Mass Spectrometry Fragmentation Mechanisms

Question 1

LO

Answer 1

• 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

Question 2

What is the molecular ion?

Answer 2

Generally, the largest m/z

Question 3

What must you look for with the molecular ion?

Answer 3

• **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)

Question 4

The molecular ion

Answer 4

Question 5

The nitrogen rule

Answer 5

Question 6

Accurate mass

Answer 6

Question 7

How to calculate the Ring and double bond equivalents

Answer 7

Question 8

Calculate the ring and double bond equivalences for the following examples

Answer 8

Question 9

Tell me about the Heterolytic and homolytic cleavage of bonds

Answer 9

Question 10

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

Answer 10

Question 11

Simple fission and hydrocarbons

Answer 11

Question 12

Identify the simple hydrocarbon

Answer 12

Question 13

Identify the simple hydrocarbon

Answer 13

Question 14

Typical features to observe in a mass spectra

Answer 14

Question 15

LO

Answer 15

• 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

Question 16

Simple fission- heteroatomic compounds

Answer 16

Question 17

Answer 17

• 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

Question 18

Fragmentation of C=X (C=O, C=S, C=NH)

Answer 18

Question 19

Aldehydes and Ketones

Answer 19

Question 20

Esters

Answer 20

Question 21

Unsaturated hydrocarbons

Answer 21

Question 22

Atomatic compounds

Answer 22

Question 23

Reversed (retro-) Diels-Alder

Answer 23

Question 24

What are the
Two general rules for fragmentation in MS?

Answer 24

*** 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

Question 25

McLafferty Re-arrangement

Answer 25

Question 26

Diethyl ether

Answer 26

Question 27

Fragmentation of diethyl ether

Answer 27

Question 28

Ether (ethyl group) re-arrangement

Answer 28

Question 29

Diethylpropion

Answer 29

Question 30

Diethylpropion metabolites I and II

Answer 30

Question 31

Reflective questions

Answer 31

* Do I understand the difference between simple fission, concerted reactions and rearrangements? * How can mass spectrometry help with the identification of drug metabolites?

Question 32

Key LO

Answer 32

* 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

Question 33

Recommended reading

Answer 33

Question 34

What is soft ionisation and what typically occurs after it?

Answer 34

* 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

Question 35

An example of ESI fragmentation is Ketamine and hydroxynorketamine, tell me about the structures

Answer 35

* 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)

Question 36

Tell me about Ketamine- accurate mass and elemental composition

Answer 36

*** 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)

Question 37

Tell me about Hydroxynorketamines- accurate mass and elemental composition

Answer 37

*** 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

Question 38

MS/MS spectra of hydroxynorketamines

Answer 38

* 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

Question 39

Fragmentation

Answer 39

*** 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

Question 40

**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?