Pharmaceutical Mass Spectrometry

Primary role of mass spectrometry in pharmaceutical analysis

Measuring mass-to-charge ratio (m/z) to identify and quantify chemical analytes.

Three essential components of a mass spectrometer

Ion source, mass analyzer, and detector.

Function of the ion source

Converts molecules into gas phase.

Function of the mass analyzer

Separates ions based on m/z.

Function of the detector

Detects ions and develops signal.

Five types of mass spectrometry ion sources

Electron impact (EI), chemical ionization (CI), MALDI, electrospray (ESI), and APCI.

Electron Impact (EI) ionization

High energy electron removes an electron; hard ionization for small volatile compounds.

Chemical Ionization (CI)

Soft ionization that transfers a proton for volatile compounds.

MALDI (Matrix-Assisted Laser Desorption/Ionization)

Uses a laser and matrix to desorb and ionize large molecules.

ESI (Electrospray Ionization)

High voltage creates droplets; used for polar nonvolatile compounds.

APCI (Atmospheric Pressure Chemical Ionization)

Ionizes a heated nebulized sample; used for small/medium less polar compounds.

Ion sources coupled to GC

EI or CI, more commonly EI.

Ion sources coupled to HPLC

ESI or APCI.

DESI and DART

Ambient ion sources used for direct sample analysis with minimal preparation.

Ion source for non-polar, volatile, thermally stable analytes

Electron impact (EI).

Ion source for polar, ionic, or large analytes

Electrospray ionization (ESI).

Molecular ion (M+•) vs Pseudo-molecular ion (MH+)

Molecular ion is a radical cation from hard ionization; pseudo-molecular is protonated (+1) from soft ionization.

m/z shifts for ion adducts in positive mode ESI-MS

+1 for H+, +23 for Na+, +39 for K+, and +18 for NH4+.

Four common mass analyzers

Q (quadrupole), IT (ion trap), TOF (time of flight), and Orbitrap.

Quadrupole (Q) mass analyzer

Observes a single ion m/z at a time; receives MS.

Ion Trap (IT) mass analyzer

Traps and fragments ions of selected polarity; receives MS and MS/MS.

Time-of-Flight (TOF) mass analyzer

Injects ions in pulses and measures flight time; small ions are faster.

Orbitrap mass analyzer

Traps ions in a barrel electrode, detecting m/z based on oscillation frequency.

Mass resolution (Rs) definition

How separated two peaks of different m/z are.

Tandem MS (MS/MS)

Technique to break down selected precursor ions into product ions.

Instruments capable of performing tandem MS

Triple quadrupole (QQQ) or ion trap (IT) MS.

Requirement for multistage fragmentation

Requires an ion trap analyzer.

Selective Reaction Monitoring (SRM/MRM) usefulness

Removes interference by tracking specific parent and product ions for sensitive quantification.

QQQ roles in an MRM experiment

Q1 selects parent ion, Q2 performs CID, Q3 selects product ion.

Nominal molar mass

Sum of integer atomic masses with no decimals.

Average molar mass

Sum of atomic weights from the periodic table including decimals.

Monoisotopic molar mass

Exact mass using the most abundant isotopes.

Nitrogen rule in EI-MS

An odd number of nitrogen atoms gives an odd mass number; an even number gives even.

Definition of an isotope

Atoms with the same number of protons/electrons but a different number of neutrons.

Calculating weighted average molar mass

Multiply isotope abundance by weight and add them together.

Stable heavy isotopic labeling

Replacing atoms with stable isotopes like 2H, 13C, 15N, or 17O.

Use of heavy isotope labeled compounds as internal standards

They co-elute in GC/HPLC but have predictable mass differences for simultaneous quantification.

Base peak in a mass spectrum

The highest peak in the spectrum.