Notes for The principle and performance of liquid chromatography–mass spectrometry

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The principle and performance of liquid chromatography–massspectrometry
(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

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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 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.

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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.
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.

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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 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 singlycharged 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,
librarysearchable mass spectra1-4.
Mass Analyzers1-7
Most commonly used mass analyzers (Slide 12) are:
a. Quadrupole analyzer

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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 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

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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.
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

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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 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 differe nt.
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

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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 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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References
1 Watson, J.T., Sparkman, O.D. Introduction to Mass Spectrometry: Instrumentation,
Applications and Strategies for Data Interpretation, 4th Edition, John Wiley & Sons, Ltd, pp173,
(2007).
2 Parasuraman, S. Rao, A., Balamurugan, S., Muralidharan, S., Kumar, K., Venugopal, V. An
overview of liquid chromatography-mass spectroscopy instrumentation. Pharmaceutical
Methods. 5, 47-55 (2014).
3 Pitt, J.J. Principles and applications of liquid chromatography-mass spectrometry in clinical
biochemistry. Clin Biochem Rev. 30 (1), 19-34, (2009).
4 Demartini, D.R. A Short Overview of the Components in Mass Spectrometry Instrumentation
for Proteomics Analyses. In Tandem Mass Spectrometry - Molecular Characterization. (Ed. A.
V. Coelho and C. de Matos Ferraz Franco), ISBN 978-953-51-1136-8, Chapter 2 (Open access)
( 2013)
5 Chan, M.H., Cheung, R.C., Law, L.K, Lit, L.C., Ng K.F., Suen, M.W., Tai, H.L. Electrospray
ionisation mass spectrometry: principles and clinical applications. Clin Biochem Rev. 24, 3-12
(2003).
6 Woźniak, B., Matraszek-Żuchowska, I., Witek, S., Posyniak, A. Development of LC-MS/MS
confirmatory method for the determination of testosterone in bovine serum. J Vet Res 61, 81-89,
(2017).
7 Keevil, B.G. LC-MS/MS analysis of steroids in the clinical laboratory. Clin Biochem. 49 (1314),
989-97. (2016), doi: 10.1016/j.clinbiochem.
8 Kauhanen, D., Sysi-Aho, M., Koistinen, K.M., Laaksonen, R., Sinisalo, J., Ekroos, K.
Development and validation of a high-throughput LC–MS/MS assay for routine measurement of
molecular ceramides. Analytical and Bioanalytical Chemistry. 408 (13), 3475–3483. (2016).
9 Rekhi, H., Rani, S., Sharma, N., Malik, A.K. A Review on Recent Applications of
HighPerformance Liquid Chromatography in Metal Determination and Speciation Analysis. Crit
Rev Anal Chem. 2;47, 524-537. (2017) doi: 10.1080/10408347.2017.1343659.
10 Kadhi,O.A., Melchini,A., Mithen,R., SahaS. Development of a LC-MS/MS Method for the
Simultaneous Detection of Tricarboxylic Acid Cycle Intermediates in a Range of Biological
Matrices. J Analyt Meth Chem. (2017). https://doi.org/10.1155/2017/5391832
11 Melanson, S.E., Ptolemy, A.S., Wasan, A.D. Optimizing urine drug testing for monitoring
medication compliance in pain management. Pain Med 14, 1813–20 (2013).

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12 Shah, P.A., Sharma, P., Shah, J.V., Sanyal, M., Shrivastav, P.S. An improved LC-MS/MS
method for the simultaneous determination of pyrazinamide, pyrazinoic acid and 5-hydroxy
pyrazinoic acid in human plasma for a pharmacokinetic study. J Chromatogr B Analyt Technol
Biomed Life Sci. 1017-1018, 52-61, (2016) doi: 10.1016/j.jchromb.2016.02.036.
13 Pohanka, A., Rosenborg, S., Lindh, J.D., Beck, O . Experiences from using LC-MS/MS for
analysis of immunosuppressive drugs in a TDM service. Clin Biochem. 49 (13-14), 1024-31,
(2016) doi: 10.1016/j.clinbiochem.2016.06.013.
14 Wan, D., Yang, J., Barnych, B., Hwang, S.H. et al. A new sensitive LC/MS/MS analysis of
vitamin D metabolites using a click derivatization reagent, 2-nitrosopyridine. J Lipid Res. doi:
10.1194/jlr.D073536 (2017)

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