Karteikarten

Mass Spectrometry

megan trent Englisch 41 Karten

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1overview• What is mass spectrometry
– Principles of ionisation and mass analysers
• Interpretation
– The mass spectrum
– Interpretation – peaks and isotopes
• Hyphenated methods GC-MS and LC-MS
– Analysis in the pharmaceutical industry
2Where is MS used?• Analysis of large molecules, like proteins and peptides, or acquisition of small, aqueous-molecules
• Searching for target compounds at a determined level of detail, or characterization of unknown samples?
• Developing separation methods from complex matrixes?
• Mass accuracy to 5 ppm, (at 400 MW that is 400.0125 MW (or 2 mDa at mass 400)
• Processing hundreds of samples a day? Thousands?
Tens of thousands? High-through-put
3Mass spectrometry in action Pharmaceutical Science• Drug metabolism studies
• Characterization of potential drugs
• Bioavailability studies
• Screening of drug candidates
• Identifying drug targets
• Bio-molecule characterization (protein
and peptides)
• High through put analysis
• Pharmacokinetics
4Mass spectrometry in action Forensics Science• Examination of evidence
– Arson, explosive, toxicology, drugs of abuse…
• Quantifications of drugs (and
metabolites) in drugs of abuse in
physiological specimens
• National security
5Mass spectrometry in action chemistry• Accurate mass
• Structural elucidation
6MS in the drug development process:
7What is Mass Spectrometry?The mass spectrometer is an instrument in which beams of ions are separated according to the mass-charge ratio (m/z), and in which the electrons are measured electrically
What can we use the mass for?
Identify, analyse, verify, quantify
8MS PrinciplesDifferent compounds can be uniquely
identified by their mass
Ethanol Mw 46.07
Salicylic acid Mw 138.12
Testosterone Mw 288.42
9Typical Mass Spectrum
10Desirable features• The mass spectrometer measures the mass better than any other
technique
• Applicable to all elements
• Applicable to all types of samples
– volatile or non-volatile
– polar or non-polar
– solids or liquids, or gaseous materials
– hydrophilic or hydrophobic
• Ultra-high detection sensitivity
• Rapid identification
• Use in conjunction with chromatographic methods (GC-MS or LCMS)
11Principles of MS
12Principles of MS InletFlow-injection
LC
GC
13Principles of MS ion source Why is ionisation required?
14Mode of ionisation
15Ion sources
16Hard versus soft ionisation• Parent or Molecular ion formation
• Hard (energetic) sources lead to excited-state
ions and fragmentation
– Good for structural information
• Soft sources causes little fragmentation
– Good for molecular weight determination
17Examples of mass
18Electron ionisation (Hard ionisation method) (Used in GC-MS) Electron Ionisation (1919 A.J. Dempster)• Routine analysis of small molecules (Mw < 400
Da)
• Excess energy absorbed by the molecules/ions
result in fragmentation
– Sometimes no molecular ion
– Good for providing structural information
• Sample introduced in the gas-phase
– Thermal desorption process (due to vacuum and heating)
– Limited to volatile, thermal stable, low mass compounds
• Inlet: gas chromatography
19Electron Ionisation
20Distortion – transfer of energy
21Mode of ionisationn (EI): Electron ejection: structural information
22EI Mass Spectrum Hexane, C6H14 MW = 86.18
23Electrospray ionisation (soft ionisation) (used in LC-MS) ESI• Atmospheric pressure ionisation
• Soft ionisation method, provides molecular
weight information
– Less fragmentation
• Suitable for analysing large bio- or synthetic polymers
• Suitable for analysing polar and even ionic lcompounds (e.g. metal complexes)
24Electrospray ionisation
25Typical mass spectrum – small molecule • Cortisol Mw 362.4 ESI+
26Proton affinity
27Multiple ion formationFor large molecules multiple protonation is possible - The ESI spectrum
therefore contains different peaks representing different charge states of
the same molecule. The same protein with a molecular weight of 10,000
can result in several peaks in the mass spectrum
28Mass spectrum of Myoglobin:
29Expected m/z• The double charged species of a peptide of MW of 3555 Da will appear in the mass
spectrum at m/z of
– 3556
– 3555
– 3554
– 1778.5
– 1777.5
30Principles of MS Mass Analyser How are the ions separated according to their mass-tocharge ratio?Quadrupole Mass Analyser
By selecting appropriate RF frequency and potential, the quadrupole
acts as a mass filter letting some m/z ions through to the detector
while other m/z ions will collide with the electrodes and cannot
reach the detector
31Quadrupole Mass Analyser
32Time of Flight (ToF)
33How do we get more structural data from soft ionisation spectra?• Coupling of 2 mass analyzers with the objective to obtain further information of a more specific nature about the analyte in question
• Generate fragmentation of molecular ion by CID (Collision Induced Dissociation)
– MS/MS
34• Though mass spectrometry is a very powerful method for analysing the structure of organic compounds, it suffers from 3 major limitations:1. Compounds cannot be characterised without clean samples
2. It does not have the ability to provide sensitive and selective analysis of complex mixture
3. MS spectra for large molecules such as peptides spectra are very complex and difficult to interpret
Solution: MS/MS
35To define the parent ion a tandem mass spectrometer is needed. Various configurations are used but all have two analysers separated by a region in which the chosen m/z ion collide with an inert gas and made to fragments
36Out putMS/MS spectrum
Spectrum of precursors
of interest and reconstructed
spectrum. Software stores
memory of the rf/dc voltages
(specific m/z values) that
coincide with fragments striking
the detector.
Spectrum of precursors
of interest and reconstructed
spectrum
37ESI spectrumconsist mainly of the protonated
molecular ion with no induced fragmentation.
However, CID can be carried out and the molecule
fragments (to give dominant alpha cleavage
fragments)
38Principles of MS Data System
39The spectrum• The mass spectrum is presented in terms of ion abundance vs. m/z ratio
• The most abundant ion formed in ionisation gives rise to the tallest peak on the mass
spectrum – this is the base peak
• All other peak intensities are relative to the base peak as a percentage
40Determination of Molecular Mass• If a molecule loses only one electron in the ionisation process, a molecular ion is
observed that gives its molecular weight – this is designated as M+ on the spectrum
• In some cases, when a molecule loses a valence electron, bonds are broken, or the ion formed quickly fragment to lower energy ions
• The masses of charged ions are recorded as fragment ions by the spectrometer
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