LCMS (LIQUID CHROMATOGRAPHY MASS SPECTROSCOPY)

1. GURU JAMBHESHWAR UNIVERSITY OF
SCIENCE AND TECHNOLOGY, HISAR
LIQUID CHROMATOGRAPHY-MASS
SPECTROSCOPY (LC-MS)
SUBMITTED BY- Shikha Kamboj
M Pharm 2ND Sem
Pharmaceutical Chemistry

2.  CONTENTS
1. CHROMATORAPHY
2. MASS SPECTROSCOPY
3. HYPHENATED TECHNIQUES
4. ADVANTAGES OF HYPHENATION
5. APPLICATIONS OF CHROMATOGRAPHY
6. LC-MS
a. Principle
b. Parts of LC-MS
7. INSTRUMENTATION
8. WORKING
9. APPLICATIONS
10. ADVANTAGES
11.DISADVATAGES

3. CHROMATOGRAPHY
• It is defined as a separation technique in which an individual
component is separated from the mixture by using stationary
phase and a suitable mobile phase. The term chromatography
is derived from the Greek words Chroma means “color” and
graphein "to write“
• The stationary phase remains fixed in place while the mobile
phase carries the components of the mixture through the
medium being used. The movement of the components in the
mobile phase is controlled by the significance of their
interactions with the mobile and/or stationary phases.
• Because of the differences in factors such as the solubility of
certain components in the mobile phase and the strength of
their affinities for the stationary phase, some components
will move faster than others, thus facilitating the separation
of the components within that mixture.

4. PRINCIPLE
• Chromatography is based on the principle where
molecules in mixture applied onto the surface or
into the solid, and fluid stationary phase (stable
phase) is separating from each other while moving
with the aid of a mobile phase.
• The factors effective on this separation process
include molecular characteristics related to
adsorption (liquid-solid), partition, and affinity.
• Because of these differences, some components of
the mixture stay longer in the stationary phase, and
they move slowly in the chromatography system,
while others pass rapidly into the mobile phase, and
leave the system faster.

5. • Stronger interaction with the stationary phase will
tend to move slower and having higher retention
time, than others which have lower or no
interactions with the stationary phase will tend to
move faster.
• Generally maximum interactions between a solute
and stationary phase takes place when both have
similar characteristics i.e. polarity
• When their properties are so different, a solute
will not tend to stay and will thus prefer to stay in
the mobile phase.

6. MASS SPECTROSCOPY
• Mass spectroscopy is an instrumental technique
in which sample is converted to rapidly moving
positive ions by electrons bombardment and
charged particles are separated according to their
masses. Mass spectrum is a plot of relative
abundance OR intensity against the ratio of
mass/charge.
• PRINCIPLE:- Conversion of neutral molecule into a
charged molecule to a positively charged
molecule. Separation of positively charged
fragments formed, based on their masses.

7. WHY WE COMBINE THESE TWO?
• Combining the two processes reduces the
possibility of error, as it is extremely unlikely that
two different molecules will behave in the same
way in both a liquid chromatography and a mass
spectrometer. While LC separates mixtures with
multiple compounds and MS provides structural
identification of the individuals.
• Therefore, when an identifying mass spectrum
appears at a characteristic retention time in a LC-
MS analysis, it typically tends to increased
certainty that the analyte of interest is in the
sample.

8. HYPHENATED TECHNIQUES
 A Hyphenated technique is combination or coupling of two
different analytical techniques with the help of proper interface.
 Hirschfield (1980) introduced the term "hyphenation" to refer to
the on-line combination of a separation technique and one or
more spectroscopic detection techniques.
 Chromatography - Produces pure or nearly pure fractions of
chemical components in a mixture. Spectroscopy – Produces
selective information for identification using standards or library
spectra.
 The hyphenated technique is developed from the coupling of a
separation technique and an online spectroscopic detection
technology.
 The number of existing techniques has been combined to expand
the utility.

9. The direct conjugation of chromatographic technique with
spectroscopic examination of separated fraction constitutes
several powerful analytical techniques.
The hyphenation does not always have to be between two
techniques; the coupling of separation or detection
techniques.
Recently, more than two techniques coupled together to form
a more powerful integrated system have revolutionized the
trace element analysis industry.
Also called double hybrid e.g. LC-PDA-MS; LC-MS-MS; LC-
NMR-MS instruments have become available and have been
applied to pharmaceutical problem solving.
Online coupling with solid phase extraction (SPE), solid phase
micro extraction or large volume injection can be
incorporated to build in a more powerful integrated system
e.g. SPE-LC-MS.

10. • Hyphenated techniques ranges from the
combination of- 1. separation-separation, 2.
separation-identification & 3. Identification-
identification techniques.

11. ADVANTAGES OF HYPHENATION
• 1. Fast and accurate analysis.
• 2. Higher degree of automation.
• 3. Higher sample throughput.
• 4. Better reproducibility.
• 5. Reduction of contamination due to its closed System.
• 6. Separation and quantification achieved at same time.
• List of Hyphenated Techniques
1. GC-MS 2. LC-MS 3. LC-NMR 4. EC-MS 5. CE-MS 6. GC-
IR 7. LC-MS-MS 8. LC-ESI-MALDI-TOF 9. GC-MS-MS 10.
GC-NMR 11. GC-AES 12. ICP-MS 13. ICP-AAS 14. ICP-
OES

12. APPLICATIONS OF CHROMATOGRAPHY
• Pharmaceutical sector
• To identify and analyze samples for the presence of trace elements
or chemicals.
• Separation of compounds based on their molecular weight and
element composition.
• Detects the unknown compounds and purity of mixture.
• In drug development.
• Food Industry
• In food spoilage and additive detection
• Determining the nutritional quality of food
• Forensic Science
• In forensic pathology and crime scene testing like analyzing blood
and hair samples of crime place.

13. • Molecular Biology Studies
• Various hyphenated techniques in chromatography such as EC-LC-
MS are applied in the study of metabolomics and proteomics along
with nucleic acid research.
• HPLC is used in Protein Separation like Insulin Purification, Plasma
Fractionation, and Enzyme Purification and also in various
departments like Fuel Industry, biotechnology, and biochemical
processes.
• Chemical industry
• In testing water samples and also checks air quality.
• HPLC and GC are very much used for detecting various
contaminants such as polychlorinated biphenyl (PCBs) in pesticides
and oils.
• In various life sciences applications.

14. LIQUID CHROMATOGRAPHY-MASS SPECTROSCOPY
• What is LC-MS?
• It is an analytical chemistry technique that combines the
physical separation capabilities of liquid chromatography
with the mass analysis capabilities of mass spectrometry.
LC-MS is a powerful technique used for many
applications which has very high sensitivity and
specificity.
• It is the combination of liquid chromatography and the
mass spectrometry. In LC-MS we are removing the detector
from the column of LC and fitting the column to interface of
MS.
•A LCMS system contains an interface that efficiently
transfer the separated components from the LC column into
the MS ion source. The interface is necessary because the

15. LC and MS devices are fundamentally incompatible.
• High sensitivity of mass spectroscopy provides the
information for identification of compounds or
structural elucidation of compounds.
• Combination of these two techniques leads the
metabolites appear from the end of the column
they enter the mass detector, where the solvent is
removed and the metabolites are ionized.
• In the most of the cases the interface used in LC-
MS are ionization source. It is application oriented
towards the specific detection and potential
identification of chemicals in the presence of
other chemicals (in a complex mixture).

16. • While the mobile phase in LC system is pressurized
liquid, the MS analyzers commonly operates under
high vacuum. Thus, it is not possible to directly pump
eluate from the LC column into the MS source.
• The interface is a mechanically simple part of the LC-
MS system that transfers the maximum amount of
analyte, removes a significant portion of the mobile
phase used in LC and preserves the chemical identity
of the chromatography products (chemically inert).
• As a requirement, the interface should not interfere
with the ionizing efficiency and vacuum conditions of
the MS system. Nowadays, most extensively applied
LC-MS interfaces are based on atmospheric pressure
ionization (API) strategies like electrospray
ionization (ESI), atmospheric-pressure chemical
ionization (APCI), and atmospheric pressure photo-
ionization (APPI).

17. PRINCIPLE OF LC-MS
• LC-MS is a hyphenated technique, combining the
separation power of LC, with the detection power of
mass spectrometry. Even with a very sophisticated
MS instrument, HPLC is still useful to remove the
interferences from the sample that would impact the
ionization.
• In LC-MS we remove the detector from the column of
LC and fit the column to interface of MS.
• The mass analyzer is then used to sort ions according
to their mass to charge ratio and detector counts the
ions emerging from the mass analyzer and may also
amplify the signal generated from each ion.

18. • As a result, mass spectrum (a plot of the ion signal
as a function of the mass-to-charge ratio) is
created, which is used to determine the elemental
nature of a sample, the masses of particles and of
molecules, and to elucidate the chemical
structures of molecules.
• In the most of the cases the interface used in LC-
MS are ionization source.

19. INSTRUMENTATION OF LC-MS
Mainly HPLC instrument contains mobile phase, pump, mixing unit
(solvent degassing system), injector (manual/auto), guard column,
analytical columns, detectors, recorder and integrators (Figure 1).

20. • The two key components in this process are the
ion source, which generates the ions, and the
mass analyzer, which sorts the ions. Several
different types of ion sources are commonly used
for LC/MS.
• Source for the production of ions OR ionization
source:-
• Ion spray, Most common ionization sources are
Electrospray ionization (ESI), Atmospheric pressure
chemical ionization (APCI) and Matrix-assisted laser
desorption/ionization (MALDI). A part from this
Electron impact (EI) and Chemical ionization (CI) or
negative chemical ionization are also used as
ionization source in MS.

21. • Interface:- most important and critical. Ions are
transferred from LC to MS region.
• MS analyzer:- Quadrupole, time of flight, ion trap
analyzer, magnetic sector mass analyzer,
• Detector:- Electron multipliers, Dynolyte
photomultiplier and Micro channel Plate.
• MOBILE PHASE:-The mobile phase is the solvent that
moves the solute through out column. General
requirements:-
• (1)low cost, high purity.
• (2)low viscosity, low toxicity, non flammability.
• (3)non corrosive to LC system component.
• Solvent strength and selectivity:- it is the ability of
solvent to elute solutes from a column.

22. • STATIONARY PHASE
• In the normal phase it contains the hydroxy group
and in the reverse phase it contains ODS (octa decyl
silane), octyl (C8), C4 (butyl), nitrile and amino
column.
• COLUMN
• Guard column and analytical column are generally
used. Column are generally made up of stainless
steel, glass, polyethylene and ploy ether ether ketone
(PEEK). Most commonly stainless steel columns are
used which can withstand with high temperature.
• Most widely used columns for LCMS are:-
• (1) fast LC column. the use of short column. (15-
50mm)
• (2) Micro LC column. the use of large column. ( 20-
150mm)

23. • PUMP- SOLVENT DELIVERY SYSTEM
• There are different types of pumps available such
as mechanical pumps and pneumatic pumps.
Mechanical pump operate with constant flow
rate and pneumatic pump operate with high
pressure.
• The solvents Or mobile phase used must be
assed through the column at high pressure.
• INJECTOR
• Septum injectors, stop flow but rheodyne
injectors are generally used.

24. • Sample preparation:-
• Sample preparation generally consists of
concentrating the analyte and removing
compounds that can cause background ion or
suppress ionization.
Example of sample preparation include:-
• (1) on –column concentration to increase analyte
concentration.
• (2) desalting to reduce the sodium and potassium
adduct formation that commonly occurs in electro
spray.
• (3) filtration to separate a low molecular-weight
drug from proteins in plasma, milk, or tissue.

26. • INTERFACES
• LC-MS systems include a device for introducing
samples (such as an HPLC) an interface for connecting
such device, an ion source that ionizes samples, an
electrostatic lens that efficiently introduces the
generated ions, a mass analyzer unit that separates ions
based on their mass-to-charge (m/z) ratio, and a
detector unit that detects the separated ions.
• In an LC-MS system, however, if the LC unit is simply
connected directly to the MS unit, the liquid mobile
phase would vaporize, resulting in large amounts of gas
being introduced into the MS unit.
• This would decrease the vacuum level and prevent
the target ions from reaching the detector. So interfaces
are to be used.

27. • The interface between a liquid phase technique
(HPLC) with a continuously flowing eluate, and a gas
phase technique carried out in a vacuum was
difficult for a long time. The advent of electrospray
ionization changed this. Currently, the most
common LC-MS interfaces are electrospray
ionization (ESI), atmospheric pressure chemical
ionization (APCI), and atmospheric pressure photo-
ionization (APPI).
• These are newer MS ion sources that facilitate the
transition from a high pressure environment (HPLC)
to high vacuum conditions needed at the MS
analyzer. Although these interfaces are described
individually, they can also be commercially available
as dual ESI/APCI, ESI/APPI, or APCI/APPI ion sources.

28. • IONIZATION
1. GAS PHASE:- Electron ionization, chemical
ionization
2. DESORPTION:- Field desorption, fast atom
bombardment (FAB), Matrix assisted laser
desorption ionization (MALDI)
3. EVAPORATIVE:- Thermospray, electrospray
ionization.
Ionization
Hard ionization soft ionization
• High energy low energy
• Increased fragmentation decreased fragmentation

29. • Electrospray ionization (ESI): Electrospray is
produced by applying a strong electric field to a
liquid passing through it.
• A solution containing the sample molecule is sprayed
through the high voltage potential capillary by the
help of nebulizer. Then the sprayed droplets are
ionized due to high voltage potential at capillary.
Heated disolvation gas will evaporate the solvent and
it will produce the molecular ion.
• It large volume to increase the sensitivity of the
detection. By using ESI high mass sample, non-
volatile molecules, liquids can be ionized and
disadvantage of this source of ionization is poor
sensitivity, low fragmentation and source is instable.

31. • Atmospheric pressure chemical ionization (APCI)
• APCI vaporizes solvent and sample molecules by
spraying the sample solution into a heater (heated to
about 400 C) using a gas, such as N2.
• Solvent molecules are ionized by corona discharge to
generate stable reaction ions.
• Principle of this technique involves nebulization of
the mobile phase with nitrogen gas and vaporization
by heating it to relatively high temperature (above
400°C). The resulting vapor is then subjected to a
corona discharge electrode to create ions.
• APCI is most commonly used ionization source used
in LC-MS. APCI are used for analysis of
pharmaceutical, environmental, toxicological, clinical
and chemical industrial/ laboratory samples.

32. • Atmospheric pressure photoionization(APPI)
• The LC eluent is vaporized using a heater at
atmospheric pressure. The resulting gas is made to
pass through a beam of photons generated by a
discharge lamp (UV lamp) which ionizes the gas
molecules.

33. • ANALYZERS
• The analyzer is component of the mass
spectrometer that takes ionized molecules and
separates them based on charge to mass ratios
and outputs them to the detector where they are
detected and later converted to a digital output.
• Quadrupole and triple quadrupole are the most
widely used analyzer because it is easy to operate
and it will cover wide mass range (10 to 4000
A.M.U./atomic mass unit). Quadrupole gives good
linearity for quantitative work and good
resolution (up to 4000), quality of mass spectra,
scanning speed (5000 A.M.U per second) and
mass accuracy

34. • A Quadrupole mass filter consists of four parallel metal
rods with different charges • Two opposite rods have an
applied + potential and the other two rods have a -
potential • The applied voltages affect the trajectory of
ions traveling down the flight path • For given DC and AC
voltages, only ions of a certain mass-to-charge ratio pass
through the quadrupole filter and all other ions are thrown
out of their original path.
• Quadrupole is composed of two pairs of metallic rods. Each
opposing rod pair is connected together electrically, and
voltage is applied between one pair of rods and the other.
A direct current voltage is then superimposed on the RF
voltage. Ions travel down the quadrupole between the
rods. Only ions of a certain mass-to-charge ratio (m/z) will
reach the detector for a given ratio of voltages. Other ions
have unstable trajectories and will collide with the rods.
This permits selection of an ion with a particular m/z and
allows the operator to scan for a range of m/z-values by
continuously varying the applied voltage.

36. • Ion trap analyzer: This analyzer is also known as the
quadrupole ion trap analyzer (QIT).Mostly it will be
used on GC/MS rather than LC/MS.
• The principle of the trap is to store the ions in a
device (ion trap) consisting of a ring electrode and
two end cap electrodes. These ions are manipulated
by using applied DC and RF fields. The amplitude of
the applied voltages enables the analyzer to trap ions
of specified mass to charge ratio within the analyzing
device. Non selected ions are given a trajectory by
the electrostatic field that causes them to exit the
trap. By filling the trap with inert gas fragmentation
of selected ions is possible. This is useful when
structural information is required.

37. • Detectors
1. Three deferent kinds of detectors are used in Mass
Spectrometry, i.e. Electron multipliers, Dynolyte
photomultiplier and Micro channel plates. Electron
multipliers dynode is used to convert either –ve, +ve
ions into electrons, that will be amplified and
detected. This will be widely used in quadrupole and
ion trap instruments.
2. The dynode of Dynolyte photomultipliers converts
the charged ions into electrons. These electrons
sticks to a phosphor and emits photons, and that
photons are made to strike the photomultiplier to
achieve multiplied signals for recording.
3. Micro channel Plate (MCP) are commonly employed
in ToF spectrometers. This will have very low time
response and high degree of sensitivity.

39. • 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. Hence the
polar components get eluted first and non-polar
component retained for the longer time.
WORKING OF LC-MS

40. • In common applications, the mobile phase is a
mixture of water and other polar solvents (e.g.,
methanol, isopropanol, and acetonitrile), and the
stationary matrix is prepared by attaching long-chain
alkyl groups (e.g., n-octadecyl or C18, C8, C4) to the
surface of irregularly or spherically shaped 5 μm
diameter silica particles.
• In HPLC, typically 20 μl of the sample of interest are
injected into the mobile phase stream delivered by a
high pressure pump. 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.

41. • The separation occurs after
repeated sorption and desorption steps occurring when
the liquid interacts with the stationary bed. The liquid
solvent (mobile phase) is delivered under high pressure
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.
• The column is the most important component of the LC
system and is designed to withstand the high pressure of
the liquid. Conventional LC columns are 100–300 mm long
with outer diameter of 6.4 mm (1/4 inch) and internal
diameter of 3.0–4.6 mm. For applications involving LC-MS,
the length of chromatography columns can be shorter (30–
50 mm) with 3–5 μm diameter packing particles.

42. • 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.
• 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 by means of electron
beams, photon beams (UV lights), laser beams.

43. • 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.

44. • Therefore, the development of devices facilitating
the transition from samples at higher pressure and
in condensed phase (solid or liquid) into a vacuum
system has been essential to develop MS as a potent
tool for identification and quantification of organic
compounds like peptides.
• MS is now in very common use in analytical
laboratories that study physical, chemical, or
biological properties of a great variety of
compounds. Among the many different kinds of
mass analyzers, the ones that find application in LC-
MS systems are the quadrupole, time-of-flight
(TOF), ion traps, and hybrid quadrupole-TOF
(QTOF) analyzers.

45. APPLICATIONS OF LCMS
• LC-MS are most widely used in food industries,
pharmaceutical and chemical industries for
quantitative and qualitative analysis.34-36
Applications of LS-MSMS are as follows.
• Molecular weight determination: Able to
determine the molecule weight of chemical
substance, pharmaceutical substances, proteins,
etc.
• Structural determination/elucidation: Tandem
mass spectrometry used to determine structural
information using mass spectral fragmentations.

46. • Pharmaceutical applications: It’s used to determine the
pharmacokinetic profile of the pharmaceuticals like drug,
drug metabolites/degradation product, impurities and
chiral impurities. The separation and detection of chiral
impurities in pharmaceuticals are of great importance
because the D-isomer of a drug can have different
pharmacological, metabolic and toxicological activity from
the L-isomer.
• Clinical and biochemical applications: MALDI-TOF MS is
used in SNP genotyping, quantification of DNA, gene
expression analysis, DNA and RNA sequencing.
• Food and Environmental applications: use to identify
aflatoxins (toxic metabolic product in certain fungi),
determine the vitamin D3 in poultry fed supplements, etc.
• Capillary electrophoresis/MS applications: Used for
analysis of peptides.

47. ADVANTAGES
• The major advantage of LC-MS is that it is the most robust
analytical technique that provides higher sensitivity and
selectivity required to detect an exact molecular weight of a
wide range of samples.
• The combination of the two analytical techniques (MS and
HPLC) reduces experimental error and improves accuracy.
• It can separate and identify solutes in low concentrations
(which are in parts per million- PPM) in a complex mixture.
• LC-MS is widely applied as regulatory compliance in the
pharmaceutical, bio-pharmaceutical, research, forensic, food,
and environmental sectors.
• The LC-MS offers high selectivity, resolution, precise mass, and
specificity as compared with other chromatography techniques.
• It is applied to a wide range of samples and able to identify the
different types of impurities.

48. DISADVATAGES
• LC-MS is an expensive technique both in terms of
capital and analysis costs.
• This is not a portable instrument; it requires special
and more space.
• To operate and data analysis of the liquid
chromatography and mass spectrometry (LC-MS)
requires a skilled and trained person.
• The LC-MS has a high maintenance cost as compared
with other analytical instruments.
• Phosphate buffer is not compatible with the LC-MS
analysis, which is the most commonly used buffer in
HPLC method development.

49. RECENT ADVANCEMENTS
• There has been a rising concern regarding the harmful
impact of biotoxins, source of origin, and the
determination of the specific type of toxin. With
numerous reports on their extensive spread, biotoxins
pose a critical challenge to figure out their parent
groups, metabolites, and concentration. In that
aspect, liquid chromatography-mass spectrometry
(LC-MS) based analysis paves the way for its accurate
identification and quantification. At the same time,
the suspect screening and nontarget screening
approach are facilitated by the HRMS platforms
during the absence of reference standards.