Introduction to TOF-MS

Interesting Analytical Features:

• Very rapid scan rates (10,000 scans per sec; however 10-100 are usually averaged to get one ‘scan’.

• Simple construction. • Unlimited mass range. • Now can provide accurate mass measurement. • Now coupled with MALDI as well as electrospray and APCI ion

sources. • Can provide ‘up-front’ CID in the single mass analyzer

configuration. • With a ‘Q-TOF’ configuration one can obtain true MS/MS data; cost

is comparable to a triple quadrupole, however.

Lecture 4, Page 45

Schematic Drawing of Time-of-Flight MS

Lecture 4, Page 46

LC-TOF-MS: Why Bother?

Better Sensitivity • 10 to 100-fold sensitivity over quadrupole systems in the full-scan mode

Improved Mass Resolution • Quadrupole resolution about 1000 • TOF resolution 10,000-15,000 and improving

Accurate Mass Measurement • Low ppm from on-line LC/MS measurements • Very helpful for qualitative analysis studies

High-Throughput LC/MS Analyses • Faster duty cycle so it can ‘keep up’ with fast chromatography • Helpful for quantitative analysis for better LC peak characterization

Lecture 4, Page 47

Courtesy Prof. Robert Cotter Lecture 4, Page 48

Agilent TOF MS Video

• FN: Agilent LC-MSD-TOF

Lecture 4, Page 49

Courtesy Prof. Robert Cotter

Lecture 4, Page 50

Courtesy Prof. Robert Cotter

Lecture 4, Page 51

Courtesy Prof. Robert Cotter

Lecture 4, Page 52

Hybrid Systems

Lecture 4, Page 53

Courtesy Prof. Robert Cotter

Lecture 4, Page 54

Courtesy Prof. Robert Cotter

Lecture 4, Page 55

Courtesy Prof. Robert Cotter

Lecture 4, Page 56

Courtesy Prof. Robert Cotter

Lecture 4, Page 57

Ions

+

+ + + + +

+

+

+

+

+

+

+

N2

Q1

+ +

+

+ +

+

+

q2

+

High Resolution and Accurate Mass

Measurements using a QTOF

Research grade QqTOF with Turbo V source

AB SCIEX

TOF MS and MS/MS ≤ 100 msec

Resolution ≥ 30’000

Mass Accuracy ≤ 5 ppm

O

NHS

OO

O

N

NN

N

O

OH551.5 552.5 553.5 554.5 555.5

m/z, Da

0

200

600

1000

1400

1600

Inte

nsty

, cp

s

552.1908

553.1931

554.1912

555.1904

FWHM : 0.0198

[M+H]+

Lecture 4, Page 58

TOF/MS of Isobaric Compounds Mix

R~32-35K

All 7 components

resolved

316.048

316.075

316.092 316.107

316.154

316.238

316.206

Name Formula MH+

Clonazepam C15H10N3O3Cl 316.0483

Oxycodone C18H21NO4 316.1543

Chlorprothixene C18H18ClNS 316.0921

Pamaquin C19H29N3O 316.2383

Flusilazol C16H15F2N3Si 316.1076

Fendiline C23H25N 316.2060

Oxfendazole C15H13N3O3S 316.0750

Lecture 4, Page 59

Courtesy Prof. Robert Cotter Lecture 4, Page 60

Ion Mobility Spectrometry

• A form of ‘gas-phase electrophoresis’ – Differential mobility of ions that differ in ‘shape’

or ionization cross section

• An older technology enjoying a rebirth of interest

• Waters first to commercialize instrument with analytical capabilities: ‘Synapt HD’

• Considerable research on-going by R. Smith, D. Clemmer, et al.

Lecture 4, Page 61

Electric Field Ion Gate Electrodes

Ion Drift / Separation

Region To Detector

From Ion

Source

Gas

Conventional ion mobility

spectrometers

Lecture 4, Page 62

GATE

From ion

source

Electric Field

Low mobility ion

High mobility ion

Lecture 4, Page 63

Conventional ion mobility

spectrometers

Synapt HDMS System

Lecture 4, Page 64

Enabling Technology

Lecture 4, Page 65

Travelling wave ion guides

• Travelling waves (T-WAVES) are employed on Waters range of Premier mass spectrometers

– Minimises ion transit times

– Enables fast switching experiments

– Virtually eliminates crosstalk issues

Lecture 4, Page 66

Raw TIC

Metabolites

Metabolites of Verapamil

NO

O

OO

CH3CH3

CH3CH3 CH3

CH3

N CH3

Lecture 4, Page 67

Use of drift time information to extract the metabolites

Separations are driven by mobility changes during

clustering and declustering. Chemical interactions

amplify the separation when polar modifiers are used.

High field declustering

mobility increases

Low field clustering-

mobility decreases

+ 3

Kv

Schneider BB, Covey TR, Coy SL, Krylov EV, and Nazarov EG, “Chemical Effects in the Separation Process of a Differential Mobility/Mass Spectrometer

System”, Anal. Chem., 2010, 82, 1867-1880.

Lecture 4, Page 68

Nitrogen Transport Gas

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Norm

aliz

ed S

ignal

-80 -60 -40 -20 0

CV (V)

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Norm

aliz

ed S

ignal

-80 -60 -40 -20 0

CV (V)

Nitrogen Transport Gas

with 2% 2-propanol modifier

Greatly increased

peak capacity

DMS with Gas Phase Modifiers

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Norm

aliz

ed S

ignal

-80 -60 -40 -20 0

CV (V)

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Norm

aliz

ed S

ignal

-80 -60 -40 -20 0

CV (V)

~15V

~100V

8x Increase

Lecture 4, Page 69

References

• Zhang, H.; Henion, J.; yang, Y.; Spooner, N., Application of API TOF coupuled with HPLC for the characterization of in vitro drug metabolites. Anal. Chem. 2000, 72 (14), 3342-3348

• Rietschel, B.; Baeumlisberger, D.; Arrey, T. N.; Bornemann, S.; Rohmer, M.; Schuerken, M.; Karas, M.; Meyer, B., The benefit of combining nLC-MALDI-Orbitrap MS data with nLC-MALDI-TOF/TOF data for proteomic analyses employing elastase. J Proteome Res 2009, 8 (11), 5317-24.

• Dale, V. C.; Speir, J. P.; Kruppa, G. H.; Stacey, C. C.; Mann, M.; Wilm, M., Applications of sustained off-resonance irradiation (SORI) and quadrupolar excitation axialization (QEA) for the characterization of biomolecules by Fourier-transform mass spectrometry (FTMS). Biochem Soc Trans 1996, 24 (3), 943-7.

• Steen, H.; Kuster, B.; Mann, M., Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning. J Mass Spectrom 2001, 36 (7), 782-90.

• Hendrickson, C. L.; Emmett, M. R., Electrospray ionization FTICR Mass Spectrometry. Rev. Phys. Chem. 1999, 50, 517-536.

Lecture 4, Page 70