B.

Nielsen

Mass Spectrometry

Pharmaceutical Analysis and Testing (OMED0104)

11.03.2009
Dr Birthe Nielsen
[email protected]
Room: G224

Learning outcomes

What is mass spectrometry (today)

 Principles of Ionization
 Principles of Mass analyzers

Interpretation (next week)

 The mass spectrum
 Qualitative analysis (Prof. Mallet)

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Applications 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
Biomolecule characterization
Proteins and peptides
Oligonucleotides
Environmental analysis
Pesticides on foods
Soil and groundwater contamination
Forensic analysis/clinical
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What is Mass Spectrometry?

The mass spectrometer is an instrument in

which beams of ions are separated according
to the mass-charge ratio (m/z), and in which
the electrons are measured electrically

What can we use the mass for?

Identify, analyze, verify, quantify

The mass spectrum

A mass spectrum is the relative abundance of different m/z in a

given sample.

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Desirable features

The mass spectrometer measures the mass better
than any other technique

Applicable to all elements

Applicable to all types of samples
 volatile or non-volatile
 polar or non-polar
 solids or liquids, or gaseous materials
 hydrophilic or hydrophobic

Ultra-high detection sensitivity

Rapid identification

Use in conjunction with chromatographic methods
(GC-MS or LC-MS)

Limitations

Instrumentation is expensive

Requires the support of trained personnel

Regular maintenance

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Principles of MS

Vacuum system

Ion Mass Data

Inlet source Analyzer Detector System

Formation of ions  separation of ions  detection of ions

Principles of MS

Vacuum system

Ion Mass Data

Inlet source Analyzer Detector System
Flow-injection
HPLC
GC
Sample plate
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Principles of MS

Vacuum system

Ion Mass Data

Inlet source Analyzer Detector System

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Why is a vacuum required?

1

The mean free path, λ, is the λ=
average distance travelled by
an ion before it collide with Nσ
an air molecule: Pressure (Torr) Gas number Mean free path
density
(molecules/cm3)
 N = gas number density
 σ = collision cross section 760 2.7 x 1019 1 µm
between the ion and the
1 3.5 x 1016 0.05 µm
molecule
0.1 3.5 x 1015 0.5 mm
0.01 3.5 x 1014 0.5 cm

If σ = 50 Å2
(typical for small
0.001 3.5 x 1013 5 cm
peptide ions) then the
following mean free paths are 1 x 10-4 3.5 x 1012 50 cm

obtained: 1 x 10-6 3.5 x 1010 50 m

1 x 10-8 3.5 x 108 5 km

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Principles of MS

Vacuum system

Ion Mass Data

Inlet source Analyzer Detector System

Why is ionization required?

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A simple point…
Ions:

M  M+ + e-

Atom Ion Electron

Ions carry a charge and can therefore be handled or their

path can be controlled through the use of electric and
magnetic fields

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Some definitions:

Mass-to-charge ratio (m/z)

Molecular ion: ion derived from the neutral molecule
by loss or gain of an electron

Pseudo-molecular ion / molecular species: ion product
from the product by abstraction or addition of a
proton or other ions

Adduct: ion formed from interaction between two
species (usually an ion and a molecule)

Isobaric ion (molecule): ions with the same nominal
mass

Isotopic ion: any ion containing one or more of the
less abundant naturally occurring isotopes of the
elements that make up its structure

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Mode of ionization
Molecular ion

Electron ejection M  M·++ e- (+) (1)

Electron capture M + e-  M·- (-) (1)

Protonation M + H+  MH+ (+) (>1)

Deprotonation MH  M- + H+ (-) (>1)

Cationization M + cation+ M(cation+)+ (+) (>1)

Some ions are already charged

Positive (+) or negative (-) mode

Number of charges depends on ion source, pH and sample

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Charged

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Protonation (cation formation)

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Deprotonation

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Cationization – adduct formation

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Electron Ejection

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Mode of ionization cont.

What would be the expected m/z values in the mass
spectrum for a peptide with the mass of 1000 Da?

Electron ejection M + e-  M·++ e- m/z = 1000

Electron capture M+ e-  M·- m/z = 1000
Protonation M + H+  MH+ m/z = 1001
Deprotonation MH  M- + H+ m/z = 999
Cationization M + cation+ m/z = 1023
M(cation+)+

Ex. Sodium (Na) 23

g/mol
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Ionization methods
Energy Sample phase
Electron Ionization (EI) Hard gas
Chemical Ionization (CI) soft gas
Desorption Ionization:
Plasma Desorption (PD) soft solid
Fast Atom Bombardment (FAB) soft solid
Matrix Assisted Laser Desorption Ionization (MALDI) soft solid
Spray techniques:
Thermospray soft solution
Electrospray ionization (ESI) soft solution
Atmospheric Pressure Chemical Ionization (APCI) soft solution

Elemental analysis (atomic ionization) solution

Inductively Coupled Plasma Ionisation (ICP)

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Differences - similarities

The difference is the source of energy
 What information do we want
 Physical state of sample

Electron beam, particles, ion beam

Same requirements
 High ionization efficiency
 A stable ion / electron beam
 Minimum background ion current
 Minimum cross-contamination between successive
samples
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Electron ionization

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Electron Ionization
(1919 A.J. Dempster)

Routine analysis of small molecules (Mw < 400 Da)

Excess energy absorbed by the molecules/ions result in
fragmentation
 Sometimes no molecular ion
 Good for providing structural information

Sample introduced in the gas-phase
 Thermal desorption process (due to vacuum and
heating)
 Limited to volatile, thermal stable, low mass
compounds

Inlet: gas chromatography

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Electron Ionization

M  M·+ Electron ejection

70 eV

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Distortion – transfer of energy

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EI Mass Spectrum
Hexane, C6H14 MW = 86.18

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Electrospray ionization

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ESI

Atmospheric pressure ionization

Soft ionization method, provides molecular
weight information

Less fragmentation

Suitable for analyzing large bio- or synthetic
polymers

Suitable for analysing polar and even ionic
compounds (e.g. metal complexes)

Enables LC-MS coupling

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Prof. J. Fenn was credited for the invention of electro

spray ionization (ESI)

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Prof. J. Fenn was credited for the invention of electro

spray ionization (ESI)

http://nobelprize.org/mediaplayer/index.php?id=904

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Electrospray ionization

Sample Inlet Nozzle Partial

Pressure = 1 atm vacuu
Inner tube diam. = (Lower Voltage)
m
100 um

N2 MH+
++
+++ + + +
++
+
++ + +
+ +
MH2+
+ ++
++ + + +
Sample in solution ++
+ + +
++
++
+ +
++ ++ +
N2 gas +
++ + +

MH3+

High voltage applied Charged droplets

to metal sheath (~4 kV)

Protonation: M + H+  MH+
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The sample solution is sprayed from a region of a strong electric field at the tip of a metal nozzel
maintained at approx 4000 V and the highly charged droplets are then electrostatically attracted to the
mass spectrometer thus causing the solvent to evaporate from the surface. As the droplets decreases in
size the electric field density on the surface increases mutual repulsion between like charges on this
surface becomes so great that it extend the forces of the surface tension and the ions begins to leave
the droplet (Taylor cone). Ions are then directed into through electrostatic lenses leading to the mass
analyzer.
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Rayleigh limit – ion ejection: favours high charge state (the

process favours the ejection of the highest charge states

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Multiple ion formation

The EI spectrum therefore contains different peaks
representing different charge states of the same molecule.

The molecular weight can be determined by:

P = m/z P = a peak in the mass spectrum

m= total mass of the ion
z= total charge
P1 = (Mr+z1)/z1 Mr = average mass of sample
P1 = m/z value for one peak in the spectrum
P2 = m/z value for a second peak in the spectrum
P2 ={Mr+(z1-1)}/(z1 - 1) z1 = charge of peak one

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Mass spectrum of Myoglobin:

Deconvolution
+14 +12
1542 z1 = Mr + z1

+11 1696 (z1 - 1) = Mr + (z1 – 1)

P1 = 1542
+10
P2 = 1696 By solving the two equations:
+16
+9
Mr = 16,951 and z1 = 11

+20

Finding the molecular weight?

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The same protein with a molecular weight of

10,000 can result in several peaks in the mass
spectrum

5+ 4+ 3+ 2+ 1+
H+
H+ H+
H+
H+
H+ H+ H+
H+ H H+
+
H+ H+ H+
H+
[M+ 5+]5+ [M+ 4+]4+ [M+ 2+]2+ [M+ 1+]1+
[M+ 3+]3+
m/z= 10,005/5 m/z= 10,004/4 m/z= 10,002/2 m/z= 10,001/1
m/z= 10,003/3
m/z= 2001 m/z= 2501 m/z= 5001 m/z= 10,001
m/z= 3334

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MALDI:
Matrix Assisted Laser
Desorption Ionization

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K. Tanaka was credited for the invention of laser

desorption ionization (leading to MALDI)

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Sample plate
Laser
hn

The sample is mixed with a

matrix. The matrix absorbs the
energy emitted by laser and
MH+ evaporates. The sample
molecules embedded in the
matrix are entrained in the
gas-phase plume. Ionization
via gas-phase proton-transfer
reactions.

+/- 20 kV Protonation:M + H+  MH+

Grid (0 V)

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The importance of the matrix

The matrix is necessary to dilute and disperse
the analyte

The matrix vaporizes carrying the sample with
it

It functions as energy mediator for ionising the
analyte itself or other neutral molecules

It serve to minimizing sample damages from
the laser radiation by absorbing most of the
incident energy

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MALDI matrices
Requirements:

Light absorption at the λ of

the laser

Formation of microcrystals
with the sample

Low sublimation
temperature

Participation in reaction to
ionize sample molecules
with high yield

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MALDI sample plate

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MALDI
Unlike ESI, MALDI forms predominantly single charged
ions:

[M+H]+

Or adducts (sodium, 23 amu, or potassium, 39 amu)

[M+Na]+ or [M+K]+

[M+H]+

[M+Na]+
[M+K]+
22 m/z
38 m/z

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Mass spectrometers can be made to record either positive or negative

ions by making the source voltage positive or negative, respectively.
The recording mode should match the charge sign of the analyte:

Peptides are generally best analyzed as positive ions, particularly those
containing arginine or lysine; because they are zwitterions they are
best analyzed at low pH

Phosphorylated or sulfated peptides may be analyzed in the -ve ion
mode

Oligonucleotides, which contain a large number of anionic phosphate
groups may nonetheless be best analyzed as positive ions at low pH, in
order to promote the formation of singly-charged species

Fatty acids are best analyzed as fatty acyl anions

Carbohydrates are more easily protonated than deprotonated,
although they may be observed as their oxyanions

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Advantages and disadvantages

Practical mass range up to 300,000 Da

Typical sensitivity on the order of femtomole to
picomole

Soft ionization – no (little) fragmentation

Suitable for complex mixtures

Tolerant to both impurities and salts

Low resolution

Matrix interference (background)- problem for
compounds below a mass of 1000 Da (‘finger-print
region’)
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Question?

What ionization technique would you
use for following compounds:

 Gasoline fractions?
 Ibuprofen?
 Insulin?

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Question

What different ionization methods can
be used to produce negative ions?

What different ionization methods can

be used to produce positive ions?

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Principles of MS

Vacuum system

Ion Mass Data

Inlet source Analyzer Detector System

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Mass Analyzers

Time of Flight (TOF)

Magnetic Sector / double focusing magnetic sector

Quadrupole mass analyzer

Quadrupole Ion Trap (QIT)

Fourier Transform Ion Cyclotron Resonance (FTICR)

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What is the difference?

Mass range = the maximum allowable m/z that can
be analysed

Scan speed = number of m/z values measured in
one time unit or the time needed to scan the complete
mass range

Resolution = The ability to distinguish between two
ions of different m/z ratios

Mass accuracy = The achievable molecular mass
(+/- Da)

Transmission efficiency = the ability to transfer the
charged species from the ion source to the mass
analyzer

Adaptability and cost

Time of Flight (TOF)

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TOF

TOF is a velocity spectrometer: the ions are
separated on basis of their velocity differences

A short pulse of ions is dispersed in time by
allowing the ions in a FFR of a long flight tube

MALDI-TOF

Q-TOF

Relative simple which makes it possible to use
them in the field
 1991 Gulf War
 Airports

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Time of Flight (ToF)

Source Detector
Field-free drift region
+

+ + + +

+ + +

+ + +
+
+
+
+ +
+
+
+ +

+ + +
+ +
+

V
Small ions reach the detector before large ones.
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Low m/z ions will reach the

detector first:
Velocity (v) is dependent upon the kinetic
1
energy (Ekinetic) and mass (m), lighter ions E kinetic = mv 2
will travel faster: 2

Ekinetic is determined by the acceleration

voltages of the instrument (V) and the 2V × e
charge of the ion (e × z) v=
m/z
The time delay (t) from the formation of
ions to the time it takes to reach the
detector depends upon the length of the L
t= m/z
flight tube (L), the m/z value of the ion, and 2V × e
the acceleration voltages in the source
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The problem: Peaks are inherently broad in

MALDI-TOF spectra (poor mass resolution)

The cause: Ions of the same mass coming from

the target have different speeds. This is due to
uneven energy distribution when the ions are
formed by the laser pulse.

Sample + matrix on target

Ions of same mass, different
velocities

+
+
+

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Reflectron TOF

A reflectron is an energy-correcting device

 Corrects for any initial position and energy

dispersion in the accelerator region of a TOF
instrument
 It is a electrostatic mirror formed by a series of
electrical lenses, each with a progressively
increasing repelling potential
 Faster-moving ions penetrates deeper in the
reflectron and spend there more time (isomers
ions are bundled together).

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A reflector focuses ions to give better

mass resolution

+
+

+20
0 V. kV

Linear TOF vs. Reflectron

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Quadrupole Mass Analyzer

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Quadrupole Mass Analyzer

4 accurately aligned parallel rods are arranged
symmetrically in a square

As the ions travel through the quadrupole they
are filtered according to their m/z value so that
a single m/z value ion can strike the detector
 Single Ion Monitoring (SIM) – a filter

This m/z value is determined by the Radio
Frequency (RF) and the Direct Current (DC)
voltages applied to the electrodes

The voltages produce an oscillating electric
field that functions as the filter to transmit
selected m/z value ions

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Quadrupole Mass Analyzer

The four electrodes are connected in pair:
 Connecting opposite pairs together generates a two-
dimensional quadrupole electric field
 The RF voltages is applied between these two pairs of
electrodes
 First part of the RF cycle: top + bottom rods are at a
positive potential, left + right rods are at a negative
potential  this squeezes positive ions into the
horizontal plane.
 The second half of the RF cycle the polarity of the
rods are reversed this changes the electrical field
and focuses the ions into the vertical plane
 The quadrupole field continues to alternate as the
ions travel through the mass analyzer (motion:
three-dimensional wave)

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Quadrupole Mass Analyzer

By selecting appropriate
RF frequency and
potential, the quadrupole
acts as a mass filter
letting some m/z ions
through to the detector
while other m/z ions will
collide with the electrodes
and cannot reach the
detector

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Where is it used?

Quadrupole mass analyzers are used
in conjunction with electrospray
ionization

Common in LC-MS and GC-MS
systems
 Electrospray – single quadrupole

 Nanospray-QQQ

QQQ - triple quadrupole mass

spectrometer

A linear series of three quadrupoles

Q1 and Q3 act as mass filters,

Q2 quadrupole is employed as a collision
cell (an RF only quadrupole (non-mass
filtering) using Ar, He or N gas to induce
collision induced dissociation of selected
parent ion(s) from Q1. Subsequent
fragments are passed through to Q3 where
they may be filtered or scanned fully
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Quadrupole ion trap mass analyzer

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Magnetic Sector

J.J.Thompson (1897) – the first mass

spectrometer used a magnet to measure
the m/z value of an electron

Ions are separated in a magnetic field
according to the momentum and charge
of the ion

Typical mass range is to m/z 5000

Electrospray and MALDI

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Ions are accelerated from the ion source into a magnetic

sector

Since the ions are charged, as they move through the
magnetic sector, the magnetic field bends the ion beam
in an arc
 The radium of this arc depends upon the momentum of the ion
and charge of the ion and the strength of the magnetic field

Ions with a greater momentum will follow an arc with a
greater radius.

Ions are separated according to their radii of curvature
and therefore only ions of a given m/z value will reach
the detector at a given magnetic field

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Magnetic sector mass

spectrometer

The low m/z ion is

separated from
the high m/z ion

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Literature
Fundamentals of Contemporary Mass Spectrometry (2007).
Chabil Dass, Wiley (New Jersey, USA). ISBN: 978-0-471-
68229-5

Introduction to Organic Spectroscopy (1997). L.M.Harwood

and T.D.W.Claridge, Oxford University Press (Oxford, UK).
ISBN: 0-19-855755-8

Pharmaceutical Analysis (2005).2nd Edition. D.G.Watson,

Elsvier. ISBN: 0-443-07445-3

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Journals

Analytical Chemistry

European Journal of Mass Spectrometry

International Journal of Mass Spectrometry

Journal of the American Society for Mass Spectrometry

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