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
Purpose of the matrix in FAB?
What if it wasn’t there?
The matrix helps to protect the analyte from fragmentation
If the matrix were absent, the direct bombardment of the analyte by the fast atoms would lead to extensive fragmentation
Summarise FAB technique
Gentle technique producing very few fragmentations
Does not require sample to be volatile
Allows the analysis of biomolecules
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)
Describe how Electrospray ionisation (ESI) occurs
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
Describe how atmospheric pressure chemical ionisation (APCI) occurs
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
Benefits of atmospheric pressure chemical ionisation (APCI) occurs?
Mass range?
Benefits:
good for less-polar compounds
excellent LC/MS interface
compatible with MS/MS methods
Mass range:
Typically less than 2000 Da
Describe the matrix assisted laser desorption ionisation technique?
molecule size?
MALDI uses a concept similar to that of the FAB technique
Suitable for biomolecules with MW up to ~500000 Da
Main differences between MALDI and FAB?
Main differences between MALDI and FAB:
In MALDI, the energy is transferred to the matrix from a laser beam
In MALDI, the matrix must have a chromophore absorbing energy at wavelength of laser
Degree of fragmentation for different techniques
EI > CI»_space; FAB, ESI, APCI, MALDI
Molecular ion [M]+• produced by ionisation is often in an excited state
This state corresponds to excess of vibrational energy leading to molecular fragmentation
The fragmentation points correspond to the weakest bonds in the molecule
What are Tandem (or hyphenated) MS Techniques?
Tandem MS techniques are based on an appropriate combination of MS with other analytical methods
Can be used to analyse a complicated mixture of different compounds and provide structural information on each component
Name the most important tandem MS techniques
The most important tandem MS techniques include:
- Gas Chromatography-MS (GC-MS)
- High-Performance Liquid Chromatography-MS (HPLC-MS or LC-MS)
- MS-MS
Describe how GC-MS is used
Gas chromatography mass spectrometry
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
Describe the LC-MS technique
Liquid chromatography mass spectrometry
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
Describe the MS-MS technique
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
Fragmentation
How does it occur?
Molecular ion [M]+• produced by ionisation is often in an excited state
This state corresponds to excess of vibrational energy leading to molecular fragmentation
The fragmentation points are associated with the weakest bonds in the molecule
Thus, 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 fragment ions seen depend upon the exact conditions used in the mass spectrometer
Possible fragment ions: B•+ (cation radical), A+ (cation), [A+H]+ (protonated ion) B•+ (cation radical), X•- (anion radical), Y- (anion), [Y-H]- (deprotonated ion)
The process of fragmentation follows simple and predictable chemical pathways
The ions which are formed will reflect the most stable cations and radical cations
The highest molecular weight peak represents the molecular ion (M•+) of the parent molecule
Rules of fragmentation
Simple alkanes tend to undergo fragmentation by the initial loss of a methyl group to form a (m-15) species
This carbocation can then undergo 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
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
Other stable carbocations are those with an adjacent heteroatom
The positive charge resulted from the loss of one electron will be normally distributed on the electronegative atom(s)
For molecules containing heteroatoms (O, N, Cl, Br, etc) a very common fragmentation is the cleavage of the a,b-bond
fragmentation of aldehydes and ketones
The predominate cleavage in aldehydes and ketones is loss of one of the side-chains to generate the substituted oxonium ion
This is an extremely favorable cleavage and this ion often represents the base peak in the spectrum
The methyl derivative (CH3C≡O+) is commonly referred to as the “acylium ion”
How does ion separation occur?
Once ionized, the analyte ions are separated by their interaction with an electric or magnetic field in high vacuum (10-9 – 10-12 bar)
High vacuum is applied to minimize the interaction of analyte ions with molecules in the air
How are the ions in a MS detected?
The positive ions and the molecular fragments produced in the ionization chamber are accelerated into an analyzing tube
The path of the charged molecules is bent by an applied magnetic field
Detection of ions
What if the ions have too much momentum or mass?
What if they don’t have enough?
High momentum ions (having high mass) will not be deflected enough and will collide with the analyzer wall
Ions having low mass (low momentum) will be deflected most by this field and will also collide with the walls of the analyzer
Ions having the proper mass-to-charge ratio will follow the path of the analyzer and collide with the Collector
This generates an electric current, which is then amplified and detected
By varying the strength of the magnetic field, the mass-to-charge ratio which is analyzed can be continuously varied.
Reassembling the fragments
Once we have a spectrum, the next stage is to determine the chemical structure of the test sample
Fragmentation of the molecular ion allows to help with this
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
When interpreting mass spectra, you need to answer at least two questions. What are these questions?
Whether the molecular ion has the same mass as we would expect?
Whether particular patterns correspond to certain structural elements of the molecule?
