MPHA4214 Pharmaceutical Analysis II

Mass Spectrometry

Audrey Yong, PhD

audrey@mahsa.edu.my
Faculty of Pharmacy,
MAHSA University
1
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What is a Mass spectrometer ?

• It is a balance that weigh ions

– m/z ratio of ions
– molecule must be ionised

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 Principles
• Charged molecule of molecular fragment are
generated in high vacuum region or immediately
prior to a sample entering a high vacuum region
• Ions are produced using a variety of methods
• The ions are generated in the gas phase so that they
can then be manipulated by the application of
either electric or magnetic fields to enable the
determination of the molecular weights

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• Vaporisation and  Mass separation/  detection
ionization selection

Source Analyzer detector

++++

Time = distance / Speed

Speed = f(mass)
Benzocaine ++
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 Mass spectrum of Benzocaine
Signal
Intensity
C9H11O2N Molecular ion
M=164 [M+H]+
165

m/z
0 50 100 150 200

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 Applications
• Mass spectrometry provides a highly specific
method for determining or confirming the identity
of structure of drugs and raw materials used in the
manufacture of drug products
• MS in conjunction with either gas chromatography
(GC-MS) of liquid chromatography (LC-MS) provides
a method for characterising impurities in drugs and
formulation excipients
• GC-MS and LC-MS provide highly sensitive and
specific methods for deter

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 Strength of MS
• The best method for getting rapid identification of
trace impurities, which should ideally be carried out
using chromatographic separation in conjuction
with high resolution mass spectrometry (HRMS) so
that elemental composition can be determined
• With the advent of electrospray mass spectrometry
and the re-emergence of time of flight mass
spectrometry, the technique will be of major use in
the quality control of therapeutic antibodies and
peptides

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 Limitations of MS
• Mass spectrometry is not currently used in routine
quality control (QC) but is place in a research and
development (R&D) environment where it is used
to solve specific problems arising from routine
processes of in process development
• The instrumentation is expensive and requires
support by highly trained personnel and regular
maintenance. However, these limitations are
gradually removed

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 Introduction
• A mass spectrometer works by generating charged
molecules or molecular fragments either in a high
vacuum or immediately prior to the sample entering
the high vacuum region
• The ionised molecules have to be generated in the gas
phase
• In classical mass spectrometry there was only one
method of producing the charged molecules but now
there are quite a number of alternatives
• Once the molecules are charged and in the gas phase
they can be manipulated by the application of either
electric of magnetic fields to enable the determination
of their molecular weight and the molecular weight of
any fragments which are produced by the molecule
breaking up
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Instrumentation

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Instrumentation

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 Magnetic sector of MS
• In magnetic sector instrument the ion generated are
pushed out of the source by a repeller potential of
same charge as the ion itself, and are then accelerated
in an electric field of ca 3-8 kV and travel through an
electrostatic field region so that they are forced to fall
into a narrow range of kinetic energies prior to entering
the field of circular magnet
• They then adopt a flight path through the magnetic
field depending on mass to charge (m/z) ratio; the large
ions are deflected less by the magnetic field

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 Magnetic sector of MS
• At a particular values for H ad V, only ions of a
particular mass adopt a flight path that enables them
to pass through the collector slit and be detected.
• If the magnetic field strength is varied, ions across a
wide mass range can be detected by the analyser, a
typical sweep time for the magnetic field across a mass
range can be detected by the analyser, a typical sweep
time for the magnetic field across a mass range of 1000
is 5-10 s but faster speeds are required if high
resolution chromatography is being used in conjunction
with mass spectrometry
• The a accelerating voltage can also be varied while the
magnetic field held constant in order to produce
separation of ions on the basis of their kinetic energies

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 Mass spectrum of Benzocaine
Signal
Intensity
C9H11O2N Molecular ion
M=164 [M+H]+
165

m/z
0 50 100 150 200

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 Type of sources  Type of analyzers

o Electrospray ionization (ESI) o Time of Flight

o Electron Impact (EI) o Quadrupole

o Chemical Ionization (CI) o Ion Trap

o Matrix Assisted Laser o Cyclotron Resonance

Desorption Ionization
(MALDI) …

o Fast Atom Bombardment

(FAB) Depends on :
... - Applications
- Type of molecule

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 The Carbon atom
 Sufficient resolution to differentiate between 1 atomic mass unit
(amu)

 13C is present in an abundance of approx. 1.1%

(12C is 98.9% abundant)

 Can usually see small peak next to major [M+H]+ peak due to
presence of 13C

 Relative height of 13C peak is proportional to the number of carbon

in a molecule

 The more carbon atoms in a molecule, the higher the chance of

finding one 13C in a molecule, the greater the % of peak at M+1

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 Mass spectrum of Benzocaine
Signal
Intensity
C9H11O2N
M=164
[M+H]+
165
% 13C
 If we have 9 carbons and there = 9 x 1.1%
is 1.1% chance of each being a = 9.9%
13C, there will be 9 x 1.1% (9.9%)

chance of finding one 13C in a

molecule.

Approx ~10%
of [M+H]+ peak
166

m/z
0 50 100 150 200

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Case of hydrophobic compound !

How would you ionize pentane for example ??

- Not miscible with water

- So you can’t even consider adjusting the pH

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 Electron Impact (EI)

• Relatively harsh ionisation technique

• Heat in vacuum, bombard with electrons
• Typical energy of each electron: 70 eV (6.75 x 103 kJ mol-1)
• Knocks an electron out of molecule
– Lone pair > π bonded pair > σ bonded pair

• Produces cation radical

Radical cation

Fragmentation

B+ C+ • Daughter ions
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 Fragmentation

With electron impact, get fragmentation of ionised molecule due to excess energy

cation radical
• The species M, B and C
are detectable by MS.

radical neutral
cation

The species B and C provide information on the molecular structure due to some
characteristic structural fragmentations

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 Alkanes

E.g. pentane

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 n-Pentane

43

M+ : Molecular ion

29 57
72

15

Source: SDBS Spectral Database for Organic Compounds

http://www.aist.go.jp/RIODB/SDBS/

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 Branched chain alkanes

Often get fragmentation next to branch site preferentially, since this forms more
stable carbocation

m/ 43
Z

Secondary carbocation
preferentially formed

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 Stability of carbocations

Tertiary > benzyl, allyl > secondary > primary > CH3+

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 Cleavage b to heteroatom
• If heteroatom in molecule, common cleavage occurs
at a,b-bond

• Lone pair on heteroatom least tightly bound

• Electron Impact (EI) ionisation causes loss of

electron from heteroatom

M+ (M-1)+

 Also observed with esters

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4-Hydroxybenzaldehyde

121 (M-1)+
O

C 122 M+
H

HO

Source: SDBS Spectral Database for Organic Compounds

http://www.aist.go.jp/RIODB/SDBS/
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 McLafferty rearrangement
Common in esters and ketones with H in g position to the C=O

Breaks C-C bond!

Enol
M+ m/Z 102

6-membered transition state Tautomerises

E.g. methyl butanoate

Stable cation
m/ 74
Z

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Methyl butanoate
CH3O

O CH3

74

102 M+

Source: SDBS Spectral Database for Organic Compounds

http://www.aist.go.jp/RIODB/SDBS/

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 Mass spectral analysis
• Sufficient resolution to differentiate between 1 atomic mass unit (amu)

• Gives mass of each individual ion with specific isotopes

(not average mass)
12C vs 13C

• Other useful isotopes

Chlorine: 35Cl / 37Cl : 75 / 25

Bromine: 79Br / 81Br : 50 / 50

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Chlorobenzene (PhCl)

M+ m/Z 112/114 112

75:25

m/ 77
Z
114

Source: SDBS Spectral Database for Organic Compounds

http://www.aist.go.jp/RIODB/SDBS/
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Bromobenzene (PhBr)

M+ m/Z 156/158
m/ 77
Z
50:50

156 158

Source: SDBS Spectral Database for Organic Compounds

http://www.aist.go.jp/RIODB/SDBS/
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Chemical Ionization (CI)
 CI is softer technique, more likely to see molecular ion

Reagent gas : Methane

(EI)

Very Ionised
strong acid Can be detected by MS

• MH+ has less energy than M+. so less likely to break down

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 The Nitrogen Rule

• For a neutral molecule made up of most abundant isotopes of elements

present, then
– odd number of N atoms  odd molecular weight
– even number of N atoms  even molecular weight

Mwt 31
Mwt 60

Mwt 139
Mwt 151
Mwt 194

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 Accurate Mass Measurement
• High resolution mass spectrometry (TOF, FT/MS analyzers)

• Mass measurement to 0.0001 amu

• Allows determination of formula CxHyOzNw

• Many molecules may have same nominal molecular mass but different formulae

e.g. formula C9H14 C8H10O C4H10S2

exact mass 122.1096 122.0732 122.0225

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 Accurate Masses of Elements

Isotope Mass
1H 1.007825 amu
12C 12.00000 amu
14N 14.00310 amu
16O 15.99490 amu
32S 31.97210 amu
35Cl 34.96890 amu
79Br 78.91830 amu

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