Medicine: Mass spectrometry - review and make questions shorter by adding more questions Flashcards
Define mass spectrometry
MS is a technique used to measure the relative mass of molecules
Requires generation of analyte ions followed by ion detection and mass analysis
Can be applied to both small organic molecules and biomolecules
Applications of mass spectrometry
Mass spectrometry 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
- Provides means for quality control of recombinant proteins (human insulin, interferons, etc)
- Helps in sequencing of proteins, peptides and oligonucleotides
- Can be used in drug discovery, e.g. for identification of expressed proteins
Main principles of mass spectrometry
The molecules of the sample are ionised and then identified according to their mass
Mass spectrum is a plot of intensity (the abundance of each ion) against mass-to-charge ratio (m/z)
Thus, the mass spectrometer is a device for producing and weighing ions
Major stages in mass spectrometry
1) Sample vaporisation
2) Ion generation
3) Ion separation according to mass/charge ratios
4) Ion detection
Where is the sample vaporised?
vacuum chamber
Where is the sample ionised?
electron beam
How are the ions separated? i.e. what by
By a magnetic field that bends the path of the charged particles
How is sample vaporisation achieved?
This can be achieved by
using heat
placing the sample in the vacuum
using fast atom bombardment (e.g. irradiation of sample by beam of Xenon atoms)
Results in even distribution of the individual molecules within the vacuum chamber
How is the sample ionised?
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
Name 6 ionisation techniques
- Electron impact ionisation (EI)
- Matrix Assisted Laser Desorption Ionisation (MALDI)
- Atmospheric pressure chemical ionisation (APCI)
- Fast atom bombardment (FAB)
- Electrospray ionisation (ESI)
- Chemical ionisation (CI)
How does EI (electron impact ionisation) occur?
Sample is vaporised by heat
Ionisation is achieved by bombarding the volatilised molecules with an electron beam
An electron beam has sufficient energy to fragment the molecule (~70 eV)
This results in molecule ionisation (~99% positively charged (cationic) radicals and ~ 1% negatively charged radicals)
The positive fragments produced (cations and radical cations) are accelerated under vacuum through a magnetic field into the deflection chamber
They are analysed on the basis of mass-to-charge ratio
EI mechanism
Rapidly moving electrons knock an electron out of the molecule (in ~99% cases)
This results in formation of cationic radicals:
Electron removal: [M] → [M]+• + 2e-
If the molecule captures an electron (~1%), this produces an anionic radical :
Electron addition: [M] → [M]-•
How does CI (chemical ionisation) occur?
CI uses a stream of electrons to ionise a reagent gas (ammonia or methane)
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
Summarise EI and CI as techniques
EI and CI are NOT gentle techniques – produce lots of fragments
Suitable for small volatile molecules with MW < 1000 Da
Cheap and relatively easy
Describe how fast atom bombardment occurs (FAB)
Bombarding an analyte sample suspended in a viscous matrix with a beam of fast moving Xe atoms
Energy transfer from Xe atoms to the matrix
Breaking of intermolecular bonds and ionisation
Desorption of analyte ions into the gas phase
note: FAB can also be used to produce anions