Mass Spectrometry

Question 1

What is Mass spectrometry?

Answer 1

MS is a technique used to measure the relative mass of molecules

Question 2

What does it require the generation of?

Answer 2

Analyte ions followed by ion detection and mass analysis

Question 3

Why is mass spectrometry useful in pharmacy?

Answer 3

Helps to:
 verify the identity of a drug substance
 confirm the presence of a particular drug in formulated products
 verify the presence of drugs and drug metabolites in clinical samples
 identify unknown drug metabolites (to allow you to see how drug acts in body)
 Provides means for quality control of recombinant proteins (human insulin, interferons, etc) It does this cheap and with good results.
 Helps in sequencing of proteins, peptides and oligonucleotides
 Can be used in drug discovery, e.g. for identification of expressed proteins – pathology in humans.

Question 4

What are the 4 major stages of MS?

Answer 4

1. ) Sample needs to be vaporised
2. ) Ion generation – need to generate ion
3. ) Ion separation according to mass-to-charge ratio
4. ) Ion Detection

Question 5

How can stage 1 (vaporise sample) be achieved?

Answer 5

Using heat
Placing the sample in the vacuum
Using fast atom bombardment (e.g. beam of xenon atoms)

Question 6

How can stage 2 (sample ionisation) be achieved?

Answer 6

This can be done by bombarding the volatilised molecules with the electrons from the electron gun. This results in molecule ionisation. Charged plates accelerate the ionised molecules into the deflection chamber.

Question 7

What ionisation techniques are there?

Answer 7

o Electron Impact Ionisation (EI)
o Chemical Ionisation (CI)
o Fast Atom Bombardment (FAB)
o Matrix Assisted Laser Desorption Ionisation (MALDI)
o Electrospray Ionisation (ESI)
o Atmospheric Pressure Chemical Ionisation (APCI)

Question 8

What is electron impact ionisation?

Answer 8

The lost electron means the analyte becomes positively charged which means they can now be accelerated in the chamber. Achieved by under vacuum or electrical waves.
An electron beam has sufficient energy to fragment the molecule (~70 eV)

Question 9

What does rapidly moving electrons knocking an electron out of a molecule lead to?

Answer 9

The formation of cationic radicals:

- Electron removal: [M] → [M]+• + 2e-

Question 10

What happens if the molecule captures an electron?

Answer 10

This produces an anionic radical:

- Electron addition: [M] → [M]-•

Question 11

What is Chemical ionisation?

Answer 11

CI uses a stream of electrons to ionise a reagent gas (ammonia or methane). The ionisation of the reagent gas results in production of strong acid (e.g. NH4+ or CH5+) Volatilised analyte molecules are ionised by strong acid (CH5+ or NH4+) via protonation.
This results in generation of an [M+H]+ ions.

Question 12

What sorts of techniques are EI and CI?

Answer 12

NOT gentle techniques - produce lots of fragments.
Suitable for small volatile molecules with mw < 1000
Cheap and easy
Not suitable for large fragments

Question 13

What is Fast Atom Bombardment?

Answer 13

1. ) Bombarding an analyte sample suspended in a viscous matrix with a beam of fast moving Xe atoms.
2. ) Energy transfer from Xe atoms to the matrix
3. ) Breaking of intermolecular bonds and ionisation
4. ) Desorption of analyte ions into the gas phase

Question 14

What does the matrix in FAB help to do?

Answer 14

The matrix helps to protect the analyte from fragmentation. If the matrix was absent, the direct bombardment of the analyte by the fast atoms would lead to extensive fragmentation. FAB can also be used to generate negatively charged ions.

Question 15

What sort of technique is FAB?

Answer 15

GENTLE technique producing very few fragments.
Does not require sample to be volatile
Suitable for molecules with MW up to ~6000
The peaks obtained in FAB are denoted as [M+H]+ (resulted from protonation) and [M-H]- (resulted from deprotonation)

Question 16

What is Electrospray ionisation (ESI)?

Answer 16

 ESI is a routine technique for the ‘soft’ ionisation
 ESI is normally applied for polar analytes (e.g. biomolecules with MW up to ~100 000 Da)
 Analyte is dissolved in (a mixture of) an organic solvent (AN or MeOH)
 pH modifier (e.g. formic, acetic acid) is used to produce ions
 Ionisation proceeds via protonation or deprotonation mechanism (depending upon the pH modifier used)
 [M+H]+ or [M-H]- are detectable ions in the ESI

Question 17

What is Atmospheric Pressure Chemical Ionisation (APCI)?

Answer 17

 Similar interface to that used for ESI
 However, the gas-phase ionisation in APCI is more effective than ESI for analyzing less-polar species
 Sample introduction is the same as for ESI (e.g. use of organic solvents (AN or MeOH) and pH modifiers (formic or acetic acid ))
 ESI and APCI are complementary methods
Mass range – typically less than 2000 Da

Question 18

What is Matrix Assisted Laser Desorption Ionisation (MALDI)?

Answer 18

 MALDI uses a concept similar to that of the FAB technique
 Main differences between MALDI and FAB:
 The energy is transferred to the matrix from a laser beam
 The matrix must have a chromophore absorbing energy at wavelength of laser
 Suitable for biomolecules with MW up to ~500000 Da
They are dissolved in matrix and then ionised. They are then draw to detector by electrical field

Question 19

What is the overall degree of fragmentation for the different techniques?

Answer 19

EI > CI&raquo_space; FAB, ESI, APCI, MALDI

Question 20

What do the fragmentation points correspond to?

Answer 20

The weakest bonds in the molecule

Question 21

What are Tandem MS techniques?

Answer 21

Based on combination of MS with other analytical methods:
 Gas Chromatography-MS (GC-MS)
 High-Performance Liquid Chromatography-MS (HPLC-MS or LC-MS)
 MS-MS

Question 22

What is GC-MS?

Answer 22

 GC-MS is used to analyse non-polar, volatile analytes
 Interfacing a GC system to an MS instrument allows to
- separate components of the analyte
- obtain mass spectrum for each point on the chromatogram
 Unique fragmentation fingerprints can be used for compound identification
 Using a library of standard MS spectra it is possible to identify an unknown analyte

Question 23

What is HPLC-MS?

Answer 23

 LC-MS is very much dependent on ionisation and ion vaporisation
 Usually ESI or Atmospheric Pressure Chemical Ionisation (APCI) are used as an ionisation method in LC-MS
 LC-MS separates out the components of a mixture and provides a MS profile on each fraction

Question 24

What is MS-MS?

Answer 24

 MS-MS is used to produce structural information about a compound
 This can be achieved by fragmenting sample ions inside the mass spectrometer and identifying the resulting fragment ions
 This information can then be pieced together to generate structural information regarding the intact molecule
 A tandem mass spectrometer has more than one analyser
 The fragmentation is often carried out within the mass analysis device (instead of MS source)
 This gives superior fragmentation results and, thus, better structural information
Complicated method which eventually gives you key structural information which helps in drug development.

Question 25

What are daughter ions?

Answer 25

the molecular ion [M]+• can generate daughter ions via the loss of either radical or neutral molecule  This produces additional peaks corresponding to the fragment ions with smaller m/z ratio  The ions which are formed will reflect the most stable cations and radical cations

Question 26

What does the highest molecular weight peak represent?

Answer 26

the molecular ion (M•+) of the parent molecule

Question 27

What fragmentation do simple alkanes tend to undergo?

Answer 27

fragmentation by the initial loss of a methyl group to form a (m-15) species

Question 28

What can this carbocation then undergo?

Answer 28

stepwise cleavage down the alkyl chain, expelling neutral fragments of general formula CnH2n+1 Fragmentations that give rise to stable carbocations will be particular favoured

Question 29

What will branched hydrocarbons form?

Answer 29

 Branched hydrocarbons form more stable secondary and tertiary carbocations  These peaks will tend to dominate the mass spectrum  In a branched chain alkanes fragmentation occurs next to the branch site

Question 30

What is a heteroatom?

Answer 30

O, N, Cl, Br, etc  For molecules containing heteroatoms (O, N, Cl, Br, etc) a very common fragmentation is the cleavage of the gammabeta bond

Question 31

What are other stable carbocations?

Answer 31

those with an adjacent heteroatom

Question 32

where will the positive charge resulted from the loss of one electron be?

Answer 32

normally distributed on the electronegative atom(s)

Question 33

What is the predominate cleavage in aldehydes and ketones?

Answer 33

loss of one of the side-chains to generate the substituted oxonium ion (RC0+) This is an extremely favorable cleavage and this ion often represents the base peak in the spectrum

Question 34

What is the methyl derivative (CH3C-=O+) commonly referred to as?

Answer 34

the acylium ion

Question 35

What is ion separation?

Answer 35

Once ionized, the analyte ions are separated by their interaction with an electric or magnetic field in high vacuum(10-9 to 10-12) High vacuum is applied to minimise the interaction of analyte with molecules in the air.

Question 36

How are molecular ions then DETECTED?

Answer 36

1. ) High momentum ions (having high mass) will not be deflected enough and will collide with the analyser wall – for these could change strength of the magnet to increase magnetic field, which means you would change angle of trajectory. 2. ) Ions having low mass (low momentum) will be deflected most by this field and will also collide with the walls of the analyser – for this you would reduce the strength of the magnet, to reduce magnetic field so trajectory would be less so you will be able to reach detector 3. ) Ions having the proper mass-to-charge ratio will follow the path of the analyser and collide with the Collector 4. ) This generates an electric current, which is then amplified and detected 5. ) By varying the strength of the magnetic field, the mass-to-charge ratio which is analysed can be continuously varied.

Question 37

What is the next stage after having a spectrum?

Answer 37

To determine the chemical structure of the test sample

Question 38

How can you identify the chemical structure?

Answer 38

 By measuring mass of these fragments, we can identify their chemical structure and reconstruct the structure of the test molecule  The analysis of mass spectra involves the re-assembling of fragments, working backwards to generate the original molecule