LC-MS:

is an analytical chemistry technique that combines the physical separation capabilities of liquid
chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS).
- While liquid chromatography separates mixtures with multiple components, mass
spectrometry provides spectral information that may help to identify (or confirm the
suspected identity of) each separated component.
- MS is not only sensitive, but provides selective detection, relieving the need for complete
chromatographic separation.

In addition to the liquid chromatography and mass spectrometry devices, an LC-MS system
contains an interface that efficiently transfers the separated components from the LC column into
the MS ion source. The interface is necessary because the LC and MS devices are fundamentally
incompatible.
- While the mobile phase in a LC system is a pressurized liquid;
- the MS analyzers commonly operate under high vacuum. Thus, it is not possible to
directly pump the eluate from the LC column into the MS source.

Currently, the most common LC-MS interfaces are electrospray ionization (ESI), atmospheric
pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI).
The ESI ion source/ interface can be used for the analysis of moderately polar molecules (e.g.,
metabolites, xenobiotics, and peptides).
- The liquid eluate coming out of the LC column is pumped through a metal capillary kept
at 3 to 5 kV.
- The liquid is nebulized at the tip of the capillary and a fine spray of charged droplets is
formed.
- To avoid contamination, this capillary is usually perpendicularly located at the
inlet of the MS system.
- The heat created by the electric potential is used to rapidly evaporate the droplets in an
atmosphere of dry nitrogen.
- Later, the ionized analytes are transferred into the high vacuum chamber of the MS as the
charged ions flow through a series of small apertures with the aid of focusing voltages.
- Positively and negatively charged ions can be detected and it is possible to switch
between the negative and positive modes of operation.
- Most ions produced in the ESI interface are multiply charged.
LC-Nuclear magnetic resonance (NMR) is also used in plant metabolomics, but this technique
can only detect and quantify the most abundant metabolites.

LC-MS has been useful to advance the field of plant metabolomics, which aims to study the
plant system at molecular level providing a non-biased characterization of the plant metabolome
in response to its environment.

NOTE: LC–MS-MS offers several advantages over GC–MS such as quicker and less extensive
extraction procedures and the ability to identify and measure a broader range of compounds.

Quality control (QC samples):

QC samples are to judge the __quality__ and assess the __analytical variance__ of the data. The
QC sample should qualitatively and quantitatively represent the entire collection of samples
included in the study, providing an average of all of the metabolomes analysed in the study. . The
QC samples are analysed intermittently for the duration of the analytical study to assess the
variance observed in the data throughout the sample preparation, data acquisition and data
pre-processing steps. Replicate injections should provide identical data for each injection,
however in reality analytical variance will be observed and the replicate QC injections can be
used to measure this variance across the analytical study.

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LIQUID CHROMATOGRAPHY:
Liquid chromatography is a method of physical separation in which the components of a liquid
mixture are distributed between two immiscible phases, i.e., stationary and mobile.
The practice of LC can be divided into five categories, i.e., adsorption chromatography, partition
chromatography, ion-exchange chromatography, size-exclusion chromatography, and affinity
chromatography.
 - Among these, the most widely used variant is the reverse-phase (RP) mode of the
partition chromatography technique, which makes use of a nonpolar (hydrophobic)
stationary phase and a polar mobile phase:

The mobile phase containing the analytes permeates through the stationary phase bed in a
definite direction. The components of the mixture are separated depending on their chemical
affinity with the mobile and stationary phases. The separation occurs after repeated sorption and
desorption steps occurring when the liquid interacts with the stationary bed.

IN HPLC:
The liquid solvent (mobile phase) is delivered under high pressure (up to 400 bar or 300.000
torr) into a packed column containing the stationary phase. The high pressure is necessary to
achieve a constant flow rate for reproducible chromatography experiments. Depending on the
partitioning between the mobile and stationary phases, the components of the sample will flow
out of the column at different times
For applications involving LC-MS, the length of chromatography columns can be shorter (30–50
mm) with 3–5 μm diameter packing particles.

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MASS SPECTROMETRY:

mass spectra (m/z information) [not considering fragmentation]

SHN: i.e. all colored peaks except purple
Peaks represent a groups of chemical features (not necessarily compounds)

base peak chromatogram showing ion current vs. intensity (most intense peak per time point)
SHN: purple line in above 3D graph

all information is compiled in one .mzML file per run

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio (m/z)
of charged particles (ions). Although there are many different kinds of mass spectrometers, all of
them make use of electric or magnetic fields to manipulate the motion of ions produced from an
analyte of interest and determine their m/z.
MS is an experiment that must take place in gas phase and under vacuum

The basic components of a mass spectrometer are the ion source, the mass analyzer, the detector,
and the data and vacuum systems. The ion source is where the components of a sample
introduced in a MS system are ionized.
In the case of electrospray ionization, the ion source moves ions that exist in liquid solution into
the gas phase. The ion source converts and fragments the neutral sample molecules into
gas-phase ions that are sent to the mass analyzer.
While the mass analyzer applies the electric and magnetic fields to sort the ions by their
masses, the detector measures and amplifies the ion current to calculate the abundances of each
mass-resolved ion.
In order to generate a mass spectrum that a human eye can easily recognize, the data
system records, processes, stores, and displays data in a computer.

The mass spectrum can be used to determine

- the mass of the analytes,
- their elemental and isotopic composition,
- or to elucidate the chemical structure of the sample

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UHPLC/qTOF-MS
We analyse chemical features (not necessarily compounds!):
● co-elution of compounds
● isotopes, adducts of a compound
● in-source fragmentation

One peak typically represents a number of features.

In order to improve separation efficiency and peak resolution, ultra performance liquid
chromatography (UHPLC) can be used instead of HPLC. This LC variant uses columns packed
with smaller silica particles (~1.7 μm diameter) and requires higher operating pressures in the
range of 310000 to 775000 torr (6000 to 15000 psi, 400 to 1034 bar).
A hybrid mass spectrometer is a device for tandem mass spectrometry that consists of a
combination of two or more m/z separation devices of different types; QTOF is one such hybrid
MS.ie
- TOF-MS: TOF is a time-of-flight mass spectrometer, where an ion's mass-to-charge ratio
is determined by a time of flight measurement. Ions are accelerated by an electric field of
known strength.This acceleration results in an ion having the same kinetic energy as any
other ion that has the same charge. The velocity of the ion depends on the mass-to-charge
ratio (heavier ions of the same charge reach lower speeds, although ions with higher
charge will also increase in velocity). The time that it subsequently takes for the ion to
reach a detector at a known distance is measured. This time will depend on the velocity
of the ion, and therefore is a measure of its mass-to-charge ratio. From this ratio and
known experimental parameters, one can identify the ion.
- Quadropole TOF: A type of mass spectrometry where the mass-to-charge ratio of the
sample ions is measured whilst the ions are held in a stable orbit by an electric field
generated by four parallel electrodes The sample ions with different masses are
accelerated to the same (known) kinetic energy and the time taken for each ion to reach a
detector at a known distance is measured.