Mass Spectrometry Part 1 — Fundamentals

Basic principle of mass spectrometry:

The basic principle of mass spectrometry (MS) is to generate ions from either inorganic or organic compounds by any suitable method, to separate ions by their mass-to-charge ratio (m/z) and detect them qualitatively and quantitatively by their respective m/z and abundance.

[Note: The term mass-to-charge ratio, m/z — (read “m over z” and write m/z)]

A mass spectrometer consists of an ion source, a mass analyzer, and a detector which are operated under high vacuum conditions. An MS device might separate the steps of sample introduction, evaporation, and successive ionization or desorption/ionization respectively. The mass spectrometers are operated under total data system control (See Figure 1)

Figure 1: General layout of a mass spectrometer [1]

Mass Spectrum:

A mass spectrum is a 2D plot of signal intensity (y-axis) vs m/z (x-axis). Each peak position gives the m/z of an ion generated from the sample analyte. The peak height corresponds to abundance of that molecular ion

Figure 2: A typical electron ionization mass spectrum of a low-mass hydrocarbon [1]

Molecular ion & fragments: The highest m/z peak may (but not always) be the molecular ion (M⁺·). Peaks at lower m/z often arise from fragment ions formed by decomposition of M⁺·

Base peak & intensities: The most intense peak is the base peak. Spectra are typically normalized so the base peak (≈ 100% relative intensity), which makes spectra comparable because relative intensities don’t depend on the absolute number of ions detected

Advantages over other analytical techniques:

1. Ultra-high sensitivity — Detection of analytes at femtomole levels for impurity/biomarker study
2. High selectivity/specificity — Measurement of precise m/z for targeted and untargeted analysis from complex matrices
3. Structural elucidation — Revealing molecular formula and structures from exact mass, isotope patterns and fragmentation spectra
4. Quantitative accuracy and wide dynamic range — Robust quantitative analysis with internal standards for bioanalysis and clinical assays
5. High-resolution/accurate-mass (HRMS) capability — Improving identification confidence with narrows mass windows (For example <5 ppm)
6. Micro-scale sample analysis requirements — Effective analysis of proteomes and metabolites at micro-scale level
7. Speed and throughput — Fast scan rates and automated workflows support high-throughput omics
8. Versatility across analyte classes — Small molecules, lipids, peptides and proteins, glycans, drugs, and metabolites
9. Isotopic information — Supports isotope tracing and formula confirmation via exact mass and isotopic patterns
10. Non-targeted discovery and targeted verification — Enabling unbiased profiling (untargeted HRMS) and highly targeted SRM/MRM in one platform family
11. Regulatory settings — Widely adopted for diagnostic biomarkers and therapeutic drug monitoring

References:

[1] Mass Spectrometry: A Textbook 3E — Jürgen H Gross, 2017