Mass Spectrometry

Hyphenated TechniquesGC-MS LC-MS

andMS-MS

Reasons for Using Chromatography with MS

• Mixture analysis by MS alone is difficult• Fragmentation from ionization (EI or CI)

• Fragments from each component in one spectrum yields intractable interpretation

• Suppression effects in soft techniques (ESI)• Analytes that are less abundant or ionize poorly

may not be detected• Quantitation of mixtures is less accurate and less

sensitive than quantitation of components that have been chromatographically separated

Chromatography -MS Data

• Rather than averaging all data into one spectrum, each full scan is saved separately to create a chromatogram– TIC - Total Ion Chromatogram– EIC - Extracted Ion Chromatogram

• Scan speed is critical for resolution of close-eluting components– peaks could be missed or averaged together– spectral tilting

• Intensities of low/high mass ions skewed

Chromatography -MS Data

• Identification of Components– Background subtraction can be useful in

cleaning up spectra• Can remove background ions• Can remove ions from co-eluting tailing compound

– EIC can support/contradict that two MS peaks are from the same/different compounds• Comparing whether time profiles overlap

– EIC can show if ions come from the peak or are in the background

Offline LCMS

• LC may also be coupled with MS by fraction collection (ion exchange, size exclusion chromatography, TLC, Flash Column)

• Fractions may be analyzed by any compatible MS technique

• Offline Automation– LC eluent can be spotted onto a MALDI target

using a robot for MS analysis

LC in line with MS

• Liquid chromatography may be directly coupled to mass spectrometry using an APCI or ESI source

• Typical flow rates:– 100 µl/min-2.5ml/min: APCI source– > 1 ml/min: ESI source, usually need splitter (≥ 4.6 mm

column)– 1 µl/min - 1 ml/min: ESI source (200 µm- 4.6 mm

column)– < 1 µl/min: nanospray source (≤ 150 µm column)

Types of LC coupled inline to MS

• Most common= reversed phase (i.e. C18, C8, C4)

• Ion exchange• Normal phase• Affinity• 2D Chromatography (Ion Exchange

followed by reversed phase)

Restrictions

• Non-volatile salts should be avoided as signal suppression will result

• Strong ion-pairing agents such as trifluoroacetate should similarly be avoided

• Need polar solvents for ESI– Normal phase not compatible with ESI (Use APCI)

• Additives must be compatible with ionization mode (i.e. pH of separation must match desired ions)– Or use post-column addition

GCMS

• With the advent of capillary columns, GC columns can be inserted directly into the ionization source

• GC is typically coupled to EI, CI, and FI• GC-MS is applicable to a narrower range of

compounds due to volatility requirements• When GC/MS is applicable, it is a fairly

trouble free technique

Tandem Mass Spectrometry (MS/MS or MSn)

• Soft ionization techniques such as ESI only result in parent ions, revealing no structural information

• Hard ionization techniques yield fragments, but specific precursor-product relationships cannot be obtained

• Isolate and fragment precursor ion and measure m/z of product ions to obtain structural information

• One mass analyzer isolates, another scans for products

Common Methods of Fragmentation

• Threshold Dissociation (Weakest bonds are broken)– Collision Induced Dissociation (CID)

• Apply an electric field to accelerate precursor ions and induce bond cleavage via collision with neutral gas molecules (Ar, N2)

– Infrared Multiphoton Dissociation (IRMPD) • Use infrared laser to break bonds

• Electron Capture Dissociation (ECD) (Random)– Multiply-charged cations capture electrons, inducing odd-

electron fragmentation

CID

AcceleratedPrecursor Ion

Fragmentationthrough one ormore collisions

Product Ions

(And Neutrals)

Ar

+

+

+

+

IRMPD

IsolatedPrecursor Ion

Fragmentationby one ormore photons

Product Ions

(And Neutrals)

hν

+

+

+

+

ECD/ETD

IsolatedPrecursor Ion

Capture of oneor more electrons

Product Ions

(And Neutrals)

e-

+

++

•

or M-

Instruments for MS/MS

• Instruments where one or more analyzers are placed in series– Triple Quadrupole (CID at eV levels)

– Four Sector (CID at keV levels)

– Quadrupole-Time-of-Flight (Q-TOF) (CID eV)

– TOF-TOF• Trapping Instruments

– Quadrupole Ion Trap (CID eV, IRMPD, ECD)

– FT-ICR (CID, SORI-CID, IRMPD, ECD)

Quadrupole Ion Trap

M+H

•MS/MS is done in one analyzer- Precursor Ion is Isolated by selective ejection- Precursor ion is given kinetic energy- Fragments are formed and trapped- Trap is scanned to detect products- Products can be isolated and fragmented etc.

Tandem Mass Spectrometry in an Ion Trap1) Measure Full Spectrum 2) Ion Isolation:

e.g., Precursor m/z 426.7 ± 1.5

300 900 1500 2000m/z

50

100

Rel

ativ

e A

bund

ance

426.7

852.3

1138.7700.2

426.7

3) Add Collision Energy

Fragmentation of 426.7

4) Measure m/z of Fragment Ions

SLNVALSLNVASLNVSLNSLS

RLR

ALRVALR

NVALRLNVALR

[For Peptide SLNVALR]

Courtesy of the MSCLS at the Univ. of Minn.

Triple Quadrupole Mass Analyzer

Sample Inlet

Q2(Collision cell)

Q3

Ion Guide

Q1EM

Detector

C+Ar Products

Product Ion Scan Mode in a Triple Quadrupole

Q1 Mass selection Q2 collision cell

Fragment Ion C

Q3 Full Scan

Ion C

Q1 only transmits

Ion C

Q3 Scans for products

A

BC

D

Ionsin

Source

Products

C+Ar C1+C2+C3

Multiple Reaction Monitoring (MRM)in a Triple Quadrupole

Q1 Mass selection Q2 collision cell

Fragment Ion C

Q3 Mass Selection

Ion C

Q1 only transmits

Ion C

Q3 only transmitsIon C2

A

BC

D

Ionsin

Source

Ion C2

Neutral Loss Scan Mode in a Triple Quadrupole

Q1 Pass A-H2O Q2 collision cell

Fragment ions one at a

time

Q3 Pass A

Ion A-H2OIon B

Ion CIon D

Ion EIon F

A-H2O A+ +H2O

Scans acrossmass range

Scans acrossmass range at18 amu lowerthan Q1

IonsA-F

A

Precursor Ion Scan Mode in a Triple Quadrupole

Q1 Mass selection Q2 collision cell

Fragment ions one at a

time

Q3 pass only 79

Ion A-PO4

Ion BIon C

Ion DIon E

Ion F

PO3- 79

Scans acrossmass range

Transmits only 79

IonsA-F

-/+ Polarity switch

Precursor ion of 79 (-)

Enhanced resolution (+)

MSMS (+)

-/+ Polarity switch

Charge state determination

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52Time, min

0.0

5.0e7

1.0e8

1.5e8

2.0e8

2.5e8

3.0e8

3.5e8

4.0e8

4.5e8

5.0e8

5.5e8

6.0e8

6.5e8

7.0e8

7.5e8

Inte

nsity

(cps

)

β-Casein Digest Total Ion Chromatogram

Courtesy of the MSCLS at the Univ. of Minn.

5 10 15 20 25 30 35 40 45 50 55 60 65Time, min

0.00

1.00e7

2.00e7

3.00e7

4.00e7

5.00e7

6.00e7

7.00e7

8.00e7

9.00e7

1.00e8

1.10e8

1.20e8

1.29e8

Inte

nsity

(cps

)Negative Precursor Ion Chromatogram

Courtesy of the MSCLS at the Univ. of Minn.

400 500 600 700 800 900 1000 1100 1200m/z, amu

100

300

500

700

900

1100

1300

1500

1700

1900

21001029.2

Inte

nsity

, cps

Precursor Ion Scan at 30 min

Courtesy of the MSCLS at the Univ. of Minn.

1031 1032 1033m/z, amu

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

1.10e5 1031.31031.7

1032.2

Inte

nsity

, cps

Positive Enhanced Resolution Scan

+2 ion, MW= 2060.6

Courtesy of the MSCLS at the Univ. of Minn.

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z, amu

2.0e5

6.0e5

1.0e6

1.4e6

1.8e6

2.2e6

2.6e6

3.0e6

3.4e6F Q pS E E Q Q Q T E D E

a1-NH3b1-NH3

I

b2

a2-NH3

b2-NH3

b3b3-NH3

b4b4-NH3 a5

b5

y12y12-NH3b6

y11y11-NH3

b7

y10y10-NH3

b8

y9

b8-NH3

y9-NH3

y8

b9-NH3y8-NH3

y7

y7-NH3

y6

y6-NH3

y5

y5-NH3

y4

y4-NH3

y3

y3-NH3

y2

y2-NH3

y1

y1-NH3

a1

Inte

nsity

, cps

MSMS on ion 1031.3 for sequence determination

Courtesy of the MSCLS at the Univ. of Minn.

Q-TOF Mass Analyzer

TOF

Sample Inlet

Q2(Collision cell)

Hexapole

Ion Guide

Q1

MCPDetector

Reflectron

Pusher

•Select 1 ion in Q1 (Product Scan)•Scan Q1 (Precursor Scan)•Cannot perform Neutral Loss Scan

FT-ICR-MS

• MS/MS is done in one analyzer– RF fields can selectively isolate one ion– RF fields (on or off resonance) can accelerate ions followed by

collisions and detection of products– IR laser or electron source can fragment isolated ions followed by

detection of products– Processes can be mixed, matched, repeated etc.

M+H

MS4 With FT-ICR

Reed et. al. JACS, 122, 10689-10696, (2000)

CO2-

-O2C

CO2-

MS6 With FT-ICRCO2

O2C-

-CID CO2

-

•CID

•

-

•

- SO2•

-O

t-BuSH-

O

CID

O•

H

--

low energy

highenergy

t-BuSH

No Reaction

Phenol

ProtonTransfer

Reed et. al. JACS, 122, 10689-10696, (2000)

Multiple ions at m/z 92

MS/MS Applications• Structural elucidation aid for unknown

compounds• Functional group confirmation• Positive Identification of known compounds

– environmental contaminants– biological metabolites– controlled substances (illegal drugs, steroids etc.)

• When Combined with Chromatography– Screening of mixtures for specific moieties– Structure-specific quantitation

• Thermochemical information (BDE etc.)• Chiral analysis