Mass Spectrometry
Dr. Peter Sharp
Brighton and Sussex University
Hospital Trust
Principles of Mass Spectrometry
Mass Spectrometers have three basic
functions
 1 Vaporisation
 2 Ionisation
 3 Ion separation according to mass /charge
m/z
Since z usually equals unity A mass
spectrometer produces and weighs ions
Ionisation - the only obstacle to a
successful analysis.
 First experiments 1930s Chapman
 late 1960s Dole developed practical method
 1980s Fenn and Mann modern techniqueion spray- multiple charging of
macromolecules-important development for
the analysis of post-translational proteins
following the human genome project.
Electron impact
ionisation/fragmentography
 This is where gaseous effluent from usually a GC
is bombarded with electrons from a filament
 Traditionally 70 eV used for ionisation and when
most chemical bonds have 4- 7 eV of energy it is
not surprising to see extensive fragmentation
producing both +ve and –ve ions.
 NBS and Pfleger libraries of fragmentation
patterns.
 Disadvantage – few Molecular ion species remain
Chemical ionisation
 This is where a secondary gas or nebulised
component is ionised first then passes that
energy of ionisation onto the target species.
 In this way the ionisation is softer than the
70 eV and considerably more molecular ions
are seen
 Reagent gases include methane, ammonia
and isobutene for positive ion ionisation.
Heated Nebulizer - APCI
Curtain Gas
.
Make-up Air
Heat
Liquid
H O
2
M
Air
for
Nebulization
Heat
+
H3O M
M+H
250u orifice
Curtain Gas
Ionisation from solids
 MALDI – Matrix assisted Laser Desorption
ionisation
 Used for larger molecules such as peptides
and proteins
API Analytical Domains
Ionic
Analyte Polarity
IonSpray
Heated
Nebulizer-APCI
GC/MS
Neutral
101
102
103
Molecular Weight
104
105
Types of ion separation
 1 using static radio frequency/magnetic
fields only and the path and/or velocity eg
sector instruments and time of flight.
 2 Using Dynamic fields of Radio frequency
with oscillating electrical fields which
selectively stabilises and destabilises the
ions eg ion trap and quadrupole instruments
Definitions
 Radio-frequency: A frequency or range of
oscillation between 3 Hz and 30 GHz
corresponding to frequency of alternating
current electrical signals used to produce
and detect radio waves. These have
electro-magnetic forces
Schematic sector mass spec
More modern instruments
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Quadrupole
Ion Trap
Time of Flight
Fourier Transform Ion Cyclotron Resonance
Tandem Mass spectrometers which may be
any combination of two of them
Quadrupole
 Uses oscillating electrical fields to stabilise
and destabilise ions passing through a RF
quadrupole field.
 Quadrupole TMS uses two analytical
quadrupole analysers separated by a
collision cell
 Usually in mass range to 1000 daltons
 Not very high resolution 0.5 Dalton
Ion Path of TMS
Ion Trap
Ion trap and quad
Ion trap
 Ions pulsed into trap with oscillating repeller
electrode –Vopen/+Vclosed
 Ions trapped and orbit in RF field set by annular
electrode
 Ions ejected from trap by increasing RF or adding
DC current via end cap electrodes
 Advantage is trapping and holding large numbers
of ions increasing signal:noise ratio.
 Disadvantage: Space-charge effect where too
many ions distort the electrical field and impair
performance
Time of Flight
 Accelerates ions using an electric field
through the same potential and measures
the time to the detector.
 Need a relatively long flight path and
therefore can be large instruments.
 Smaller ions go faster
 Can measure mass to 4 decimal places!
Therefore good for isotope identification
Fourier Transform ion cyclotron
resonance
 Measures mass by detection of the image current of ions
cyclotroning in a magnetic field
 The ions are injected into a Penning Trap(a static
electric/magnetic ion trap) where they effectively form part
of a circuit. Detectors at fixed positions measure the
electrical signal(weak AC ) when the ions pass near them.
Since the frequency of cycling is a function of
mass/charge, this can be deconvoluted by performing a
Fourier transform on the signal.
 Since a single ion is counted more than once this
technique has high sensitivity, high resolution and
precision.
Ion cyclotron resonance
 Ions detected without colliding with detector via
the image current
 Ions can be excited by pulses of RF which boosts
the image current which then fades back to the
unexcited state
 Through Fourier transformation this fade in current
yields increased information on the mass
spectrum
 Good for accurate mass measurement of proteins
MS/MS: Modes of Operation
Product Ion Scan
Select Precursor
Ion
CAD
Scan Products
Precursor Ion
Scan
ScanPrecursor
Ion
CAD
Select Product Ion
Constant
Neutral Loss
Scan precursor and product ions at a constant mass off-set between Q1 and Q3
Type of MS/MS Scans
Product Ion Scan
m1+ set
Product ion spectrum of a particular compound
m2+
m2+
+
m2 scan
m1+
m2+
Precursor Ion Scan
m1+ scan
A set of compounds with a common product ion
m2+
m1+
m1+
m1+
m2+ set
Constant Neutral Loss Scan
m1+ scan
A set of compounds with a common neutral fragment
+
m
m2+ scan
m2+
m2+
m1
-m
-m
m1+
Multiple reaction monitoring
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Morphine
codeine
dihydrocodeine
6-acetyl morphine
methadone
dipipanone
moramide
pentazocine
propoxyphene
paracetamol
amphetamine
286 -152
300 -152
302 -199
328 -165
310 -265
350 - 265
393 - 306
286 - 218
340 - 58
152 - 110
136 - 91
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Mdma
cocaine
benzoylecgonine
chlormethiazole
tramadol
phenethylamine
methaqualone
nalbuphine
buprenorphine
noscapine
hydroxyzine
194 - 163
304 - 182
290 -168
162 - 113
264 - 58
122 - 77
251 - 132
358 - 161
468 - 414
413 - 220
375 - 201
The molecular ion species of syringe
contents
Peter Sharp:
heroin
noscapine
Acetyl codeine
papaverine
paracetamol
Acetyl morphine
cocaine
Problems with mass spectrometry
 Ionisation suppression – some molecules ionise
much easier than others and those with a greater
avidity for protonation may cause suppression of
ionisation of those less polar compounds.
 Use standard addition quantitation techniques to
minimise effects of ionisation suppression
 Specificity of ions – nortriptyline, norvenlafaxine
and Tramadol all have same MW 263 and all can
produce the same daughter ion mass 58
More drawbacks
 Too many ions cause interference –
especially in ion traps – like all forms of
separation/chromatography any system can
be under/overloaded. Good results
obtained with optimal ion concentrations
 Isotope c-12, c-13 and Cl-35 and Cl-37
produce reduced specificity with machines
of lower resolution eg unity MW resolution
 Low MW daughter ions have less specificity