Quattro microTM Operator Training Course MS/MS Theory Presentation (Neonatal application focus)

Topics
Introduction to Mass Spectrometry Electrospray Ionization (ESI)

Ion travel through the Mass Spectrometer

Quadrupole Theory Mass Spectra and MS/MS Data Acquisition Mode

What is Mass Spectrometry?

An analytical technique in which:
Gaseous Ions are produced from neutral molecules Ions are separated according to their mass-to-charge (m/z) ratio Ions are detected and recorded as a plot of ion abundance vs. m/z (mass spectrum)

m2

m1

m4 m4
m3

m3 m2

m1

What is MS/MS or Tandem Mass Spectrometry?

An analytical technique in which: Several mass spectrometers are serially linked
MS 1 MS 2

Ion Source

Collision Cell

Conventionally, MS/MS consists of two mass

analyzers separated by a collision cell

How Does MS/MS Work?

Sample must be first ionized

Ionized sample is sent to first mass analyzer Ionized sample mixture is sorted by m/z A single ion can be selected Selected ions are sent to a collision cell Fragmentation of selected ions takes place
Fragment ions are sent to second mass analyzer Fragment ions are sorted by m/z A single fragment ion can be selected

What is an Ion?
Mass Analysis
9 of C (12.000) = 108.000
1 of N (14.003) = 14.003 11 of H (1.008) = 2 of O (15.995) = 11.088 31.990 165.081

Phe : Molecular Weight = 165.08 PheH+ : m/z = 166.08

Protonated (Ionized) Phe

Gas Phase Ion Fragmentation

AB +
Precursor or molecular Ion

CAD

A + B+
Neutral Loss Product Ion

This fragmentation behavior allows for several types of scanning modes

Tandem Quadrupole Instruments

In a triple quadrupole or tandem mass spectrometer, MS1 and MS2 are mass analyzers that filter ions.

MS1
MS1 is used to scan a range of precursor ion masses or to select a particular precursor ion mass and pass the ions to the collision cell

Collision Cell
In the collision cell, the ions from MS1 collide with Argon atoms and fragment into daughter (product) ions

MS2
MS2 is used to scan a range of daughter ion masses or to select a particular daughter ion mass and pass the ion(s) on to the detector

A Generic LC/MS Configuration

Data Ion Source Sample Introduction AtmosIon Transport Mass Analyzer Detector
1.00 2.00 3.00 4.00
228 m/z 162 m/z

5.00 Minutes

198 m/z

6.00

7.00

8.00

9.00

10.00

Liquid from LC

pheric Pressure Create Gas Phase Ions

Low Vacuum Region Get Ions into the Mass Spec

High Vacuum Region

Mass Analyze Ions

Schematic Overview of the Quattro microTM

Removable Sample Cone Transfer Optics RF Lens Quadrupole MS1 Hexapole Collision Quadrupole Cell MS2

ESI Probe

Conversion Dynode

Phosphor

Pre Filter Post Filter 10-1 mbar Rotary Pump 10-3 to 10-4 mbar Turbo Pump 10-5 mbar Turbo Pump

Pre Filter

PMT

Electrospray Ionization (ESI)

Electrospray Ionization (ESI)

Liquid is sprayed out of a capillary tube to which a high voltage is applied to form a spray of charged droplets. ESI is a type of Atmospheric Pressure Ionization (API) since the ions are formed at atmospheric pressure

2004 Waters Corporation

4H08

Electrospray Probe Tip

Stainless Steel Tube
Stainless Steel Capillary

Liquid

Electrospray Plume

Electrospray Probe Tip

Nebulizer Gas

Liquid

Nebulizer Gas

Electrospray Plume

When a nebilizer gas (usually nitrogen) is used, the electrospray process is often referred to as Pneumatically Assisted Electrospray

Electrospray Probe Tip

Desolvation Gas Nebulizer Gas

Liquid

Nebulizer Gas Desolvation Gas

Electrospray Plume

Desolvation Gas Flow

Nitrogen Heater Wires

Heater Wires Nitrogen

API Probes for Z SPRAYTM Source

Electrospray Probe

Electrospray Ionisation
Example of Positive Electrospray

2.5-4.0 kV

High Voltage Power Supply

Counter Electrode

Droplet Formation in Positive Ion Electrospray

More Negative Ions + + + + - + - + -+ - - - - - -+ - + than Positive Ions

+ +-+ + + + + - + + +

Liquid

+ +- + + - + + - + +- + +- +- - + + + + - + + -+ + + + + ++ + + + -

--+ + - - + -- + +- - - + + + + Taylor Cone

Positively Charged Droplets More Positive Ions

High Voltage

Electrospray Probe Tip

than Negative Ions

Electrospray Droplet Undergoing Fission

+ +
Solvent Coulombic + + Evaporation + + Fission

+ + +

+ + +

+ +

+ +

+ +

+ +

+ +

+ +

+ + + + +

Charge resides on the surface of the droplet. Solvent evaporates from the droplet and the droplet shrinks until the charge density on the surface reaches a point where the repulsive force between charges exceeds the liquid surface tension that holds the drop together. At that point, the drop fissions and a set of small droplets are expelled from the main droplet.

Electrospray Ions
Positive Electrospray Ions are produced by the addition of a positively charged ion (e.g H+, NH4+, Na+) to a molecule. These positively charged ions that are added are often referred to as adducts.
H N H H N O C H3 CH 3

N + H+
O

C H3 CH 3

Lidocaine

Negative Electrospray Ions are most often produced by the removal of a proton (hydrogen ion) from a molecule.
Ibuprofen
C H3 H 3C C H3 C H3

O OH
H 3C

C H3

O O

+ H+

Ions Enter the Z SPRAYTM Source

Ions

ESI Probe

Ions created by the Electrospray probe are drawn in through the sample cone along with nitrogen gas (and some other gases from the mobile phase).

RF Lens

Quads

Cone Gas Cone Function

ESI Probe
Plume of Ions, Clusters, Neutrals and Stuff The cone gas helps create ions with fewer clusters and helps keep out neutrals which yields better S/N. Thus, less stuff collects on the inner Orifice Cone

Cone Gas

N2

N2

Quattro microTM: Sample Cone and Cone Gas Nozzle

Z SPRAYTM Source - Electrospray

Z SprayTM
Exhaust Cone Gas Sample Cone Isolation Valve Nebulizer Gas Desolvation Gas Rotary Pump Turbomolecular Pump

Sample

RF Lens
Quads

Extraction Cone

Why ZSpray?
Spray = Neutral solvent Evaporating (cone-shaped region from initial spray)

Spray To analyzer Solution inlet Skimmer Spray Ion beam

In ZSpray
Solution inlet Sample Cone
Extraction cone

In a Z-Spray source, more ions make it into the mass spec

To analyzer of mass spectrometer

Ion beam

Quattro microTM Z-Spray Source

Desolvation Heater Electrospray Probe

Isolation Valve

Sample Cone

Ions Enter the RF Lens Region

As the ions pass by the ESI entrance to the RF Lens Probe region, the ions are extracted from the gas flow and accelerated by the voltage difference and pressure difference between the source region and the RF Lens region. The source region is both at a higher potential (cone voltage) and pressure than the RF Lens region.
Ions at Atmospheric Pressure Sample Cone and Source Block at Cone Voltage

RF Lens

Quads

To Rough Pump

Extraction Cone and RF Lens at Lower Voltages and Lower Pressure

Ions Pass Through the Quadrupoles

ES or APcI Probe Sample Cone and Source Block at Cone Voltage

Ions RF Lens

Analyzer Section (at Lowest Pressure (Best Vacuum))

Quads, Collision Cell and Detector To Rough Pump Extraction Cone and RF Lens at Lower Voltages and Lower Pressure

As the ions pass from the source region, through the RF Lens and quadrupoles, a series of potential differences and pressure drops (better vacuum) help propel the ions through the middle of the RF Lens and quadrupoles and on to the detector.

Quadrupole Theory

Quadrupole Theory

Quadrupole Theory

End View A quadrupole mass analyzer is an assembly of four parallel rods arranged equidistantly from a central (imaginary) axis. Through the application of DC and RF (radio frequency) voltages, ions can be filtered along the central axis and their mass measured to yield a mass spectrum. Depending upon the exact potential applied to the quadrupole, ions with masses too large or too small will not pass through the quadrupole. These ions will strike the rods and be lost.

Quattro microTM - RF Transfer Lens

RF Lens - Hexapole
RF Lens
Hexapole Assembly

Radio frequency plus a small bias voltage transports all masses. Designed to insure ion focusing in a relatively poor vacuum. Delivers the ions in a tightly focused beam to the quadrupole where they can be analyzed.

End View Note Voltage Polarities

Ions

Trajectories of the Ions

As the ions pass down the middle of the quadruple assembly, the ions are either pulled towards a rod or pushed away from the rod depending on the voltages applied to the rod The trajectories of ions as they pass through the quadrupole assembly can be calculated Since voltage applied to a quadrupole rod is a combination of a constant voltage and an oscillating RF voltage and there are two pairs of these rods, this calculation is very complicated so only a qualitative description of the calculation will be given

Quadrupole Mass Analyzer: RF Voltage

+V 0 -V +V 0 -V

Each rod in a quadrupole is connected to the rod on the opposite side. The RF voltage is applied 180 degrees out of phase to each pair of rods.

Quadrupole Analyzer
Pre-filter Quadrupole Mass Filter
Rejected Ions

Ion with Stable Trajectory

-1

If the correct voltages (DC and RF) are applied to the rods, ions with the desired mass can pass through the rod assembly down the middle of the quadrupoles and reach the detector. All other ions will spiral out and be lost. By changing the voltages, different masses can be filtered through the system to produce a mass spectrum.

Quattro microTM - Ion Optical Rail

Applied Potential to Rods

The voltage applied to an opposing pair of rods is given by:

f =
DC Voltage

U - V cos wT
RF Voltage

The voltage applied to the other pair of opposing rods is:

f = - U + V cos wT

Typically:

DC Voltages (U) are in the range of 1000 V RF Voltages (V) range from 1000 to 6000 V RF frequencies (w) are around 1 MHz and fixed

Quadrupole Operated as a Mass Filter Optimum Quadrupole Operational Line

m1 m2 m3

m/z: m3 > m2 > m1

1

1.2 1
0 200 250 300 350

U (DC) 0.6
0.4 0.2 0 0 0.2

0.8

m/z
400

m3
500 550 600

450

m2

m1

0.4

0.6 (RF) 0.8 V

1.2

Derivatization
A chemical modification during sample preparation to aid in data acquisition
The addition of a butyl group to the carboxylate functionality of the analyte

The butyl ester that is formed aids in sample analysis by forcing a permanent positive charge (acylcarnitines) or by making the charging process more efficient (amino acids)
Butylation increases the non-polar character of the analytes and lower polarity =better desolvation and better sample introduction to the vacuum environment

Derivatization of Amino Acids and Acylcarnitines

Amino acids
O H2N CH C R Am i n o a c i d ( Fr e e a c id ) H C l, H eat R = Amino acid Side Chain H2 C O C H2 H2 C H CH3 + O H H3C H2 H2 O + + O C C H3 C N C C C C O C CH3 H2 H H2 H2 H3C Acylcarnitine butyl ester O CR O OH

Acylcarnitines
H2 C C H2 CH3 O R = Acyl Chain CR H3C O O + H2 H2 H3C N C C C C OH + C C H2 H H2 CH3 C H O H3C H2
Acylcarnitine (Free acid) HCl, Heat

+
HO

H2 C

1-Butanol

1-Butanol

O H2N CH C R

Amino acid butyl ester

MS/MS Data Acquisition Modes

Mass Spectra
and MS/MS Data Acquisition Modes

2004 Waters Corporation

4H08

MS/MS Data Acquisition Modes

MS Mode
MS1 Scan

MS/MS Modes
Daughter/Product Ion Scan Parent/Precursor Ion Scan MRM Constant Neutral Loss
4H08

How Does MS/MS Work?

Sample ionization and introduction Ion fragmentation Ion detection

MS 1

MS 2

Ion Source Ion sorting and selection

Collision Cell
Fragment Ion sorting and selection

MS1 Scan
MS1 Scanning Collision Cell (No Argon) RF (+ DC) MS2 RF

10 -100V m1 m2 m3

MS1 Scan

MS/MS Modes
MS1

Collision Cell (w/Argon)

MS2

MS1 is used as a mass selector and allows ions of a particular mass to pass into the collision cell

In the collision cell, the ions from MS1 collide with Ar atoms and fragment into daughter (product) ions.

MS2 is used as a mass selector and allows daughter ions of a particular mass to pass on to the detector

In the collision cell, a potential is applied (typically 5-40 eV) to control the energy of the collisions between the ions and Ar atoms.

MS/MS Modes

Quattro microTM MS/MS

Low energy collisions (simple fragmentation pathways) Collision gas of choice is Argon Collision gas pressure is normally fixed while the collision energy is used to alter the degree of fragmentation

Daughter/Product Ion Scan

MS1 Fixed Collision Cell (w/Argon) 5-40 eV MS2 Scanning

-5V m1

1V

m2
m1 m3

Determines Collision Induced Dissociation (CID) produced daughter ions of a particular parent ion

Daughter/Product Ion Scan

Daughter ion scan- common MS/MS mode Select one mass in MS1 and send into the collision cell and fragment, MS1 is fixed MS2 scans the fragments for a given mass range Used for structural information gathering and identification of product ions First step to developing quantitative method

Daughter/Product Ion Scan

m1+ set

Product Ion Scan

Product ion spectrum of a particular compound

m2+

m1+

m2+ m2+

m2+ scan

Daughter Ion Scan Effect of Changing Collision Energy

100 % 0 100 % 0 100
230

MS/MS Spectra of Chlorpheniramine (MW=274) Daughter Ions of m/z=275 M+H Collision Energy = 5 eV

275

Collision Energy = 10 eV

275 230

% 0 100

Collision Energy = 12 eV

275

230

% 0 150

Collision Energy = 17 eV
160 170 180 190 200 210 220 230 240 250 260 270 280 m/z 290

Daughter Ion Scan Effect of Changing Collision Energy(Cont.)

100

MS/MS Spectra of Chlorpheniramine (MW=274) Daughter Ions of m/z=275

230

Collision Energy = 17 eV M+H

0 100

Collision Energy = 30 eV
%
167 180 201 202 230

0 100
167

Collision Energy = 38 eV
180 194 201 230

0 150

160

170

180

190

200

210

220

230

240

250

260

270

280

m/z 290

Parent/Precursor Ion Scan

Consider a class of compounds that are similar in structure:

Different Compounds That Are Somewhat Similar In Structure

Different Neutral Fragments

Same Charged Fragment

CID

+
CID

Parent Ion Scans can be used to detect those compounds whose molecular ions produce the same charge fragment.

Parent/Precursor Ion Scan

MS1
Scanning

Collision Cell (w/Argon) 5-40 eV

MS2 Fixed

m1 m2

5V

5V
m3

Find ions that will produce via CID, daughter ions with a particular m/z

Parent/Precursor Ion Scan

Select fragment at m/z x on MS2 (fixed) Scan MS1 for a given mass range Observe signal from ions giving fragment of m/z x (selected in MS2) Used to determine the origin of particular product ion(s) created in the collision cell Seen in newborn screening literature for acylcarnitines

Parent/Precursor Ion Scan

m1+ scan

Precursor Ion Scan

A set of compounds with a common product ion

m2+ m1+ m1+

m1+

m2+ set

Parent Ion of 85 scan: Acylcarnitines

O CR CH3 H 3C N O C C H2 H

Acylcarnitine butyl ester derivative

O H2 C O C H2 H2 C CH3

Underivatized Acylcarnitine free acid

CH3 O C C H2 H H3 C N

O CR O C C H2 OH

CH3

C C H2

R = Acyl Chain

CH3

CAD
O O CR

CAD
Loss of 1-butene by 1,4 Hydrogen rearrangement
CH3 H2 C H 2C C H H3C CH3

RC H 3C N O H CH C OH O

CH3 H3C N

O C C H2 H

O C C H2 O

H2 C CH C H2 H

CH

CH3

C CH3 H2 O

1-Butene

Faty acid
RC

Oxonium Ion

OH

Loss of faty acid functionality by 1,4 Hydrogen rearrangement

+
H 2C HC

H C CH O H 2C

Carbonium Ion

+
C H H C C OH O

Loss of trimethyl amine by -cleavage (heterolytic cleavage)

H 3C CH3 H 3C N

H3C N

H C

H C C

O OH

C CH3 H2

Common Fragment Ion m/z 85

Trimethyl amine
CH3

Analysis of Acylcarnitines by MS/MS - Parent Scan

Neutral Loss (NL) Scan

Different Compounds That Are Somewhat Similar In Structure Same Neutral Fragment Different Charged Fragments

CID
CID

+
+

Neutral Loss Scans can be used to detect those compounds whose molecular ions produce the same neutral fragment.

Neutral Loss (NL) Scan

MS1 Scanning Collision Cell (w/Argon) 5-40 eV MS2 Scanning

-5V m1
m2

1V

m1 - offset m2 - offset

Q1 and Q2 scan together. m/z of Q2 is m/z of Q1 minus an offset.

Neutral Loss (NL) Scan

Neutral loss scan (Example NL102) MS1 & MS2 both scan a given mass range but with a constant offset (difference) between ranges scanned Spectra indicate which ions lose a neutral species equal to MS1 MS2 difference Seen in newborn screening literature for amino acids Complement to Parent/Precursor Ion Scan

Neutral Loss (NL) Scan

m1+ scan

Constant Neutral Loss Scan

A set of compounds with a common neutral fragment

m2+

m1+ - m m2+ - m m1+

m2+ scan

NL of 102 Scan: Amino Acids

AA-Butyl ester Delivered to MS by LC System Protonated Precursor Ion Mass Selected by Q1

H2N CH R O C O C H2

H2 C C H2

CH3

H3N

+
O C O C H2

+ H+
Protonation, Takes place in Ion Source

CH R

H2 C C H2

CH3

Take place inside collision cell Fragment Ion Mass selected by Q3

Collisional Activation with N2

CO

+
O H C H2

H2N H2 C C H2
Fragmentation

H O C O C H2

CH3

CH

H2 C C H2

CH3

H2N R

CH

Neutral loss of 28 + 74 = 102

Collisionally Activated Precursor Ion (transition State)

Analysis of Amino Acids by MS/MS NL

O N OH N O O N

Phenylalanine
O N O N O O N

HO

HO

HO

Tyrosine

Analysis of Amino Acids by MS/MS NL

O N OH N O O N

Deriv
m/z = 222 Phenylalanine
O N O N O O N

Deriv
HO HO

m/z = 238

HO

Tyrosine

Analysis of Amino Acids by MS/MS NL

O N OH N O O N

Deriv
m/z = 222 Phenylalanine
O N OH N O O

C.I.D. m/z = 120

Deriv
HO HO

C.I.D. m/z = 238

HO

Tyrosine CID Results in the Loss of 102 Da

m/z = 136

Analysis of Amino Acids by MS/MS NL

Derivitzed Amino Acid Mix Infused 10 L/min
NeoLynxTestMix_CNL 1 (0.510) 188.0 191.1 100

Quattro micro
Neutral Loss 102ES+ 4.28e7

Leucine Phenylalanine
222.1

Tyrosine
227.2

Methionine

238.1240.1 209.0 206.1

0 180

185

190

195

200

205

210

215

220

225

230

235

240

245

m/z 250

Other peaks are from deuterated forms of these amino acids

Multiple Reaction Monitoring (MRM)

MS1 Fixed Collision Cell (w/Argon) 5-40 eV MS2 Fixed

-5V m1

1V mx

MRMs are used to monitor selected analyte(s) via their daughter ions

Multiple Reaction Monitoring (MRM)

Select a particular mass transition, both MS1 and MS2 are fixed Measure ion intensity from that single mass transition Can cycle through many transitions during course of measurement Measure only what you set up to see Time is spent measuring only desired signals More signal per transition, best way to maximize signal intensity of product ions

Multiple Reaction Monitoring (MRM)

Precursor ion set

Fragmentation (CID)

Product ion set