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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5405_Библиотеки_им_академика_М_И_Перельмана.pdf
Comprehensive Insights into Mass Spectrometry
Abstract
Mass spectrometry (MS) is a powerful analytical technique used in various
scientific disciplines. Operating on the principle of ion separation by mass-to-
charge ratio, MS offers deep insights into diverse compounds. It encompasses
several types and components, such as the ion source, mass analyzer, and
detector. MS spectra are central to compound identification, quantification, and
structural elucidation. Strategies such as electrospray ionization (ESI) and matrix-
assisted laser desorption/ionization (MALDI) are critical in proteomics,
metabolomics, and lipidomics, aiding the characteri zation of proteins,
metabolites, and lipids. In pharmaceutical, biomedical, and clinical sciences,
MS is fundamental for biomarker discovery, clinical diagnostics, and drug
analysis. It plays a pivotal role in forensic investigations for drug and trace
evidence identification. Additionally, it contributes to environmental and agricul-
tural sciences by monitoring contaminants, pesticide residues, and livestock
health. MS is an indispensable tool that has revolutionized research and analysis
across diverse fields, enabling precise and comprehensive exploration of matter.
9
Keywords
Mass spectrometry · Ionization techniques · Mass analyzers · Tandem mass
spectrometry · High-resolution mass spectrometry · Mass spectra types
9.1 Introduction
A mass spectrometer is a device that determines the mass of molecules by measuring
the mass-to-charge ratio (m/z) of ions. In this technique, molecules are bombarded
with electrons, resulting in the production of highly energized positive ions that
further fragment into smaller fragments. These ions are then separated in a magnetic
361
362 9 Comprehensive Insights into Mass Spectrometry
Fig. 9.1 Schematic representation of workflow of mass spectrometry. This figure illustrates the
working principle of MS. The process begins with the ionization of a sample composed of particles
with varying masses (represented by circles of different sizes). The sample is first introduced into
the ion source, where molecules are ionized, as indicated in the top left corner. In the first step (top
middle), the ions are directed toward a mass analyzer, where they are separated based on their massto-charge (m/z) ratio. The larger ions (blue circle) are shown moving more slowly and are detected
later than smaller ions (pink circles). As shown in the middle section, the ions are separated in the
mass analyzer, and their intensities are recorded. The graph on the right shows the corresponding
MS spectrum, where each peak corresponds to the number of ions with specific masses. The smaller
ions (12 units) produce an earlier peak, while larger ions (14 units) are detected later in the
spectrum. In the final step (bottom section), a comprehensive MS spectrum is produced,
representing the detected ions and their relative abundances, allowing for the identification and
quantification of the sample components
or electric field based on their mass-to-charge ratio. Figure 9.1 illustrates how a mass
spectrometer works. This technique is used for studying the impact of ionization
energy on molecules. It depends on the type of chemical reactions that occur in the
sample, which typically exists in the gaseous phase. The function of a mass
spectrometer is associated with its components. For example, the ion source is
responsible for generating ions, the mass analyzer separates ions based on their
mass-to-charge ratio, and the detector measures th
displayed on
a mass spectrum, which is a plot showing the relationship between ion
e separated ions. The results are
abundance and mass-to-charge ratio. Mass spectrometry is also used for the detection of isotopes based on their masses. Today, mass spectrometry is often used in
conjunction with gas chromatography to estimate the quantities of contaminants and
toxins.
9.3 Instrumentation 363
9.2 Principle
The principle of mass spectrometry involves the precise measurement of the massto-charge ratio (m/z) of ions. A sample is first introduced into the mass spectrometer
and ionized, meaning its molecules are converted into charged particles (ions). The
ionization proces s can be done using different techniques, such as electron ionization
(EI), matrix-assisted laser desorption/ionization (MALDI), or electrospray ionization
(ESI), depending on the sample’s nature. Once ionized, these ions are accelerated
through an electric or magnetic field and directed into a mass analyzer. The mass
analyzer separates the ions based on their mass-to-charge ratio (m/z), with lighter
ions moving faster than heavier ions. Various types of mass analyzers are used,
including time-of-flight (TOF), quadrupole, or Fourier-transform ion cyclotron
resonance (FT-ICR). Finally, the separated ions reach a detector, which measures
their abundance and produces a spectrum. This spectrum represents the mass-tocharge ratio of the ions and provides valuable information about the molecular
weight and structure of the sample.
9.3 Instrumentation
The instrumentation of mass spectrometry is a critical component of the technique,
and it involves various essential elements and components that work together to
analyze the mass-to-charge ratio (m/z) of ions. The following sections provide an
overview of the key components and instrumentation in mass spectrometry
(Fig.
9.2).
9.3.1 Inlet System
The inlet system introduces the sample into the mass spectrometer, and it may
involve a sample introduction method such as direct injection, gas chromatography
(GC), liquid chromatography (LC), or other techniques depending on the
application.
9.3.2 Ionization Source
This is the initial component where the sample is ionized. It is a crucial component
used to convert the sample into ions through a process known as ionization. In mass
spectrometry, three main sample types are typically analyzed: solid, liquid, and/or
gas samples. Various methods are employed to ionize the molecules, and their
schematic representations are illustrated in Fig.
9.3.
364 9 Comprehensive Insights into Mass Spectrometry
Fig. 9.2 Schematic representation of the components of a mass spectrometer. This figure outlines
the major components of a mass spectrometer and their respective functions. The process begins
with the inlet system, which introduces the sample into the instrument via direct introduction or
infusion. The ion source then ionizes the sample using various techniques, including electron
ionization (EI), chemical ionization (CI), electrospray ionization (ESI), atmospheric pressure
chemical ionization (APCI), atmospheric pressure photoionization (APPI), and matrix-assisted
laser desorption/ionization (MALDI). Once ionized, the ions are directed to the separator, which
utilizes different technologies such as sector field analyzer (SFA), double focusing analyzer (DFA),
time-of-flight (TOF), Fourier-transform ion cyclotron resonance (FT-ICR), or quadrupole analyzers
to separate ions based on their mass-to-charge ratios. After separation, the ions are sent to the
collector, where detection occurs using Faraday cylinders, electrometers, electron multipliers, or
photographic plates to record the data. The entire system operates under high vacuum, maintained
by oil diffusion or mercury pumps, as indicated by the vacuum system
9.3.2.1 Electron Ionization (EI)
Electron ionization (EI) is one of the earliest and most widely used ionization
techniques in mass spectrometry, particularly for the analysis of small, volatile,
and thermally stable compounds. In EI, a beam of high-energy electrons (usually
at 70 electron volts, or eV) is directed at a gas-phase sample. The electron beam
interacts with the neutral analyte molecules, knocking off an electron from the
molecule and generating positively charged molecular ions (M
+•
).
The high energy of the electrons also causes the molecular ions to fragment into
smaller ionized fragments, creating a rich fragmentation pattern that can be used to
identify the structure of the molecule. This fragmentation is both an advantage and a
limitation: while the pattern is often highly reproducible and useful for structural
elucidation, it can make the identification of the molecular ion (which represents the
intact molecule) more challenging.
9.3.2.1.1 Key Features of EI
• Hard ionization
technique: EI imparts a high amount of energy, leading to
extensive fragmentation.
9.3 Instrumentation 365
Fig. 9.3 Schematic representation of sources of ionization. This figure demonstrates the different
ionization methods used in mass spectrometry based on the physical state of the sample (solid,
liquid, or gas). For solid samples, ionization can be achieved directly or by transforming the sample
into a solid phase, typically using matrix-assisted laser desorption/ionization (MALDI) technique.
Liquid samples are made into a solution before ionization, where the chemical properties of the
analyte in solution determine the appropriate ionization technique. The figure shows that for liquid
samples, ionization methods such as atmospheric pressure chemical ionization (APCI), atmospheric
pressure photoionization (APPI), and electrospray ionization (ESI) are used. For gas samples, the
analyte is converted into a gaseous phase before ionization. In this case, methods such as chemical
ionization (CI) and electron ionization (EI) are used, depending on the chemical properties of the
analyte in the gas phase. The arrows illustrate the relationships between sample states and their
respective ionization techniques
• Fragmentation patterns: These patterns are often characteristic and can be com-
pared to reference spectra for compound identification.
• Best suited for small, volatile molecules: EI works well for compounds with low
molecular weights and high volatility, such as organic compounds and small
drugs.
• Mass spectral libraries: EI spectra are commonly cataloged in large mass spectral
libraries, making it easier to match experimental spectra to known compounds.
9.3.2.1.2 Applications
• Commonly used in GC-MS due
ability to vaporize and ionize small
to the
molecules.
• Ideal for compounds with molecular weights below 1000 Da.
• Not suitable for large biomolecules, which tend to fragment excessively under EI
conditions.
9.3.2.2 Electrospray Ionization (ESI)
ESI is a “soft” ionization technique widely used in mass spectrometry for analyzing
large, polar, and nonvolatile molecules such as proteins, peptides, nucleic acids, and
366 9 Comprehensive Insights into Mass Spectrometry
other biomolecules. ESI is particularly known for its ability to ionize large molecules
without causing significant fragmentation, preserving the molecular integrity of the
analyte. In ESI, a liquid sample is infused through a very fine needle at high voltage,
typically 1–5 kV, which creates an aerosol of tiny, charged droplets. As these
droplets pass through a drying gas or under vacuum, solvent evaporation reduces
the droplet size, eventually leading to the generatio
n of gas-phase ions. These ions
are then analyzed by the mass spectrometer.
9.3.2.2.1 Key Features of ESI
• Soft ionization technique: ESI does not impa rt enough energy to cause extensive
fragmentation of the analyte, which makes it ideal for detecting intact
molecular ions.
• Multiple charging: ESI can generate ions with multiple charges (protonation or
deprotonation), which is particularly useful for large biomolecules. This results
es of peaks corresponding to different charge states, allowing large
a seri
molecules to be detected within the mass range of most instruments.
• Sensitivity to polar molecules: ESI is highly effective for ionizing polar and ionic
compounds, which might not ionize well with other methods such as EI.
• Analyzing macr omolecules: The technique is especially suited for
macromolecules such as proteins, peptides, oligonucleotides, and
preserving
their molecular integrity.
polymers,
• Coupling with LC-MS: ESI is often paired with liquid chromatography (LC) in
LC-MS setups, enabling the separation and identification of complex mixtures.
in
9.3.2.2.2 Mechanism
• Sample introduction: The liquid sample is introduced through a fine capillary
needle, which is subjected to a high voltage (usually positive or negative).
• Formatio n of charged droplets: As the sample exits the needle, the high voltage
creates a fine spray of charged droplets.
• Solvent evaporation: The charged droplets undergo solvent evaporation, aided by
a drying gas or vacuum, leading to a reduction in droplet size.
• Ion desorption: Eventually, the electrostatic repulsion between the charges causes
the droplets to disintegrate, releasing gas-phase ions.
• Mass analysis: The resulting ions are introduced into the mass analyzer, where
they are detected and analyzed based on their mass-to-charge ratio (m/z).
9.3.2.2.3 Applications
• Proteomics: ESI is commonly used
for prote
in identification and quantification,
as it can handle large and complex biomolecules.
• Metabolomics: In metabolite profiling, ESI is highly effective for detecting polar
metabolites.
• Pharmaceutical
analysis: It
is frequently used in the analysis of drug compounds
and their metabolites.
• Peptide sequencing: ESI,
coupled with tandem mass spectrometry (MS/MS),
allows for sequencing of peptides and proteins.
9.3 Instrumentation 367
• Nucleic acid analysis: ESI can be used to analyze nucleic acids and
oligonucleotides, preserving their mass and structure.
9.3.2.2.4 Advantages
• Preserva tion of molecular structure: ESI does not fragment delicate molecules,
making it ideal for the study of biomolecules.
• Multiple charge states: The ability to generate multiply charged ions allows large
biomolecules to fall within the mass range of typical mass spectrometers.
• Compatibility with liquid chromatography (LC-MS): ESI is commonly used in
conjunction with liquid chromatography, allowing separation and analysis of
complex biological samples.
9.3.2.3 Chemical Ionization (CI)
CI is a “soft” ionization technique in mass spectrometry that invol ves the use of
reagent gases to ionize the analyte molecules. In contrast to electron ionization (EI),
CI tends to cause less fragmentation of the analyte, making it more useful for
identifying molecular ions and obtaining molecular weight information. In CI, a
reagent gas (such as methane, ammonia, or isobutane) is introduced into the ion
source, where it is ionized by electrons from an electron beam. These reagent gas
ions then interact with the analyte molecules, transferring charge through protonation or other chemical reactions, leading to the formation of positively charged ions
of the analyte, which are subsequently analyzed by the mass spectrometer.
9.3.2.3.1 Key Features of CI
• Soft ionization technique: Similar to ESI, CI results in less fragmentation com-
pared to EI, making it more suitable for identifying intact molecular ions.
• Proton transfer: In positive CI, the most common ionization mechanism involves
the transfer of a proton to the analyte molecule from the reagent gas, resulting in
[M + H]
• Reagent gas: Different reagent gases can be used, with
(C
+
ions (molecular ions with an added proton).
methane (CH
), and ammonia (NH3) being the most common. The choice of gas
4H10
), isobutane
4
influences the ionization process and the mass spectrum obtained.
• Analyzing less volatile compounds: CI can be used for relatively nonvolatile and
thermally unstable compounds that may not ionize well with EI.
9.3.2.3.2 Mechanism
1. Reagent gas ionization: In the CI source, electrons from a filament ionize the
reagent gas, producing positive reagent gas ions. For example, when methane is
used, it forms CH
+
5
, CH
+
, or C2H
4
+
.
5
2. Interaction with analyte molecule: These reagent gas ions collide with the analyte
molecules, transferring charge. In positive CI, a proton is often transferred,
forming a [M + H]
3. Mass analysis: The
+
ion of the analyte.
protonated analyte ions or other ionized species are then
introduced into the mass analyzer for detection and analysis.
368 9 Comprehensive Insights into Mass Spectrometry
9.3.2.3.3 Types of Reagent Gases
• Methane (CH
(e.g., CH
• Ammonia (NH
): Commonly used in CI, methane creates a series of reactive ions
4
+
) that can protonate analyte molecules.
5
): Ammonia as a reagent gas leads to less fragmentation and often
3
provides cleaner mass spectra, useful for more delicate analytes.
• Isobutane (C
): Another reagent gas option that is particularly useful for
4H10
producing less complex spectra.
9.3.2.3.4 Ionization Process
• Positive CI: In positive chemical ionization, the reagent gas donates a proton to
the analyte, forming a positively
charged [M
+
ion. This method is typically
+ H]
used for detecting molecules with basic sites or those that can accept a proton.
• Negative CI: In negative chemical ionization, electron capture is the dominant
process, leading to negatively charged ions, particularly useful for analytes that
contain electronegative groups like halogens or nitro groups.
9.3.2.3.5 Applications
• Molecular weight determination: CI is ideal for determining the molecular
weights of molecules by generating mol
ecular ions
([M + H]
+
) with minimal
fragmentation.
• Pharmaceuticals: CI is widely used in pharmaceutical analysis, particularly for
drugs and metabolites
where preser
ving molecular integrity is important.
• Environmental and forensic analysis: In negative ion CI, the technique is partic-
ularly useful for detecting compounds with high electron affinities, such as
halogenated environmental pollutants or explosives.
• Complex organic molecules: CI is also useful for analyzing complex organic
molecules that may fragment too much under EI conditions.
9.3.2.3.6 Advantages
• Less fragmentation: CI causes signi ficantly less fragmentation compared to EI,
which makes it easier to identify the molecular ion of the analyte.
• Flexibility with reagent gases: The ability to choose
different
reagent gases
allows flexibility in optimizing the ionization for different analytes.
• Soft ionization for thermally labile compounds: CI is better suited for analyzing
compounds that may degrade or fragment under the harsh conditions of EI.
9.3.2.3.7 Limitations
• Complexity of
spectra: Depending
on the reagent gas used, the spectra can
sometimes be more complex due to additional ion species (such as adducts with
the reagent gas).
• Lower sensitivity compared to EI: CI typically produces fewer ions than EI,
resulting in lower sensitivity for some applications.
9.3 Instrumentation 369
9.3.2.4 Atmospheric Pressure Ionization (API)
Atmospheric pressure ionization (API) refers to a category of ionization techniques
in mass spectrometry that operate under atmospheric pressure, as opposed to the
vacuum conditions required by many other ionization methods such as electron
ionization (EI) and chemical ionization (CI). API is a “soft” ionization method,
meaning it causes minimal fragmentation of the analyte, preserving the molecular
ion and allowing for accurate molecular mass analysis. API is widely used in
conjunction with liquid chromatography (LC-MS) for the analysis of biomolecules,
pharmaceuticals, and other complex mixtures. These methods are used for ionizing
compounds in the liquid phase and are highly efficient for large biomolecules and
polar analytes.
9.3.2.4.1 Key Features of API
• Operation at atmospheric pressure: API techniques ionize samples at atm o-
spheric pressure,
facilitates easier interfacing with liquid chromatography
which
systems and reduces the need for compl ex vacuum setups.
• Soft ionization: API methods such as ESI and APCI result in minimal fragmenta-
tion, preserving the molecular ion and aiding in the accurate identification of
analytes.
• High sensitivity: API techniques, especially ESI, are highly sensitive and can
detect analytes at very low concentrations, making them suitable for trace
analysis.
• Suitability for large and polar molecules: API techniques are particularly well-
suited for large biomolecules (e.g., proteins, peptides) and polar compounds,
which may not ionize well using traditional techniques such as EI.
9.3.2.4.2 Types of API
Electrospray ionization: ESI is one of the most widely used ionization techniques in
API. It is particularly suited for polar and large biomolecules such as proteins,
peptides, and nucleotides.
• Mechanism: In ESI, the
liquid sample
is passed through a fine needle at high
voltage, creating a fine spray of charged droplets. As the solvent evaporates, the
analyte molecules are left charged and enter the mass spectrometer as ions.
• Applications: ESI is used extensively in biological and pharmaceutical studies,
especially for analyzing large biomolecules and complex mixtures such as
proteins and peptides.
Atmospheric pressure chemical ionization (APCI): APCI is another API method
used for relatively small and less polar compounds.
• Mechanism: In APCI,
the liquid sample is nebulized and vaporized in a heated
region, then ionized using a corona discharge. The ionized reagent gas interacts
with the analyte molecules, transferring charge through protonation (positive ion
mode) or electron capture (negative ion mode).
370 9 Comprehensive Insights into Mass Spectrometry
• Applications: APCI is well-suited for smaller molecules and less polar
compounds, including lipids, steroids, and small drug molecules.
9.3.2.4.3 General API Process
• Sample intr oduction: A liquid sample is introduced into the ion source (typically
after passing throu gh a liquid chromatography system).
• Ionization: The analyte molecules are ionized at atmospheric pressure using an
electrical field (ESI) or a chemical reagent (APCI).
• Desolvation: The solvent surrounding the ionized analyte evaporates,
behind charged
analyte ions.
• Ion transfer: The charged analyte ions are guided into the mass spect
leaving
rometer for
analysis.
4.4 Applications of API
9.3.2.
• Proteomics: API methods, especially ESI, are crucial in the field of proteomics
for analyzing large , complex
proteins
and peptides.
• Pharmaceuticals: Both ESI and APCI are widely used in the pharmaceutical
industry for drug discovery, metabolite identificatio
n, and
quality control.
• Metabolomics: API methods facilitate the analysis of small molecules and
metabolites in biological syst
ems, playing
a key role in metabolomics.
• Environmental analysis: API is used for detecting trace levels of pollutants and
contaminants in environmental samples.
9.3.2.4.5 Advantages of API
• Soft ionization: API techniques cause minimal fragmentation, preserving molec-
ular ions for better molecular mass determination.
• Wide range of analytes: API is versatile and can ionize a wide range of analytes,
from large biomolecules (e.g., proteins) to small polar molecules (e.g., drug
compounds).
• High sensitivity: API techniques, especially ESI, offer high sensitivity, allowing
the detection of analytes at very low concentrations.
• Compatibility with LC-MS: API techniques are ideally suited for coupling with
liquid chromatography, making them highly useful in fields such as proteomics,
metabolomics, and pharmaceutical analysis.
9.3.2.4.6 Limitations
• Matrix effects: In complex sample matrices, API can experience ion suppression
or enhancement effects, leading to inaccurate quantification.
• Limited to polar compounds: API, particularly ESI, is more effective for polar
and ionizable compounds and may not perform well with nonpolar analytes.
9.3.2.5 Fast Atom Bombardment (FAB)
Fast atom bombardment (FAB) is an ionization technique used in mass spectrometry, particularly suited for the analysis of nonvolatile and thermally labile
compounds, such as peptides, nucleotides, and other polar organic molecules.