LC - MS

Liquid Chromatography - Mass Spectrometry

Brahmesh Reddy B R and Aishwarya G

PhD Scholars
Department of Plant Physiology
 PRESENTATION REPORT

on

LC-MS
LIQUID CHROMATOGRAPHY - MASS SPECTROSCOPY

submitted for the partial fulfilment of the requirements of the course

MBB 504 (1+2)

TECHNIQUES IN MOLECULAR BIOLOGY - I

offered by
Dr. Nagesh N and Dr. Poornima R

02:00 pm on 20/01/2023
Department of Plant Biotechnology

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What is LC-MS?

LC-MS is an analytical technique that involves the physical separation of target

compounds (or analytes) followed by their mass-based detection. Although relatively
new, its sensitivity, selectivity and accuracy have made it a technique of choice for
detecting microgram or nanogram quantities of various analytes ranging from drug
metabolites, pesticides and food contaminants, to natural product extracts.

How does LC-MS work?

LC separation

LC brings about a physical separation of the analytes in a liquid sample or a

solution of a solid sample. A few microliters of sample solution are injected into a
flowing stream of a solvent, called the mobile phase. While the optimal injection volume
is dependent on the experimental conditions, it is possible to inject as little as 0.1 µL up
to as much as 100 µL of the sample accurately using an autosampler.1 The mobile phase
is continuously pumped through a column (a stainless-steel tube) usually filled with silica
particles coated with another liquid, the stationary phase. When the sample
solution-mobile phase mix reaches the column, its components will differentially interact
with the stationary phase (which remains in the column) depending upon their chemical
composition or physical properties. Based on the mechanism of interaction between the
analyte and the stationary phase, LC separations have been classified into different
modes, such as:

- Partition chromatography – based on the differing solubility and hydrophobicity of the

analytes in the stationary phase as compared to the mobile phase.

- Ion-exchange chromatography – separates the analytes on the basis of their ionic

charges.

- Size-exclusion chromatography – exploits the differences in the sizes of the analyte

molecules to separate them.

- Affinity chromatography – separates the analytes based on their ability to bond with the
stationary phase.

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LC detection

The mobile phase flowing out of the column (the eluent) passes through a detector
that “responds” to a certain physical or chemical property, such as refractive index or
light absorption, of the analytes within it. This response is captured as a signal or a
“peak” whose intensity (peak area or peak height) corresponds to the amount of the
component present in the sample. The time at which the detector “sees” the analyte is its
RT. The identity of a compound in a sample can be confirmed by comparing its RT with
the RT of a known compound. While this is not an accurate method of compound
identification, it helps when some information about the sample is known a priori.

What is mass spectrometry and how does mass spectrometry work?

MS is a form of chemical analysis used to measure the mass-to-charge ratio (m/z)

of atoms and/or molecules in a sample. It is also capable of distinguishing between
different isotopes of the same element. Depending upon the type of mass spectrometer,
these measurements can often be used to determine the exact molecular weight of the
sample components and to identify unknown compounds.

There are many different types of mass spectrometers, but they all have three
features in common. The first is some means by which atoms or molecules from the
sample can be ionized. Neutral species cannot be steered by electric fields used in mass
spectrometers, and thus it is necessary to produce ions. There are many different means

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by which this can be accomplished, and they are collectively referred to as ion sources.
The second component of all mass spectrometers is the mass analyzer itself. There are
several different means by which the m/z ratio of ions can be measured. Time-of-flight
(ToF), magnetic sector and quadrupole mass analyzers are the most common, each with
its own set of strengths and limitations. The final component common to all mass
spectrometer systems is a means of detecting or counting the number of ions of a specific
m/z value. These devices are called detectors and they too come in several different
forms with the most common being electron multipliers, Faraday cups, channeltrons and
channel plates. Again, each has its own particular strength and weakness.

A final factor that needs consideration is how to couple the ion source to the
sample so as to produce the ions for measurement, especially in light of the fact that all
mass spectrometers must be operated under vacuum. In some cases, the sample will also
be housed under vacuum, in others the sample will be at atmospheric pressure
(generally referred to as the ambient MS techniques) and some may incorporate some
other form of separation technology prior to introduction to the ionization chamber. The
following sections will go over these three common components to mass spectrometers
in more detail.

Ion sources for mass spectrometry

Ionization is essential for any MS analysis, for which there are many methods suited to
different sample types and applications. Broadly, these can be broken down into gas
phase methods, desorption methods, and spray methods. An outline of each is given
below.

Gas phase methods

Electron ionization (EI) - analyte molecules must be in the vapor phase to allow
effective interaction with the energetic electrons produced in a vacuum by a heated
filament. EI can be considered a fairly harsh method of molecule fragmentation and
ionization and is most commonly used when samples are relatively volatile and have low
molecular weight.

Chemical ionization (CI) - a gas is introduced into an EI ionization chamber at a

concentration higher than the analyte. The interaction of the carrier gas with the
electrons will produce several molecular ions, which will subsequently react further
with the excess carrier gas and form different molecular ions. These ions will then react
with the analyte molecules to form analyte molecular ions through several different

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mechanisms. CI is a very soft ionization technique and does not lead to extensive
fragmentation.

Direct analysis in real time (DART) – plasma is created, producing ions, electrons and
excited-state species. Interaction of the excited state species with a liquid, solid or vapor
phase sample is then responsible for the ionization of the analyte molecule. DART is able
to analyze materials of different shapes and sizes with no prior sample preparation and
in ambient conditions.

Inductively coupled plasma (ICP) – a prepared liquid containing the analyte is

aerosolized and converted to gas phase ions using plasma. ICP has the ability to ionize
almost all elements.

Desorption methods

Matrix assisted laser desorption ionization (MALDI) – a “matrix”, dictated by the type
of molecule to be detected, is added in excess to the sample to be analyzed. The sample is
then irradiated by a laser, vaporizing the analyte molecules with little to no
fragmentation or decomposition. Both positively and negatively charged ions can be
created. MALDI is one of the major “soft” ionization methods, particularly useful for the
analysis of large or labile molecules.

Fast atom bombardment (FAB) - a beam of accelerated ionized atoms is focused onto
the sample to be analyzed, ejecting and ionizing target analyte. This is a soft ionization
technique, able to produce positively and negatively charged ions.

Thermal ionization sources – heated Cs, producing positive ions, is the most common
primary ion source and can be focused with electrostatic ion optics for secondary ion
MS.

Plasma ionization sources – commonly used to produce beams of gaseous ions,

electrons are emitted into a gas, often pure oxygen, ionizing it and creating a plasma. The
ions can then be filtered by charge and accelerated into a beam.

Liquid metal ion sources (LMIS) – sources are low melting point metals, often Ga, to
which the application of heat and an electric field produces ions at a small point source.
Ion beams produced by LIMS are characterized by the smallest spot sizes and highest
brightness, particularly advantageous in MS imaging where high spatial resolution is
required.

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Spray methods

Electrospray ionization (ESI) – a mist of charged droplets are reduced in size through
solvent evaporation until gas phase ions are ejected. This soft ionization technique
suitable for analysis of large molecules and macromolecules.

Desorption electrospray ionization (DESI) - very similar to ESI except the charged
droplets formed in the ESI source are directed to a sample held at ambient pressure.
Reflected droplets then carry the desorbed and ionized sample.

Types of mass analyzers

Following sample ionization, the ions must be separated and this occurs in the mass
analyzer. Commonly used mass analyzers include:

Time-of-flight (ToF) – ions are separated according to their m/z ratio based on the length
of time it takes them to travel through a flight tube of known length to reach a detector.

Quadrupole - ions entering the quadrupole have their trajectory deflected by electrical
potential in a manner that is proportional to their m/z value. Changing the potential
allows only ions of specific m/z values to reach the chamber end and be detected.

Magnetic sector - magnetic fields disperse ions in trajectories according to their m/z
ratios in a manner that is analogous to the way a glass prism disperses light into its
various wavelengths or colors.

Ion trap – works similarly to a quadrupole but the electrodes are ring shaped and ions
are separated and detected by discharging ions with unstable oscillations from the
system and into the detector rather than detecting those with stable oscillations.

Orbitrap - borrows technology from many of the other types of mass analyzers. Two
electrically isolated cup-shaped outer electrodes face each other with a spindle-like
central electrode around which ions of a specific mass-to-charge ratio spread into
orbiting rings. The conical shape of electrodes pushes ions toward the widest part of the
trap and the outer electrodes are then used for current detection. It is the only method
described here that uses an image current rather than some detection device to detect
the ions.

Tandem mass spectrometry (tandem MS) - refers to hybrid methods involving more
than one type of mass spectrometer to increase specificity and mass resolving capability.

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Types of ion detector

A key element to all MS systems is the type of detector used to convert a current of mass
separated ions into measurable signal. Different types of detectors are used depending
upon factors including dynamic range, spatial information retention, noise and
suitability to the mass analyzer.

Commonly used detectors include the:

Electron multiplier (EM) - a serial connection of discrete metal plates that amplify a
current of ions by a factor of ~108 into a measurable current of electrons

Faraday cup (FC) - ions hitting the collector cause a flow of electrons from ground
through the resistor and the resulting potential drop across the resistor is amplified.

Photomultiplier conversion dynode - ions initially strike a dynode, resulting in electron

emission. The electrons produced then strike a phosphor screen which in turn releases
photons. The photons then pass into the multiplier where amplification occurs in a
cascade fashion – much like the EM.

Array detectors (including detectors for simultaneous measurement of several ions of

different m/z and detectors for position-sensitive ion detection) - covers a broad range of
detector types and systems that may combine multiple detection technologies

Using MS for LC detection

Although a wide variety of detectors of differing technologies and sensitivities have been
coupled with LC for analyzing different sample types, the mass spectrometer has
emerged as a selective, sensitive and universal detector.

Unlike other detectors, the LC eluent carrying the separated analytes is not
allowed to flow into the mass spectrometer. While the LC system is operated at ambient
pressures, the mass spectrometer is operated under vacuum and the two are coupled
through an interface. As the column eluent flows into the interface, the solvent is
evaporated by applying heat and the analyte molecules are vaporized and ionized. This
is a crucial step as the mass spectrometer is only capable of detecting and measuring the
gas phase ions. As the analyte ions are generated at atmospheric pressure in the
interface, the process is called atmospheric pressure ionization (API) and the interface is

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known as the API source. Electrospray ionization (ESI) and atmospheric pressure
chemical ionization (APCI) are the most commonly used sources in LC-MS analysis.

The analyte ions are drawn into the mass spectrometer where they are subjected
to electric fields and/or magnetic fields. The flight paths of the ions are altered by varying
the applied fields which ensures their separation from one another on the basis of their
mass-to-charge (m/z) values. Post-separation, the ions can be collected and detected by a
variety of mass detectors,2 of which the most common one is the electron-multiplier.
When the separated ions strike the surface of the electron-multiplier (a dynode),
secondary electrons are released. These secondary electrons are multiplied by cascading
them through a series of dynodes. The amplified current generated by the flow of the
secondary electrons is measured and correlated to the ion concentrations in the mass
spectrometer at any given instant in time

Plotting LC-MS data

The abundances of the ions measured during the analysis of a sample by LC-MS
are plotted as a total ion chromatogram (TIC). This plot displays the peak intensities of
the analyte ions versus their RT. Further, each point in the chromatogram is associated
with a mass spectrum. The mass spectrum depicts the ion abundances versus the
measured m/z values. The mass spectrum of a compound not only provides information
about the mass of the parent compound (from the m/z value of its ion), but also helps to

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elucidate the structure of the compound from the relative abundances of isotopic mass
peaks. The area of the analyte peak is used for its quantification.

The mass spectrometer can be operated in two modes, a) scan and b) selected ion
monitoring (SIM). In the scan mode, it is set to detect all the ions from low m/z to high
m/z values within a specified time period. This mode is used when analyzing unknown
samples or when there is no available information about the ions present in a sample.
When operating in SIM mode, the mass spectrometer is set to measure specific m/z
values. This is the preferred mode of operation for accurate quantification of known
compounds in a sample.

How does liquid chromatography work?

The mobile phase, typically a solvent, is used to transport the sample through the
system with the aid of a high-pressure pump. However, it also plays a critical role in the
separation process. A small volume of sample (1-100 µL) is loaded into a sample loop
(Figure 1 (2)), and is then injected into the mobile phase flow by means of a six-port valve
and this triggers the start of the chromatographic run. Once the sample has been
injected, the mobile phase is pumped through to the column. A variety of column lengths

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(30 to 250 mm) and internal diameters (1 to 4.6 mm) are available, packed with
stationary phase adsorbent materials of differing activities and particle sizes (1.5 to
10-micron diameter) that together define the column efficiency and selectivity. The
column is located in a column oven; at higher temperatures (45 ºC) the viscosity of the
mobile phase decreases which increases its linear velocity. This in turn reduces the run
time and also improves the chromatographic resolution. Components in the mixture that
have a higher affinity to the mobile phase will migrate through the column quickly with
little interaction with the stationary phase. As the band of the component leaves or elutes
from the column, the detector, will give a response that is proportional to the
concentration of the component. The time taken between injection and detection is
known as the retention time. The retention time for a component will be very specific for
a given set of chromatographic conditions and may be compared with that of a standard
for identification.

After the autosampler injects the sample into the mobile phase the separation
process is carried out in the column. The selectivity of the chromatographic system has
the largest influence over the chromatographic resolution and should be tailored for the
application and components under investigation. The selectivity may be modified by
changing the eluotropic strength of the mobile phase (different solvents) or the specific

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chemical functional groups present in the stationary phase (changing the column type).

After the autosampler injects the sample into the mobile phase the separation
process is carried out in the column. The selectivity of the chromatographic system has
the largest influence over the chromatographic resolution and should be tailored for the
application and components under investigation. The selectivity may be modified by
changing the eluotropic strength of the mobile phase (different solvents) or the specific
chemical functional groups present in the stationary phase (changing the column type).

The hyphenation of mass spectrometry to liquid chromatography (LC-MS)

Mass spectrometry is arguably the best detector that can be hyphenated to a

liquid chromatograph due to its high sensitivity, linear dynamic range, selectivity and
even specificity when using instrumentation with a very high mass resolving power. The
technique of mass spectrometry is used to determine the mass-to-charge ratio (m/z) of a
component or analyte. Unlike gas chromatography-mass spectrometry (GC-MS), the
hyphenation of an LC system to MS was not easy and took many years to develop.
Electrospray ionization (ESI) is the most common ionization technique used in LC-MS
today, where the ionization process takes place at atmospheric pressure. It was the
development of an atmospheric pressure inlet to the high vacuum required within the
mass spectrometry system that was difficult to achieve. Microflow and low-flow LC-MS
are proving useful in areas including biomarker detection and biopharmaceutical
analysis.

How do you read an LC-MS mass spectrum and what does it tell you?

Electrospray ionization is a very soft ionization technique meaning that there is

very little fragmentation observed during the formation of ions. An LC-MS system may
be run in either positive ion mode for basic analytes generating protonated molecules
[M+H]+, or negative ion mode for acidic analytes generating deprotonated molecules
[M-H]-.

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Case studies

1. Schuster O, Zvi A, Rosen O, Achdout H, Ben-Shmuel A, Shifman O, Yitzhaki S,

Laskar O, Feldberg L. Specific and Rapid SARS-CoV-2 Identification Based on
LC-MS/MS Analysis. ACS Omega. 2021 Jan 26;6(5):3525-3534. doi:
10.1021/acsomega.0c04691. PMID: 33585737; PMCID: PMC7857140.
2. Mosaad I. Morsy, Eman G. Nouman, Youmna M. Abdallah, Mourd A. Zainelabdeen,
Mohamed M. Darwish, Ahmed Y. Hassan, Amira S. Gouda, Mamdouh R. Rezk,
Ahmed M. Abdel-Megied, Hoda M. Marzouk, A novel LC-MS/MS method for
determination of the potential antiviral candidate favipiravir for the emergency
treatment of SARS-CoV-2 virus in human plasma: Application to a bioequivalence
study in Egyptian human volunteers,Journal of Pharmaceutical and Biomedical
Analysis, Volume 199, 2021,114057,ISSN 0731-7085,
https://doi.org/10.1016/j.jpba.2021.114057.
3. Jebaseelan, S. & Jose, B. & Meera, Dr.R.. (2021). Phytochemical Investigation Using
LC-MS Analysis and Antimicrobial Activities of Leaf Extract of Muntingia calabura
Linn. International Journal of Pharmaceutical Sciences Review and Research. 69.
10.47583/ijpsrr.2021.v69i02.006.
4. Wang, Guodong & Wang, G.-D. (2014). Applications of LC-MS in Plant
Metabolomics. 10.1007/978-94-017-9291-2_9.

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REFERENCES

1. How a Mass Spectrometer Works, Types of Instrumentation and Interpreting Mass

Spectral Data | Technology Networks
2. Liquid Chromatography – Including HPLC, UHPLC and LCxLC | Technology
Networks
3. LC-MS – What Is LC-MS, LC-MS Analysis and LC-MS/MS | Technology Networks
4. Types of Ion Detector for Mass Spectrometry | Technology Networks
5. Mass Analyzers for Mass Spectrometry | Technology Networks
6. Mass Spectrometry Ionization | Technology Networks

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