MS - The effect of drug structure on fragmentation DBE and the nitrogen rule

What is mass spectrometry?

Process of ionization, separation and detection if ions according to their mass-to-charge (m/z) ratio

What are the uses of mass spec?

• Molecular weight – how big?

• Fragmentation/isomers

• Isotope analysis

• Drug metabolite analysis

What are the advantages and disadvantages of mass spec?

• Advantages - smallest sample size 10-12 g, accurate mwt to 4 dec. pl, exact molecular formulae

• Disadvantages - destructive technique, no sample recovery, requires expert operator, and high-cost equipment

What is ionisation?

High energy electrons impact on neutral drug to remove e- from the drug to give molecular ion = molecular weight (M)

What is fragmentation?

Charged molecular ion is a high energy radical cation that can break/fragment to give daughter ions that are separated according to mass-to-charge (m/z) ratio (M^+.)

What is detected in mass spec?

Only charged structures detected, not radicals or neutrals

• High vacuum required because of the highly reactive structures formed

• M^- and M⁺ or M^+. and M

What is the base peak in mass spec?

The tallest, most intense peak in the spectrum, representing the most abundant ion.

• It is assigned a relative abundance/intensity of 100%

Is the M^+. always the base peak and why?

No

• Branching gives a weak M^+. as it can fragment and break

Why does CH₃CH₂Cl have 2 peaks for M^+. ?

Due to Cl having 35 and 37 isotopes

• In a 3:1 ratio

What are the 3 fragments CH₃CH₂Cl can break into?

• Homolytic cleavage - radical formation, breaking C-C

  • CH₃CH₂Cl^+. → CH₂=Cl⁺ → m/z 49,51

• Heterolytic cleavage - carbocation formation, breaking C-Cl

  • CH₃CH₂Cl^+. → CH₃CH₂⁺ → m/z 29

• Loss of neutral fragment - lose uncharged, stable molecule, HCl

  • CH₃CH₂Cl^+. → CH₂CH₂⁺ → m/z 28

Give examples of neutral molecules that can be lost

• H₂ — loss of hydrogen

• H₂O — dehydration (common in alcohols)

• CO — carbonyl-containing compounds

• CO₂ — carboxylic acids, esters

• NH₃ — amines, amides

• HCl, HBr, HI — haloalkanes (e.g., chloroethane)

• CH₄ — alkanes

• C₂H₄ (ethene) — common in alkyl chain rearrangements

How many chlorines coresspond to number of molecular ions?

• 1 Chlorine Atom: Produces two molecular ion peaks with a 3:1 intensity ratio. M and M+2

• 2 Chlorine Atoms: Produce three molecular ion peaks with a 9:6:1 intensity ratio. M and M+2 and M+4

• 3 Chlorine Atoms: Produce four molecular ion peaks with a 27:27:9:1 ratio. M and M+2 and M+4 and M+6

Why does nicotinamide have 2 fragmentation pathways?

• Proportion of molecular ions fragment at the C-C bond

• Other molecular ions fragment at the amide C-N bond

Why does amphetamine give m/z = 91 not 77 in the MS?

The fragmentation process leads to the formation of a highly stable benzyl carbocation (tropylium ion)

What is Mclafferty rearrangement?

When the molecular ion forms, the carbonyl oxygen abstracts the γ‑hydrogen. This occurs through a six‑membered cyclic transition state, leading to:

1. Transfer of the γ‑H to the carbonyl oxygen

2. Cleavage of the α–β bond

3. Formation of:

   • A charged enol radical cation (detected in MS)

   • A neutral alkene (not detected)

Happens in ketones, aldehydes, esters, and carboxylic acids that have a γ‑hydrogen.

What is combustion analysis?

Gives drug composition % C,H,N …also F,Cl,P…..

• O measured by difference few mg sample needed

What is the empirical and molecular formula?

• Empirical formula - gives ratio of each element using % composition from combustion analysis

• Molecular formula - calculate exact number of each atom present using empirical formula and molecular weight from MS

Check if % add up to 100 to see if O is present

How do you know a drug is pure using mass spec?

For any new drug, % for all elements measured must be
within +/- 0.3 of the calculated value

What is the double bond equivalent and equation?

Total number of double bonds + rings.

• Large DBE value but small number of C suggest rings present in the drug

• Divalent sulphur - oxidation state = 2 → makes 2 bonds

• Tetravalent sulphur - makes 4 bonds

• Hexavalent sulphur - 6 covalent bonds → add 4x number of sulphurs

Calculate the empirical formula, molecular formula and DBE of nicotinamide

• Molecular formula, M+ = 122
  Empirical formula = C6H6N2O = 122
  Empirical formula x 1 = Molecular formula

• DBE

½ (2N_C - N_H + N_N + 2) = ½ ((2 x 6) – 6 + 2 + 2) = ½ (10) = 5

What is the nitrogen rule?

• Even number of nitrogens, even molecular weight

• Odd number of nitrogens, odd molecular weight (0 is even)

E