Mass Spectrometry 3 Flashcards
MS Interpretation
- General inspection
- Identify base peak (can be (quasi-)molecular ion!)
- Lot of fragmentation or limited number of stable ions? - Identify highest m/z (take isotopes into account!)
- Are you certain this is M+●?
- If so then mass odd/even → nr. of N - Are there “A + 2” elements?
- Use above & find all potential molecular formulae (may help to use 12C/13C calculation)
- Check if formulae can explain OE+.
- Derive structure using all information incl.
- Tables (peaks and losses) - Basic principles
Common Fragmentation Patterns
• Electronegative atom tends to carry + charge
• α-cleavage (bond in α position to site of ionisation):
Fission of bond at atom adjacent to charged atom
– Homolytic
– Heterolytic
• Protontransfer
• Ring structure fragmentation
α Cleavage: EtOH
• Homolytic: 1 electron remains on each atom • O electronegative • 2 electrons: 1 to radical 1 to C=O • Loss of largest radical favoured • + on electroneg atom
• Heterolytic
• Minor here
• Homolytic favoured
if hetero atoms
Homolytic cleavages
- Homolytic α-cleavages:
- OH
- C=O
- C-O-C
- C-NH2
Self-test
• Butan-2-ol. • Indicate the – type(s) of cleavage – fragments lost for the major ions
Common losses for EI
H radical - often a major ion in amines, alcohols and aldehydes
CH3 radical - most readily lost from quaternary C
H2O - readily lost from secondary or tertiary alcohols
Ring structures: Cyclohexanol
Molecular rearrangement prior to fragmentation more likely if ring
Base peak: C3H5O+
- O+•
- Homolytic cleavage 3. H-transfer
Diels-Alder fragmentation
- Can occur with ring systems
• Example: Limonene
Drug molecules
- Abundant molecular ion (codeine)
- Homolytic α-cleavage (bupivacaine)
- Isotope peaks (chloroquinone)
- Tropylium ion (L-dopa)
- McLafferty rearrangement (apronal)
Codeine
- Ring -> fragmentation complex
• 229 closely related to codeine structure
– Does require re- arrangement of ring
• Drugs with extensive ring structures tend to have large molecular ion peaks if…
• No side chains
• Side chains without hetero atoms to direct cleavage
Bupivacaine
• No obvious molecular ion • Homolytic α-cleavage • Directed by N in ring • m/z 140 dominates spectrum – Note that even mass rule does not apply to fragments
Isotope peaks: Chloroquinone
- (A):EI
- α-Homo cleavage
- Adjacent N in chain
- (B):NICI (NI = neg ion CI = chem. ionisation)
Tropylium ion: L-dopa
• Benzyl group • Very stable cation • Other processes may compete • Here: homolytic cleavage
McLafferty rearrangement
• -COOH, Esters • Ketones • Amides • side chain ≥ 3C-atoms • Unusual for drugs – α-cleavage – Long chain lipid (such as fatty acid esters)
More than just MS…
• GC-MS – Ionisation techniques: • EI / PICI / NICI – Applications: • Impurity profiling • LC-MS • Tandem-MS
Enhancing the “molecular” ion
• Electrospray Ionisation (ESI):
– Add or remove H+ (using acid-base chemistry)
– Add Na+, K+ (naturally in solution / dopant) – adducts
– Crucial: also better to get large (bio-) molecules into vapour and couples well with LC
Enhancing the “molecular” ion
• Chemical Ionisation (CI): PICI / NICI
– Target functionality with choice of reagents
– Tweak energy to get desired level of fragmentation
– PICI: adducts; NICI: M-
Enhancing the “molecular” ion
• Laser-ablation (MALDI)
– (Deposit sample onto solid substrate)
– Add matrix
Positive Ion Chemical Ionisation
- PICI
* CH4
Negative Ion “Chemical Ionisation”
- NICI requires electron-capturing compound to analyse
• Generate low energy electrons by collisions with reagent gas: < 10 eV
– Resonance electron capture: AB-
– Dissociative electron capture: A- + B
• Psoralen
GC-MS: Impurity profiling
- FDA: identify if > 0.1%
- Propanolol
- Commercial sample
Collision-Induced Dissociation (CID)
-EI = hard ionisation -> extensive (too much?) fragmentation
-PICI, NICI much “softer”
-ESI often used in combination with LC
– Very soft
-clear molecular ion, but not much fragmentation
Need fragmentation to solve molecular structure!
CID in tandem-MS (or MS/MS or MS-MS or MSn)
- Often using 3 quadrupole mass filters in sequence
- Molecular ion selected (Q1)
- fragmented using Ar-gas in collision cell (Q2)
- Separated (Q3)
Tandem-MS (MS/MS)
- Lidocaine
- ESI
- Ketamine
Example: Kanamycin
- Extremely polar
- 4 basic centres
- ESI: Unfragmented
- CID of m/z 485
ESI & Multiple Charge States
• Highest charged state ~ # basic functionalities (not precise, often works…)
• Extends mass range for same analyser: m/z
• Molecular weight ↔ nr. of “molecular ion” peaks
- calculate the molecular weight with ease & precision
MS of Proteins
-Most instruments: m/z < 3000 Da
-Proteins > 3000 Da -need multiple charges!
• n = charge on MB (≈ z)
• MA, MB adjacent ions
• Mass: MA>MB
MS of Proteins
mass =
mass = (m/z) x z
MS of Proteins
n =
MA - 1/ MA- MB
Applications of LC-MS in Pharmacy
- Quality control: Determination of impurities
• Use in drug discovery
• Drug (formulation) degradation
• Profiling impurities & degradants
QC: Impurities
- insulin-like growth factor
- ESI-MS
- Protein mass
- Identify impurities
MS in drug discovery
• Leuenkephalin – opioid – peptide – ESI – m/z 556
LC-MS: Degradant of famotidine
- Effect of stress conditions in pack
* LC-MS
• Famotidine
- Famotidine MS-MS
* Same for degradant
