Mass Spectrometry Flashcards
For mass spectrometry;
A) What is it
B) Qualitative or quantitative?
C) What is it used for?
D) Describe how it is operated?
A)
- Analytical technique that assesses a compound/mixture via the conversion of its component molecules into ions which are subsequently analysed according to their mass
- Identity (ID) of a compound/drug can be determined or confirmed by means of its molecular weight i.e. MW/Mr
B)
QUALITATIVE and quantitative analytical method
C)
- MS ROUTINELY APPLIED IN ANALYSIS/ID OF DRUGS, DRUG METABOLITES AND DRUG IMPURITIES
- represents a highly sensitive technique utilised for trace analysis i.e. 10-3g to 10-12g (milli- to picogram level)
D)
- Very sophisticated and expensive instrument requiring a high level of operator skill
- Operates under high vacumn (<10-5 mBar i.e. very low pressure) in order to avoid any sample ion collisions/reactions with atmospheric molecules
- Basic system requires the sample to be presented in the vapour (gaseous) state prior to ion generation
- Molecular ion generated is normally a cationic species (+ve ions) which if unstable may undergo fragmentation i.e. decomposes into smaller fragments
- Molecular ion (M +) and any fragments are analysed to provide a MASS SPECTRUM i.e. the MS output
- MS represents a DESTRUCTIVE analytical technique
What are some pharmaceutical applications for MS?
Drug Bioanalysis: For TDM, research and forensic testing purposes. Normally utilised for qualitative/ID and/or ‘confirmatory’ purposes. But due to costs may not represent ‘routine’ analytical approach in some facilities for standard TDM and forensic work
Drug Quality Control: For analysis of drug substances and their formulations. Due to its high sensitivity is commonly applied for detection/ID of drug impurities (impurity profiling) and also drug formulation stability/stress testing
Drug Discovery and Development: Routinely employed analytical tool for several aspects/stages of drug candidate ‘preclinical’ studies. Most notably isynthesis, screening, stability and biological studies (e.g. drug metabolite ID) of drug candidates.
Ion generation/Ionisation method used depends on the compound and mass spectrometer type
What are some common ionisation methods?
- Electron Ionisation (EI)
- Chemical Ionisation (CI)
- Fast Atom Bombardment (FAB)
- Matrix Assisted Laser Desorption Ionisation (MALDI)
For Electron Ionisation (EI) –> hard ionisation technique, but most basic and common;
A) What is the process?
B) Which equation describes it?
C) What is the significance of M+
D) What is fragmentation? Describe what fragmentation pattern is
E) What are the apllications of EI?
A)
- Sample must be vaporised prior to its ionisation thus sample compound(s) must be vaproisable
- Sample vapour is subsequently ionised with a high energy electron beam (via W or Re filament)
- Electron (e-) collision with sample molecule (M) leads to an e- being ‘knocked off’ the molecule
- Induces the formation of a positively charged MOLECULAR ION/CATION i.e. M⨥or M+
B)
See attached image with example
C)
- M + formed represents a high energy species hence usually very unstable in nature
- Energy transferred into M + can induce covalent bond breakage in the molecule leading to its extensive decomposition or FRAGMENTATION
D)
- FRAGMENTATION is a unimolecular process resulting in the formation of smaller molecular ions known as fragment or daughter ions e.g.
E)
- Fragmentation patterns are typically complex resulting in several fragment ions
- Each molecular ion has its own characteristic and unique fragmentation pattern i.e. it serves as a ‘Fragmentation Fingerprint’
- M+ and its fragment ions are repelled from the ion chamber, accelerated then focussed by an array of charged slits onto a detector/analyser
F)
- Application: Analysis of relatively low MW organic compounds i.e. <500 Da (500 amu)
For Chemical Ionisation (CI) –> soft ionisation technique;
A) What does it involve
B) Describe the process
C) Is it a lower energy process than EI?
D) Application
A)
- Involves the use of an ionised reagent gas e.g. ammonia (NH3), methane (CH4)
B)
- Reagent gas is initially ionised by EI to yield reagent plasma (ionised gas) gives CH5+ and C2H5+
- Sample vapour (M) is then added and becomes ionised by interaction with reagent plasma via a series of intermolecular reactions
- CI predominantly generates a protonated molecular ion [M+H]+ (Quasimolecular ion)
C)
- CI is a lower energy process than EI and hence a less destructive ionisation method
D)
- Like IE for analysis of relatively low MW organic compounds i.e. <500 Da
For Fast Atom Bombardment (FAB) –> soft ionisation technique;
A) Describe the process
B) What is formed
C) Application
A)
- Sample is dissolved in a viscous liquid matrix (e.g. glycerol) and placed on metal target
- Target is then bombarded with high energy beam of inert neutral atoms e.g. Xenon, Argon
- Sample molecules are slowly ‘spluttered off’ the target and then collide with each other
- Collisions lead to sample ‘self-ionisation’ via a series of matrix induced protonation and deprotonation processes
B)
- Stable ‘low energy’ [M+H] + are formed mostly and minimal fragmentation is observed (more stable molecular ion usually permits the more rapid sample compound identification)
C)
- Analysis of organic salts and high MW polar (non-volatile) organic compounds i.e. <10,000 Da (e.g. peptides, small proteins)
For Matrix Assisted Laser Desorption Ionisation (MALDI) –> soft ionisation method;
A) How is the sample setup?
B) Describe the process
C) Application
A)
- Sample is embedded in an organic crystalline matrix (e.g. carboxylic acids, urea) and dispersed onto a steel target
B)
- Target is bombarded with a pulsed laser beam
- Matrix absorbs photons and then ionises the sample molecules i.e. indirect sample ionisation
- Ions formed are slowly ‘desorbed off’ the target i.e. escape from the organic matrix
- Very minimal fragmentation is observed
- Generally only M+ are detected (as Base peak)
C)
- Analysis of large high MW polar organic compounds i.e. <100,000 Da (e.g. proteins, oligonucleotides, oligosaccharides)
- Very sensitive technique (up to 1000 sensitivity of FAB) and useful for drug bioanalysis
What is Mass Spectrum? Outline its properties
Represents the spectral output of the mass spectrometer
- Presented as a HISTOGRAM of ‘Ion m/z value (Mass) vs % Relative ion abundance (% Intensity)’
- Ions represented as PEAKS (lines) along x-axis
- BASE PEAK is the most intense peak and is arbitrarily assigned an intensity of 100% –> denotes most stable ion
- Other ion peaks on the spectrum are reported as a % of the base peak
- Presence and intensity of a M + peak depends on its stability and the ionisation method
How does the molecular ion peak (M+) change throughout the different ionisation methods
Typically in EI mass sepctra:
- M+ peak = very low intensity or absent
Typically in CI and FAB mass spectra:
- Molecular ion + H [M +1]+ peak = Base peak
Typically in MALDI mass spectra:
- [M + H]+ or M+ peak = Base peak
> ID of an unknown M+ requires determination of molecular formula - necessitates ‘peak fitting’ to a rational combination of elements (e.g. C, H, O, N)
> Databases of ‘Mass/MW vs Molecular Formula’ correlation data (for varying elemental combinations) are available for compound ID purposes
What are isotope peaks? Give an example.
- Atomic elements exist in various isotopic forms that give rise to isotope peaks in the mass spectrum
- Isotope peaks are typically low intensity and found as ‘paired’ or ‘shoulder’ peaks adjacent to larger intensity ‘major isotope’ peaks in the spectrum
- Isotope peak intensity is proportional to no. of isotope atoms present AND their natural isotopic abundance
- Certain elements can give rise to a characteristic ‘set of peaks’ with a readily identifiable isotope peak ‘intensity ratio’ between the major and minor isotopic forms
- Certain isotope peaks that arise due to the presence of specific elements may serve an important diagnostic function and prove beneficial in compound identification
For Ion Fragmentation;
A) What is it?
B) What it dependent on
C) What is it influenced by
D) What type of fragmentation occurs?
E) How does the fragmentation occur? What are the two types
A)
Represents the decomposition of the molecular ion M + via a series of covalent bond breakages
B)
- Dependent on the compound structure/type and ionisation method e.g. often extensive in EI-MS
C)
Degree and complexity of ion fragmentation is inlfuenced primarily by:
- COVALENT BOND STRENGTH
- M+ AND FRAGMENT ION STABILITY
D)
Molecules, ions and radicals undergo fragmentation patterns
- UNIMOLECULAR fragmentations predominate due to the very low sample ion concentration
E)
Ion fragmentation usually occurs via α-BOND CLEAVAGE
- HOMOLYTIC α-BOND CLEAVAGE
- HETEROLYTIC α-BOND CLEAVAGE
Both processes are driven by the formation of stable species (especially neutral species)
What is HOMOLYTIC α-CLEAVAGE?
Involves bond breakage where one ‘single’ electron is ‘returned’ to each of the two atoms
- For M+ (an odd e- ion) results in the formation of a free radical and cation fragments (See attached image)
- Process is usually initiated by a heteroatom i.e. X = O, N, S, Halogen
- Base peak and the major fragments usually arise from homolytic α-cleavage
What is HETEROLYTIC α-CLEAVAGE?
- Involves α-transfer of an ‘electron pair’ towards the charged atom (most e- deficient)
- For M+ results in the formation of a cation and free radical fragments e.g.
- Analyte/Sample molecule dependent process and majority of MINOR intensity ions typically result from heterolytic α-cleavage
Discuss some fragmentation rules. Provide the table of common fragments
Bond breakage(s) usually:
- Promoted by ‘electron rich’ group(s) and/or atom(s)
- Occurs at ‘single’ covalent bonds
- Favoured at substituted alkyl C-atoms
- Accompanied by loss of small, neutral molecules
1 x Bond Cleavage –> Cation + Radical
2 x Bond Cleavage (simultaneous) –> Cation + Neutral molecule
Unsaturated bonds stabilise ions and radicals
Also certain cyclic, aromatic and heteroaromatic ring systems assist in ion/radical stabilisation
What is Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS/HPLC-MS) used for?
- Basically involves a GC or HPLC system fitted with a mass spectrometric (MS) detector
- Couples the high separative capability of a chromatographic system with the identification (detection) capability of MS
- Involves use of hybrid/hyphenated instruments
- Employed extensively for the identification of components within various mixtures
- Important analytical tool for the detection/ID of drugs and their metabolites in biological media
Magnetic sector analyser, quadrupole analyser, time of flight (TOF) analyser
What is isotpic abundance? Give an exmaple.
Abundance (amount) of isotopes for a chemical element as naturally found on a planet (Earth)
