Mass spectrometry

Why it matters

Mass spectrometry (MS) measures the mass-to-charge ratio (m/z) of ions. It identifies unknown compounds, confirms the identity of peaks in a chromatogram, and, as LC-MS/MS, quantifies drugs, pesticides, metabolites and proteins at trace levels with high selectivity. It is now standard equipment in pharmaceutical, environmental, forensic, clinical and biochemistry labs, so analysts are expected to read a simple spectrum even if they do not run the instrument.

The core idea: ionize, separate, detect

1. Ion source: turns molecules into gas-phase ions.
   • Electron ionization (EI), used with GC: a 70 eV electron beam knocks out an electron, giving the molecular ion M+. and many reproducible fragments. EI spectra can be matched against libraries such as NIST's.
   • Electrospray ionization (ESI), used with LC: a "soft" method that usually gives [M+H]+ (positive mode) or [M-H]- (negative mode) with little fragmentation. Large molecules such as proteins pick up many charges.
2. Mass analyser: separates ions by m/z.
   • Quadrupole: a mass filter; robust and quantitative. Triple quadrupoles (QqQ) are the workhorse of LC-MS/MS quantitation.
   • Time-of-flight (TOF): measures flight time; high resolution and accurate mass.
   • Orbitrap and FT-ICR: very high resolution and mass accuracy.
3. Detector: counts ions. The spectrum plots relative abundance against m/z. The tallest peak is the base peak (set to 100 %).

Reading a simple EI spectrum

• Molecular ion: usually the highest-m/z peak in the cluster (if it survives). Its m/z gives the molecular mass (for singly charged ions, m/z = mass).
• Nitrogen rule: an odd molecular mass suggests an odd number of nitrogen atoms.
• Isotope patterns:
  • Chlorine (35Cl and 37Cl about 3:1) gives an M+2 peak about one third the height of M.
  • Bromine (79Br and 81Br about 1:1) gives M and M+2 peaks of nearly equal height.
  • Carbon-13 gives a small M+1 peak, about 1.1 % per carbon atom.
• Fragments: losses from M point to structure: M - 15 (loss of CH3), M - 18 (water, from alcohols), M - 29 (CHO or C2H5), M - 45 (COOH). Common fragment ions include m/z 43 (CH3CO+ or C3H7+), 77 (phenyl, C6H5+) and 91 (tropylium, C7H7+, typical of benzyl groups).

Worked example: two unknowns

Unknown 1 shows peaks at m/z 156 and 158 of almost equal height, and a base peak at 77.

• Equal M and M+2: one bromine.
• 77: a phenyl group.
• 156 - 79 = 77, so the molecule is bromobenzene, C6H5Br.

Unknown 2 (ESI positive mode) shows a strong peak at m/z 152.07.

• In ESI the main ion is usually [M+H]+, so the neutral mass is about 151.06.
• Paracetamol (acetaminophen), C8H9NO2, has a monoisotopic mass of 151.063: a plausible match, to be confirmed with a reference standard's retention time and MS/MS fragments. The odd mass is consistent with one nitrogen.

Quantitation with LC-MS/MS

A triple quadrupole selects a precursor ion in the first quadrupole, fragments it in the collision cell, and selects a product ion in the third. Monitoring a specific precursor-to-product pair is called multiple reaction monitoring (MRM). It filters out almost all background, which is why LC-MS/MS can measure nanograms per liter. Quantitation uses calibration curves and, ideally, a stable-isotope-labelled internal standard (the same molecule with 13C or deuterium), which corrects for matrix effects: other compounds in the sample that suppress or enhance ionization.

Common mistakes

• Assuming the highest peak is the molecular ion (it is the base peak, which is often a fragment).
• Forgetting that ESI gives [M+H]+, so the neutral mass is 1.007 lower.
• Ignoring isotope patterns that reveal Cl or Br immediately.
• Quantifying in complex matrices without an appropriate internal standard.

Quick recap

• MS measures m/z: ion source (EI hard, ESI soft), mass analyser (quadrupole, TOF, Orbitrap), detector.
• Molecular ion gives the mass; isotope patterns reveal Cl (3:1) and Br (1:1); fragments reveal structure.
• ESI usually gives [M+H]+ or [M-H]-.
• LC-MS/MS with MRM and isotope-labelled internal standards is the standard for trace quantitation.

Try it

Open the Do tab for the quiz: interpret four small spectra.