ion Scans may be Software Controlled

• Goal: collect MS/MS spectra for complex mixtures• Complex mixture can be separated by HPLC• HPLC linked directly to analyzer by ESI source• Mass analyzer collects continuous MS spectra • At pre-determined intensity of a precursor ion, MS/MS

spectra acquired– Data Dependent acquisition – Dynamic Exclusion = exclude repeats

Ion Currentover 60 min

MS

MS/MS

26.47

571.29

13

Advantages for product ion scan

NOTES

QQQ

Q-trap

Q-TOF

TOF-TOF

Ion Trap (3D, LT)

ICR

Q or Trap-ICR

LT-Orbitrap

Select ScanDissociate

Orbitrap Animation

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MS/MS Scan Modes

Select

Select

Select Select

Scan

Scan

Scan Scan

Dissociate

Dissociate

Dissociate

Dissociate

Product Ion Scan

Neutral Loss Scan

Precursor Ion Scan


Δ

• Screen for precursor ions that produce a given product ion• Q1 is scanned• All precursor ions collide with target gas (in CID)• Fragments (product ions) are formed• Q3 allows transmission of one fragment ion m/z• Run as HPLC-MS/MS experiment• Prerequisite: Determine expected product ions by MS/MS • Output = chromatogram showing time/intensity of precursors

of interest and reconstructed spectrum

Precursor Ion Scan

Q1 Q3q2 (gas)

SelectScan Dissociate

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Precursor Ion Scan – Detection of


Scan Precursors

(sequential rf/dc)



rf/dc 1

Dissociate

(collide with gas)

at rf/dc 1

16



Select fragment

(fixed rf/dc )

at rf/dc 1



rf/dc 2

Dissociate

(collide with gas)

at rf/dc 2

17



(fixed rf/dc )

Select fragment

at rf/dc 2



rf/dc 3

Dissociate

(collide with gas)

at rf/dc 3

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(fixed rf/dc )

Select fragment

at rf/dc 3

Precursor Ion SpectrumReconstructed by software

200 300 400 500 600

0

20

40

60

80

100

Rel

ativ

e In

tens

ity

m/z

Q1 rf/dc 2

Q1 rf/dc 3

Software stores memory of the rf/dc voltages that coincidewith fragments striking the detector!

These rf/dc voltages equal specific

m/z values

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Coming into Q1

all ions In mixture(TIC)

In Q1, at one rf/dc ratio, m/z = 842.5

m/z = 842.5

Q3 fixed to detect 436.2

m/z = 436.2 total ion Currentm/z 842.5

Reconstructed chromatogram

Precursor ion result – precursor of 436.2

NOT DETECTED

hits the detector

Precursor ion isFragmented in q2

• Consider identification of a mixture of halogenated compounds by MS/MS

• Describe a Precursor Ion Scan that might be used to identify all monohalogenated benzenes in a sample

• What is the m/z that hits the detector?

• What happens in Q1, q2, Q3? • Draw the spectrum

Br I ClF

C 12 Cl 35/37

H 1 Br 79/81

F 19 I 127

Learning Check: FACT SHEETPrecursor Ion Scan

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Learning Check: PROBLEM SOLVERPrecursor Ion Scan

1) Calculate the mass of one precursor ion, for example, fluorobenzene

2) Draw a likely fragment ion common to all of these analytes? (assume a simple fragment from M+ is formed)

3) Calculate the mass of the common fragment

_____ carbon @ 12 = __________ hydrogen @ 1 = __________ fluorine @ 19 = _____

Total = _____

_____ carbon @ 12 = __________ hydrogen @ 1 = _____

Total = _____

Learning Check: PROBLEM SOLVERPrecursor Ion Scan

1) Calculate the mass of one precursor ion, for example, fluorobenzene

2) Draw a likely fragment ion common to all of these analytes? (assume a simple fragment from M+ is formed)

3) Calculate the mass of the common fragment

_____ carbon @ 12 = __________ hydrogen @ 1 = __________ fluorine @ 19 = _____

Total = _____

_____ carbon @ 12 = __________ hydrogen @ 1 = _____

Total = _____

HH

H H

H

+.F

6 725 5

96

6 725 5

77

1 19

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Learning Check: Precursor Ion Scan

• What m/z hits the detector?

• What happens in Q1 q2 Q3?

• Draw the spectrum

Q1 q2 Q3

RelativeIntensity

0 50 100 150 200m/z

Scan all ions sequential CID Fix: m/z 77

96 112

114

156/158 204

6 Carbon @ 12 = 725 hydrogen @ 1 = 5

F

77 + 19 = 96

Cl

77 + 35 = 11277 + 79 = 156

Br

77 + 127 = 204

I

m/z = 77

+HH

H H

H

.

Precursor Ion Scan: A literature exampleCombinatorial Chemistry

• Combinatorial libraries result from the simultaneous synthesis of a great number of compounds.– analytical challenge to characterize

• Purpose: Determine purity and identity of pooled library

• QQQ mass spectrometerPROBLEM:

MS SCAN IS COMPLEXAND PROVIDES LITTLE

INFORMATION

Triolo, 2001

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Precursor Ion Scan: A literature exampleCombinatorial Chemistry

• The compound components X, Y, Z are not identified– mass of X = 299– mass of Y = 40– mass of Z = 100

• Library compounds, example if AA1 = Arg, AA2 = Ala:– X-Arg-Y-Ala-Z [mass of Arg = 156, Ala = 71]– mass: 299 + 156 + 40 + 71 + 100 = 666– for mass spectrometry, add 1 proton to form ion: 666 + 1 = 667

• When AA1 = Arg, a fragment will form, m/z = 455

Library compounds:X-AA1-Y-AA2-Z

Triolo, 2001

X-Arg-Y-AA2-Zm/z = 455

Learning Check: FACT SHEETPrecursor Ion Scan in Combinatorial Chemistry


Alanine ALA 71Arginine ARG 156Asparagine ASN 114Aspartic Acid ASP 115Cystein CYS 103Glutamic Acid GLU 129Glutamine GLN 128Glycine GLY 57Histidine HIS 137Isoleucine ILE 113Leucine LEU 113Lysine LYS 128Methionine MET 131Phenylalanine PHE 147Proline PRO 97Serine SER 87Threonine THR 101Tryptophan TRP 186Tyrosine TYR 163Valine VAL 99

X-AA1-Y-AA2-Z

A fragment ion will form for cleavage at this bond when

aa1 = Argininem/z = 455

R ?

X AA1 Y AA2 Z

299 156 40 ? 100

Sum 455 140 + ?

595 + AA2 + 1 = Precursor Mass

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Precursor ARG ?

[M+H]+ X AA1 Y H+ AA2 Z653 299 156 40 1 100667 299 156 40 1 100683 299 156 40 1 100693 299 156 40 1 100695 299 156 40 1 100697 299 156 40 1 100709 299 156 40 1 100710 299 156 40 1 100711 299 156 40 1 100724 299 156 40 1 100725 299 156 40 1 100727 299 156 40 1 100733 299 156 40 1 100743 299 156 40 1 100752 299 156 40 1 100759 299 156 40 1 100

Learning Check: precursor scan results

The precursor ion results of this experiment are shown

in the left column

find the amino acidfor each of these

compounds

• Consider identification of a mixture of these library compounds by MS/MS

• Describe a Precursor Ion Scan that might be used to determine that all amino acids are represented in position 2in the compounds (AA2) if position 1 = Arg




X-AA1-Y-AA2-Z

A fragment ion will form for cleavage at this bond when

aa1 = Argininem/z = 455

Learning Check: FACT SHEETPrecursor Ion Scan in Combinatorial Chemistry

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• What happens in Q1, q2, Q3?

• Draw the spectrumfor a fewcompounds

Q1 q2 Q3

600 650 700 750 800m/z

Learning Check: Precursor Ion Scan in Combinatorial Chemistry

Scan all ions sequential CID Fix: m/z 455

Gly Pro Phe

X-Arg-Y-Gly-Z(299+156+40+57+100 +1 = 653)

X-Arg-Y-Pro-ZX-Arg-Y-Phe-Z

m/z = 455 X-Arg-+

(299) + (156)

Precursor Ion Scan of m/z 455 of a pooled library

MS

Precursor Scan

Ref: Triolo, 2001

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Advantages for precursor scan

NOTES

QQQ

Q-trap

Q-TOF

TOF-TOF

Ion Trap (3D, LT)

ICR

Q or Trap-ICR

LT-Orbitrap

SelectScan Dissociate

MS/MS Scan Modes

Select

Select

Select Select

Scan

Scan

Scan Scan

Dissociate

Dissociate

Dissociate

Dissociate

Product Ion Scan

Neutral Loss Scan

Precursor Ion Scan


Δ

26

• Screen for ions that undergo a common loss • Q1 and Q3 are both scanned• Q3 is offset by the neutral loss selected• The precursor ion collides in q2 forming fragments• Compounds providing the selected loss are detected • Run as HPLC-MS/MS experiment• Prerequisite: Determine expected loss by MS/MS • Output = chromatogram showing time/intensity of precursors

of interest and reconstructed spectrum

Neutral Loss Scan

Q1 Q3q2 (gas)

Scan Scan(offset from Q1)

DissociateΔ

Neutral Loss Scan Loss of m/z =


Scan Precursors

(sequential rf/dc)

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rf/dc 1

Dissociate

(collide with gas)



(Offset rf/dc)

Scan for offset m/z

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rf/dc 2

Dissociate

(collide with gas)



(Offset rf/dc)

Scan for offset m/z

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rf/dc 3

Dissociate

(collide with gas)



(Offset rf/dc)

Scan for offset m/z

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Neutral Loss SpectrumReconstructed by software

200 300 400 500 600

0

20

40

60

80

100R

elat

ive

Inte

nsity

m/z

Q1 offset rf/dc 2

Software stores memory of the rf/dc voltages that coincidewith fragments striking the detector!

The rf/dc voltages equals a specific

m/z value

Learning Check: Neutral Loss Scan


• Describe a Neutral Loss Scanthat might be used to identify all Chlorine containing compounds



Br I ClF

C 12 Cl 35/37

H 1 Br 79/81

F 19 I 127

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Q1 q2 Q3

RelativeIntensity

0 50 100 150 200m/z





Q1 q2 Q3

RelativeIntensity

0 50 100 150 200m/z

Scan all ions sequential CID scan offset35 amu

112-35

m/z of Q1 less 35for example:

chloro-benzene:112-35 = 77

Neutral loss of 35 or 37

Cl+

+HH

H H

H

.

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Neutral Loss Scan: A literature exampleDrug Metabolite

• Early stages in design of a drug metabolism study• Want to “Fish out” relevant metabolites• Metabolites are in human urine after administration of tolcapone

– tolcapone is a catechol-O-methyl transferase inhibitor• Possible metabolite is a glucoronide of tolcapone

– metabolites are structurally related to parent drug– but, product ion spectra may be energy dependent

tolcapone Hopfgartner, 2003

Neutral Loss Scan: A literature exampleDrug Metabolite

glucuronideconjugates

commonly providemass loss of 176

researchers expect a metabolite that is a glucuronide of tolcapone

O

OH

HO

HO

OH

O

OH

Expected conjugate:mass273 = tolcapone176 = glucuronide add’n449tolcapone

Mass = 273

tolcapone glucoronideMass = 449

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Neutral Loss Scan: A literature exampleQ-trap (Q3 = Linear ion trap)

Hopfgartner, 2003

Metabolite of tolcapone:LC-MS/MS Analysis of human urine

Hopfgartner, 2003

TIC of neutral lossof 176 Da

neutral loss spectrumat t = 5.8 min

MS/MS spectrum 30 eV

MS/MS spectrum50 eVsame fragments as

MS/MS of tolcapone

m/z (Neg ion):tolcaponeconjugate = 449

loss of H = -1

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Advantages for neutral loss scan

NOTES

QQQ

Q-trap

Q-TOF

TOF-TOF

Ion Trap (3D, LT)

ICR

Q or Trap-ICR

LT-Orbitrap

Scan ScanDissociate

MS/MS Scan Modes

Select

Select

Select Select

Scan

Scan

Scan Scan

Dissociate

Dissociate

Dissociate

Dissociate

Product Ion Scan

Neutral Loss Scan

Precursor Ion Scan


Δ

35

• Single (SRM) or Multiple (MRM) reaction monitoring• Quantitative target analyte scan• Q1 is fixed to allow transmission of one precursor m/z• This precursor ion collides in q2 forming fragments• Q3 is fixed to allow transmission of one fragment m/z• Run as HPLC-MS/MS experiment• Prerequisite: Determine expected product ions by MS/MS • Output = chromatogram showing time/intensity of

precursors of interest and reconstructed spectrum

Selected Reaction Monitoring (SRM or MRM)

Q1 Q3q2 (gas)

Select SelectDissociate

Selected Reaction Monitoring


Select one m/z

(fixed Vac/Vdc)

36



(collide with gas)

Dissociate



(Fixed rf/dc)

Select one m/z

37

Learning Check: Selected Ion Monitoring


• Describe a SRM Scan that might be used to identify fluorobenzene



Br I ClF

C 12 Cl 35/37

H 1 Br 79/81

F 19 I 127

Learning Check: Selected Ion Monitoring




Q1 q2 Q3

RelativeIntensity

0 50 100 150 200m/z

m/z = 77

Fix: m/z 96 CID Fix: m/z 77

96F

+HH

H H

H

.

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MRM example: Detection of an antiviral drug and it’s metabolite in human plasma

Ref: Yadav, 2009

• herpes virus replication inhibited by action of acyclovir but low bioavailability

• valacyclovir metabolizes to acylovir with high bioavailability• Goal: accurate detection in plasma

• QQQ mass spectrometer, MDS SCIEX API-4000• Studied fragmentation of compounds by CID

acyclovir (ACV)valacyclovir (VCV)

fluconazole (internal std - IS)

Product ion mass spectra

VCV 325.2/152.2

ACV 226.2/152.2

IS 307.1/220.3

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MRM chromatograms VCV & IS in plasma

VCV ACV IS

blank

IS only

VCV & ISplasma

VCV & ISplasma(subject)

Mean pharmacokinetic profile afteroral administration of 1000 mg VCV tablet

to 41 healthy subjects

Ref: Yadav, 2009

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MRM example: Improve Sensitivity for Corticosteroid Detection

Ref: Antignac, 2000

• Used illegally as growth promoters in cattle• Purpose: detect low residue levels in biological matrices

• QQQ mass spectrometer (QuattroLC, Micromass)• Studied fragmentation of corticosteroids by CID

– Determined negative mode to produce more specific ions

• Evaluated 3 acquisition methods in negative mode– Product ion – Neutral loss– Multiple reaction monitoring

Improving Sensitivity for Corticosteroid Detection

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Comparison: Product Ion, Neutral Loss, MRM

1 ng

10 pg

100 pg

blank

Total ion currentChromatograms

Neutral Loss10X more sensitivethan MS/MS

MRM10X more sensitivethan N.Loss

Ref: Antignac, 2000

Improving Sensitivity for Corticosteroid Detection

MRM = best methodrequires setting many transitionsfor mixture analysisQ1 set for multiple [M+acetate]-Q3 set for 2 products of each

(-60 and -30 from M+acet]-

42

MRM chromatograms of mixture of 11 steroids

Advantages for selected ion monitoring

NOTES

QQQ

Q-trap

Q-TOF

TOF-TOF

Ion Trap (3D, LT)

ICR

Q or Trap-ICR

LT-Orbitrap

Select SelectDissociate

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MS/MS Scan Modes Summary

Select

Select

Select Select

Scan

Scan

Scan Scan

Dissociate

Dissociate

Dissociate

Dissociate

Product Ion Scan

Neutral Loss Scan

Precursor Ion Scan


Qualitative Structural Information

Screen for compound types that lose a detectable fragment

Screen for compound types that lose a neutral

Identify specific compounds

serine

threonine

C 12

H 1

O 16

P 31

16+16+16+31 = 79

phosphorylationadded to serine:79-1+2 = 80

could be lost from serine(as an ion):79 (PO3

-)

could be lost from serine(as a neutral):80 + 18 (H2O)= 98

MS/MS Scan Modes Strategy: Phosphorylationof serine, threonine or tyrosine

tyrosine

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Precursor Ion and Neutral Loss ScansHopfgartner G., Husser C., Zell M.; Rapid Screening and characterization of drug

metabolites using a new quadrupole-linear ion trap mass spectrometer, JMS, 2003; 38: 138-150.

Triolo A, Altamura, M., Cardinali, F., Sisto, A., Maggi C., Mass spectrometry and combinatorial chemistry: a short outline, JMS, 2002; 36:1249-1259.

Chemushevich, I.V., Loboda A.V., Thomson B.A., An introduction to quadrupole –time-of-flight mass spectrometry, JMS, 2001, 36:849-865.

Multiple Reaction MonitoringAntignac, J.P., Bizec, B.L., Monteau, F., Poulain, F., Andre, F., Collision-induced

dissociation of corticosteroids in electrospray tandem mass spectrometry and development of a screening method by high performance liquid chromatography/tandem mass spectrometry, RCMS, 2000, 14, 33-39.

Yadav, M., Upadhyay, V., Singhal, P., Goswami, S., Shhrivastav, P.S., Stability evaluation and sensitive determination of antiviral drug, valacyclovir and its metabolite acyclovir in human plasma by a rapid liquid chromatography–tandem mass spectrometry method, J.Chrom.B, 2009, 877(8-9), 680-688

Suggested Reading List & References

ms/ms scan modes - elte

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