Mass Spectrometry Fragmentation Mechanisms Flashcards
LO
- Revision of key learning outcomes
o Ionisation
o Mass spectrum
Molecular ion, isotopic mass
o General principles of fragmentation - Accurate mass
- Fragmentation
o Simple fission
Charge localised on heteroatom
Cleavage beta-position from heteroatom - To be able to interpret the fragmentation of simple mass spectra
- To be able to confirm the identity of substances
- To be able to facilitate the identification of unknown substances
What is the molecular ion?
Generally, the largest m/z
What must you look for with the molecular ion?
- **Look for unusual losses M+.-4 to M+.-14 **
o Dot indicates odd number of electrons
o .H, H2, .H & H2,…
o (Loss of He, Li, Be, B, C, …CH, ..CH2 does not occur due to the high amount of energy required for this)
o .CH3 this is a common loss (loss of 15 mass units)
The molecular ion
The nitrogen rule
Accurate mass
How to calculate the Ring and double bond equivalents
Calculate the ring and double bond equivalences for the following examples
Tell me about the Heterolytic and homolytic cleavage of bonds
How would the following circumstances be written following the principles of fragmentation:
* Loss of radical from molecular ion
* Loss of neutral molecules from molecular ions
* Loss of radical from fragment ion
* Loss of neutral molecule from fragment ion
Simple fission and hydrocarbons
Identify the simple hydrocarbon
Identify the simple hydrocarbon
Typical features to observe in a mass spectra
LO
- Fragmentation- simple fission
o Charge localised on heteroatom
o Cleavage beta-position from heteroatom - Fragmentation rearrangements
o Ether re-arrangement
o McLafferty re-arrangement - Summary
Simple fission- heteroatomic compounds
- if have N and O in same molecules
o Localise charge on N or O
o If on N then end up with species a and if charge on O then end up with species b
o Both can occur but N is more stable so a is more abundant
o Mass spec looks at relative abundance
Fragmentation of C=X (C=O, C=S, C=NH)
Aldehydes and Ketones
Esters
Unsaturated hydrocarbons
Atomatic compounds
Reversed (retro-) Diels-Alder
What are the
Two general rules for fragmentation in MS?
*** McLafferty **
o Stabilisation of charge is the most important factor for determining the fragmentation of a molecules
o Even electron species are most stable than those with an odd number of electrons
*** Stevenson **
o In a fragmentation the charge will go with the fragment with the lower ionisation potential
McLafferty Re-arrangement
Diethyl ether
Fragmentation of diethyl ether
Ether (ethyl group) re-arrangement
Diethylpropion
Diethylpropion metabolites I and II
Reflective questions
- Do I understand the difference between simple fission, concerted reactions and rearrangements?
- How can mass spectrometry help with the identification of drug metabolites?
Key LO
- MS is destructive but very sensitive
- MS is not inherently quantitative
- MS may be used quantitatively with internal standards that maybe isotopically labelled
- Spectrum may be library searches and or interpreted
- Molecular ion
o Isotopic mass, accurate mass- elemental composition - Fragmentation
o Simple fission, charge localised on heteroatom
o Re-arrangement, ether, McLafferty
Recommended reading
What is soft ionisation and what typically occurs after it?
- In soft ionisation, such as occurs under electrospray ionisation, the ions produced are even electron species such as protonated molecular ions
- After soft ionisation, fragmentation is induced (typically in a tandem MS instrument i.e., MS/MS) by accelerating the ions into a collision gas, a process known as collision induced dissociation (CID)
- After CIS these even electron species mainly fragment by loss of neutrals so that the fragment is also an even electron species
- An example of ESI fragmentation will be explained with ketamine and hydroxynotketamine
An example of ESI fragmentation is Ketamine and hydroxynorketamine, tell me about the structures
- Ketamine has benzene ring with chlorine attached
- And cyclohexone with methyl amine attached
- Hydroxynorketamines made by metabolism of ketamine
- The hydroxy group can be in 4 positions –> 4 possible isomers (phenols)
- If hydroxyl in cyclohexanone group, they can also be in 4 positions –> 4 isomers
- Methyl amine is demethylated in the hydroxynorketamines (nor as lost the methyl group)
Tell me about
Ketamine- accurate mass and elemental composition
*** Ketamine **
o Elemental composition C13H16CINO
o Exact mass=237.09204
o Chlorine-37 isotope to be considered alongside Cl35
*** Protonated ketamine- **when ionised in the MS
protonated ketamine is formed
o Elemental composition C13H17CINO+
o Exact mass= 238.09932 (as protonated the nitrogen rule doesn’t follow)
o 37Cl exact mass 240.09637 (32% relative abundance)
Tell me about
Hydroxynorketamines- accurate mass and elemental composition
*** Protonated ketamine **
o Elemental composition C13H17CINO+
o Exact mass= 238.09932
o 37Cl exact mass 240.09637 (32% relative abundance)
*** Protonated hydroxynorketamines **
o Elemental composition C12H15CINO2+
o Exact mass= 240.07858 (this is different from integer difference)
o 37Cl exact mass 242.07563 (32% relative abundance)- 2 mass units up from ketamine its self
MS/MS spectra of hydroxynorketamines
- A- alcohol hydroxynorketamine
- B- Phenolic hydroxynorketamine
- MS/MS can choose the precursor ion to go on and fragment further in second MS
- This means that what we are looking at is fragments of 240
- C- instead of 240, collect Cl37 isotope and look at alcohol
- D- Cl37 isotope and look at phenol
- C and D have some similarities and differences between isotopes. No 179 and 127 in D and no 143 in D
Fragmentation
* some fragment of alcohol metabolite in A
o Benzene ring does not contain OH
o Fragment as m/z 125 (as seen in bottom RHS)
o Another ion m/z 222 seen is where the OH has been eliminated by water
o Elemental composition worked out from mass to 5 dp
*** B **
o Phenol in the benzene ring
o Odd m/z as no N so the rule gets reversed from the fragments
o Ion at 141 which did not appear in alcoholic version
**Mass spectrometry- fragmentation mechanisms
Relative topics and questions **
* Do I understand the difference between simple fission, concerted reactions, and rearrangements?
* How can mass spectrometry help with the identification of drug metabolites?
