This document summarizes the principles and components of liquid chromatography-mass spectrometry (LC-MS). LC-MS combines liquid chromatography with mass spectrometry to separate and analyze compounds. Key components discussed include the liquid chromatograph, which separates compounds using a mobile and stationary phase; the mass spectrometer, which ionizes the separated compounds and measures their mass-to-charge ratios; and the interface between the two, which introduces the separated compounds into the mass spectrometer. Common ionization sources, mass analyzers, detectors, and data recording methods used in LC-MS are also described.

1. 1
The principle and performance of liquid chromatography–mass spectrometry
(LC-MS)
Ljubica Glavaš-Obrovac
Introduction
Chromatography is a separation technique used to separate the individual compound from
a mixture using a stationary and mobile phase. Chromatographic separation is based on the
principles of chirality, ion exchange, molecular exclusion, affinity, adsorption and partition.
According to the state of the mobile phase, chromatography can be divided into gas
chromatography, liquid chromatography, and supercritical fluid chromatography. According to
the geometric forms of the stationary phase, chromatography can be divided into column
chromatography and planar chromatography (paper chromatography and thin layer
chromatography).
Combination of chromatography with spectrometry is first reported in 1967 and first
liquid chromatography–mass spectrometry (LC-MS) system was introduced in 1980s. LC-MS is
an analytical chemistry technique that combines the physical separation capabilities of liquid
chromatography with the mass analysis and mass spectrometry. LC-MS is now a routine
technique providing a simple and robust interface to determine a wide range of compounds in
biological samples in the research and clinical laboratory setting1
. Fast scanning speeds allow a
high degree of multiplexing and many compounds can be measured in a single analytical run
(Slide 2).
Liquid chromatography–mass spectrometry (LC-MS) system
The components of LC-MS are a liquid chromatograph (LC) and mass spectrometer (MS) that
are interconnected by interface, which has a multiple role: liquid release, neutralization of neutral
molecules and introduction of analytes into the analyzer (Slide 3). Cross-flow transitions
occurring in the intermediate are vaporization and desorption2
.
Liquid chromatography (LC)
The most commonly used liquid chromatography (LC) method is column chromatography
which regards liquid as a mobile phase. A basic LC system consists of (a) a solvent inlet filter,
(b) pump, (c) inline solvent filter, (d) injection valve, (e) pre-column filter, (f) column, (g)
detector, (h) recorder, (i) backpressure regulator, and (j) waste reservoir. As shown (Slide 4), the
solvent inlet brings in the mobile phase which is then pumped through the inline solvent filter and
passed through the injection valve. This is where the sample is introduced in the mobile phase
flow path. It then gets passed through another filter and then passed through the column where
the sample will be separated into its components. The detector detects the separation of the
analytes and the recorder, usually a computer, will record this information. The sample then goes

2. 2
through a backpressure filter and into waste. LC has a great advantage on the capability of
separating complex samples, so it is the most effective option when mixtures separation is
needed, but is not suitable to obtain structural information of the material3
. High performance
liquid chromatography (HPLC) is modified based on the classical LC. It is a form of column
chromatography that pumps analyte in a mobile phase at a high pressure through the column with
chromatographic packing material (stationary phase). HPLC has the ability to separate, and
identify compounds, that are present in any sample that can be dissolved in a liquid in trace
concentrations as low as parts per trillion. These separations are useful in the proteomics area
where high sensitivity and resolution are required to identify as many components as possible1-3
.
Mass spectrometry (MS)
Mass spectrometry is an analytical technique widely used to quantify known materials, to
identify unknown compounds within a sample, and to elucidate the structure and chemical
properties of different molecules. MS is widely used due to its high selectivity, high sensitivity,
and capability of providing information including relative molecular mass and structural
characteristics. This technique basically studies the effect of ionizing energy on molecules4
.
Mass Spectrometry Instrumentation
Mass spectrometers operate by converting the analyte to a charged (ionized) state, with
subsequent analysis of the ions and any fragment ions that are produced during the ionization
process, on the basis of their mass to charge ratio (m/z) (Slide 5). Several different technologies
are available for both ionization and ion analysis, resulting in many different types of mass
spectrometers with different combinations of these two processes. Schematic view of basic
components of mass spectrometer is shown on Slide 6.
The mass spectrometer consists of:
1. Sample Injection Unit: To introduce the samples to be studied to the ion source
2. Ion generation unit or Ionization Source: For producing ions from the tested analyte.
3. Mass Analyzer: For resolving the ions into their characteristics mass components
according to their mass-to-charge ratio.
4. Detector System: For detecting the ions and recording the relative abundance of each of
the resolved ionic species.
5. Data System: To control the instrument, acquire and manipulate data, and compare
spectra to reference libraries.
For the proper MS function, the mass analyzer, and the mass detector must be kept under a high
vacuum condition of 3×10-4
to 1.3 ×10-5
Pa. This high vacuum in spectrometer requires two
pumping stages. The first stage is a mechanical pump which provides rough vacuum down to
1x10-1
Pa and the second stage uses turbo molecular pumps or diffusion pumps to provide desired
high vacuum.

3. 3
Ion Sources
Current ion sources are capable of handling a wide range of flow rates and mobile phase
compositions so existing LC separations can often be directly coupled to the mass spectrometer.
The most widely used ion sources (Slide 7) are:
a. Electrospray Ionization (ESI)
b. Atmospheric Pressure Chemical Ionization Source (APCI)
c. Atmospheric Pressure Photo-Ionization (APPI)
d. Thermospray Ionization (TSI)
e. Particle Beam Ionization (PBI)
a. Electrospray Ionization (ESI) is one of the most widely used ionization methods in an LC-MS
system that is fully compatible with analyzer5
(Slide 8). While standard electrospray ionization
sources in mass spectrometer can generally handle flow rates up to 1 mL/min lower flow rates
result in improved sensitivity. ESI is considered a “soft” ionization source, meaning that
relatively little energy is imparted to the analyte, and hence little fragmentation occurs. ESI uses
electrical energy to assist the transfer of ions from solution into the gaseous phase before they are
subjected to mass spectrometric analysis. The use of a nebulizing gas (e.g. nitrogen), which
shears around the eluted sample solution, enhances a higher sample flow rate. In ESI an analyte is
introduced to the source at flow rates as low as 1 µl min-1
. As shown in the Slide 8 the analyte
solution flow passes through the electrospray needle that has a high potential difference with
respect to the counter electrode, typically in the range from 1 to 6 kV. With the aid of an elevated
ESI-source temperature and/or another stream of nitrogen drying gas, the charged droplets are
continuously reduced in size by evaporation of the solvent, leading to an increase of surface
charge density and a decrease of the droplet radius. As the droplets traverse the space between the
needle tip and the cone, solvent evaporation occurs and the droplet shrinks until it reaches the
point that the surface tension can no longer sustain the charge (the Rayleigh limit) at which point
a Coulombic explosion occurs and the droplet is ripped apart. Finally, the electric field strength
within the charged droplet reaches a critical point at which it is kinetically and energetically
possible for ions at the surface of the droplets to be ejected into the gaseous phase. The emitted
ions are sampled by a sampling skimmer cone and are then accelerated into the mass analyzer for
subsequent analysis of molecular mass and measurement of ion intensity.
With ESI-MS is possible to analyze moderately polar molecules and is well suited to the
analysis of many metabolites, xenobiotics and peptides. Although neutral and low polarity
molecules such as lipids can also be converted to ionic form in solution or in gaseous phase by
protonation or cationization (e.g. metal cationization) can be studied by ESI-MS, this may not be
efficiently ionized by this method1-3
.
b. Atmospheric Pressure Chemical Ionization Source (APCI). In APCI, as with ESI, liquid is
pumped through a capillary and nebulized at the tip (Slide 9). A corona discharge takes place
near the tip of the capillary, initially ionizing gas and solvent molecules present in the ion
source1-3
. These ions then react with the analyte and ionize it via charge transfer. This technique

4. 4
is useful for small, thermally stable molecules that are not well ionized by ESI such free steroid,
lipids and fat soluble vitamins6, 7
.
c. Atmospheric Pressure Photo-Ionization (APPI) uses photons to excite and ionize molecules
after nebulization (Slide 9). The energy of the photons is chosen to minimize concurrent
ionization of solvents and ion source gases. The technique also gives predominantly singly-
charged ions and has been used for the analysis of neutral compounds such as steroids6,8
.
d. Thermospray Ionization (TSI) is a rapid, highly specific and sensitive combined high
performance liquid LC-MS method in which a liquid is flowed through a heated capillary to
produce a spray of droplets and solvent vapor (Slide 10). Ions are formed due to the imbalance of
charges in the droplets or by a heated filament1-4
.
e. Particle Beam Ionization (PBI) is a LC-MS method in which the effluent is passed through a
heated capillary to form an expanding jet of vapor and aerosol particles. After passing through a
skimmer that acts as a momentum separator, the beam impinges on a heated surface to form ions
through chemical ionization at the surface or ionization of the resulting vapor in a chemical
ionization or electron ionization source (Slide 11). Electron impact ionization following gas
chromatography or particle beam introduction typically generates very reproducible, library-
searchable mass spectra1-4
.
Mass Analyzers1-7
Most commonly used mass analyzers (Slide 12) are:
a. Quadrupole analyzer
b. The time-of-flight (TOF) analyzer
c. Ion trap analyzers
d. Hybrid analyzers
Quadrupole analyzer consists of a set of four parallel metal rods. A combination of constant and
varying (radio frequency) voltages allows the transmission of a narrow band of m/z values along
the axis of the rods (Slide 13). By varying the voltages with time it is possible to scan across a
range of m/z values, resulting in a mass spectrum. Most quadrupole analyzers operate at more
than 4000 m/z and scan speeds up to 1000 m/z per sec or more. They usually operate at unit mass
resolution meaning that the mass accuracy is seldom better than 0.1 m/z. As an alternative to
scanning, the quadrupoles can be set to monitor a specific m/z value. This technique is useful in
improving the detection limits of targeted analytes because more detector time can be devoted to
detecting specific ions instead of scanning across ions that are not produced by the analyte.
Stepping can be carried out in a few milliseconds and a panel of m/z values can be stepped
through for the detection of several analytes. Ions can be induced to undergo fragmentation by

5. 5
collisions with an inert gas such as nitrogen or argon, by a process called collision induced
dissociation. One type of collision cell is a quadrupole that has been designed to maintain the low
pressure of the collision gas required for dissociation and transmit most of the fragment ions that
are produced. A particularly useful mass spectrometer configuration is obtained by placing a
collision cell between two quadrupole mass analyzers. This combination is called a triple
quadrupole mass spectrometer and is an example of tandem MS in which two or more stages of
mass analysis are independently applied. Quadrupole analyzers, either in the single or triple
quadrupole configuration, are widely used in clinical LC-MS applications owing to the ease of
scanning and the good quality quantitative data obtained.
b. The time-of-flight (TOF) analyzer operates by accelerating ions through a high voltage (Slide
14). The velocity of the ions, and hence the time taken to travel down a flight tube to reach the
detector, depends on their m/z values. If the initial accelerating voltage is pulsed, the output of the
detector as a function of time can be converted into a mass spectrum. The TOF analyzer can
acquire spectra extremely quickly with high sensitivity. It also has high mass accuracy, which
allows molecular formulas to be determined for small molecules.
c. Ion trap analyzers use three hyperbolic electrodes to trap ions in a three-dimensional space
using static and radio frequency voltages (Slide 14). Ions are then sequentially ejected from the
trap on the basis of their m/z values to create a mass spectrum. Alternatively, a specific ion can be
isolated in the trap by the application of an exciting voltage while other ions are ejected. An inert
gas can also be introduced into the trap to induce fragmentation. An interesting feature of these
ion trap analyzers is the ability to fragment and isolate ions several times in succession before the
final mass spectrum is obtained, resulting in so-called MSn
capabilities.
d. Hybrid analyzers. Tandem mass spectrometers that use combinations of different mass
analyzers are useful for LC-MS. The third quadrupole of a triple quadrupole MS can be replaced
by a TOF analyzer to produce a hybrid quadrupole time-of-flight (QTOF) mass spectrometer.
QTOF instruments have been used extensively in the proteomics field but are more limited in
their scanning functions than triple quadrupole instruments. It is also possible to design
instruments in which the third quadrupole of a triple quadrupole MS operates in a different mode
in which ions are trapped and then sequentially ejected on the basis of their m/z values. This is
known as a linear ion trap and the overall configuration is often referred to as a QTrap
instrument. The end quadrupole can be switched between ion trap mode and conventional
quadrupole mode so the instrument combines useful features of both triple quadrupole and ion
trap analyzers. When used in ion trap mode, sensitivity in product ion scanning is considerably
enhanced, and additional fragmentation can be induced within the ion trap allowing an additional
stage of fragmentation and mass analysis.

6. 6
Detectors1-7
When ions are separated by a mass analyzer it is necessary to qualitatively and
quantitatively determine them. Detection is most commonly performed electrically, by taking
abundance - the total ionic current, and some type of electron multiplier is frequently used (Slide
15). The Faraday cup is more an ion collector than detector (Slide 16). It collect entered ions and
transfers their charge to the cup. Charge is usually transferred to electronics outside the vacuum
system. Type of electronics determines whether measured as charge, current or voltage. The
Faraday cup seems simple but in practice becomes quite complicated. The first and major
complication is that the ions entering have energies significantly higher than the work function of
the cup material (stainless steel, carbon, graphite) what cause the generation of free electrons,
known as secondary electrons.
When small power abundances (10-9
-10-6
A) are needed, various single-cell electric
amplifiers (DC-Amplifiers), photomultiplier conversion dynodes, electron multiplier, and
vibrating reed electrometer are used.
The principle of the electron multiplier function is based on the use of several
consecutive dynodes with a growing potential (Slide 17). Ionic air from the mass analyzer falls
on the multiplier electrode and sparks electrons, usually one to two electrons per ion. They are
accelerated on the way to the next Faraday cup which has higher potential than the previous one,
so that even more electrons are emitted and so in the order of 8 to 20 times. In this way, the input
signal strengthens up to 1012
times, which is why it has a high sensitivity. The highest
susceptibility is achieved at a voltage of about 3000 V, but such a high voltage shortens the life of
the detector.
In the photomultiplier (Slide 18), ions are emitted from a mass analyzer, translated into photons,
and detect. This device has a lower sensitivity, but it is much longer lasting.
Data Recording1-7
Multiple reactions monitoring by computers is commonly used in LC-MS assays. Data collecting
during MS analysis (Slide 19) can be performed by:
1. Capturing a complete mass spectrum - SCAN technique
2. Selected ion monitoring - SIM technique
3. Probability Based Matching system
1. SCAN technique
SCAN technique implies mass scanning in the given range while simultaneously
monitoring the retention time which allows identification of the analyte. The default volume
range and the chromatographic scan rate determine the duration of the dwell time. During each
cycle, each mass of the given range is recorded only once, and the cycles are repeated during
chromatography. The total chromatogram of ions represents the graph of the dependence of the

7. 7
total abundances collected during the analysis, from time to time. The data are obtained on the
quality (time retention) and the quantity (peak area), and by constant length chromatography as
well. On the basis of these data, ionic chromatogram can be displayed. By its use, the selectivity
of peaks that overlaps to a great extent is increased, if the characteristic properties of the
overlapped components are different. Scanning is usually performed at a speed of 0.5 to 1 scan/s.
SCAN technique is used more in qualitative analysis.
2. SIM technique
It is used in quantitative analysis. Prior to its use, in order to achieve optimal conditions,
analysis must be performed by the SCAN method. The SIM technique detects the values of m/z
only of the representative ions of the observed molecule. Tracking time is bigger, so it increases
the sensitivity even 100 to 1000 times. Characteristic junctions start time, and dwell times as well
are selected based on the data obtained through the SCAN technique. The chromatogram is
obtained as a dependence on the total abundance collected during the time analysis, and gives
data on the quality (retention time) and the quantity (point surface) of the observed compounds.
Each point in the chromatogram represents a sum of abundances of observed ions. SIM
techniques can also be used for qualitative determination of trace components.
3. Probability Based Matching system
It is very useful in compounds identification because it identifies the component by
dividing the spectra in the existing database with an unknown spectrum of the test compound.
McLafferety's algorithm-based probability-based evaluation method has been used to identify a
component. This system applies a retrieval method, so the entire content of the library can be
compared to an unknown spectrum. When choosing the most significant peaks of the reference
mass spectrum, both mass and abundance are equally valued. Reverse search determines whether
peaks in the reference mass spectrum are present in the spectrum of the test substance. If an
excess of peaks appears in the examined spectrum, they are ignored so that the mass spectra of
the analyte mixture and the impurities present can be analyzed. In most other systems, the mass
spectrum of an unknown compound is compared with already known spectra.
Most MS instruments have the capability of data-dependent acquisition, meaning that it is
possible to switch between the different modes of operation within a run based on the results that
are being acquired. The computer also captures spectra, primarily processes them, recognizes and
performs computations related to the application of MS. In addition to the MS data, the computer
requires data from other analytical procedures, from documentation as well, and commercial and
proprietary data libraries too.
Applications9,10,11, 12, 13, 14
Mass spectrometry using electrospray ionization and other ionization methods as well,
can be applied to a much wide range of biological molecules and will thus find greater

8. 8
application in the laboratory medicine (Slide 20). Direct injection methods can determine many
analytes with high through-put when highly specific tandem MS is used for detection. LC-MS
provides superior specificity and sensitivity compared to direct injection methods. When
combined with stable isotope dilution, LC-MS can be used to develop highly accurate and
reproducible assays. Modern mass spectrometers are highly sensitive and LC-MS assays are in
the use for pharmaceutical analysis, bioavailability studies, drug metabolism studies,
pharmacokinetics, characterization of potential drugs, drug degradation product analysis,
screening of drug candidates, identifying drug targets, biomolecule characterization, proteins,
peptides and oligonucleotides analyses, environmental analysis such as determination of
pesticides on foods, soil and groundwater contamination, and forensic analysis as well.
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