MS and MS/MS

Mass Spectrometry

MS is a wide-ranging analytical technique that relates to the production and detection of charged species.
The basic principle of MS is to generate ions from compounds by a method that would be able to separate these ions by their mass-to-charge ratio (m/z) and to detect them by their respective m/z and abundances.
Ionization is the process whereby electrons are either removed or added to atoms or molecules to produce ions.

Structure of a Mass Spectrometer

Some basic concepts…

A simple mass spectrum is represented by a two dimensional bar plot:

Some basic concepts…

Usually each compound produce >1 ion

Points-per-peak

Enough data points per chromatographic peak
(i.e., at least 7–8)

Points-per-peak

Mass resolution

Ability of a mass analyzer to separate two adjacent peaks:
- Low-resolution mass spectrometers (LRMS) can separate sufficiently two ions differing by one mass unit
- High-resolution mass instruments (HRMS) can discriminate between isobaric species, even with in complex matrices
HRMS instruments are required to determine the elemental composition.

Resolving power

RP = M / d(M)

Isotopic patterns

Isotope	Mass	d(Mass)	Abundance
C(12)	12.000000	0.000000	98.900
C(13)	13.003355	1.003355	1.100

N(14)	14.003074	0.000000	99.630
N(15)	15.000109	0.997035	0.370

O(16)	15.994915	0.000000	99.760
O(17)	16.999131	1.004216	0.038
O(18)	17.999159	2.004244	0.200

H(1)	1.007825	0.000000	99.900
H(2)	2.014102	1.006277	0.015

Isotopic patterns

Theoretical isotopic pattern calculator: enviPat
Check examples with: C7H6O5 and C8H12N4O3

Mass resolution & Isotopic patterns

Isotope	d(Mass)
C(12)	0.000000
C(13)	1.003355

N(14)	0.000000
N(15)	0.997035

O(16)	0.000000
O(17)	1.004216
O(18)	2.004244

H(1)	0.000000
H(2)	1.006277

Mass resolution

Acquiring data at higher resolving power results in better mass accuracy, but the scan duration increases with increasing resolving power, thus diminish number of scan/peak

Mass resolution

Full Scan Mode

Only one MS function without induced fragmentation is acquired to generate ions of the molecular species, adducts and in-source fragments.

Full Scan Mode

Acquisition performed as a full scan over a wide m/z range, depending on the particular application and/or compounds of interest.

Lipidomics: 100 - 2,000 Da
Metabolomics on biofluids: 70-800 Da

Data Dependent Acquisition Mode

The DDA mode allows the production of fragmentation spectra for structure elucidation.

The instrument acquires a full scan MS1 and, when certain criteria are met, performs a specified number of MS/MS acquisitions on the most intense ions before switching back to full scan MS1.

Data Dependent Acquisition Mode

The fragmentation is only performed on the signals that meet specified and user-guided criteria:

Signal threshold to switch from FS to MS/MS
Number of MS/MS spectra per cycle
Exclusion lists: static and dynamic
Inclusion list (optional)