LC_MS.pptx

Department of Plant Biotechnology
MBB 504 (2+1) / Techniques in Molecular Biology - I
Liquid chromatography
Mass spectrometry (LC-MS)
Brahmesh Reddy B R and Aishwarya G
I year Ph.D
Department of Plant Physiology

What is LC-MS?
LC-MS is an analytical technique that involves 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 even nanogram quantities of a variety of analytes ranging
from drug metabolites, pesticides and food adulterants, to natural product extracts.
Brahmesh Reddy B R
Aishwarya G
Liquid Chromatography
Mass spectrometry

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.
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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

LC separation
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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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.
LC separation
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Liquid
Chromatography
A multi-component mixture that is
soluble in the liquid mobile phase is
separated due to the individual
components’ unique partitioning
between the mobile phase (Figure 1
(1)) and the stationary phase
(column) (Figure 1 (3)).
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Figure 1: A simplified diagram of a liquid chromatograph hyphenated to a mass spectrometer (LC-MS) showing: (1) binary pump for mobile phase, (2)
autosampler 6-port valve and injector loop, (3) column heater with column, (4) mass spectrometer detector, (5) PC
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

The mobile phase, typically a
solvent, is used to transport the
sample through the system with
the aid of a high-pressure pump
(Figure 1 (1)). However, it also
plays a critical role in the
separation process.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Once the sample has been
injected, the mobile phase is
pumped through to the column
(Figure 1 (3)).
A variety of column lengths (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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

As the band of the component
leaves or elutes from the
column, the detector
(Figure 1 (4)), will give a
response that is proportional to
the concentration of the
component.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

The data acquisition system
(Figure 1 (5)) records the
detector response as a function
of retention time in a
chromatogram.
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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Chromatogram
Figure 2: Chromatogram output from an HPLC or LC-MS
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Chromatogram
The peaks recorded in the
chromatogram (Figure 2) are
usually integrated to determine the
peak area
Peak area is proportional to the
concentration of the component
present in the sample.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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.
Chromatogram
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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Modes of operation
● Isocratic
● Gradient
An isocratic method will use the same
mobile phase composition for the
duration of the chromatographic run
with no change in selectivity.
A gradient method will enable the
mobile phase composition to be changed
as a function of time, which is usually
optimized to either increase the
chromatographic resolution or shorten
run times.
Considering the mobile phase, there
are two main modes of operation to
choose from when running a liquid
chromatograph, namely,
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

The hyphenation of mass spectrometry
to liquid chromatography
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

LS is best
hyphenated to MS
● High sensitivity
● Linear dynamic range
● Selectivity
● High specificity
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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Mass Spectrometry
Wilhelm Wien, J.J. Thomson and Francis Aston
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Mass Spectrometry
Figure 1: Outline of the main steps of MS and common variants available at each step
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

There are many different types of mass spectrometers, but they all have three features in common (Figure 1).
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 by which this can be accomplished, and they are collectively referred to as ion sources.
Mass Spectrometry
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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.
Mass Spectrometry
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Demo of m/z ratio of 2,3 - dichloro toluene
m/z
Ratio
Mass to charge ratio
m / z =
Mass number
Charge number
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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, channel trons and channel plates.
Mass Spectrometry
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Mass Spectrometry
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Ion sources
Component 1
1. Gas phase methods
a. Electron Ionization (EI)
b. Chemical ionization (CI)
c. Direct Analysis in Real Time (DART)
d. Inductively coupled plasma
2. Desorption methods
a. Matrix assisted Laser DI (MALDI)
b. Fast Atom Bombardment (FAB)
c. Thermal Ionization Sources
d. Plasma Ionization Sources
e. Liquid Metal Ion Sources (LIMS)
3. Spray methods
a. Electrospray Ionization (ESI)
b. Desorption Electrospray Ionization (DESI)
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Electrospray Ionization (ESI)
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

The sample is delivered
into a capillary held at
high voltage (a few kV).
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

This produces a mist of
charged droplets of the
same polarity.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

By using a drying gas or
elevated temperatures,
the charged droplets
move through the source
and are gradually
reduced in size through
evaporation of the
solvent,
leading to an increased
surface charge density.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

At a certain point, the
electric field strength
within the droplet will be
large enough for ions at
the surface of the
droplet to eject into the
gaseous phase
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Mass Analyzers
Component 2
1. Time of flight (ToF) - time required
2. Quadrupole - trajectory deflection
3. Magnetic sensor - dispersion lll prism
4. Ion trap - quadrupole with ringed electrodes
5. Orbitrap - opposite cups and imagery
6. Tandem MS - hybrid
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Quadrupole mass analyzers
● Quadrupole mass analyzers consist of two pairs of metal rods equidistant from each other and biased
at equal and opposite potentials.
● These twin potentials contain a fixed direct current (DC) and alternating radio frequency (RF)
component, where the strength of the RF component can be varied.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Quadrupole mass analyzers
● Any ion entering the quadrupole will have its trajectory deflected by the potential in a manner that is
proportional to its m/z value.
● At specific RF values, only one specific m/z value will resonate with the field and be able to navigate to
the end of the quadrupole and be detected. Ions with other m/z values will collide with the
quadrupoles, lose their charge and not be detected.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Ion Detectors
Component 3
1. Electron multiplier -> x 108
2. Faraday cup (FC) -> potential drop amplified
3. Photomultipler conversion dynode
4. Array detectors - hybrid
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Electron multipliers (EM)
The essence of an EM is a serial connection
of discrete metal plates called dynodes that
amplifies a current of ions by a factor of ~108 into
a measurable current of electrons.
When a single secondary ion enters the EM,
it is stopped by the first conversion dynode. The
energy of impact is dissipated in part by ejection
of electrons from the dynode material, creating
an electrical charge. Additional electrons are
ejected by a cascade process through subsequent
dynodes. At the final dynode the accumulated
charge is measured as a voltage pulse. Schematic diagram illustrating how a detected incoming ion is converted into a
measurable signal using an EM detector
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Faraday cup (FC)
It consists of a hollow conducting electrode
connected to ground through a high resistance.
The ions hitting the collector cause a flow of
electrons from ground through the resistor and
the resulting potential drop across the resistor is
amplified.
The elementary charge on a single ion is
1.6 x 10-19 C. Therefore, a count rate of 1 x 106 c/s
(about the upper realistic limit for EM detector
usage) would produce a current of 1.6 x 10-13 A.
Schematic diagram of a Faraday cup ion detector
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Photomultiplier conversion
dynode
The ions initially strike a dynode which
results 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 with the electron
multiplier
Schematic diagram of a photomultiplier conversion dynode detector
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Array detectors
Schematic diagram of double focusing magnetic sector mass spectrometer
incorporating a multicollector system and static magnetic field – the nanoscale
secondary ion mass spectrometer (NanoSIMS).
Array detectors can cover a broad range of
detector types and systems but can be generally
broken down into two categories:
● detectors that can measure many ions of
differing mass-to-charge ratio (m/z) values
simultaneously
● detectors that are position sensitive
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

LC - MS
IN A NUTSHELL
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

● 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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

● As the analyte ions are generated at atmospheric pressure in the interface, the process is
called atmospheric pressure ionization (API) and the interface is known as the API source.
● Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the
most commonly used sources in LC-MS analysis.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

● 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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

● 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
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Plotting LC-MS data
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

How do you read an LC-MS
mass spectrum and what does
it tell you?
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

How do you read an LC-MS mass spectrum and what does it
tell you?
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 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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

tell you?
The mass spectrometer can be operated in two
modes,
a) scan
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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

tell you?
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]-.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

LC-MS analysis
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

LC-MS analysis
● quantification of genotoxic impurities in active pharmaceutical ingredients
● detection of doping agents, such as anabolic agents and simulants, in exhaled breath
● quantification of drug metabolites in biological fluids
● detection of adulterants in food materials and dietary supplements
● determination of alkylphenol ethoxylates (APEOs) in tannery sediments
● quantification of personal care products in swimming pool and river water samples
● quantification of nucleotides and their derivatives in bacterial cells
● quantification of the proteome
● as a rapid assay for the detection of SARS-CoV-2
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Case Study 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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Schuster O, Zvi A, Rosen O, Achdout H, Ben-Shmuel A, Shifman O, Yitzhaki S,Laskar
O, Feldberg L. Specificand 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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Case Study 2
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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Plants and LC-MS
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Wang, Guodong & Wang, G.-D. (2014). Applications of LC-MS in Plant Metabolomics. 10.1007/978-94-017-9291-2_9.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

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.
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

Thank you
Brahmesh Reddy B R
Aishwarya G
Mass spectrometry

LC_MS.pptx

Recommended

Recommended

More Related Content

Similar to LC_MS.pptx

Similar to LC_MS.pptx (20)

More from Brahmesh Reddy B R

More from Brahmesh Reddy B R (10)

Recently uploaded

Recently uploaded (20)

LC_MS.pptx